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Sample records for acids including dna

  1. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication.

    PubMed Central

    Koonin, E V

    1993-01-01

    A new superfamily of (putative) DNA-dependent ATPases is described that includes the ATPase domains of prokaryotic NtrC-related transcription regulators, MCM proteins involved in the initiation of eukaryotic DNA replication, and a group of uncharacterized bacterial and chloroplast proteins. MCM proteins are shown to contain a modified form of the ATP-binding motif and are predicted to mediate ATP-dependent opening of double-stranded DNA in the replication origins. In a second line of investigation, it is demonstrated that the products of unidentified open reading frames from Marchantia mitochondria and from yeast, and a domain of a baculovirus protein involved in viral DNA replication are related to the superfamily III of DNA and RNA helicases that previously has been known to include only proteins of small viruses. Comparison of the multiple alignments showed that the proteins of the NtrC superfamily and the helicases of superfamily III share three related sequence motifs tightly packed in the ATPase domain that consists of 100-150 amino acid residues. A similar array of conserved motifs is found in the family of DnaA-related ATPases. It is hypothesized that the three large groups of nucleic acid-dependent ATPases have similar structure of the core ATPase domain and have evolved from a common ancestor. PMID:8332451

  2. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Namhai Chua; Kush, A.

    1993-02-16

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids.

  3. Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

    PubMed

    Major, G N; Gardner, E J; Carne, A F; Lawley, P D

    1990-03-25

    DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).

  4. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1993-02-16

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a pu GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  5. cis-acting DNA regulatory elements, including the retinoic acid response element, are required for tissue specific laminin B1 promoter/lacZ expression in transgenic mice.

    PubMed

    Sharif, K A; Li, C; Gudas, L J

    2001-05-01

    The LAMB1 gene encodes the laminin beta1 subunit of laminin, an extracellular matrix protein. Using several transgenic mouse lines containing various lengths of the LAMB1 promoter driving lacZ reporter gene expression, regions of LAMB1 promoter that contain cis-acting DNA regulatory element(s) have been identified. The 3.9LAMB1betagal transgene is expressed in various tissues during development. LAMB1 transgene expression is observed in a selective set of nephrons of the neonatal and adult kidneys. The cis-acting DNA regulatory elements responsible for LAMB1 transgene expression in ovaries and in juvenile kidneys are present between -'1.4 and -0.7 kb relative to the transcription start site, while those of adult kidneys are located between -2.5 and -1.4 kb. The LAMB1 transgene is also expressed in the epididymis of 1 week old transgenic mice. Mutation of the retinoic acid response element (RARE) in the context of the 3.9LAMB1betagal transgene results in loss of LAMB1 transgene expression in all tissues. Thus, sequences between -2.5 and -0.7 kb plus the RARE are required for appropriate expression of the LAMB1 transgene in mice.

  6. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

  7. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

  8. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  9. cDNA encoding a polypeptide including a hevein sequence

    SciTech Connect

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  10. DNA extraction from rice endosperm (including a protocol for extraction of DNA from ancient seed samples).

    PubMed

    Mutou, Chiaki; Tanaka, Katsunori; Ishikawa, Ryuji

    2014-01-01

    Deoxyribonucleic acid (DNA) extracted from endosperm can be effectively used for rapid genotyping using seed tissue, to evaluate seed quality from packaged grains and to determine the purity of milled grains. Methods outlined here are optimal procedures to isolate DNA from endosperm tissue of modern rice grains and of aged rice remains preserved between 50 and 100 years. The extracted DNA can be used to amplify regions of chloroplast genomic DNA (ctDNA), mitochondrial genomic DNA (mtDNA), and nuclear genomic DNA using standard PCR protocols. In addition, we describe an optimal procedure to process archaeological grain specimens, aged for a couple of thousand years, to isolate DNA from these ancient samples, referred to here as ancient DNA (aDNA). The aDNA can be successfully amplified by PCR using appropriate primer pairs designed specifically for aDNA amplification.

  11. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  12. cDNA encoding a polypeptide including a hev ein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  13. DNA Damage Response Factors from Diverse Pathways, Including DNA Crosslink Repair, Mediate Alternative End Joining

    PubMed Central

    Howard, Sean M.; Yanez, Diana A.; Stark, Jeremy M.

    2015-01-01

    Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection. PMID:25629353

  14. Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

    PubMed Central

    Romanowski, Gerd; Lorenz, Michael G.; Sayler, Gary; Wackernagel, Wilfried

    1992-01-01

    The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [3H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca2+-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ. Images PMID:16348772

  15. Amino Acid Racemization and the Preservation of Ancient DNA

    NASA Technical Reports Server (NTRS)

    Poinar, Hendrik N.; Hoss, Matthias

    1996-01-01

    The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber.

  16. Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids.

    PubMed

    Olson, Xiaoping; Kotani, Shohei; Yurke, Bernard; Graugnard, Elton; Hughes, William L

    2017-03-30

    Locked nucleic acids (LNAs) are conformationally restricted RNA nucleotides. Their increased thermal stability and selectivity toward their complements make them well-suited for diagnostic and therapeutic applications. Although the structural and thermodynamic properties of LNA-LNA, LNA-RNA, and LNA-DNA hybridizations are known, the kinetic effects of incorporating LNA nucleotides into DNA strand displacement systems are not. Here, we thoroughly studied the strand displacement kinetics as a function of the number and position of LNA nucleotides in DNA oligonucleotides. When compared to that of an all-DNA control, with an identical sequence, the leakage rate constant was reduced more than 50-fold, to an undetectable level, and the invasion rate was preserved for a hybrid DNA/LNA system. The total performance enhancement ratio also increased more than 70-fold when calculating the ratio of the invading rate to the leakage rate constants for a hybrid system. The rational substitution of LNA nucleotides for DNA nucleotides preserves sequence space while improving the signal-to-noise ratio of strand displacement systems. Hybrid DNA/LNA systems offer great potential for high-performance chemical reaction networks that include catalyzed hairpin assemblies, hairpin chain reactions, motors, walkers, and seesaw gates.

  17. Extraterrestrial Amino Acids in Ureilites Including Almahata Sitta

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Glavin, D. P.; Callahan, M. P.; Dworkin, J. P.

    2011-01-01

    Ureilites are a class of meteorites that lack chondrules (achondrites) but have relatively high carbon abundances, averaging approx.3 wt %. Using highly sensitive liquid chromatography coupled with UV fluorescence and time-of-flight mass spectrometry (LC-FD/ToF-MS), it was recently determined that there are amino acids in. fragment 94 of the Almahata Sitta ureilite[l]. Based on the presence of amino acids that are rare in the Earth's biosphere, as well as the near-racemic enantiomeric ratios of marry of the more common amino acids, it was concluded that most of the detected amino acids were indigenous to the meteorite. Although the composition of the Almahata Sitta ureilite appears to be unlike other recovered ureilites, the discovery of amino acids in this meteorite raises the question of whether other ureilites rnav also contain amino acids. Herein we present the results of LC-FDlTo.F-MS analyses of: a sand sample from the Almahata Sitta strewn held, Almahata Sitta fragments 425 (an ordinary H5 chondrite) and 427 (ureilite), as well as an Antarctic ureilite (Allan lulls, ALHA 77257).

  18. Flexibility of nucleic acids: From DNA to RNA

    NASA Astrophysics Data System (ADS)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  19. Interactions of carcinogens with DNA (deoxyribonucleic acid)

    SciTech Connect

    Broyde, S.; Shapiro, R.

    1989-10-01

    The principal goal of this research has been the determination of the conformational changes produced in DNA by the covalent binding of a carcinogenic aromatic amine, and the correlation of these changes with the mutations and carcinogenic effects initiated by the same substances. To this end, we have devised new synthetic methods for the preparation of oligonucleotides modified by derivatives af 4-aminobiphenyl and aniline. We have also performed potential energy minimization studies on the above substances and on single and double stranded DNA fragments bearing the above amines as well as acetylaminofluorene, aminofluorene, aminopyrene and the antibiotic mitomycin. Our computations have been carried out on DOE supercomputers using our program, DUPLEX. We have defined a number of novel structures for these modified DNAs, including Hoogsteen, wedge'' (see below) denatured, cross-linked and intercalated forms. Some suggestions have been made about the relation of these forms to mutagenesis. 7 refs.

  20. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  1. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  2. A DNA origami nanorobot controlled by nucleic acid hybridization.

    PubMed

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

    2014-07-23

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

  3. Improved DNA hybridization parameters by Twisted Intercalating Nucleic Acid (TINA).

    PubMed

    Schneider, Uffe Vest

    2012-01-01

    This thesis establishes oligonucleotide design rules and applications of a novel group of DNA stabilizing molecules collectively called Twisted Intercalating Nucleic Acid - TINA. Three peer-reviewed publications form the basis for the thesis. One publication describes an improved and rapid method for determination of DNA melting points and two publications describe the effects of positioning TINA molecules in parallel triplex helix and antiparallel duplex helix forming DNA structures. The third publication establishes that TINA molecules containing oligonucleotides improve an antiparallel duplex hybridization based capture assay's analytical sensitivity compared to conventionel DNA oligonucleotides. Clinical microbiology is traditionally based on pathogenic microorganisms' culture and serological tests. The introduction of DNA target amplification methods like PCR has improved the analytical sensitivity and total turn around time involved in clinical diagnostics of infections. Due to the relatively weak hybridization between the two strands of double stranded DNA, a number of nucleic acid stabilizing molecules have been developed to improve the sensitivity of DNA based diagnostics through superior binding properties. A short introduction is given to Watson-Crick and Hoogsteen based DNA binding and the derived DNA structures. A number of other nucleic acid stabilizing molecules are described. The stabilizing effect of TINA molecules on different DNA structures is discussed and considered in relation to other nucleic acid stabilizing molecules and in relation to future use of TINA containing oligonucleotides in clinical diagnostics and therapy. In conclusion, design of TINA modified oligonucleotides for antiparallel duplex helixes and parallel triplex helixes follows simple purpose dependent rules. TINA molecules are well suited for improving multiplex PCR assays and can be used as part of novel technologies. Future research should test whether combinations of TINA

  4. Catalysis of the Carbonylation of Alcohols to Carboxylic Acids Including Acetic Acid Synthesis from Methanol.

    ERIC Educational Resources Information Center

    Forster, Denis; DeKleva, Thomas W.

    1986-01-01

    Monsanto's highly successful synthesis of acetic acid from methanol and carbon monoxide illustrates use of new starting materials to replace pretroleum-derived ethylene. Outlines the fundamental aspects of the acetic acid process and suggests ways of extending the synthesis to higher carboxylic acids. (JN)

  5. Nucleic Acid Engineering: RNA Following the Trail of DNA.

    PubMed

    Kim, Hyejin; Park, Yongkuk; Kim, Jieun; Jeong, Jaepil; Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2016-02-08

    The self-assembly feature of the naturally occurring biopolymer, DNA, has fascinated researchers in the fields of materials science and bioengineering. With the improved understanding of the chemical and structural nature of DNA, DNA-based constructs have been designed and fabricated from two-dimensional arbitrary shapes to reconfigurable three-dimensional nanodevices. Although DNA has been used successfully as a building block in a finely organized and controlled manner, its applications need to be explored. Hence, with the myriad of biological functions, RNA has recently attracted considerable attention to further the application of nucleic acid-based structures. This Review categorizes different approaches of engineering nucleic acid-based structures and introduces the concepts, principles, and applications of each technique, focusing on how DNA engineering is applied as a guide to RNA engineering.

  6. Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.

    PubMed

    Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

    2017-03-22

    A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the

  7. Evolution and Phylogeny of Large DNA Viruses, Mimiviridae and Phycodnaviridae Including Newly Characterized Heterosigma akashiwo Virus

    PubMed Central

    Maruyama, Fumito; Ueki, Shoko

    2016-01-01

    Nucleocytoplasmic DNA viruses are a large group of viruses that harbor double-stranded DNA genomes with sizes of several 100 kbp, challenging the traditional concept of viruses as small, simple ‘organisms at the edge of life.’ The most intriguing questions about them may be their origin and evolution, which have yielded the variety we see today. Specifically, the phyletic relationship between two giant dsDNA virus families that are presumed to be close, Mimiviridae, which infect Acanthamoeba, and Phycodnaviridae, which infect algae, is still obscure and needs to be clarified by in-depth analysis. Here, we studied Mimiviridae–Phycodnaviridae phylogeny including the newly identified Heterosigma akashiwo virus strain HaV53. Gene-to-gene comparison of HaV53 with other giant dsDNA viruses showed that only a small proportion of HaV53 genes show similarities with the others, revealing its uniqueness among Phycodnaviridae. Phylogenetic/genomic analysis of Phycodnaviridae including HaV53 revealed that the family can be classified into four distinctive subfamilies, namely, Megaviridae (Mimivirus-like), Chlorovirus-type, and Coccolitho/Phaeovirus-type groups, and HaV53 independent of the other three groups. Several orthologs found in specific subfamilies while absent from the others were identified, providing potential family marker genes. Finally, reconstruction of the evolutionary history of Phycodnaviridae and Mimiviridae revealed that these viruses are descended from a common ancestor with a small set of genes and reached their current diversity by differentially acquiring gene sets during the course of evolution. Our study illustrates the phylogeny and evolution of Mimiviridae–Phycodnaviridae and proposes classifications that better represent phyletic relationships among the family members. PMID:27965659

  8. Evolution and Phylogeny of Large DNA Viruses, Mimiviridae and Phycodnaviridae Including Newly Characterized Heterosigma akashiwo Virus.

    PubMed

    Maruyama, Fumito; Ueki, Shoko

    2016-01-01

    Nucleocytoplasmic DNA viruses are a large group of viruses that harbor double-stranded DNA genomes with sizes of several 100 kbp, challenging the traditional concept of viruses as small, simple 'organisms at the edge of life.' The most intriguing questions about them may be their origin and evolution, which have yielded the variety we see today. Specifically, the phyletic relationship between two giant dsDNA virus families that are presumed to be close, Mimiviridae, which infect Acanthamoeba, and Phycodnaviridae, which infect algae, is still obscure and needs to be clarified by in-depth analysis. Here, we studied Mimiviridae-Phycodnaviridae phylogeny including the newly identified Heterosigma akashiwo virus strain HaV53. Gene-to-gene comparison of HaV53 with other giant dsDNA viruses showed that only a small proportion of HaV53 genes show similarities with the others, revealing its uniqueness among Phycodnaviridae. Phylogenetic/genomic analysis of Phycodnaviridae including HaV53 revealed that the family can be classified into four distinctive subfamilies, namely, Megaviridae (Mimivirus-like), Chlorovirus-type, and Coccolitho/Phaeovirus-type groups, and HaV53 independent of the other three groups. Several orthologs found in specific subfamilies while absent from the others were identified, providing potential family marker genes. Finally, reconstruction of the evolutionary history of Phycodnaviridae and Mimiviridae revealed that these viruses are descended from a common ancestor with a small set of genes and reached their current diversity by differentially acquiring gene sets during the course of evolution. Our study illustrates the phylogeny and evolution of Mimiviridae-Phycodnaviridae and proposes classifications that better represent phyletic relationships among the family members.

  9. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    PubMed Central

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  10. A new class of DNA photolyases present in various organisms including aplacental mammals.

    PubMed Central

    Yasui, A; Eker, A P; Yasuhira, S; Yajima, H; Kobayashi, T; Takao, M; Oikawa, A

    1994-01-01

    DNA photolyase specifically repairs UV light-induced cyclobutane-type pyrimidine dimers in DNA through a light-dependent reaction mechanism. We have obtained photolyase genes from Drosophila melanogaster (fruit fly), Oryzias latipes (killifish) and the marsupial Potorous tridactylis (rat kangaroo), the first photolyase gene cloned from a mammalian species. The deduced amino acid sequences of these higher eukaryote genes show only limited homology with microbial photolyase genes. Together with the previously cloned Carassius auratus (goldfish) gene they form a separate group of photolyase genes. A new classification for photolyases comprising two distantly related groups is proposed. For functional analysis P.tridactylis photolyase was expressed and purified as glutathione S-transferase fusion protein from Escherichia coli cells. The biologically active protein contained FAD as light-absorbing cofactor, a property in common with the microbial class photolyases. Furthermore, we found in the archaebacterium Methanobacterium thermoautotrophicum a gene similar to the higher eukaryote photolyase genes, but we could not obtain evidence for the presence of a homologous gene in the human genome. Our results suggest a divergence of photolyase genes in early evolution. Images PMID:7813451

  11. Gas chromatographic analysis of infant formulas for total fatty acids, including trans fatty acids.

    PubMed

    Satchithanandam, Subramaniam; Fritsche, Jan; Rader, Jeanne I

    2002-01-01

    Twelve powdered and 13 liquid infant formulas were analyzed by using an extension of AOAC Official Method 996.01 for fat analysis in cereal products. Samples were hydrolyzed with 8 N HCl and extracted with ethyl and petroleum ethers. Fatty acid methyl esters were prepared by refluxing the mixed ether extracts with methanolic sodium hydroxide in the presence of 14% boron trifluoride in methanol. The extracts were analyzed by gas chromatography. In powdered formulas, saturated fatty acid (SFA) content (mean +/- SD; n = 12) was 41.05 +/- 3.94%, monounsaturated fatty acid (MUFA) content was 36.97 +/- 3.38%, polyunsaturated fatty acid (PUFA) content was 20.07 +/- 3.08%, and total trans fatty acid content was 1.30 +/- 1.27%. In liquid formulas, SFA content (mean +/- SD; n = 13) was 42.29 +/- 2.98%, MUFA content was 36.05 +/- 2.47%, PUFA content was 20.65 +/- 2.40%, and total trans fatty acid content was 0.88 +/- 0.54%. Total fat content in powdered formulas ranged from 4.4 to 5.5 g/100 kcal and linoleic acid content ranged from 868 to 1166 mg/100 kcal. In liquid formulas, total fat content ranged from 4.1 to 5.1 g/100 kcal and linoleic acid content ranged from 820 to 1100 mg/100 kcal. There were no significant differences between powdered and liquid infant formulas in concentrations of total fat, SFA, MUFA, PUFA, or trans fatty acids.

  12. Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology.

    PubMed

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J; Walter, Nils G

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA 'staples'. This revolutionary approach has led to the creation of a multitude of two-dimensional and three-dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy.

  13. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

    DOEpatents

    Nasarabadi, Shanavaz [Livermore, CA

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reaction chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.

  14. The energetics of tightly bent DNA: a composite elastica model including local melting

    NASA Astrophysics Data System (ADS)

    Evans, Arthur; Levine, Alex

    2012-02-01

    Melting transitions are well-known to be affected by the application of mechanical stress. Motivated by the experiments of Zocchi and collaborators (Qu and Zocchi 2011, EPL 94 18003), we explore the effect of the application of mechanical stress on DNA melting in a particular composite of a stiff double stranded piece of DNA (dsDNA), shorter than its own persistence length, whose ends are linked by a flexible single stranded piece of DNA (ssDNA). The flexible ssDNA acts as a Gaussian polymer coil bending the stiff dsDNA through an elastic force that is controllable by the length of the ssDNA chain. In this talk we present theoretical predictions for two experimentally accessible features: the degree of local dsDNA melting and the local elastic energy of the dsDNA/ssDNA construct both as a function of the length of the attached ssDNA. We also address the effect of introducing a nick (broken covalent bond) in the dsDNA backbone on these results and discuss the implications of such data on the relative importance of backbone elasticity versus base stacking and base pairing interactions in determining the elasticity of dsDNA. This work also addresses open questions in the nonlinear elasticity of DNA in tightly bent curves.

  15. Supplemental safflower oil affects the fatty acid profile, including conjugated linoleic acid, of lamb.

    PubMed

    Boles, J A; Kott, R W; Hatfield, P G; Bergman, J W; Flynn, C R

    2005-09-01

    The objective of this study was to determine whether increasing levels of dietary safflower oil would alter unsaturated fat (especially CLA) and tocopherol content of lamb, animal performance, carcass characteristics, or color stability of lamb muscle tissue. Targhee x Rambouillet wethers (n = 60) were assigned to one of three diets (four pens per treatment with five lambs per pen) in a completely random design. Diets were formulated with supplemental safflower oil at 0 (control), 3, or 6% (as-fed basis) of the diet. Diets containing approximately 80% concentrate and 20% roughage were formulated, on a DM basis, to be isocaloric and isonitrogenous and to meet or exceed NRC requirements for Ca, P, and other nutrients. A subsample of 12 wethers per treatment was selected based on average BW (54 kg) and slaughtered. Carcass data (LM area, fat thickness, and internal fat content) and wholesale cut weight (leg, loin, rack, shoulder, breast, and foreshank), along with fatty acid, tocopherol, and color analysis, were determined on each carcass. The LM and infraspinatus were sampled for fatty acid profile. Increasing safflower oil supplementation from 0 to 3 or 6% increased the proportion of linoleic acid in the diet from 49.93 to 55.32 to 62.38%, respectively, whereas the percentage of oleic acid decreased from 27.94 to 23.80 to 20.73%, respectively. The percentage of oil in the diet did not (P > or = 0.11) alter the growth and carcass characteristics of lambs, nor did it alter the tocopherol content or color stability of meat. Increasing levels of safflower oil in lamb diets decreased (P < 0.01) the weight percentage of oleic acid in the infraspinatus and LM, and increased linoleic acid (P < 0.01). Oil supplementation increased (P < 0.01) the weight percentage of various isomers of CLA in muscle, with the greatest change in the cis-9,trans-11 isomer. Supplementation of sheep diets with safflower oil, up to 6% of the diet, resulted in increasing levels of unsaturated fatty

  16. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  17. Protective Effect of Folic Acid on Oxidative DNA Damage

    PubMed Central

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

    2015-01-01

    Abstract Although previous reports have linked DNA damage with both transmissions across generations as well as our own survival, it is unknown how to reverse the lesion. Based on the data from a Randomized, Double-blind, Placebo Controlled Clinical Trial, this study aimed to assess the efficacy of folic acid supplementation (FAS) on DNA oxidative damage reversal. In this randomized clinical trial (RCT), a total of 450 participants were enrolled and randomly assigned to 3 groups to receive folic acid (FA) 0.4 mg/day (low-FA), 0.8 mg/day (high-FA), or placebo (control) for 8 weeks. The urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) and creatinine (Cr) concentration at pre- and post-FAS were measured with modified enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. A multivariate general linear model was applied to assess the individual effects of FAS and the joint effects between FAS and hypercholesterolemia on oxidative DNA damage improvement. This clinical trial was registered with ClinicalTrials.gov, number NCT02235948. Of the 438 subjects that received FA fortification or placebo, the median (first quartile, third quartile) of urinary 8-OHdG/Cr for placebo, low-FA, and high-FA groups were 58.19 (43.90, 82.26), 53.51 (38.97, 72.74), 54.73 (39.58, 76.63) ng/mg at baseline and 57.77 (44.35, 81.33), 51.73 (38.20, 71.30), and 50.65 (37.64, 76.17) ng/mg at the 56th day, respectively. A significant decrease of urinary 8-OHdG was observed after 56 days FA fortification (P < 0.001). Compared with the placebo, after adjusting for some potential confounding factors, including the baseline urinary 8-OHdG/Cr, the urinary 8-OHdG/Cr concentration significantly decreased after 56 days FAS [β (95% confidence interval) = −0.88 (−1.62, −0.14) and P = 0.020 for low-FA; and β (95% confidence interval) = −2.68 (−3.42, −1.94) and P < 0.001 for high-FA] in a dose-response fashion (Ptrend

  18. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli.

  19. Switchable reconfiguration of nucleic acid nanostructures by stimuli-responsive DNA machines.

    PubMed

    Liu, Xiaoqing; Lu, Chun-Hua; Willner, Itamar

    2014-06-17

    CONSPECTUS: The base sequence in DNA dictates structural and reactivity features of the biopolymer. These properties are implemented to use DNA as a unique material for developing the area of DNA nanotechnology. The design of DNA machines represents a rapidly developing research field in the area of DNA nanotechnology. The present Account discusses the switchable reconfiguration of nucleic acid nanostructures by stimuli-responsive DNA machines, and it highlights potential applications and future perspectives of the area. Programmed switchable DNA machines driven by various fuels and antifuels, such as pH, Hg(2+) ions/cysteine, or nucleic acid strands/antistrands, are described. These include the assembly of DNA tweezers, walkers, a rotor, a pendulum, and more. Using a pH-oscillatory system, the oscillatory mechanical operation of a DNA pendulum is presented. Specifically, the synthesis and "mechanical" properties of interlocked DNA rings are described. This is exemplified with the preparation of interlocked DNA catenanes and a DNA rotaxane. The dynamic fuel-driven reconfiguration of the catenane/rotaxane structures is followed by fluorescence spectroscopy. The use of DNA machines as functional scaffolds to reconfigurate Au nanoparticle assemblies and to switch the fluorescence features within fluorophore/Au nanoparticle conjugates between quenching and surface-enhanced fluorescence states are addressed. Specifically, the fluorescence features of the different DNA machines are characterized as a function of the spatial separation between the fluorophore and Au nanoparticles. The experimental results are supported by theoretical calculations. The future development of reconfigurable stimuli-responsive DNA machines involves fundamental challenges, such as the synthesis of molecular devices exhibiting enhanced complexities, the introduction of new fuels and antifuels, and the integration of new payloads being reconfigured by the molecular devices, such as enzymes or

  20. DNA sequence analyses of blended herbal products including synthetic cannabinoids as designer drugs.

    PubMed

    Ogata, Jun; Uchiyama, Nahoko; Kikura-Hanajiri, Ruri; Goda, Yukihiro

    2013-04-10

    In recent years, various herbal products adulterated with synthetic cannabinoids have been distributed worldwide via the Internet. These herbal products are mostly sold as incense, and advertised as not for human consumption. Although their labels indicate that they contain mixtures of several potentially psychoactive plants, and numerous studies have reported that they contain a variety of synthetic cannabinoids, their exact botanical contents are not always clear. In this study, we investigated the origins of botanical materials in 62 Spice-like herbal products distributed on the illegal drug market in Japan, by DNA sequence analyses and BLAST searches. The nucleotide sequences of four regions were analyzed to identify the origins of each plant species in the herbal mixtures. The sequences of "Damiana" (Turnera diffusa) and Lamiaceae herbs (Mellissa, Mentha and Thymus) were frequently detected in a number of products. However, the sequences of other plant species indicated on the packaging labels were not detected. In a few products, DNA fragments of potent psychotropic plants were found, including marijuana (Cannabis sativa), "Diviner's Sage" (Salvia divinorum) and "Kratom" (Mitragyna speciosa). Their active constituents were also confirmed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), although these plant names were never indicated on the labels. Most plant species identified in the products were different from the plants indicated on the labels. The plant materials would be used mainly as diluents for the psychoactive synthetic compounds, because no reliable psychoactive effects have been reported for most of the identified plants, with the exception of the psychotropic plants named above.

  1. DNA Tetrominoes: The Construction of DNA Nanostructures Using Self-Organised Heterogeneous Deoxyribonucleic Acids Shapes

    PubMed Central

    Ong, Hui San; Rahim, Mohd Syafiq; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

    2015-01-01

    The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner. PMID:26258940

  2. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  3. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  4. Effect of omega-3 fatty acids on the modification of erythrocyte membrane fatty acid content including oleic acid in peritoneal dialysis patients.

    PubMed

    An, W S; Lee, S M; Son, Y K; Kim, S E; Kim, K H; Han, J Y; Bae, H R; Park, Y

    2012-01-01

    Erythrocyte membrane fatty acids (FA), such as oleic acid, are related to acute coronary syndrome. There is no report about the effect of omega-3 FA on oleic acid in peritoneal dialysis (PD) patients. We hypothesized that omega-3 FA can modify erythrocyte membrane FA, including oleic acid, in PD patients. In a double-blind, randomized, placebo-controlled study, 18 patients who were treated with PD for at least 6 months were randomized to treatment for 12 weeks with omega-3 FA or placebo. Erythrocyte membrane FA content was measured by gas chromatography at baseline and after 12 weeks. The erythrocyte membrane content of eicosapentaenoic acid and docosahexaenoic acid was significantly increased and saturated FA and oleic acid were significantly decreased in the omega-3 FA supplementation group after 12 weeks compared to baseline. In conclusion, erythrocyte membrane FA content, including oleic acid, was significantly modified by omega-3 FA supplementation for 12 weeks in PD patients.

  5. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    PubMed

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

  6. Mammalian cell DNA damage and repair kinetics of monohaloacetic acid drinking water disinfection by-products.

    PubMed

    Komaki, Yukako; Pals, Justin; Wagner, Elizabeth D; Mariñas, Benito J; Plewa, Michael J

    2009-11-01

    Haloacetic acids (HAAs) are the second most common class of chlorinated water disinfection by-products (DBPs). The single cell gel electrophoresis genotoxicity assay using Chinese hamster ovary (CHO) cells was modified to include liquid holding recovery time to measure genomic DNA damage and repair kinetics of three monoHAAs: chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA). The rank order of genotoxic potency was IAA > BAA > CAA from previous research. The concentration of each HAA was chosen to generate approximately the same level of genotoxic damage. No cytotoxicity was expressed during the 24 h liquid holding period. Nuclei from CHO cells treated with BAA showed the lowest rate of DNA repair (t(50) = 296 min) compared to that of CAA or IAA (t(50) = 134 and 84 min, respectively). The different rates of genomic repair expressed by IAA or CAA versus BAA suggest that different distributions of DNA lesions are induced. The use of DNA repair coupled with genomic technologies may lead to the understanding of the biological and genetic mechanisms involved in toxic responses induced by DBPs.

  7. Assignment of the Perfluoropropionic Acid-Formic Acid Complex and the Difficulties of Including High K_a Transitions.

    NASA Astrophysics Data System (ADS)

    Obenchain, Daniel A.; Lin, Wei; Novick, Stewart E.; Cooke, S. A.

    2016-06-01

    We recently began an investigation into the perfluoropropionic acid\\cdotsformic acid complex using broadband microwave spectroscopy. This study aims to examine the possible double proton transfer between the two interacting carboxcyclic acid groups. The spectrum presented as a doubled set of lines, with spacing between transitions of < 1 MHz. Transitions appeared to be a-type, R branch transitions for an asymmetric top. Assignment of all K_a=1,0 transitions yields decent fits to a standard rotational Hamiltonian. Treatment of the doubling as either a two state system (presumably with a double proton transfer) or as two distinct, but nearly identical conformations of the complex produce fits of similar quality. Including higher K_a transitions for the a-type, R-branch lines greatly increases the error of these fits. A previous study involving the trifluoroacetic acid\\cdotsformic acid complex published observed similar high K_a transitions, but did not include them in the published fit. We hope to shed more light on this conundrum. Similarities to other double-well potential minimum systems will be discussed. Martinache, L.; Kresa, W.; Wegener, M.;, Vonmont, U.; and Bauder, A. Chem. Phys. 148 (1990) 129-140.

  8. Interaction of photosensitive surfactant with DNA and poly acrylic acid

    SciTech Connect

    Zakrevskyy, Yuriy Paasche, Jens; Lomadze, Nino; Santer, Svetlana; Cywinski, Piotr; Cywinska, Magdalena; Reich, Oliver; Löhmannsröben, Hans-Gerd

    2014-01-28

    In this paper, we investigate interactions and phase transitions in polyelectrolyte-surfactant complexes formed between a cationic azobenzene-containing surfactant and two types of polyelectrolytes: natural (DNA) or synthetic (PAA: poly acrylic acid). The construction of a phase diagram allowed distancing between four major phases: extended coil conformation, colloidally stable compacted globules, colloidal instability range, and surfactant-stabilized compact state. Investigation on the complexes’ properties in different phases and under irradiation with UV light provides information about the role of the surfactant's hydrophobic trans isomers both in the formation and destruction of DNA and PAA globules as well as in their colloidal stabilization. The trans isomer shows much stronger affinity to the polyelectrolytes than the hydrophilic cis counterpart. There is no need for complete compensation of the polyelectrolyte charges to reach the complete compaction. On contrary to the findings previously reported in the literature, we demonstrate – for the first time – complete polyelectrolyte compaction which occurs already at 20% of DNA (and at 50% of PAA) charge compensation. The trans isomer plays the main role in the compaction. The aggregation between azobenzene units in the photosensitive surfactant is a driving force of this process. The decompaction can be realized during UV light irradiation and is strongly influenced by the interplay between surfactant-surfactant and surfactant-DNA interactions in the compacted globules.

  9. Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

    PubMed

    Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

    2014-05-01

    The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

  10. A plea for modern botanical collections to include DNA-friendly material.

    PubMed

    Gaudeul, Myriam; Rouhan, Germinal

    2013-04-01

    Botanists have long collected herbarium specimens during their expeditions, and the importance of such collections is broadly acknowledged nowadays. It is largely recognized that material for molecular studies must be accompanied by herbarium material to be deposited in a recognized herbarium (vouchers). By contrast, the collection of herbarium specimens with no material for genetic analyses is unfortunately still common. The evolution of science and the need to face new environmental challenges require some changes in the way science is planned and performed. Here, we highlight some key scientific areas which could greatly benefit from such DNA-friendly collections, and we make a plea - and a call to all botanists - for the routine collection of DNA-friendly material together with herbarium specimens.

  11. Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens.

    PubMed

    Yang, Genyan; Erdman, Dean E; Kodani, Maja; Kools, John; Bowen, Michael D; Fields, Barry S

    2011-01-01

    This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner.

  12. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    PubMed

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process.

  13. Selection and characterization of single stranded DNA aptamers for the hormone abscisic Acid.

    PubMed

    Grozio, Alessia; Gonzalez, Victor M; Millo, Enrico; Sturla, Laura; Vigliarolo, Tiziana; Bagnasco, Luca; Guida, Lucrezia; D'Arrigo, Cristina; De Flora, Antonio; Salis, Annalisa; Martin, Elena M; Bellotti, Marta; Zocchi, Elena

    2013-10-01

    The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 μM and 0.80 ± 0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.

  14. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  15. Evaluation of DNA typing as a positive identification method for soft and hard tissues immersed in strong acids.

    PubMed

    Robino, C; Pazzi, M; Di Vella, G; Martinelli, D; Mazzola, L; Ricci, U; Testi, R; Vincenti, M

    2015-11-01

    Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes.

  16. Clustered DNA damages induced by high and low LET radiation, including heavy ions

    NASA Technical Reports Server (NTRS)

    Sutherland, B. M.; Bennett, P. V.; Schenk, H.; Sidorkina, O.; Laval, J.; Trunk, J.; Monteleone, D.; Sutherland, J.; Lowenstein, D. I. (Principal Investigator)

    2001-01-01

    Clustered DNA damages--here defined as two or more lesions (strand breaks, oxidized purines, oxidized pyrimidines or abasic sites) within a few helical turns--have been postulated as difficult to repair accurately, and thus highly significant biological lesions. Further, attempted repair of clusters may produce double strand breaks (DSBs). However, until recently, there was no way to measure ionizing radiation-induced clustered damages, except DSB. We recently described an approach for measuring classes of clustered damages (oxidized purine clusters, oxidized pyrimidine clusters, abasic clusters, along with DSB). We showed that ionizing radiation (gamma rays and Fe ions, 1 GeV/amu) does induce such clusters in genomic DNA in solution and in human cells. These studies also showed that each damage cluster results from one radiation hit (and its track), thus indicating that they can be induced by very low doses of radiation, i.e. two independent hits are not required for cluster induction. Further, among all complex damages, double strand breaks comprise--at most-- 20%, with the other clustered damages being at least 80%.

  17. Karyotypic features including organizations of the 5S, 45S rDNA loci and telomeres of Scadoxus multiflorus (Amaryllidaceae)

    PubMed Central

    Monkheang, Pansa; Chaveerach, Arunrat; Sudmoon, Runglawan; Tanee, Tawatchai

    2016-01-01

    Abstract Scadoxus multiflorus Martyn, 1795 is an ornamental plant with brilliantly colored flowers. Even though its chromosomes are rather large, there is no karyotype description reported so far. Therefore, conventional and molecular cytogenetic studies including fluorescence in situ hybridization (FISH) with 45S and 5S rDNA, and human telomere sequence (TTAGGG)n probes (Arabidopsis-type telomere probes yielded negative results) were carried out. The chromosome number is as reported previously, 2n = 18. The nine chromosome pairs include two large submetacentric, five large acrocentric, one medium acrocentric, two small metacentric and eight small submetacentric chromosomes. Hybridization sites of the 45S rDNA signals were on the short arm ends of chromosomes #1, #3 and #8, while 5S rDNA signals appeared on the long arm of chromosome 3, in one homologue as a double signal. The telomere signals were restricted to all chromosome ends. Three chromosome pairs could be newly identified, chromosome pair 3 by 5S rDNA and chromosomes #1, #3 and #8 by 45S rDNA loci. In addition to new information about rDNA locations we show that the ends of Scadoxus multiflorus chromosomes harbor human instead of Arabidopsis-type telomere sequences. Overall, the Scadoxus multiflorus karyotype presents chromosomal heteromorphy concerning size, shape and 45S and 5S rDNA positioning. As Scadoxus Rafinesque, 1838 and related species are poorly studied on chromosomal level the here presented data is important for better understanding of evolution in Amaryllidaceae. PMID:28123684

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

    PubMed

    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.

  19. Inhibiting DNA methylation causes an interferon response in cancer via dsRNA including endogenous retroviruses

    PubMed Central

    Chiappinelli, Katherine B.; Strissel, Pamela L.; Desrichard, Alexis; Li, Huili; Henke, Christine; Akman, Benjamin; Hein, Alexander; Rote, Neal S.; Cope, Leslie M.; Snyder, Alexandra; Makarov, Vladimir; Buhu, Sadna; Slamon, Dennis J.; Wolchok, Jedd D.; Pardoll, Drew M.; Beckmann, Matthias W.; Zahnow, Cynthia A.; Mergoub, Taha; Chan, Timothy A.; Baylin, Stephen B.; Strick, Reiner

    2015-01-01

    Summary We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a Type I Interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response twofold, and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model. PMID:26317466

  20. CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

    PubMed

    Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

    2011-04-25

    We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

  1. Amino acid composition, including key derivatives of eccrine sweat: potential biomarkers of certain atopic skin conditions.

    PubMed

    Mark, Harker; Harding, Clive R

    2013-04-01

    The free amino acid (AA) composition of eccrine sweat is different from other biological fluids, for reasons which are not properly understood. We undertook the detailed analysis of the AA composition of freshly isolated pure human eccrine sweat, including some of the key derivatives of AA metabolism, to better understand the key biological mechanisms governing its composition. Eccrine sweat was collected from the axillae of 12 healthy subjects immediately upon formation. Free AA analysis was performed using an automatic AA analyser after ninhydrin derivatization. Pyrrolidine-5-carboxylic acid (PCA) and urocanic acid (UCA) levels were determined using GC/MS. The free AA composition of sweat was dominated by the presence of serine accounting for just over one-fifth of the total free AA composition. Glycine was the next most abundant followed by PCA, alanine, citrulline and threonine, respectively. The data obtained indicate that the AA content of sweat bears a remarkable similarity to the AA composition of the epidermal protein profilaggrin. This protein is the key source of free AAs and their derivatives that form a major part of the natural moisturizing factor (NMF) within the stratum corneum (SC) and plays a major role in maintaining the barrier integrity of human skin. As perturbations in the production of NMF can lead to abnormal barrier function and can arise as a consequence of filaggrin genotype, we propose the quantification of AAs in sweat may serve as a non-invasive diagnostic biomarker for certain atopic skin conditions, that is, atopic dermatitis (AD).

  2. The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation

    PubMed Central

    Kaufman-Szymczyk, Agnieszka; Majewski, Grzegorz; Lubecka-Pietruszewska, Katarzyna; Fabianowska-Majewska, Krystyna

    2015-01-01

    Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review. PMID:26703571

  3. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    PubMed

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

  4. Prooxidant action of chebulinic acid and tellimagrandin I: causing copper-dependent DNA strand breaks.

    PubMed

    Yi, Zong-Chun; Liu, Yan-Ze; Li, Hai-Xia; Wang, Zhao

    2009-04-01

    The prooxidant activity of two hydrolysable tannins, chebulinic acid and tellimagrandin I, on plasmid DNA and genomic DNA of cultured MRC-5 human embryo lung fibroblasts was assessed. The results revealed that both hydrolysable tannins in combination with Cu(II) induced DNA strand breaks in pBR322 plasmid DNA in a concentration-dependent manner. Chebulinic acid and tellimagrandin I also induced genomic DNA strand breaks of MRC-5 human embryo lung fibroblasts in the presence of Cu(II). After treatment with chebulinic acid or tellimagrandin I alone, the pBR322 plasmid DNA and genomic DNA in MRC-5 cells kept intact. In addition, addition of Cu(I) reagent bathocuproinedisulfonic acid or catalase markedly inhibited the copper-dependent DNA strand breaks by both tannins. However, three typical hydroxyl radical scavengers, DMSO, ethanol and mannitol, did not inhibit the DNA strand breaks. Both tannins were able to reduce Cu(II) to Cu(I). These results indicated that chebulinic acid and tellimagrandin I induced the copper-dependent strand breaks of pBR322 plasmid DNA and MRC-5 genomic DNA with prooxidant action, in which Cu(II)/Cu(I) redox cycle and H(2)O(2) were involved and hydroxyl radical formation is important in the hypothetical mechanism by which DNA strand breaks are formed.

  5. DNA adsorption to and elution from silica surfaces: influence of amino acid buffers.

    PubMed

    Vandeventer, Peter E; Mejia, Jorge; Nadim, Ali; Johal, Malkiat S; Niemz, Angelika

    2013-09-19

    Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers. Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction.

  6. Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum.

    PubMed

    Kitamura, Takuya; Seki, Naoya; Kihara, Akio

    2017-03-28

    Although normal fatty acids (FAs) are degraded via β-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2-deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

  7. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    ABSTRACT

    Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  8. Fatty acid composition including cis-9, trans-11 CLA of cooked ground lamb

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little information is available on effect of cooking on beneficial fatty acids such as conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids (PUFA). The objective of this study was to examine impact of cooking on the FA composition of ground lamb of two different muscles. Samples were p...

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

    PubMed

    Tang, M; Subbiah, M T

    1996-01-19

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

  10. [Antiinflammatory therapy in ostheoarthritis including omega 3 and omega 6 fatty acids].

    PubMed

    Dzielska-Olczak, Małgorzata; Nowak, Jerzy Z

    2012-05-01

    Osteoarthritis (ostheoarthrosis, OA) is characterized by progressive destruction of articular cartilage, remodeling of the periarticular bone and inflammation of the synovial membrane. In patients occur joints pain, impaired joints motion and disability. The results of many studies indicate an inflammation as foundation of this disease. The management of OA include a combination of pharmacological treatments and nonpharmacological interventions. Pharmacological treatments include used paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs) and chondroprotectives (glucosamine, chondroitin sulfate and so on). NSAIDs long-term use associated with serious adverse effects. OA symptoms are effectively reduced by nutrients such omega 3 and omega 6 fatty acids (PUFAs as EPA, DHA), which decrease the need for non-steroidal drugs and may less adverse events. They exerts, particularly EPA, anti-inflammatory effect, inhibit catabolic processes, stimulate the anabolic process in the cartilage in the joint. Many different evidence validate that omega 3 alleviate the progression of osteoarthritis and have exciting therapeutic potential for preventing cartilage degradation associated with chronic inflammatory in joints.

  11. Role of Amino Acid Insertions on Intermolecular Forces between Arginine Peptide Condensed DNA Helices

    PubMed Central

    DeRouchey, Jason E.; Rau, Donald C.

    2011-01-01

    In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads. PMID:21994948

  12. Spherical Nucleic Acids: A New Form of DNA

    NASA Astrophysics Data System (ADS)

    Cutler, Joshua Isaac

    Spherical Nucleic Acids (SNAs) are a new class of nucleic acid-based nanomaterials that exhibit unique properties currently being explored in the contexts of gene-based cancer therapies and in the design of programmable nanoparticle-based materials. The properties of SNAs differ from canonical, linear nucleic acids by virtue of their dense packing into an oriented 3-dimensional array. SNAs can be synthesized from a number of useful nanoparticle templates, such as plasmonic gold and silver, magnetic oxides, luminescent semi-conductor quantum dots, and silica. In addition, by crosslinking the oligonucleotides and dissolving the core, they can be made in a hollow form as well. This dissertation describes the evolution of SNAs from initial studies of inorganic nanoparticle-based materials densely functionalized with oligonucleotides to the proving of a hypothesis that their unique properties can be observed in a core-less structure if the nucleic acids are densely packed and highly oriented. Chapter two describes the synthesis of densely functionalized polyvalent oligonucleotide superparamagnetic iron oxide nanoparticles using the copper-catalyzed azide-alkyne cycloaddition reaction. These particles are shown to exhibit cooperative binding in a density- and salt concentration-dependent fashion, with nearly identical behaviors to those of SNA-functionalized gold nanoparticles. Importantly, these particles are the first non-gold particles shown to be capable of entering cells in high numbers via the SNA-mediated cellular uptake pathway, and provided the first evidence that SNA-mediated cellular uptake is core-independent. In the third chapter, a gold nanoparticle catalyzed alkyne cross-linking reaction is described that is capable of forming hollow organic nanoparticles using polymers with alkyne-functionalized backbones. With this method, the alkyne-modified polymers adsorb to the particle surfaces, cross-link on the surface, allowing the gold nanoparticle to be

  13. A fluorescence-based analysis of aristolochic acid-derived DNA adducts.

    PubMed

    Romanov, Victor; Sidorenko, Victoria; Rosenquist, Thomas A; Whyard, Terry; Grollman, Arthur P

    2012-08-01

    Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.

  14. DNA-LCEB: a high-capacity and mutation-resistant DNA data-hiding approach by employing encryption, error correcting codes, and hybrid twofold and fourfold codon-based strategy for synonymous substitution in amino acids.

    PubMed

    Hafeez, Ibbad; Khan, Asifullah; Qadir, Abdul

    2014-11-01

    Data-hiding in deoxyribonucleic acid (DNA) sequences can be used to develop an organic memory and to track parent genes in an offspring as well as in genetically modified organism. However, the main concerns regarding data-hiding in DNA sequences are the survival of organism and successful extraction of watermark from DNA. This implies that the organism should live and reproduce without any functional disorder even in the presence of the embedded data. Consequently, performing synonymous substitution in amino acids for watermarking becomes a primary option. In this regard, a hybrid watermark embedding strategy that employs synonymous substitution in both twofold and fourfold codons of amino acids is proposed. This work thus presents a high-capacity and mutation-resistant watermarking technique, DNA-LCEB, for hiding secret information in DNA of living organisms. By employing the different types of synonymous codons of amino acids, the data storage capacity has been significantly increased. It is further observed that the proposed DNA-LCEB employing a combination of synonymous substitution, lossless compression, encryption, and Bose-Chaudary-Hocquenghem coding is secure and performs better in terms of both capacity and robustness compared to existing DNA data-hiding schemes. The proposed DNA-LCEB is tested against different mutations, including silent, miss-sense, and non-sense mutations, and provides substantial improvement in terms of mutation detection/correction rate and bits per nucleotide. A web application for DNA-LCEB is available at http://111.68.99.218/DNA-LCEB.

  15. Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Mitochondrial DNA Replication and PGC-1α Gene Expression in C2C12 Muscle Cells

    PubMed Central

    Lee, Mak-Soon; Shin, Yoonjin; Moon, Sohee; Kim, Seunghae; Kim, Yangha

    2016-01-01

    Mitochondrial biogenesis is a complex process requiring coordinated expression of nuclear and mitochondrial genomes. The peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis, and it controls mitochondrial DNA (mtDNA) replication within diverse tissues, including muscle tissue. The aim of this study was to investigate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mtDNA copy number and PGC-1α promoter activity in C2C12 muscle cells. mtDNA copy number and mRNA levels of genes related to mitochondrial biogenesis such as PGC-1α, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam) were assayed by quantitative real-time PCR. The PGC-1α promoter from −970 to +412 bp was subcloned into the pGL3-basic vector, which includes a luciferase reporter gene. Both EPA and DHA significantly increased mtDNA copy number, dose and time dependently, and up-regulated mRNA levels of PGC-1α, NRF1, and Tfam. Furthermore, EPA and DHA stimulated PGC-1α promoter activity in a dose-dependent manner. These results suggest that EPA and DHA may modulate mitochondrial biogenesis, which was partially associated with increased mtDNA replication and PGC-1α gene expression in C2C12 muscle cells. PMID:28078253

  16. Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Mitochondrial DNA Replication and PGC-1α Gene Expression in C2C12 Muscle Cells.

    PubMed

    Lee, Mak-Soon; Shin, Yoonjin; Moon, Sohee; Kim, Seunghae; Kim, Yangha

    2016-12-01

    Mitochondrial biogenesis is a complex process requiring coordinated expression of nuclear and mitochondrial genomes. The peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis, and it controls mitochondrial DNA (mtDNA) replication within diverse tissues, including muscle tissue. The aim of this study was to investigate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mtDNA copy number and PGC-1α promoter activity in C2C12 muscle cells. mtDNA copy number and mRNA levels of genes related to mitochondrial biogenesis such as PGC-1α, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam) were assayed by quantitative real-time PCR. The PGC-1α promoter from -970 to +412 bp was subcloned into the pGL3-basic vector, which includes a luciferase reporter gene. Both EPA and DHA significantly increased mtDNA copy number, dose and time dependently, and up-regulated mRNA levels of PGC-1α, NRF1, and Tfam. Furthermore, EPA and DHA stimulated PGC-1α promoter activity in a dose-dependent manner. These results suggest that EPA and DHA may modulate mitochondrial biogenesis, which was partially associated with increased mtDNA replication and PGC-1α gene expression in C2C12 muscle cells.

  17. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

    PubMed Central

    Law, S W; Grant, S M; Higuchi, K; Hospattankar, A; Lackner, K; Lee, N; Brewer, H B

    1986-01-01

    Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias. PMID:3464946

  18. Acidity constant determination of novel drug precursor benzothiazolon derivatives including acyl and piperazine moieties

    NASA Astrophysics Data System (ADS)

    Sıdır, İsa; Gülseven Sıdır, Yadigar; Berber, Halil

    2013-07-01

    In this study, protonation and deprotonation behaviors of eight new drug precursor benzothiazolon derivatives in all of acidic and basic scale (super acidic, pH, super basic regions) are analyzed by using UV-visible spectrophotometric technique. Acidity constants (pKa), elucidation of the structure and protonation mechanisms of the studied molecules are obtained. Substituent effect on acidity constant values is discussed. These molecules are protonated from oxygen atom of acetamide group in the keto form. The protonation is found to be considerably contributed by the keto form.

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

    PubMed

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

    2005-01-01

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

  20. Inhibition of N-methyl-N-nitrosourea-induced mutagenicity and DNA methylation by ellagic acid.

    PubMed Central

    Dixit, R; Gold, B

    1986-01-01

    Ellagic acid, a naturally occurring plant phenol, inhibits the activity of the direct-acting mutagen N-methyl-N-nitrosourea (MeNU) in Salmonella typhimurium TA100. Ellagic acid at 0.10, 0.25, 0.50, and 1.00 mM inhibited the mutagenicity of MeNU (0.40 mM) by 3%, 13%, 45%, and 60%, respectively. Ellagic acid (3 mM) also inhibited the mutagenic activity of N,N-dimethylnitrosamine (25-200 mM) in the presence of pyrazole-induced rat liver fraction S-9. The effect of ellagic acid on DNA methylation was studied by incubating 0, 0.72, 1.32, 2.64, and 6.60 mM ellagic acid with DNA (0.9 mM nucleotide) and [3H]MeNU (0.66 mM). HPLC analysis of DNA hydrolysates showed that ellagic acid caused a dose-dependent 36-84% decrease in O6-methylguanine but only a 20% decrease in the 7-methylguanine adduct. Under conditions where methylation at the O6 position of guanine in double-stranded DNA was inhibited 65% by ellagic acid, no significant inhibition of either O6- or 7-methylguanine formation was detected in single-stranded DNA. Affinity-binding studies revealed that [3H]ellagic acid binds equally to double-stranded or single-stranded DNA but that poly(dA X dT) binds 1.5 times as much ellagic acid as does poly(dG X dC). The binding of ellagic acid to DNA is dependent on the concentration of both ellagic acid and DNA. The specific inhibition of O6-methylguanine formation only in double-stranded DNA and the relatively low inhibition of 7-methylguanine formation rule out the possibility that ellagic acid prevents DNA alkylation by scavenging the electrophilic intermediate generated in the hydrolysis of MeNU. The results suggest that ellagic acid inhibition of MeNU-induced mutagenicity is due to specific inhibition of methylation at the O6 position of guanine through an ellagic acid-duplex DNA affinity-binding mechanism. PMID:3464940

  1. Influence of Fatty Acid Precursors, Including Food Preservatives, on the Growth and Fatty Acid Composition of Listeria monocytogenes at 37 and 10°C ▿

    PubMed Central

    Julotok, Mudcharee; Singh, Atul K.; Gatto, Craig; Wilkinson, Brian J.

    2010-01-01

    Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives, on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C4, C5, and C6 branched-chain carboxylic acid, and C3 and C4 straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein. PMID:20048057

  2. Chromosome alterations in breast carcinomas: frequent involvement of DNA losses including chromosomes 4q and 21q.

    PubMed Central

    Schwendel, A.; Richard, F.; Langreck, H.; Kaufmann, O.; Lage, H.; Winzer, K. J.; Petersen, I.; Dietel, M.

    1998-01-01

    Comparative genomic hybridization was applied to map DNA gains and losses in 39 invasive ductal breast carcinomas. Frequent abnormalities included gains on chromosomal regions 1q, 8q, 11q12-13, 16p, 19, 20q and X as well as frequent losses on 1p, 5q, 6q, 9p, 11q, 13q and 16q. Furthermore, frequent losses on 4q (20 cases) and 21q (14 cases) were found for the first time in this tumour type. High copy number amplifications were observed at 8q12-24, 11q11-13 and 20q13-ter. Highly differentiated tumours were associated with gains on 1q and 11q12-13 along with losses on 1p21-22, 4q, 13q, 11q21-ter. Undifferentiated breast carcinomas were characterized by additional DNA imbalances, i.e. deletions of 5q13-23, all of chromosome 9, the centromeric part of chromosome 13 including band 13q14 and the overrepresentation of chromosome X. We speculate that these changes are associated with tumour progression of invasive ductal breast cancer. Images Figure 2 Figure 3 PMID:9743305

  3. Identification and DNA sequence analysis of 15 new {alpha}{sub 1}-antitrypsin variants, including two PI*QO alleles and one deficient PI*M allele

    SciTech Connect

    Faber, J.P.; Kirchgesser, M.; Schwaab, R.; Bidlingmaier, F.; Poller, W.; Weidinger, S.; Olek, K. |

    1994-12-01

    The authors have investigated the molecular basis of 15 new {alpha}{sub 1}-antitrypsin ({alpha}1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect {alpha}1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promotor region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of {alpha}1AT: the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3{prime} frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr{sup 68}(ACC){yields}Ile(ATC); and an in-frame trinucleotide deletion {Delta}Phe{sup 51} (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal {alpha}1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.

  4. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.

    PubMed

    Mittra, Indraneel; Khare, Naveen Kumar; Raghuram, Gorantla Venkata; Chaubal, Rohan; Khambatti, Fatema; Gupta, Deepika; Gaikwad, Ashwini; Prasannan, Preeti; Singh, Akshita; Iyer, Aishwarya; Singh, Ankita; Upadhyay, Pawan; Nair, Naveen Kumar; Mishra, Pradyumna Kumar; Dutt, Amit

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  5. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase

    PubMed Central

    Sahonero-Canavesi, Diana X.; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M.; Geiger, Otto

    2016-01-01

    Summary Phospholipids are well known for their membrane forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth. PMID:25711932

  6. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.

    PubMed

    Sahonero-Canavesi, Diana X; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M; Geiger, Otto

    2015-09-01

    Phospholipids are well known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth.

  7. Real-time monitoring of matrix acidizing including the effects of diverting agents

    SciTech Connect

    Hill, A.D.; Zhu, D.

    1996-05-01

    Real-time monitoring of the injection rate and pressure during matrix acidizing provides operators with a way to determine the changing skin factor as stimulation proceeds. Current methods are based either on the assumption of steady-state flow in the region around the wellbore affected by acid injection or on computer solution of the transient flow equations describing the unsteady reservoir flow process occurring during acidizing. In this paper, a new method for real-time monitoring of matrix acidizing, the inverse injectivity vs. superposition time function plot, is presented. This new method can be applied with a spreadsheet computer program or a programmable calculator and accounts for the transient flow effects occurring during matrix acidizing at multiple rates and injection pressures. The evolving skin factor during a matrix treatment is readily obtained from the diagnostic plot. Hypothetical examples show how the inverse injectivity plot can be used to assess the efficiency of stimulation and diversion. Comparisons with previously presented field cases show the new method to be a simple and accurate means of monitoring the evolving skin factor during matrix acidizing.

  8. Nucleic acid chemistry in the organic phase: from functionalized oligonucleotides to DNA side chain polymers.

    PubMed

    Liu, Kai; Zheng, Lifei; Liu, Qing; de Vries, Jan Willem; Gerasimov, Jennifer Y; Herrmann, Andreas

    2014-10-08

    DNA-incorporating hydrophobic moieties can be synthesized by either solid-phase or solution-phase coupling. On a solid support the DNA is protected, and hydrophobic units are usually attached employing phosphoramidite chemistry involving a DNA synthesizer. On the other hand, solution coupling in aqueous medium results in low yields due to the solvent incompatibility of DNA and hydrophobic compounds. Hence, the development of a general coupling method for producing amphiphilic DNA conjugates with high yield in solution remains a major challenge. Here, we report an organic-phase coupling strategy for nucleic acid modification and polymerization by introducing a hydrophobic DNA-surfactant complex as a reactive scaffold. A remarkable range of amphiphile-DNA structures (DNA-pyrene, DNA-triphenylphosphine, DNA-hydrocarbon, and DNA block copolymers) and a series of new brush-type DNA side-chain homopolymers with high DNA grafting density are produced efficiently. We believe that this method is an important breakthrough in developing a generalized approach to synthesizing functional DNA molecules for self-assembly and related technological applications.

  9. Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

    PubMed Central

    Prat, S; Cortadas, J; Puigdomènech, P; Palau, J

    1985-01-01

    The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins. Images PMID:3839076

  10. DNA Methylation Perturbations in Genes Involved in Polyunsaturated Fatty Acid Biosynthesis Associated with Depression and Suicide Risk

    PubMed Central

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-yu; Cooper, Thomas B.; Burke, Ainsley K.; Oquendo, Maria A.; Mann, J. John; Sublette, M. Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention. PMID:25972837

  11. Universal Dynamic DNA Assembly-Programmed Surface Hybridization Effect for Single-Step, Reusable, and Amplified Electrochemical Nucleic Acid Biosensing.

    PubMed

    Liu, Shufeng; Fang, Li; Wang, Yanqun; Wang, Li

    2017-03-07

    The traditional sensitive electrochemical biosensors are commonly confronted with the cumbersome interface operation and washing procedures and the inclusion of extra exogenous reagents, which impose the challenge on the detection simplicity, reliability, and reusability. Herein, we present the proof-of-principle of a unique biosensor architecture based on dynamic DNA assembly programmed surface hybridization, which confers the single-step, reusable, and enzyme-free amplified electrochemical nucleic acid analysis. To demonstrate the fabrication universality three dynamic DNA assembly strategies including DNA-fueled target recycling, catalytic hairpin DNA assembly, and hybridization chain reaction were flexibly harnessed to convey the homogeneous target recognition and amplification events into various DNA scaffolds for the autonomous proximity-based surface hybridization. The current biosensor architecture features generalizability, simplicity, low cost, high sensitivity, and specificity over the traditional nucleic acid-related amplified biosensors. The lowest detection limit of 50 aM toward target DNA could be achieved by hybridization chain reaction-programmed surface hybridization. The reliable working ability for both homogeneous solution and heterogeneous inteface facilitates the target analysis with a robust reliability and reproducibility, also making it to be readily extended for the integration with the kinds of detecting platforms. Thus, it may hold great potential for the biosensor fabrication served for the point-of-care applications in resource constrained regions.

  12. Case Studies in Systems Chemistry. Final Report. [Includes Complete Case Study, Carboxylic Acid Equilibria

    ERIC Educational Resources Information Center

    Fleck, George

    This publication was produced as a teaching tool for college chemistry. The book is a text for a computer-based unit on the chemistry of acid-base titrations, and is designed for use with FORTRAN or BASIC computer systems, and with a programmable electronic calculator, in a variety of educational settings. The text attempts to present computer…

  13. Kinetic model of water disinfection using peracetic acid including synergistic effects.

    PubMed

    Flores, Marina J; Brandi, Rodolfo J; Cassano, Alberto E; Labas, Marisol D

    2016-01-01

    The disinfection efficiencies of a commercial mixture of peracetic acid against Escherichia coli were studied in laboratory scale experiments. The joint and separate action of two disinfectant agents, hydrogen peroxide and peracetic acid, were evaluated in order to observe synergistic effects. A kinetic model for each component of the mixture and for the commercial mixture was proposed. Through simple mathematical equations, the model describes different stages of attack by disinfectants during the inactivation process. Based on the experiments and the kinetic parameters obtained, it could be established that the efficiency of hydrogen peroxide was much lower than that of peracetic acid alone. However, the contribution of hydrogen peroxide was very important in the commercial mixture. It should be noted that this improvement occurred only after peracetic acid had initiated the attack on the cell. This synergistic effect was successfully explained by the proposed scheme and was verified by experimental results. Besides providing a clearer mechanistic understanding of water disinfection, such models may improve our ability to design reactors.

  14. Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

    PubMed Central

    Kasarda, D D; Okita, T W; Bernardin, J E; Baecker, P A; Nimmo, C C; Lew, E J; Dietler, M D; Greene, F C

    1984-01-01

    The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently. Images PMID:6589619

  15. Genomic DNA Methylation Changes in Response to Folic Acid Supplementation in a Population-Based Intervention Study among Women of Reproductive Age

    PubMed Central

    Berry, Robert J.; Hao, Ling; Li, Zhu; Maneval, David; Yang, Thomas P.; Rasmussen, Sonja A.; Yang, Quanhe; Zhu, Jiang-Hui; Hu, Dale J.; Bailey, Lynn B.

    2011-01-01

    Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (−14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation. PMID:22163281

  16. DNA Diagnostics: Nanotechnology-enhanced Electrochemical Detection of Nucleic Acids

    PubMed Central

    Wei, Fang; Lillehoj, Peter B.; Ho, Chih-Ming

    2010-01-01

    The detection of mismatched base pairs in DNA plays a crucial role in the diagnosis of genetic-related diseases and conditions, especially for early stage treatment. Among the various biosensors that have been employed for DNA detection, electrochemical sensors show great promise since they are capable of precise DNA recognition and efficient signal transduction. Advancements in micro- and nanotechnologies, specifically fabrication techniques and new nanomaterials, have enabled for the development of highly sensitive, highly specific sensors making them attractive for the detection of small sequence variations. Furthermore, the integration of sensors with sample preparation and fluidic processes enables for rapid, multiplexed DNA detection for point-of-care (POC) clinical diagnostics. PMID:20075759

  17. Export of aquatic productivity, including highly unsaturated fatty acids, to terrestrial ecosystems via Odonata.

    PubMed

    Popova, Olga N; Haritonov, Anatoly Y; Sushchik, Nadezhda N; Makhutova, Olesia N; Kalachova, Galina S; Kolmakova, Anzhelika A; Gladyshev, Michail I

    2017-03-01

    Based on 31-year field study of the abundance and biomass of 18 species of odonates in the Barabinsk Forest-Steppe (Western Siberia, Russia), we quantified the contribution of odonates to the export of aquatic productivity to surrounding terrestrial landscape. Emergence varied from 0.8 to 4.9g of wet biomass per m(2) of land area per year. Average export of organic carbon was estimated to be 0.30g·m(-2)·year(-1), which is comparable with the average production of herbivorous terrestrial insects in temperate grasslands. Moreover, in contrast to terrestrial insects, emerging odonates contained high quantities of highly unsaturated fatty acids (HUFA), namely eicosapentaenoic acid (20:5n-3, EPA), and docosahexaenoic acid (22:6n-3, DHA), which are known to be essential for many terrestrial animals, especially for birds. The export of EPA+DHA by odonates was found to be 1.92-11.76mg·m(-2)·year(-1), which is equal to an average general estimation of the export of HUFA by emerging aquatic insects. Therefore, odonates appeared to be a quantitatively and qualitatively important conduit of aquatic productivity to forest-steppe ecosystem.

  18. [Studies on the interaction of the metal complex of hydrazide of podophyllic acid with DNA].

    PubMed

    Wang, Ping-Hong; Zhang, Qi; Wang, Liu-Fang; Song, Yu-Min; Qu, Jian-Qiang; Liu, Ying-Qian

    2006-05-01

    The interaction between the metal complex of hydrazide of podophyllic acid and calf thymus (CT) DNA was studied by using absorption spectra, fluorescence spectra and DNA heat denaturation. It was found that the intensity of the maximal absorption peaks from absorption spectra is weakened in the presence of the metal complex of hydrazide of podophyllic acid compared with that in the absence of the metal complex. All the experimental results show that the intercalation mode was proved to exist between HDPP-Ni complexes and CT DNA.

  19. The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence

    PubMed Central

    Lorenc, Michał T.; Dudley, Kevin J.; Hellens, Roger P.

    2017-01-01

    Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5’ terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker. PMID:28231340

  20. The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence.

    PubMed

    Philips, Joshua G; Naim, Fatima; Lorenc, Michał T; Dudley, Kevin J; Hellens, Roger P; Waterhouse, Peter M

    2017-01-01

    Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5' terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker.

  1. A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes.

    PubMed Central

    Sturrock, S S; Dryden, D T

    1997-01-01

    The S subunits of type I DNA restriction/modification enzymes are responsible for recognising the DNA target sequence for the enzyme. They contain two domains of approximately 150 amino acids, each of which is responsible for recognising one half of the bipartite asymmetric target. In the absence of any known tertiary structure for type I enzymes or recognisable DNA recognition motifs in the highly variable amino acid sequences of the S subunits, it has previously not been possible to predict which amino acids are responsible for sequence recognition. Using a combination of sequence alignment and secondary structure prediction methods to analyse the sequences of S subunits, we predict that all of the 51 known target recognition domains (TRDs) have the same tertiary structure. Furthermore, this structure is similar to the structure of the TRD of the C5-cytosine methyltransferase, Hha I, which recognises its DNA target via interactions with two short polypeptide loops and a beta strand. Our results predict the location of these sequence recognition structures within the TRDs of all type I S subunits. PMID:9254696

  2. Spermatozoa bound to solid state hyaluronic acid show chromatin structure with high DNA chain integrity: an acridine orange fluorescence study.

    PubMed

    Yagci, Artay; Murk, William; Stronk, Jill; Huszar, Gabor

    2010-01-01

    During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% ± 2.0% and 45.0% ± 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection.

  3. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  4. Amino acid racemization in amber-entombed insects: implications for DNA preservation

    NASA Technical Reports Server (NTRS)

    Bada, J. L.; Wang, X. S.; Poinar, H. N.; Paabo, S.; Poinar, G. O.

    1994-01-01

    DNA depurination and amino acid racemization take place at similar rates in aqueous solution at neutral pH. This relationship suggests that amino acid racemization may be useful in accessing the extent of DNA chain breakage in ancient biological remains. To test this suggestion, we have investigated the amino acids in insects entombed in fossilized tree resins ranging in age from <100 years to 130 million years. The amino acids present in 40 to 130 million year old amber-entombed insects resemble those in a modern fly and are probably the most ancient, unaltered amino acids found so far on Earth. In comparison to other geochemical environments on the surface of the Earth, the amino acid racemization rate in amber insect inclusions is retarded by a factor of >10(4). These results suggest that in amber insect inclusions DNA depurination rates would also likely be retarded in comparison to aqueous solution measurements, and thus DNA fragments containing many hundreds of base pairs should be preserved. This conclusion is consistent with the reported successful retrieval of DNA sequences from amber-entombed organisms.

  5. Protection of folic acid through encapsulation in mesoporous silica particles included in fruit juices.

    PubMed

    Ruiz-Rico, María; Pérez-Esteve, Édgar; Lerma-García, María J; Marcos, María D; Martínez-Máñez, Ramón; Barat, José M

    2017-03-01

    Folic acid (FA) is a synthetic vitamin commonly used for food fortification. However, its vulnerability to processing and storage implies loss of efficiency, which would induce over-fortification by processors to obtain a minimum dose upon consumption. Recent studies have indicated potential adverse effects of FA overdoses, and FA protection during processing and storage could lead to more accurate fortification. In addition, sustained vitamin release after consumption would help improve its metabolism. The objective of this work was to study controlled FA delivery and stability in fruit juices to reduce potential over-fortification risks by using gated mesoporous silica particles (MSPs). The obtained results indicated that FA encapsulation in MSPs significantly improved its stability and contributed to controlled release after consumption by modifying vitamin bioaccessibility. These results confirmed the suitability of MSPs as support for controlled release and protection of bioactive molecules in food matrices in different food production and storage stages.

  6. Method Optimization of Deoxyribonucleic Acid (DNA) Thin Films for Biotronics

    DTIC Science & Technology

    2011-09-01

    Added to the Spin-coater ......................................................................4 3.3 Comparison of Spin - Coating Speed and Sample...precipitate after centrifugation. ..............................3 Figure 3. Diagram of spin - coating method. First, the DNA-CTMA solution was pipetted onto... spin - coating speeds. ...................................................................................................................6 Figure 5

  7. Uracil misincorporation into DNA and folic acid supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE: We evaluated the relation between UrMis in different ...

  8. Fate of N-nitrosodimethylamine, trihalomethane and haloacetic acid precursors in tertiary treatment including biofiltration.

    PubMed

    Farré, Maria José; Reungoat, Julien; Argaud, Francois Xavier; Rattier, Maxime; Keller, Jürg; Gernjak, Wolfgang

    2011-11-01

    The presence of disinfection by-products (DBPs) such as trihalomethanes (THMs), haloacetic acids (HAAs) and N-nitrosamines in water is of great concern due to their adverse effects on human health. In this work, the removal of N-nitrosodimethylamine (NDMA), total THM and five HAA precursors from secondary effluent by biological activated carbon (BAC) is investigated at full and pilot scale. In the pilot plant two filter media, sand and granular activated carbon, are tested. In addition, we evaluate the influence of ozonation prior to BAC filtration on its performance. Among the bulk of NDMA precursors, the fate of four pharmaceuticals containing a dimethylamino moiety in the chemical structure are individually investigated. Both NDMA formation potential and each of the studied pharmaceuticals are dramatically reduced by the BAC even in the absence of main ozonation prior to the filtration. The low removal of NDMA precursors at the sand filtration in comparison to the removal of NDMA precursors at the BAC suggests that adsorption may play an important role on the removal of NDMA precursors by BAC. Contrary, the precursors for THM and HAA formation are reduced in both sand filtration and BAC indicating that the precursors for the formation of these DBPs are to some extent biodegradable.

  9. RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

    PubMed Central

    Sirois, Cherilyn M.; Jin, Tengchuan; Miller, Allison L.; Bertheloot, Damien; Nakamura, Hirotaka; Horvath, Gabor L.; Mian, Abubakar; Jiang, Jiansheng; Schrum, Jacob; Bossaller, Lukas; Pelka, Karin; Garbi, Natalio; Brewah, Yambasu; Tian, Jane; Chang, ChewShun; Chowdhury, Partha S.; Sims, Gary P.; Kolbeck, Roland; Coyle, Anthony J.; Humbles, Alison A.

    2013-01-01

    Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo. PMID:24081950

  10. [The effect of spermine on acid-base equilibrium in DNA molecule].

    PubMed

    Slonitskiĭ, S V; Kuptsov, V Iu

    1990-01-01

    The influence of spermine (Sp) on the acid-induced predenaturational and denaturational transitions in the DNA molecule structure has been studied by means of circular dichroism, spectrophotometric and viscometric titration at supporting electrolyte concentration 10 mM NaCl. The data available indicate that at [N]/[P] less than or equal to 0.60 (here [N] and [P] are molar concentrations of Sp nitrogen and DNA phosphours, respectively) the cooperative structural B----B(+)----S transitions are accompanied by the DNA double-helice winding. No competition for proton acceptor sites in the DNA molecule between H+ and Sp4+ cations has been observed when binding to neutral macromolecule. At 0.60 less than or equal to [N]/[P] less than or equal to 0.75 the displacement of the B----B(+)----S transitions midpoints to acidic pH region has been established. This is accompanied by DNA condensation and the appearance of differential scattering of circularly polarized light. The calculations carried out in the framework of the two-variable Manning theory have shown that the acid-induced reduction of the effective polyion charge density facilitates the Sp-induced DNA condensation. It has been shown that the acid-base equilibrium in the DNA molecule is determined by local [H+] in the 2-3 A hydrated monolayer of the macromolecule. An adequate estimation of [H+] can be obtained on the basis of the Poisson-Boltzman approach. The data obtained are consistent with recently proposed hypothesis of polyelectrolyte invariance of the acid-base equilibrium in the DNA molecule.

  11. The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

    PubMed Central

    Senthong, Pattama; Millington, Christopher L.; Wilkinson, Oliver J.; Marriott, Andrew S.; Watson, Amanda J.; Reamtong, Onrapak; Eyers, Claire E.; Williams, David M.; Margison, Geoffrey P.; Povey, Andrew C.

    2013-01-01

    The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O6-carboxymethylguanine (O6-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O6-CMG is not a substrate for the human version of the DNA damage reversal protein O6-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O6-alkylguanine lesions by removing alkyl groups from the O6-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O6-methylguanine (O6-MeG) or O6-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O6-alkyl group and its transfer to the active-site cysteine residue of MGMT. O6-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O6-CMG is a potential causative agent. PMID:23335782

  12. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes.

    PubMed

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2'-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of -22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL(-1). In the presence of divalent Ca(2+), the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca(2+)/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca(2+) and the synthesized copolymer on DNA transfection was observed. The use of Ca(2+) or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca(2+)/DNA polyplex could serve as a promising candidate for gene delivery.

  13. Identification of dairy lactic acid bacteria by tRNAAla-23S rDNA-RFLP.

    PubMed

    Mancini, Andrea; Lazzi, Camilla; Bernini, Valentina; Neviani, Erasmo; Gatti, Monica

    2012-12-01

    The aim of this study was to evaluate the potential of target tRNA(Ala)-23S ribosomal DNA for identification of lactic acid bacteria strains associated with dairy ecosystem. For this purpose tRNA(Ala)-23S ribosomal DNA Restriction Fragment Length Polymorphism (tRNA(Ala)-23S rDNA-RFLP) was compared with two widely used DNA fingerprinting methods - P1 Random Amplified Polymorphic DNA (RAPD), (GTG)5 repetitive extragenic palindromic PCR (rep-PCR) - for their ability to identify different species on a set of 10 type and 34 reference strains. Moreover, 75 unknown isolates collected during different stages of Grana Padano cheese production and ripening were identified using tRNA(Ala)-23S rDNA-RFLP and compared to the RFLP profiles of the strains in the reference database. This study demonstrated that the target tRNA(Ala)-23S rDNA has high potential in bacterial identification and tRNA(Ala)-23S rDNA-RFLP is a promising method for reliable species-level identification of lactic acid bacteria (LAB) in dairy products.

  14. Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen.

    PubMed

    Suzukawa, Andréia Akemi; Vieira, Alessandra; Winnischofer, Sheila Maria Brochado; Scalfo, Alexsandra Cristina; Di Mascio, Paolo; Ferreira, Ana Maria da Costa; Ravanat, Jean-Luc; Martins, Daniela de Luna; Rocha, Maria Eliane Merlin; Martinez, Glaucia Regina

    2012-05-01

    Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen ((1)O(2)), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with (1)O(2), they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT.

  15. Deoxyribonucleic acid (DNA)-Ni-nanostrands composites for EMI shielding

    NASA Astrophysics Data System (ADS)

    Ouchen, Fahima; Wilson, Benjamin G.; Yaney, Perry P.; Salour, Michael M.; Grote, James G.

    2016-09-01

    In this study, we demonstrated the use of DNA-CTMA (DC) in combination with Nickel Nanostrands (NiNs) for application in Electromagnetic Interference (EMI) shielding. The addition of NiNs fillers to DC led to films with higher shielding effectiveness (SE) than when Silver nanoparticles were used. An enhanced EMI shielding effectiveness (SE) was also achieved by the fabrication of the DC-NiNs shielding film structure in a layered architecture. Very thin layer of Guanine ( 60 nm) were inserted between layers of DNA-NiNs ( 100um each) to total a thickness of 500um of the shielding film. An increase of the SE by 6-8 dB for the layered structure as compared to the bulk thick film with NiNs loadings up to 10 wt%. At higher loadings (>10 wt. %), a significant physical degradation of the films was observed for all films regardless of the thickness or the process of fabrication.

  16. Simultaneous optimization of monolayer formation factors, including temperature, to significantly improve nucleic acid hybridization efficiency on gold substrates.

    PubMed

    Pris, Andrew D; Ostrowski, Sara G; Garaas, Sarah D

    2010-04-20

    Past literature investigations have optimized various single factors used in the formation of thiolated, single stranded DNA (ss-DNA) monolayers on gold. In this study a more comprehensive approach is taken, where a design of experiment (DOE) is employed to simultaneously optimize all of the factors involved in construction of the capture monolayer used in a fluorescence-based hybridization assay. Statistical analysis of the fluorescent intensities resulting from the DOE provides empirical evidence for the importance and the optimal levels of traditional and novel factors included in this investigation. We report on the statistical importance of a novel factor, temperature of the system during monolayer formation of the capture molecule and lateral spacer molecule, and how proper usage of this temperature factor increased the hybridization signal 50%. An initial theory of how the physical factor of heat is mechanistically supplementing the function of the lateral spacer molecule is provided.

  17. Role of amino acid insertions on intermolecular forces between arginine peptide condensed DNA helices: implications for protamine-DNA packaging in sperm.

    PubMed

    DeRouchey, Jason E; Rau, Donald C

    2011-12-09

    In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.

  18. Longitudinal distributions of dicarboxylic acids, ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids in the marine aerosols from the central Pacific including equatorial upwelling

    NASA Astrophysics Data System (ADS)

    Hoque, Mir Md. Mozammal; Kawamura, Kimitaka

    2016-03-01

    Remote marine aerosol samples (total suspended particles) were collected during a cruise in the central Pacific from Japan to Mexico (1°59'N-35°N and 171°54'E-90°58'W). The aerosol samples were analyzed for dicarboxylic acids (C2-C11), ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids as well as organic and elemental carbon, water-soluble organic carbon, and total nitrogen (WSTN). During the study, diacids were the most abundant compound class followed by fatty acids, ω-oxoacids, and α-dicarbonyls. Molecular compositions of diacids showed a predominance of oxalic (C2) acid followed by malonic (C3) and succinic (C4) acids. Oxalic acid comprises 74% of total diacids. This result suggests that photochemical production of oxalic acid is significant over the central Pacific. Spatial distributions of diacids, ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids together with total carbon and WSTN showed higher abundances in the eastern equatorial Pacific where the upwelling of high-nutrient waters followed by high biological productivity is common, indicating that their in situ production is important in the warmer central Pacific through photochemical oxidation from their gaseous and particulate precursors. This study demonstrates that there is a strong linkage in biogeochemical cycles of carbon in the sea-air interface via ocean upwelling, phytoplankton productivity, sea-to-air emissions of organic matter, and formation of secondary organic aerosols in the eastern equatorial Pacific.

  19. Switchable mechanical DNA ``arms'' operating on nucleic acid scaffolds associated with electrodes or semiconductor quantum dots

    NASA Astrophysics Data System (ADS)

    Pelossof, Gilad; Tel-Vered, Ran; Liu, Xiaoqing; Willner, Itamar

    2013-09-01

    Functional footholds linked to DNA scaffolds associated with surfaces provide nano-engineered assemblies acting as switching devices. By the assembly of a β-cyclodextrin receptor on one foothold, and a ferrocene-modified nucleic acid on a second foothold, the switchable and reversible, fuel-driven activation of ``molecular arms'' proceeds, transduced by electrochemical or optical signals.Functional footholds linked to DNA scaffolds associated with surfaces provide nano-engineered assemblies acting as switching devices. By the assembly of a β-cyclodextrin receptor on one foothold, and a ferrocene-modified nucleic acid on a second foothold, the switchable and reversible, fuel-driven activation of ``molecular arms'' proceeds, transduced by electrochemical or optical signals. Electronic supplementary information (ESI) available: Experimental procedures, time-dependent deactivation of a DNA ``arm'' using a DNA anti-fuel, and control experiments, excluding β-cyclodextrin from the systems. See DOI: 10.1039/c3nr02653a

  20. Molecular phylogenetics of subfamily Ornithogaloideae (Hyacinthaceae) based on nuclear and plastid DNA regions, including a new taxonomic arrangement

    PubMed Central

    Martínez-Azorín, Mario; Crespo, Manuel B.; Juan, Ana; Fay, Michael F.

    2011-01-01

    Background and Aims The taxonomic arrangement within subfamily Ornithogaloideae (Hyacinthaceae) has been a matter of controversy in recent decades: several new taxonomic treatments have been proposed, based exclusively on plastid DNA sequences, and these have resulted in classifications which are to a great extent contradictory. Some authors have recognized only a single genus Ornithogalum for the whole subfamily, including 250–300 species of variable morphology, whereas others have recognized many genera. In the latter case, the genera are inevitably much smaller and they are better defined morphologically. However, some are not monophyletic as circumscribed. Methods Phylogenetic analyses of Ornithogaloideae were based on nucleotide sequences of four plastid regions (trnL intron, trnL-F spacer, rbcL and matK) and a nuclear region (ITS). Eighty species covering all relevant taxonomic groups previously recognized in the subfamily were sampled. Parsimony and Bayesian analyses were performed. The molecular data were compared with a matrix of 34 morphological characters. Key Results Combinations of plastid and nuclear data yielded phylogenetic trees which are better resolved than those obtained with any plastid region alone or plastid regions in combination. Three main clades are found, corresponding to the previously recognized tribes Albuceae, Dipcadieae and Ornithogaleae. In these, up to 19 clades are described which are definable by morphology and biogeography. These mostly correspond to previously described taxa, though some need recircumscription. Morphological characters are assessed for their diagnostic value for taxonomy in the subfamily. Conclusions On the basis of the phylogenetic analyses, 19 monophyletic genera are accepted within Ornithogaloideae: Albuca, Avonsera, Battandiera, Cathissa, Coilonox, Dipcadi, Eliokarmos, Elsiea, Ethesia, Galtonia, Honorius, Loncomelos, Melomphis, Neopatersonia, Nicipe, Ornithogalum, Pseudogaltonia, Stellarioides and

  1. Liver protective effect of ursodeoxycholic acid includes regulation of ADAM17 activity

    PubMed Central

    2013-01-01

    Background Ursodeoxycholic acid (UDCA) is used to treat primary biliary cirrhosis, intrahepatic cholestasis, and other cholestatic conditions. Although much has been learned about the molecular basis of the disease pathophysiology, our understanding of the effects of UDCA remains unclear. Possibly underlying its cytoprotective, anti-apoptotic, anti-oxidative effects, UDCA was reported to regulate the expression of TNFα and other inflammatory cytokines. However, it is not known if this effect involves also modulation of ADAM family of metalloproteinases, which are responsible for release of ectodomains of inflammatory cytokines from the cell surface. We hypothesized that UDCA modulates ADAM17 activity, resulting in amelioration of cholestasis in a murine model of bile duct ligation (BDL). Methods The effect of UDCA on ADAM17 activity was studied using the human liver hepatocellular carcinoma cell line HepG2. Untransfected cells or cells ectopically expressing human ADAM17 were cultured with or without UDCA and further activated using phorbol-12-myristate-13-acetate (PMA). The expression and release of ADAM17 substrates, TNFα, TGFα, and c-Met receptor (or its soluble form, sMet) were evaluated using ELISA and quantitative real-time (qRT) PCR. Immunoblotting analyses were conducted to evaluate expression and activation of ADAM17 as well as the level of ERK1/2 phosphorylation after UDCA treatment. The regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by UDCA was studied using zymography and qRT-PCR. A mouse model of acute cholestasis was induced by common BDL technique, during which mice received daily orogastric gavage with either UDCA or vehicle only. Liver injury was quantified using alkaline phosphatase (ALP), relative liver weight, and confirmed by histological analysis. ADAM17 substrates in sera were assessed using a bead multiplex assay. Results UDCA decreases amount of shed TNFα, TGFα, and sMet in cell culture media and the phosphorylation of

  2. Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology

    PubMed Central

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

  3. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  4. Calculation on spectrum of direct DNA damage induced by low-energy electrons including dissociative electron attachment.

    PubMed

    Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

    2017-03-01

    In this work, direct DNA damage induced by low-energy electrons (sub-keV) is simulated using a Monte Carlo method. The characteristics of the present simulation are to consider the new mechanism of DNA damage due to dissociative electron attachment (DEA) and to allow determining damage to specific bases (i.e., adenine, thymine, guanine, or cytosine). The electron track structure in liquid water is generated, based on the dielectric response model for describing electron inelastic scattering and on a free-parameter theoretical model and the NIST database for calculating electron elastic scattering. Ionization cross sections of DNA bases are used to generate base radicals, and available DEA cross sections of DNA components are applied for determining DNA-strand breaks and base damage induced by sub-ionization electrons. The electron elastic scattering from DNA components is simulated using cross sections from different theoretical calculations. The resulting yields of various strand breaks and base damage in cellular environment are given. Especially, the contributions of sub-ionization electrons to various strand breaks and base damage are quantitatively presented, and the correlation between complex clustered DNA damage and the corresponding damaged bases is explored. This work shows that the contribution of sub-ionization electrons to strand breaks is substantial, up to about 40-70%, and this contribution is mainly focused on single-strand break. In addition, the base damage induced by sub-ionization electrons contributes to about 20-40% of the total base damage, and there is an evident correlation between single-strand break and damaged base pair A-T.

  5. Superimposed Code Theoretic Analysis of Deoxyribonucleic Acid (DNA) Codes and DNA Computing

    DTIC Science & Technology

    2010-01-01

    hybridization that occurs between a DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research addresses how the...are 5′→3′ and strands with strikethrough are 3′→5′. A dsDNA duplex formed between a strand and its reverse complement is called a Watson - Crick (WC...3’ 5’ 3’ 5’TACGCGACTTTC3’ 5’GAAAGTCGCGTA3’ ATCAAACGATGC GCATCGTTTGAT Watson Crick (WC) Duplexes TACGCGACTTTC

  6. Associations between whole peripheral blood fatty acids and DNA methylation in humans

    PubMed Central

    de la Rocha, Carmen; Pérez-Mojica, J. Eduardo; León, Silvia Zenteno-De; Cervantes-Paz, Braulio; Tristán-Flores, Fabiola E.; Rodríguez-Ríos, Dalia; Molina-Torres, Jorge; Ramírez-Chávez, Enrique; Alvarado-Caudillo, Yolanda; Carmona, F. Javier; Esteller, Manel; Hernández-Rivas, Rosaura; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio; Lund, Gertrud

    2016-01-01

    Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5’UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage. PMID:27181711

  7. Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

    PubMed Central

    Fojta, M; Vetterl, V; Tomschik, M; Jelen, F; Nielsen, P; Wang, J; Palecek, E

    1997-01-01

    Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. PMID:9129832

  8. Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

    PubMed

    Yao, Dongbao; Song, Tingjie; Sun, Xianbao; Xiao, Shiyan; Huang, Fujian; Liang, Haojun

    2015-11-11

    Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures.

  9. Binding of retinoic acid receptor heterodimers to DNA. A role for histones NH2 termini.

    PubMed

    Lefebvre, P; Mouchon, A; Lefebvre, B; Formstecher, P

    1998-05-15

    The retinoic acid signaling pathway is controlled essentially through two types of nuclear receptors, RARs and RXRs. Ligand dependent activation or repression of retinoid-regulated genes is dependent on the binding of retinoic acid receptor (RAR)/9-cis-retinoic acid receptor (RXR) heterodimers to retinoic acid response element (RARE). Although unliganded RXR/RAR heterodimers bind constitutively to DNA in vitro, a clear in vivo ligand-dependent occupancy of the RARE present in the RARbeta2 gene promoter has been reported (Dey, A., Minucci, S., and Ozato, K. (1994) Mol. Cell. Biol. 14, 8191-8201). Nucleosomes are viewed as general repressors of the transcriptional machinery, in part by preventing the access of transcription factors to DNA. The ability of hRXRalpha/hRARalpha heterodimers to bind to a nucleosomal template in vitro has therefore been examined. The assembly of a fragment from the RARbeta2 gene promoter, which contains a canonical DR5 RARE, into a nucleosome core prevented hRXRalpha/hRARalpha binding to this DNA, in conditions where a strong interaction is observed with a linear DNA template. However, histone tails removal by limited proteolysis and histone hyperacetylation yielded nucleosomal RAREs able to bind to hRXRalpha/hRARalpha heterodimers. These data establish therefore the role of histones NH2 termini as a major impediment to retinoid receptors access to DNA, and identify histone hyperacetylation as a potential physiological regulator of retinoid-induced transcription.

  10. Fecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular single-stranded DNA viruses.

    PubMed

    Lima, Diane; Cibulski, Samuel Paulo; Finkler, Fabrine; Teixeira, Thais; Varela, Ana Paula; Cerva, Cristine; Loiko, Márcia; Scheffer, Camila; Dos Santos, Helton; Mayer, Fabiana; Roehe, Paulo

    2017-01-18

    This study is focused on the identification of the fecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7,743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with BLASTx revealed that 279 contigs (4%) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes. In addition, a range of circular replication-associated protein encoding (CRESS) DNA viruses, were also identified. The characterization of the fecal virome of healthy chickens described here provides not only a description of the viruses encountered in such niche but should also represent a baseline for future studies comparing viral populations in healthy and diseased chicken flocks. Moreover, it may also be relevant for human health, since chickens represent a significant proportion of the animal protein consumed worldwide.

  11. Molecular dynamics simulations of G-DNA and perspectives on the simulation of nucleic acid structures

    PubMed Central

    šponer, Jiří; Cang, Xiaohui; Cheatham, Thomas E.

    2013-01-01

    The article reviews the application of biomolecular simulation methods to understand the structure, dynamics and interactions of nucleic acids with a focus on explicit solvent molecular dynamics simulations of guanine quadruplex (G-DNA and G-RNA) molecules. While primarily dealing with these exciting and highly relevant four-stranded systems, where recent and past simulations have provided several interesting results and novel insight into G-DNA structure, the review provides some general perspectives on the applicability of the simulation techniques to nucleic acids. PMID:22525788

  12. Zinc complexes of the antibacterial drug oxolinic acid: structure and DNA-binding properties.

    PubMed

    Tarushi, Alketa; Psomas, George; Raptopoulou, Catherine P; Kessissoglou, Dimitris P

    2009-06-01

    The neutral mononuclear zinc complexes with the quinolone antibacterial drug oxolinic acid in the absence or presence of a nitrogen donor heterocyclic ligand 2,2'-bipyridine or 1,10-phenanthroline have been synthesized and characterized. The experimental data suggest that oxolinic acid is on deprotonated mode acting as a bidentate ligand coordinated to the metal ion through the ketone and one carboxylato oxygen atoms. The crystal structures of (chloro)(oxolinato)(2,2'-bipyridine)zinc(II), 2, and bis(oxolinato)(1,10-phenanthroline)zinc(II), 3, have been determined with X-ray crystallography. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the DNA-binding constants have been calculated. Competitive studies with ethidium bromide (EB) have shown that complex 3 exhibits the ability to displace the DNA-bound EB indicating that it binds to DNA in strong competition with EB.

  13. In silico comparative analysis of DNA and amino acid sequences for prion protein gene.

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences.

  14. Mechanism of action of nalidixic acid: Purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme

    PubMed Central

    Sugino, Akio; Peebles, Craig L.; Kreuzer, Kenneth N.; Cozzarelli, Nicholas R.

    1977-01-01

    A target protein for nalidixic and oxolinic acids in Escherichia coli, the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A polypeptide of this molecular weight is uniquely induced by a λ nalA transducing phage, thereby showing that the purified Pnal is a product of the nalA gene. Nalidixic and oxolinic acids inhibit DNA gyrase activity and induce formation of a relaxation complex analogue. Treatment of the complex with sodium dodecyl sulfate causes a doublestrand break in the DNA substrate and the resulting linear molecule seems covalently bound to protein. Complex formation, unlike the introduction of supertwists, does not require ATP or relaxed circular DNA and is insensitive to novobiocin. DNA gyrase from a strain with a nalA mutation conferring drug resistance (nalAr) is 1/100 as sensitive to oxolinic and nalidixic acids with respect to inhibition of supertwisting and induction of the pre-linearization complex. Addition of Pnal restores drug sensitivity and stimulates DNA gyrase activity. DNA gyrase preparations and Pnal catalyze a third reaction sensitive to nalidixic and oxolinic acids, the ATP-independent relaxation of supertwister DNA. Relaxation by gyrase from nalAr cells is drug resistant. The nicking-closing activity is distinct from E. coli ω protein in several properties, including the ability to relax positively supertwisted DNA. We postulate that the nalA gene product occurs in two molecular forms, as Pnal and as a gyrase component. Both forms catalyze nicking-closing, and inhibition of this activity by nalidixic and oxolinic acids may account for the inhibition of DNA synthesis by these drugs. Images PMID:200930

  15. Endosomal Escape and Transfection Efficiency of PEGylated Cationic Lipid–DNA Complexes Prepared with an Acid-Labile PEG-Lipid

    PubMed Central

    Chan, Chia-Ling; Majzoub, Ramsey N.; Shirazi, Rahau S.; Ewert, Kai K.; Chen, Yen-Ju; Liang, Keng S.

    2012-01-01

    Cationic liposome–DNA (CL–DNA) complexes are being pursued as nonviral gene delivery systems for use in applications that include clinic trials. However, to compete with viral vectors for systemic delivery in vivo, their efficiencies and pharmacokinetics need to be improved. The addition of poly (ethylene glycol)-lipids (PEGylation) prolongs circulation lifetimes of liposomes, but inhibits cellular uptake and endosomal escape of CL–DNA complexes. We show that this limits their transfection efficiency (TE) in a manner dependent on the amount of PEG-lipid, the lipid/DNA charge ratio, and the lipid membrane charge density. To improve endosomal escape of PEGylated CL–DNA complexes, we prepared an acid-labile PEG-lipid (HPEG2K-lipid, PEG MW 2000) which is designed to lose its PEG chains at the pH of late endosomes. The HPEG2K-lipid and a similar but acid-stable PEG-lipid were used to prepare PEGylated CL–DNA complexes. TLC and dynamic light scattering showed that HPEG2K-CL–DNA complexes are stable at pH 7.4 for more than 24 hours, but the PEG chains are cleaved at pH 5 within one hour, leading to complex aggregation. The acid-labile HPEG2K-CL–DNA complexes showed enhanced TE over complexes stabilized with the acid-stable PEG-lipid. Live-cell imaging showed that both types of complexes were internalized to quantitatively similar particle distributions within the first 2 hours of incubation with cells. Thus, we attribute the increased TE of the HPEG2K-CL–DNA complexes to efficient endosomal escape, enabled by the acid-labile HPEG2K-lipid which sheds its PEG chains in the low-pH environment of late endosomes, effectively switching on the electrostatic interactions that promote fusion of the membranes of complex and endosome. PMID:22469293

  16. DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Brown, W. Ted; Junaid, Mohammed A.

    2016-01-01

    It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases. PMID:27199632

  17. Potential cytoprotection: antioxidant defence by caffeic acid phenethyl ester against free radical-induced damage of lipids, DNA, and proteins.

    PubMed

    Wang, Ting; Chen, Lixiang; Wu, Weimin; Long, Yuan; Wang, Rui

    2008-05-01

    Oxidative stress is considered to be a major cause of cellular injuries in a variety of chronic health problems, such as carcinogenesis and neurodegenerative disorders. Caffeic acid phenethyl ester (CAPE), derived from the propolis of honeybee hives, possesses a variety of biological and pharmacological properties including antioxidant and anticancer activity. In the present study, we focused on the diverse antioxidative functionalities of CAPE and its related polyphenolic acid esters on cellular macromolecules in vitro. The effects on human erythrocyte membrane ghost lipid peroxidation, plasmid pBR322 DNA, and protein damage initiated by the water-soluble initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and hydrogen peroxide (H(2)O(2)) were monitored by formation of hydroperoxides and by DNA nicking assay, single-cell alkaline electrophoresis, and SDS-polyacrylamide gel electrophoresis. Our results showed that CAPE and its related polyphenolic acid esters elicited remarkable inhibitory effects on erythrocyte membrane lipid peroxidation, cellular DNA strand breakage, and protein fragmentation. The results suggest that CAPE is a potent exogenous cytoprotective and antigenotoxic agent against cell oxidative damage that could be used as a template for designing novel drugs to combat diseases induced by oxidative stress components, such as various types of cancer.

  18. DNA base dimers are stabilized by hydrogen-bonding interactions including non-Watson-Crick pairing near graphite surfaces.

    PubMed

    Shankar, Akshaya; Jagota, Anand; Mittal, Jeetain

    2012-10-11

    Single- and double-stranded DNA are increasingly being paired with surfaces and nanoparticles for numerous applications, such as sensing, imaging, and drug delivery. Unlike the majority of DNA structures in bulk that are stabilized by canonical Watson-Crick pairing between Ade-Thy and Gua-Cyt, those adsorbed on surfaces are often stabilized by noncanonical base pairing, quartet formation, and base-surface stacking. Not much is known about these kinds of interactions. To build an understanding of the role of non-Watson-Crick pairing on DNA behavior near surfaces, one requires basic information on DNA base pair stacking and hydrogen-bonding interactions. All-atom molecular simulations of DNA bases in two cases--in bulk water and strongly adsorbed on a graphite surface--are conducted to study the relative strengths of stacking and hydrogen bond interactions for each of the 10 possible combinations of base pairs. The key information obtained from these simulations is the free energy as a function of distance between two bases in a pair. We find that stacking interactions exert the dominant influence on the stability of DNA base pairs in bulk water as expected. The strength of stability for these stacking interactions is found to decrease in the order Gua-Gua > Ade-Gua > Ade-Ade > Gua-Thy > Gua-Cyt > Ade-Thy > Ade-Cyt > Thy-Thy > Cyt-Thy > Cyt-Cyt. On the other hand, mutual interactions of surface-adsorbed base pairs are stabilized mostly by hydrogen-bonding interactions in the order Gua-Cyt > Ade-Gua > Ade-Thy > Ade-Ade > Cyt-Thy > Gua-Gua > Cyt-Cyt > Ade-Cyt > Thy-Thy > Gua-Thy. Interestingly, several non-Watson-Crick base pairings, which are commonly ignored, have similar stabilization free energies due to interbase hydrogen bonding as Watson-Crick pairs. This clearly highlights the importance of non-Watson-Crick base pairing in the development of secondary structures of oligonucleotides near surfaces.

  19. Selected nutrient contents, fatty acid composition, including conjugated linoleic acid, and retention values in separable lean from lamb rib loins as affected by external fat and cooking method.

    PubMed

    Badiani, Anna; Montellato, Lara; Bochicchio, Davide; Anfossi, Paola; Zanardi, Emanuela; Maranesi, Magda

    2004-08-11

    Proximate composition and fatty acid profile, conjugated linoleic acid (CLA) isomers included, were determined in separable lean of raw and cooked lamb rib loins. The cooking methods compared, which were also investigated for cooking yields and true nutrient retention values, were dry heating of fat-on cuts and moist heating of fat-off cuts; the latter method was tested as a sort of dietetic approach against the more traditional former type. With significantly (P < 0.05) lower cooking losses, dry heating of fat-on rib-loins produced slightly (although only rarely significantly) higher retention values for all of the nutrients considered, including CLA isomers. On the basis of the retention values obtained, both techniques led to a minimum migration of lipids into the separable lean, which was higher (P < 0.05) in dry heating than in moist heating, and was characterized by the prevalence of saturated and monounsaturated fatty acids. On the whole, the response to cooking of the class of CLA isomers (including that of the nutritionally most important isomer cis-9,trans-11) was more similar to that of the monounsaturated than the polyunsaturated fatty acids.

  20. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin.

    Arsenic causes cancer in human skin, urinary bladder, lung, liver and kidney and is a significant world-wide public health problem. Although the metabolism of inorganic arsenic is ...

  1. Design and synthesis of N-benzoyl amino acid derivatives as DNA methylation inhibitors.

    PubMed

    Garella, Davide; Atlante, Sandra; Borretto, Emily; Cocco, Mattia; Giorgis, Marta; Costale, Annalisa; Stevanato, Livio; Miglio, Gianluca; Cencioni, Chiara; Fernández-de Gortari, Eli; Medina-Franco, José L; Spallotta, Francesco; Gaetano, Carlo; Bertinaria, Massimo

    2016-11-01

    The inhibition of human DNA Methyl Transferases (DNMT) is a novel promising approach to address the epigenetic dysregulation of gene expression in different diseases. Inspired by the validated virtual screening hit NSC137546, a series of N-benzoyl amino acid analogues was synthesized and obtained compounds were assessed for their ability to inhibit DNMT-dependent DNA methylation in vitro. The biological screening allowed the definition of a set of preliminary structure-activity relationships and the identification of compounds promising for further development. Among the synthesized compounds, L-glutamic acid derivatives 22, 23, and 24 showed the highest ability to prevent DNA methylation in a total cell lysate. Compound 22 inhibited DNMT1 and DNMT3A activity in a concentration-dependent manner in the micromolar range. In addition, compound 22 proved to be stable in human serum and it was thus selected as a starting point for further biological studies.

  2. DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

    PubMed

    Li, Yueran; Chen, Xifeng; Wang, Bidou; Liu, Guangxing; Tang, Yuguo; Miao, Peng

    2016-06-07

    Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection.

  3. A Concentrated Hydrochloric Acid-based Method for Complete Recovery of DNA from Bone.

    PubMed

    Huynen, Leon; Lambert, David M

    2015-11-01

    The successful extraction of DNA from historical or ancient animal bone is important for the analysis of discriminating genetic markers. Methods used currently rely on the digestion of bone with EDTA and proteinase K, followed by purification with phenol/chloroform and silica bed binding. We have developed a simple concentrated hydrochloric acid-based method that precludes the use of phenol/chloroform purification and can lead to a several-fold increase in DNA yield when compared to other commonly used methods. Concentrated hydrochloric acid was shown to dissolve most of the undigested bone and allowed the efficient recovery of DNA fragments <100 bases in length. This method should prove useful for the recovery of DNAs from highly degraded animal bone, such as that found in historical or ancient samples.

  4. Multiple hypothesis correction is vital and undermines reported mtDNA links to diseases including AIDS, cancer, and Huntingdon's.

    PubMed

    Johnston, Iain G

    2016-09-01

    The ability to sequence mitochondrial genomes quickly and cheaply has led to an explosion in available mtDNA data. As a result, an expanding literature is exploring links between mtDNA features and susceptibility to, or prevalence of, a range of diseases. Unfortunately, this great technological power has not always been accompanied by great statistical responsibility. I will focus on one aspect of statistical analysis, multiple hypothesis correction, that is absolutely required, yet often absolutely ignored, for responsible interpretation of this literature. Many existing studies perform comparisons between incidences of a large number (N) of different mtDNA features and a given disease, reporting all those yielding p values under 0.05 as significant links. But when many comparisons are performed, it is highly likely that several p values under 0.05 will emerge, by chance, in the absence of any underlying link. A suitable correction (for example, Bonferroni correction, requiring p < 0.05/N) must, therefore, be employed to avoid reporting false positive results. The absence of such corrections means that there is good reason to believe that many links reported between mtDNA features and various diseases are false; a state of affairs that is profoundly negative both for fundamental biology and for public health. I will show that statistics matching those claimed to illustrate significant links can arise, with a high probability, when no such link exists, and that these claims should thus be discarded until results of suitable statistical reliability are provided. I also discuss some strategies for responsible analysis and interpretation of this literature.

  5. Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase.

    PubMed

    Nabel, Christopher S; Lee, Jae W; Wang, Laura C; Kohli, Rahul M

    2013-08-27

    Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID's functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID's selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2'-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID's reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2'-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID's closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2'-fluoro-RNA substrates, AID's deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID's DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA.

  6. Plasma ω-3 fatty acid levels negatively and ω-6 fatty acid levels positively associated with other cardiovascular risk factors including homocysteine in severe obese subjects.

    PubMed

    Mehmetoglu, Idris; Yerlikaya, F Hümeyra; Kurban, Sevil; Polat, Hakkı

    2012-01-01

    Obesity and homocysteine (tHcy) are important risk factors for cardiovascular diseases (CVD). Plasma omega-3 fatty acids (ω-3 FAs) and omega-6 fatty acids (ω-6 FAs) are essential fatty acids with diverse biological effects in human health and disease. We have investigated the relation of plasma ω-3 FAs and ω-6 FAs levels with other cardiovascular risk factors including tHcy in severe obese subjects. This study was performed on 96 severe obese and 65 normal weight subjects. Plasma fatty acid composition was measured by GC/MS and serum tHcy level was measured by HPLC methods. There were no differences between groups in terms of concentrations of serum tHcy, plasma ω-3 FAs, ω-6 FAs and ω-3/ω-6 ratio, whereas serum vitamin B-12 (p<0.01) and folic acid (p<0.05) levels were lower than those of the normal weight subjects. Homocysteine positively correlated with ω-6 FAs and negatively correlated with ω-3 FAs in severe obese and normal weight subjects. Serum vitamin B-12 positively correlated with ω-3 FAs (p<0.01) and ω-3/ω-6 ratio (p<0.01) and negatively correlated with ω-6 FAs (p<0.05) in severe obese subjects. Serum folic acid positively correlated with ω-3 FAs (p<0.01) in severe obese subjects. Our results suggest an association between the plasma ω-3 FAs and ω-6 FAs and serum tHcy concentrations in severe obese and normal weight subjects. Low levels vitamin B-12 and folic acid may have been responsible for the elevated tHcy levels in severe obese subjects, increasing the risk for future development of cardiovascular diseases.

  7. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes

    PubMed Central

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2′-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of −22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL−1. In the presence of divalent Ca2+, the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca2+/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca2+ and the synthesized copolymer on DNA transfection was observed. The use of Ca2+ or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca2+/DNA polyplex could serve as a promising candidate for gene delivery. PMID:25767385

  8. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

    PubMed

    Wagler, Patrick; Minero, Gabriel Antonio S; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S

    2015-10-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems.

  9. DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase.

    PubMed

    Sabouri, Nasim; McDonald, Karin R; Webb, Christopher J; Cristea, Ileana M; Zakian, Virginia A

    2012-03-15

    Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

  10. FT-midIR determination of fatty acid profiles, including trans fatty acids, in bakery products after focused microwave-assisted Soxhlet extraction.

    PubMed

    Ruiz-Jiménez, J; Priego-Capote, F; Luque de Castro, M D

    2006-08-01

    A study of the feasibility of Fourier transform medium infrared spectroscopy (FT-midIR) for analytical determination of fatty acid profiles, including trans fatty acids, is presented. The training and validation sets-75% (102 samples) and 25% (36 samples) of the samples once the spectral outliers have been removed-to develop FT-midIR general equations, were built with samples from 140 commercial and home-made bakery products. The concentration of the analytes in the samples used for this study is within the typical range found in these kinds of products. Both sets were independent; thus, the validation set was only used for testing the equations. The criterion used for the selection of the validation set was samples with the highest number of neighbours and the most separation between them (H<0.6). Partial least squares regression and cross validation were used for multivariate calibration. The FT-midIR method does not require post-extraction manipulation and gives information about the fatty acid profile in two min. The 14:0, 16:0, 18:0, 18:1 and 18:2 fatty acids can be determined with excellent precision and other fatty acids with good precision according to the Shenk criteria, R (2)>/=0.90, SEP=1-1.5 SEL and R (2)=0.70-0.89, SEP=2-3 SEL, respectively. The results obtained with the proposed method were compared with those provided by the conventional method based on GC-MS. At 95% significance level, the differences between the values obtained for the different fatty acids were within the experimental error.

  11. DNA damage and oxidative stress induced by acetylsalicylic acid in Daphnia magna.

    PubMed

    Gómez-Oliván, Leobardo Manuel; Galar-Martínez, Marcela; Islas-Flores, Hariz; García-Medina, Sandra; SanJuan-Reyes, Nely

    2014-08-01

    Acetylsalicylic acid is a nonsteroidal anti-inflammatory widely used due to its low cost and high effectiveness. This compound has been found in water bodies worldwide and is toxic to aquatic organisms; nevertheless its capacity to induce oxidative stress in bioindicators like Daphnia magna remains unknown. This study aimed to evaluate toxicity in D. magna induced by acetylsalicylic acid in water, using oxidative stress and DNA damage biomarkers. An acute toxicity test was conducted in order to determine the median lethal concentration (48-h LC50) and the concentrations to be used in the subsequent subacute toxicity test in which the following biomarkers were evaluated: lipid peroxidation, oxidized protein content, activity of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, and level of DNA damage. Lipid peroxidation level and oxidized protein content were significantly increased (p<0.05), and antioxidant enzymes significantly altered with respect to controls; while the DNA damage were significantly increased (p<0.05) too. In conclusion, acetylsalicylic acid induces oxidative stress and DNA damage in D. magna.

  12. Novel molecular beacon DNA probes for protein-nucleic acid interaction studies

    NASA Astrophysics Data System (ADS)

    Li, Jianwei J.; Perlette, John; Fang, Xiaohong; Kelley, Shannon; Tan, Weihong

    2000-03-01

    We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 107 M-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

  13. Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics.

    PubMed

    Rauch, J; Massicotte, H; Tannenbaum, H

    1985-01-01

    Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.

  14. Anti-Fatigue Effect by Peptide Fraction from Protein Hydrolysate of Croceine Croaker (Pseudosciaena crocea) Swim Bladder through Inhibiting the Oxidative Reactions including DNA Damage

    PubMed Central

    Zhao, Yu-Qin; Zeng, Li; Yang, Zui-Su; Huang, Fang-Fang; Ding, Guo-Fang; Wang, Bin

    2016-01-01

    The swim bladder of the croceine croaker (Pseudosciaena crocea) was believed to have good curative effects in various diseases, including amnesia, insomnia, dizziness, anepithymia, and weakness after giving birth, in traditional Chinese medicine. However, there is no research focusing on the antioxidant and anti-fatigue peptides from croceine croaker swim bladders at present. Therefore, the purpose of this study was to investigate the bioactivities of peptide fractions from the protein hydrolysate of croceine croaker related to antioxidant and anti-fatigue effects. In the study, swim bladder peptide fraction (SBP-III-3) was isolated from the protein hydrolysate of the croceine croaker, and its antioxidant and anti-fatigue activities were measured using in vitro and in vivo methods. The results indicated that SBP-III-3 exhibited good scavenging activities on hydroxyl radicals (HO•) (EC50 (the concentration where a sample caused a 50% decrease of the initial concentration of HO•) = 0.867 mg/mL), 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) (EC50 = 0.895 mg/mL), superoxide anion radical (O2−•) (EC50 = 0.871 mg/mL), and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical (ABTS+•) (EC50 = 0.346 mg/mL). SBP-III-3 also showed protective effects on DNA damage in a concentration-effect manner and prolonged the swimming time to exhaustion of Institute of Cancer Research (ICR) mice by 57.9%–107.5% greater than that of the control. SBP-III-3 could increase the levels of muscle glucose (9.4%–115.2% increase) and liver glycogen (35.7%–157.3%), and decrease the levels of blood urea nitrogen (BUN), lactic acid (LA), and malondialdehyde (MDA) by 16.4%–22.4%, 13.9%–20.1%, and 28.0%–53.6%, respectively. SBP-III-3 also enhanced the activity of lactic dehydrogenase to scavenge excessive LA for slowing the development of fatigue. In addition, SBP-III-3 increased the activities superoxide dismutase, catalase, and glutathione peroxidase to reduce the

  15. Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

    PubMed

    Bai, Xiaoguang; Talukder, Poulami; Daskalova, Sasha M; Roy, Basab; Chen, Shengxi; Li, Zhongxian; Dedkova, Larisa M; Hecht, Sidney M

    2017-03-17

    Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

  16. Chase the direct impact of rainfall into groundwater in Mt. Fuji from multiple analyses including microbial DNA

    NASA Astrophysics Data System (ADS)

    Kato, Kenji; Sugiyama, Ayumi; Nagaosa, Kazuyo; Tsujimura, Maki

    2016-04-01

    A huge amount of groundwater is stored in subsurface environment of Mt. Fuji, the largest volcanic mountain in Japan. Based on the concept of piston flow transport of groundwater an apparent residence time was estimated to ca. 30 years by 36Cl/Cl ratio (Tosaki et al., 2011). However, this number represents an averaged value of the residence time of groundwater which had been mixed before it flushes out. We chased signatures of direct impact of rainfall into groundwater to elucidate the routes of groundwater, employing three different tracers; stable isotopic analysis (delta 18O), chemical analysis (concentration of silica) and microbial DNA analysis. Though chemical analysis of groundwater shows an averaged value of the examined water which was blended by various water with different sources and routes in subsurface environment, microbial DNA analysis may suggest the place where they originated, which may give information of the source and transport routes of the water examined. Throughout the in situ observation of four rainfall events showed that stable oxygen isotopic ratio of spring water and shallow groundwater obtained from 726m a.s.l. where the average recharge height of rainfall was between 1500 and 1800 m became higher than the values before a torrential rainfall, and the concentration of silica decreased after this event when rainfall exceeded 300 mm in precipitation of an event. In addition, the density of Prokaryotes in spring water apparently increased. Those changes did not appear when rainfall did not exceed 100 mm per event. Thus, findings shown above indicated a direct impact of rainfall into shallow groundwater, which appeared within a few weeks of torrential rainfall in the studied geological setting. In addition, increase in the density of Archaea observed at deep groundwater after the torrential rainfall suggested an enlargement of the strength of piston flow transport through the penetration of rainfall into deep groundwater. This finding was

  17. Evaluation of deoxyribonucleic acid (DNA) isolated from human bloodstains exposed to ultraviolet light, heat, humidity, and soil contamination

    SciTech Connect

    McNally, L.; Shaler, R.C.; Baird, M.; Balazs, I.; De Forest, P.; Kobilinsky, L. )

    1989-09-01

    This study was designed to analyze the effects of common environmental insults on the ability to obtain deoxyribonucleic acid (DNA) restriction fragment-length polymorphisms (RFLP) patterns from laboratory prepared specimens. The environmental conditions studied include the exposure of dried bloodstains to varying amounts of relative humidity (0, 33, 67, and 98%), heat (37{degree}C), and ultraviolet light for periods of up to five days. In addition, the effect of drying over a four-day period in whole blood collected with and without ethylenediaminetetraacetate (EDTA) was examined. The results of the study showed that, under the conditions studied, the integrity of DNA is not altered such that false RFLP patterns are obtained. The only effect observed was that the overall RFLP pattern becomes weaker, but individual RFLP fragments are neither created nor destroyed.

  18. Fatty acid and DNA analyses of Permian bacteria isolated from ancient salt crystals reveal differences with their modern relatives.

    PubMed

    Vreeland, Russell H; Rosenzweig, William D; Lowenstein, Tim; Satterfield, Cindy; Ventosa, Antonio

    2006-02-01

    The isolation of living microorganisms from primary 250-million-year-old (MYA) salt crystals has been questioned by several researchers. The most intense discussion has arisen from questions about the texture and age of the crystals used, the ability of organisms to survive 250 million years when exposed to environmental factors such as radiation and the close similarity between 16S rRNA sequences in the Permian and modern microbes. The data in this manuscript are not meant to provide support for the antiquity of the isolated bacterial strains. Rather, the data presents several comparisons between the Permian microbes and other isolates to which they appear related. The analyses include whole cell fatty acid profiling, DNA-DNA hybridizations, ribotyping, and random amplified polymorphic DNA amplification (RAPD). These data show that the Permian strains, studied here, differ significantly from their more modern relatives. These differences are accumulating in both phenotypic and molecular areas of the cells. At the fatty acid level the differences are approaching but have not reached separate species status. At the molecular level the variation appears to be distributed across the genome and within the gene regions flanking the highly conserved 16S rRNA itself. The data show that these bacteria are not identical and help to rule out questions of contamination by putatively modern strains.

  19. One-stop genomic DNA extraction by salicylic acid-coated magnetic nanoparticles.

    PubMed

    Zhou, Zhongwu; Kadam, Ulhas S; Irudayaraj, Joseph

    2013-11-15

    Salicylic acid-coated magnetic nanoparticles were prepared via a modified one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by nonspecific binding of the particles as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared with traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally friendly.

  20. Stable Valence Anions of Nucleic Acid Bases and DNA Strand Breaks Induced by Low Energy Electrons

    SciTech Connect

    Rak, Janusz; Mazurkiewicz, Kamil; Kobylecka, Monika; Storoniak, Piotr; Haranczyk, Maciej; Dabkowska, Iwona; Bachorz, Rafal A.; Gutowski, Maciej S.; Radisic, Dunja; Stokes, Sarah T.; Eustis, Soren; Wang, Di; Li, Xiang; Ko, Yeon J.; Bowen, Kit H.

    2008-05-08

    The investigation of structures and properties of nucleic acids has fascinated and challenged researchers ever since the discovery of their relation to genes. Extensive studies have been carried out on these species to unravel the mystery behind the selection of these molecules as genetic material by nature and to explain various physico-chemical properties. However, a vast pool of information is yet to be discovered. DNA constituents, mainly aromatic purine and pyrimidine bases, absorb ultraviolet irradiation efficiently, but the absorbed energy is quickly released in the form of ultrafast nonradiative decays. Recently impressive progress has been made towards the understanding of photophysical and photochemical properties of DNA fragments.

  1. Acetylsalicylic acid, aging and coronary artery disease are associated with ABCA1 DNA methylation in men

    PubMed Central

    2014-01-01

    Background Previous studies have suggested that DNA methylation contributes to coronary artery disease (CAD) risk variability. DNA hypermethylation at the ATP-binding cassette transporter A1 (ABCA1) gene, an important modulator of high-density lipoprotein cholesterol and reverse cholesterol transport, has been previously associated with plasma lipid levels, aging and CAD, but the association with CAD has yet to be replicated. Results ABCA1 DNA methylation levels were measured in leucocytes of 88 men using bis-pyrosequencing. We first showed that DNA methylation at the ABCA1 gene promoter locus is associated with aging and CAD occurrence in men (P < 0.05). The latter association is stronger among older men with CAD (≥61 years old; n = 19), who showed at least 4.7% higher ABCA1 DNA methylation levels as compared to younger men with CAD (<61 years old; n = 19) or men without CAD (n = 50; P < 0.001). Higher ABCA1 DNA methylation levels in older men were also associated with higher total cholesterol (r = 0.34, P = 0.03), low-density lipoprotein cholesterol (r = 0.32, P = 0.04) and triglyceride levels (r = 0.26, P = 0.09). Furthermore, we showed that acetylsalicylic acid therapy is associated with 3.6% lower ABCA1 DNA methylation levels (P = 0.006), independent of aging and CAD status of patients. Conclusions This study provides new evidence that the ABCA1 epigenetic profile is associated with CAD and aging, and highlights that epigenetic modifications might be a significant molecular mechanism involved in the pathophysiological processes associated with CAD. Acetylsalicylic acid treatment for CAD prevention might involve epigenetic mechanisms. PMID:25093045

  2. Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

    PubMed

    Göder, Anja; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-08-01

    Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.

  3. Auto-assembly of nanometer thick, water soluble layers of plasmid DNA complexed with diamines and basic amino acids on graphite: Greatest DNA protection is obtained with arginine.

    PubMed

    Khalil, T T; Boulanouar, O; Heintz, O; Fromm, M

    2017-02-01

    We have investigated the ability of diamines as well as basic amino acids to condense DNA onto highly ordered pyrolytic graphite with minimum damage after re-dissolution in water. Based on a bibliographic survey we briefly summarize DNA binding properties with diamines as compared to basic amino acids. Thus, solutions of DNA complexed with these linkers were drop-cast in order to deposit ultra-thin layers on the surface of HOPG in the absence or presence of Tris buffer. Atomic Force Microscopy analyses showed that, at a fixed ligand-DNA mixing ratio of 16, the mean thickness of the layers can be statistically predicted to lie in the range 0-50nm with a maximum standard deviation ±6nm, using a simple linear law depending on the DNA concentration. The morphology of the layers appears to be ligand-dependent. While the layers containing diamines present holes, those formed in the presence of basic amino acids, except for lysine, are much more compact and dense. X-ray Photoelectron Spectroscopy measurements provide compositional information indicating that, compared to the maximum number of DNA sites to which the ligands may bind, the basic amino acids Arg and His are present in large excess. Conservation of the supercoiled topology of the DNA plasmids was studied after recovery of the complex layers in water. Remarkably, arginine has the best protection capabilities whether Tris was present or not in the initial solution.

  4. Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

    PubMed Central

    Sharrocks, A D; Gille, H; Shaw, P E

    1993-01-01

    The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure. Images PMID:8417320

  5. Changes in SCD gene DNA methylation after bariatric surgery in morbidly obese patients are associated with free fatty acids

    PubMed Central

    Morcillo, Sonsoles; Martín-Núñez, Gracia Mª; García-Serrano, Sara; Gutierrez-Repiso, Carolina; Rodriguez-Pacheco, Francisca; Valdes, Sergio; Gonzalo, Montserrat; Rojo-Martinez, Gemma; Moreno-Ruiz, Francisco J.; Rodriguez-Cañete, Alberto; Tinahones, Francisco; García-Fuentes, Eduardo

    2017-01-01

    Stearoyl CoA Desaturase-1 (SCD) is considered as playing an important role in the explanation of obesity. The aim of this study was to evaluate whether the DNA methylation SCD gene promoter is associated with the metabolic improvement in morbidly obese patients after bariatric surgery. The study included 120 subjects with morbid obesity who underwent a laparoscopic Roux-en Y gastric by-pass (RYGB) and a control group of 30 obese subjects with a similar body mass index (BMI) to that found in morbidly obese subjects six months after RYGB. Fasting blood samples were obtained before and at six months after RYGB. DNA methylation was measured by pyrosequencing technology. DNA methylation levels of the SCD gene promoter were lower in morbidly obese subjects before bariatric surgery but increased after RYGB to levels similar to those found in the control group. Changes of DNA methylation SCD gene were associated with the changes of free fatty acids levels (r = −0.442, p = 0.006) and HOMA-IR (r = −0.249, p = 0.035) after surgery. RYGB produces an increase in the low SCD methylation promoter levels found in morbidly obese subjects. This change of SCD methylation levels is associated with changes in FFA and HOMA-IR. PMID:28393901

  6. Incorporation and/or adduction of formic acid with DNA in vivo studied by HPLC-AMS

    NASA Astrophysics Data System (ADS)

    Zhu, Jiadan; Cheng, Yan; Sun, Hongfang; Wang, Haifang; Li, Yuankai; Liu, Yuanfang; Ding, Xingfang; Fu, Dongpo; Liu, Kexin; Wang, Deqing; Deng, Xiaoyong

    2010-04-01

    The contribution of incorporation and/or adduction of formic acid with liver DNA in mouse was investigated using accelerator mass spectrometry (AMS) associated with high performance liquid chromatography (HPLC). Four kinds of 5'-formylated adducts, which were prepared by the reaction of formic acid and deoxyribonucleosides in vitro, were used as references for the HPLC-AMS analysis of in vivo adduction. After the administration of sodium 14C-formate to mice, the liver DNA pellets were isolated and enzymatically digested to deoxyribonucleosides. A precise analysis of the hydrolysate by HPLC-AMS indicates that a majority of formic acid incorporates directly into DNA, whereas less than 1.5% might form instable formylated DNA adducts in vivo. The results greatly support the important perspective that formic acid is not carcinogenic. Moreover, this study demonstrates that a combination of HPLC with AMS is an essential means for the evaluation of DNA adduction.

  7. Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

    SciTech Connect

    Wnek, S.M.; Medeiros, M.K.; Eblin, K.E.; Gandolfi, A.J.

    2009-12-01

    Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA{sup III}); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA{sup III} [URO-MSC(+)] and after subsequent removal of MMA{sup III} [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA{sup III}, URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA{sup III}. URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA{sup III} exposure, suggesting the presence of MMA{sup III} is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA{sup III} results in: the induction of DNA damage that remains elevated upon removal of MMA{sup III}; increased levels of ROS that play a role in MMA{sup III} induced-DNA damage; and decreased PARP activity in the presence of MMA{sup III}.

  8. Ascorbic acid extends replicative life span of human embryonic fibroblast by reducing DNA and mitochondrial damages.

    PubMed

    Hwang, Won-Sang; Park, Seong-Hoon; Kim, Hyun-Seok; Kang, Hong-Jun; Kim, Min-Ju; Oh, Soo-Jin; Park, Jae-Bong; Kim, Jaebong; Kim, Sung Chan; Lee, Jae-Yong

    2007-01-01

    Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 microM) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-beta-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic) sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 microM of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.

  9. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

    PubMed

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-10-01

    We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.

  10. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

    PubMed Central

    2010-01-01

    Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

  11. The epsilon subunit of DNA polymerase III Is involved in the nalidixic acid-induced SOS response in Escherichia coli.

    PubMed

    Pohlhaus, Jennifer Reineke; Long, David T; O'Reilly, Erin; Kreuzer, Kenneth N

    2008-08-01

    Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in Escherichia coli. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing--cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a dinD::lacZ reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that dnaQ insertion mutants are specifically deficient in the SOS response to nalidixic acid. dnaQ encodes DNA polymerase III epsilon subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type dnaQ gene, confirming involvement of the epsilon subunit. To further characterize the SOS deficiency of dnaQ mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the dnaQ mutant strain, while two tested SOS genes (recA and recN) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.

  12. High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes.

    PubMed

    Igoudjil, Anissa; Massart, Julie; Begriche, Karima; Descatoire, Véronique; Robin, Marie-Anne; Fromenty, Bernard

    2008-06-01

    The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c

  13. Optoelectronic studies on heterocyclic bases of deoxyribonucleic acid for DNA photonics.

    PubMed

    El-Diasty, Fouad; Abdel-Wahab, Fathy

    2015-10-01

    The optoelectronics study of large molecules, particularly π-stacking molecules, such as DNA is really an extremely difficult task. We perform first electronic structure calculations on the heterocyclic bases of 2'-deoxyribonucleic acid based on Lorentz-Fresnel dispersion theory. In the UV-VIS range of spectrum, many of the optoelectronic parameters for DNA four bases namely adenine, guanine, cytosine and thymine are calculated and discussed. The results demonstrate that adenine has the highest hyperpolarizability, whereas thymine has the lowest hyperpolarizability. Cytosine has the lower average oscillator energy and the higher lattice energy. Thymine infers the most stable nucleic base with the lower phonon energy. Thymine also has the highest average oscillator energy and the lower lattice energy. Moreover, the four nucleic acid bases have large band gap energies less than 5 eV with a semiconducting behavior. Guanine shows the smallest band gap and the highest Fermi level energy, whereas adenine elucidates the highest band gap energy.

  14. Probing the Influence of Amino Acids on Photoluminescence from Carbon Nanotubes Suspended with DNA.

    PubMed

    Kurnosov, N V; Leontiev, V S; Karachevtsev, V A

    2016-11-01

    The quantitative analysis of amino acid levels in the human organism is required for the early clinical diagnosis of a variety of diseases. In this work the influence of 13 amino acid doping on the photoluminescence (PL) from the semiconducting single-walled carbon nanotubes (SWNTs) suspended with single-stranded DNA (ssDNA) in water has been studied. Amino acid doping leads to the PL enhancement and the strongest increase was found after cysteine doping of the nanotube suspension while addition of other amino acids yielded the significantly smaller effect. The emphasis of cysteine molecules is attributed to presence of the reactive thiol group that turns cysteine into reducing agent that passivates the p-defects on the nanotube sidewall and increases the PL intensity. The reasons of PL enhancement after doping with other amino acids are discussed. The response of nanotube PL to cysteine addition depends on the nanotube aqueous suspension preparation with tip or bath sonication treatment. The enhancement of the emission from different nanotube species after cysteine doping was analyzed too. It was shown that the increase of the carbon nanotube PL at addition of cysteine allows successful monitoring of the cysteine concentration in aqueous solution in the range of 50-1000 μM.

  15. DNA methylation landscape of fat deposits and fatty acid composition in obese and lean pigs

    PubMed Central

    Zhang, Shunhua; Shen, Linyuan; Xia, Yudong; Yang, Qiong; Li, Xuewei; Tang, Guoqing; Jiang, Yanzhi; Wang, Jinyong; Li, Mingzhou; Zhu, Li

    2016-01-01

    Obese and lean type pig breeds exhibit differences in their fat deposits and fatty acid composition. Here, we compared the effect of genome-wide DNA methylation on fatty acid metabolism between Landrace pigs (LP, leaner) and Rongchang pigs (RP, fatty). We found that LP backfat (LBF) had a higher polyunsaturated fatty acid content but a lower adipocyte volume than RP backfat (RBF). LBF exhibited higher global DNA methylation levels at the genome level than RBF. A total of 483 differentially methylated regions (DMRs) were located in promoter regions, mainly affecting olfactory and sensory activity and lipid metabolism. In LBF, the promoters of genes related to ATPase activity had significantly stronger methylation. This fact may suggest lower energy metabolism levels, which may result in less efficient lipid synthesis in LBF. Furthermore, we identified a DMR in the miR-4335 and miR-378 promoters and validated their methylation status by bisulfite sequencing PCR. The hypermethylation of the promoters of miR-4335 and miR-378 in LBF and the resulting silencing of the target genes may result in LBF’s low content in saturated fatty acids and fat deposition capacity. This study provides a solid basis for exploring the epigenetic mechanisms affecting fat deposition and fatty acid composition. PMID:27721392

  16. Potent protection of gallic acid against DNA oxidation: results of human and animal experiments.

    PubMed

    Ferk, Franziska; Chakraborty, Asima; Jäger, Walter; Kundi, Michael; Bichler, Julia; Mišík, Miroslav; Wagner, Karl-Heinz; Grasl-Kraupp, Bettina; Sagmeister, Sandra; Haidinger, Gerald; Hoelzl, Christine; Nersesyan, Armen; Dušinská, Maria; Simić, Tatjana; Knasmüller, Siegfried

    2011-10-01

    Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-π) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against γ-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of formation

  17. Impact of hedonic evaluation on consumers' preferences for beef attributes including its enrichment with n-3 and CLA fatty acids.

    PubMed

    Baba, Yasmina; Kallas, Zein; Costa-Font, Montserrat; Gil, José María; Realini, Carolina E

    2016-01-01

    The impact of hedonic evaluation on consumers' preferences for beef attributes was evaluated (origin, animal diet, fat content, color, price) including its enrichment with omega-3 (n-3) and conjugated linoleic acid (CLA) fatty acids. One group of consumers (n=325) received information about n-3 and CLA, while the other group (n=322) received no information. Consumers conducted a Discrete Choice Experiment (DCE), using the recently developed Generalized Multinomial Logit model; followed by a blind hedonic evaluation of beef samples, which were identified after tasting, and finally repeated the DCE. Results showed that hedonic evaluation had a significant impact on consumers' preferences, which were similar after tasting for all consumers, with less emphasis on the fat content, color, and origin attributes and greater emphasis on animal diet. Preference for n-3 enriched beef increased, while preference for CLA enriched beef was still not significant after tasting. The information provided had a significant effect on consumers' beef preferences, but no significant impact on beef liking scores.

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

    PubMed Central

    Kirillova, Yuliya; Boyarskaya, Nataliya; Dezhenkov, Andrey; Tankevich, Mariya; Prokhorov, Ivan; Varizhuk, Anna; Eremin, Sergei; Esipov, Dmitry; Smirnov, Igor; Pozmogova, Galina

    2015-01-01

    New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA. PMID:26469337

  19. Boric Acid Reduces the Formation of DNA Double Strand Breaks and Accelerates Wound Healing Process.

    PubMed

    Tepedelen, Burcu Erbaykent; Soya, Elif; Korkmaz, Mehmet

    2016-12-01

    Boron is absorbed by the digestive and respiratory system, and it was considered that it is converted to boric acid (BA), which was distributed to all tissues above 90 %. The biochemical essentiality of boron element is caused by boric acid because it affects the activity of several enzymes involved in the metabolism. DNA damage repair mechanisms and oxidative stress regulation is quite important in the transition stage from normal to cancerous cells; thus, this study was conducted to investigate the protective effect of boric acid on DNA damage and wound healing in human epithelial cell line. For this purpose, the amount of DNA damage occurred with irinotecan (CPT-11), etoposide (ETP), doxorubicin (Doxo), and H2O2 was determined by immunofluorescence through phosphorylation of H2AX((Ser139)) and pATM((Ser1981)) in the absence and presence of BA. Moreover, the effect of BA on wound healing has been investigated in epithelial cells treated with these agents. Our results demonstrated that H2AX((Ser139)) foci numbers were significantly decreased in the presence of BA while wound healing was accelerated by BA compared to that in the control and only drug-treated cells. Eventually, the results indicate that BA reduced the formation of DNA double strand breaks caused by agents as well as improving the wound healing process. Therefore, we suggest that boric acid has important therapeutical effectiveness and may be used in the treatment of inflammatory diseases where oxidative stress and wound healing process plays an important role.

  20. Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase alpha activity.

    PubMed

    Simbulan, C M; Taki, T; Tamiya-Koizumi, K; Suzuki, M; Savoysky, E; Shoji, M; Yoshida, S

    1994-03-16

    The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase alpha were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase alpha, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 microM of CSE, 50 microM of SPG or GM3, and 80 microM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elongating the sugar chain diminished it. It was clearly shown that the N-acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase alpha activity.

  1. Leading-edge forensic DNA analyses and the necessity of including crime scene investigators, police officers and technicians in a DNA elimination database.

    PubMed

    Lapointe, Martine; Rogic, Anita; Bourgoin, Sarah; Jolicoeur, Christine; Séguin, Diane

    2015-11-01

    In recent years, sophisticated technology has significantly increased the sensitivity and analytical power of genetic analyses so that very little starting material may now produce viable genetic profiles. This sensitivity however, has also increased the risk of detecting unknown genetic profiles assumed to be that of the perpetrator, yet originate from extraneous sources such as from crime scene workers. These contaminants may mislead investigations, keeping criminal cases active and unresolved for long spans of time. Voluntary submission of DNA samples from crime scene workers is fairly low, therefore we have created a promotional method for our staff elimination database that has resulted in a significant increase in voluntary samples since 2011. Our database enforces privacy safeguards and allows for optional anonymity to all staff members. We also offer information sessions at various police precincts to advise crime scene workers of the importance and success of our staff elimination database. This study, a pioneer in its field, has obtained 327 voluntary submissions from crime scene workers to date, of which 46 individual profiles (14%) have been matched to 58 criminal cases. By implementing our methods and respect for individual privacy, forensic laboratories everywhere may see similar growth and success in explaining unidentified genetic profiles in stagnate criminal cases.

  2. Information transfer from DNA to peptide nucleic acids by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

  3. Inhibition of N-nitrosamine carcinogenesis and aflatoxin DNA damage by ellagic acid

    SciTech Connect

    Mandal-Chaudhuri, S.

    1988-01-01

    The effect of ellagic acid (EA), on the tumorigenicity of N-nitrosobenzylmethylamine (NBMA) in the rat esophagus was investigated. Groups of 30 male F-344 rats were fed a semipurified diet containing EA for 27 weeks. N-nitrosobenzylmethylamine was administered subcutaneously, once a week for 18 weeks. Ellagic acid produced a significant inhibition in the average number of esophageal tumors at both 20 weeks and 27 weeks. To investigate the mechanism(s) of this inhibition, EA was tested for its effect on the metabolism, DNA-binding and DNA-adduct formation of NBMA in cultured explants of rat esophagus. Explants were incubated in medium containing EA at concentrations of 10, 50, and 100 {mu}M for 16 hours, followed by the addition of 1{mu}M ({sup 3}H)NBMA and EA for 12 hours. Explant DNA was isolated by phenol extraction and hydroxylapatite chromatography, and benzaldehyde formation was determined by h.p.l.c. analysis of the culture medium. Finally, EA was examined for its ability to inhibit DNA damage induced by aflatoxin B{sub 1} (AFB{sub 1}) in cultured explants of rat trachea and esophagus, and human tracheobronchus.

  4. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  5. Influence of cold stress on contents of soluble sugars, vitamin C and free amino acids including gamma-aminobutyric acid (GABA) in spinach (Spinacia oleracea).

    PubMed

    Yoon, Young-Eun; Kuppusamy, Saranya; Cho, Kye Man; Kim, Pil Joo; Kwack, Yong-Bum; Lee, Yong Bok

    2017-01-15

    The contents of soluble sugars (sucrose, fructose, glucose, maltose and raffinose), vitamin C and free amino acids (34 compounds, essential and non-essential) were quantified in open-field and greenhouse-grown spinaches in response to cold stress using liquid chromatography. In general, greenhouse cultivation produced nutritionally high value spinach in a shorter growing period, where the soluble sugars, vitamin C and total amino acids concentrations, including essential were in larger amounts compared to those grown in open-field scenarios. Further, low temperature exposure of spinach during a shorter growth period resulted in the production of spinach with high sucrose, ascorbate, proline, gamma-aminobutyric acid, valine and leucine content, and these constitute the most important energy/nutrient sources. In conclusion, cultivation of spinach in greenhouse at a low temperature (4-7°C) and exposure for a shorter period (7-21days) before harvest is recommended. This strategy will produce a high quality product that people can eat.

  6. "Nano-oddities": unusual nucleic acid assemblies for DNA-based nanostructures and nanodevices.

    PubMed

    Yatsunyk, Liliya A; Mendoza, Oscar; Mergny, Jean-Louis

    2014-06-17

    CONSPECTUS: DNA is an attractive polymer building material for nanodevices and nanostructures due to its ability for self-recognition and self-assembly. Assembly relies on the formation of base-specific interactions that allow strands to adopt structures in a controllable fashion. Most DNA-based higher order structures such as DNA cages, 2D and 3D DNA crystals, or origamis are based on DNA double helices stabilized by Watson-Crick complementarity. A number of nonclassical pairing patterns are possible between or among DNA strands; these interactions result in formation of unusual structures that include, but are not limited to, G-quadruplexes, i-motifs, triplexes, and parallel-stranded duplexes. These structures create greater diversity of DNA-based building blocks for nanomaterials and have certain advantages over conventional duplex DNA, such as enhanced thermal stability and sensitivity to chemical stimuli. In this Account, we briefly introduce these alternative DNA structures and describe in detail their utilization in a variety of nanomaterials and nanomachines. The field of DNA "nano-oddities" emerged in the late 1990s when for the first time a DNA nanomachine was designed based on equilibrium between B-DNA and noncanonical, left-handed Z-DNA. Soon after, "proof-of-principle" DNA nanomachines based on several DNA "oddities" were reported. These machines were set in motion by the addition of complementary strands (a principle used by many B-DNA-based nanodevices), by the addition of selected cations, small molecules, or proteins, or by a change in pH or temperature. Today, we have fair understanding of the mechanism of action of these devices, excellent control over their performance, and knowledge of basic principles of their design. pH sensors and pH-controlled devices occupy a central niche in the field. They are usually based on i-motifs or triplex DNA, are amazingly simple, robust, and reversible, and create no waste apart from salt and water. G

  7. DNA damage in lymphocytes of benzene exposed workers correlates with trans,trans-muconic acids and breath benzene levels.

    PubMed

    Sul, Donggeun; Lee, Eunil; Lee, Mi-Young; Oh, Eunha; Im, Hosub; Lee, Joohyun; Jung, Woon-Won; Won, Namhee; Kang, Hyung-Sik; Kim, Eun-Mi; Kang, Seong-Kyu

    2005-04-04

    Benzene causes many kinds of blood disorders in workers employed in many different environments. These diseases include myelodisplastic syndrome and acute and chronic myelocytic leukemia. In the present study, five occupational work places, including six industrial process types, namely, printing, shoe-making, methylene di-aniline (MDA), nitrobenzene, carbomer, and benzene production were selected, and the levels of breath benzene, and trans,trans-muconic acids (t,t-MA) and phenol in urine were evaluated, as well as hematological changes and lymphocyte DNA damage. The concentration of benzene in breath was less than 3 ppm in the workplaces, and benzene exposure was found to be higher in work places where benzene is used, than in those where benzene is produced. At low levels of benzene exposure, urinary t,t-MA correlated strongly with benzene in air. Highest Olive tail moments were found in workers producing carbomer. Levels of breathzone benzene were found to be strongly correlated with Olive tail moment values in the lymphocytes of workers, but not with hematological data in the six workplaces types. In conclusion, the highest benzene exposures found occurred in workers at a company, which utilized benzene in the production of carbomer. In terms of low levels of exposure to benzene, urinary t,t-MA and DNA damage exhibited a strong correlation with breath benzene, but not with hematological data. We conclude that breath benzene, t,t-MA and lymphocytic DNA damage are satisfactory biomonitoring markers with respect to benzene exposure in the workplace.

  8. Nucleotide sequence of Marchantia polymorpha chloroplast DNA: a region possibly encoding three tRNAs and three proteins including a homologue of E. coli ribosomal protein S14.

    PubMed Central

    Umesono, K; Inokuchi, H; Ohyama, K; Ozeki, H

    1984-01-01

    The nucleotide sequence of a region of Marchantia polymorpha chloroplast DNA was determined. On this DNA sequence (3.38kb), three open reading frames (ORFs) and three putative tRNA genes were detected in the following order: -ORF701-tRNASer(UGA)-ORF702-tRNAGly(GCC)-initiator tRNAMet(CAU)-ORF703-. The ORF703 is composed of 100 codons in which those for lysine (15%) and arginine (11%) are abundant, and could be accounted for as a counterpart of E. coli ribosomal protein S14 since they share 45% homology in the amino acid sequences. The ORF701 appears to code for a membrane protein, showing a periodic appearance of seven clusters of hydrophobic amino acids. Although the mechanisms remain unknown, the ORF701 causes a streptomycin-sensitive phenotype in resistant mutants of E. coli. The ORFs and tRNA genes are separated from each other by extremely AT-rich spacers containing sequences of dyad symmetry. The third letter positions of the codons in the ORFs are also rich in A and T residues. PMID:6393057

  9. Translocation of single stranded DNA through the α-hemolysin protein nanopore in acidic solutions

    PubMed Central

    de Zoysa, Ranulu Samanthi S.; Krishantha, D.M. Milan; Zhao, Qitao; Gupta, Jyoti; Guan, Xiyun

    2012-01-01

    The effect of acidic pH on the translocation of single-stranded DNA through the α-hemolysin pore is investigated. Two significantly different types of events, i.e., deep blockades and shallow blockades, are observed at low pH. The residence times of the shallow blockades are not significantly different from those of the DNA translocation events obtained at or near physiological pH, while the deep blockades have much larger residence times and blockage amplitudes. With a decrease in the pH of the electrolyte solution, the percentage of the deep blockades in the total events increases. Furthermore, the mean residence time of these long-lived events is dependent on the length of DNA, and also varies with the nucleotide base, suggesting that they are appropriate for use in DNA analysis. In addition to be used as an effective approach to affect DNA translocation in the nanopore, manipulation of the pH of the electrolyte solution provides a potential means to greatly enhance the sensitivity of nanopore stochastic sensing. PMID:21997574

  10. Proposed binding mechanism of galbanic acid extracted from Ferula assa-foetida to DNA.

    PubMed

    Ahmadi, F; Shokoohinia, Y; Javaheri, Sh; Azizian, H

    2017-01-01

    Recently, galbanic acid (GA), a sesquiterpenoid coumarin, has been introduced as an apoptotic and geno/cytotoxicity agent. In the present study, GA has been extracted from Ferula assa-foetida, a native medicinal plant in Iran, and characterized by (1)H NMR, mass spectroscopy. Additionally, spectroscopic studies have been performed in order to investigate its DNA-interaction mode. The electrochemical behavior of GA has been studied by cyclic voltammetry (CV) in various scan rates. In neutral media (pH=7.3) one irreversible cathodic peak was obtained at -1.46 V, while in higher scan rates an irreversible one was determined at -1.67 V. According to the voltametric data GA can be easily reduced by 2e(-)/2H(+) mechanism at hanging mercury drop electrode (HMDE). The interaction of GA with ct-DNA was evaluated by CV, differential pulse voltammetry (DPV), enhancement fluorescence, UV-Vis, FT-IR spectroscopy and molecular docking. The molecular docking study shows that the GA interacts to DNA on partial intercalation mode via DNA groove binding and forms a complex by van der Waals and electroastatic interactions. In addition, the thermodynamic parameters of GA-DNA complex were investigated with ΔH°, ΔS° and ΔG° values of 15.81KJmol(-1), 133.95Jmol(-1) and -23.10KJmol(-1), respectively. All data revealed that the GA is binding to DNA by van der Waals and electrostatic interactions through the partial intercalations from the DNA's grooves.

  11. Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples

    PubMed Central

    Fanaei, Hamed; Khayat, Samira; Halvaei, Iman; Ramezani, Vahid; Azizi, Yaser; Kasaeian, Amir; Mardaneh, Jalal; Parvizi, Mohammad Reza; Akrami, Maryam

    2014-01-01

    Background: Oxidative stress in teratozoospermic semen samples caused poor assisted reproductive techniques (ART) outcomes. Among antioxidants, ascorbic acid is a naturally occurring free radical scavenger and as such its presence assists various other mechanisms in decreasing numerous disruptive free radical processes. Objective: The main goal of this study was to evaluate potential protective effects of ascorbic acid supplementation during in vitro culture of teratozoospermic specimens. Materials and Methods: Teratozoospermic semen samples that collected from 15 volunteers were processed, centrifuged and incubated at 37oC until sperm swimmed-up. Supernatant was divided into four groups and incubated at 37oC for one hour under different experimental conditions: Control, 10 µm A23187, 600µm ascorbic acid and 10 µm A23187+600 µm ascorbic acid. After incubation sperm motility, viability, acrosome reaction, DNA damage and malondialdehyde levels were evaluated. Results: Our results indicated that after one hour incubation, ascorbic acid significantly reduced malondialdehyde level in ascorbic acid group (1.4±0.11 nmol/ml) compared to control group (1.58±0.13 nmol/ml) (p<0.001). At the end of incubation, progressive motility and viability in ascorbic acid group (64.5±8.8% and 80.3±6.4%, respectively) were significantly (p<0.05 and p<0.001, respectively) higher than the control group (54.5±6.8% and 70.9±7.3%, respectively). A23187 significantly (p<0.0001) increased acrosome reaction in A23187 group (37.3±5.6%) compared to control group (8.5±3.2%) and this effect of A23187 attenuated by ascorbic acid in ascorbic acid+A23187 group (17.2±4.4%). DNA fragmentation in ascorbic acid group (20±4.1%) was significantly (p<0.001) lower than controls (28.9±4.6%). Conclusion: In vitro ascorbic acid supplementation during teratozoospermic semen processing for ART could protect teratozoospermic specimens against oxidative stress, and it could improve ART outcome. PMID

  12. Differentiation of strains in Mycobacterium tuberculosis complex by DNA sequence polymorphisms, including rapid identification of M. bovis BCG.

    PubMed Central

    Frothingham, R

    1995-01-01

    The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. microti, and M. africanum. Seven strains of the M. tuberculosis complex were sequenced in a region of about 300 bp which contains multiple 15-bp tandem repeats and which is part of a 1,551-bp open reading frame. Four distinct sequences were obtained, each defining a sequevar. A sequevar includes the strain or strains with a given sequence. The type strain M. tuberculosis TMC 102 (H37Rv) was designated sequevar MED-G. When compared to MED-G, sequevar LONG had an insertion of one 15-bp tandem repeat and sequevar SHORT had a deletion of one tandem repeat. Sequevar MED-C had a G-->C substitution, coding for the conservative change Ser-->Thr. BanI cuts only sequevar MED-C at the site of the substitution. PCR-restriction enzyme analysis was used to determine the sequevars of 92 M. tuberculosis complex strains. All 23 M. bovis BCG strains belonged to sequevar MED-C. The M. africanum type strain was sequevar SHORT. The remaining 68 strains of M. tuberculosis, M. bovis (not BCG), and M. microti were sequevars LONG (3 strains) or MED-G (65 strains). PCR-restriction enzyme analysis was applied to reference strains and clinical isolates with a worldwide distribution. This method provides rapid, sensitive, and specific identification of the important vaccine strain M. bovis BCG. PMID:7790448

  13. Mitochondrial DNA sequence evolution and phylogeny of the Atlantic Alcidae, including the extinct great auk (Pinguinus impennis).

    PubMed

    Moum, Truls; Arnason, Ulfur; Arnason, Einar

    2002-09-01

    The Atlantic auk assemblage includes four extant species, razorbill (Alca torda), dovekie (Alle alle), common murre (Uria aalge), and thick-billed murre (U. lomvia), and one recently extinct species, the flightless great auk (Pinguinus impennis). To determine the phylogenetic relationships among the species, a contiguous 4.2-kb region of the mitochondrial genome from the extant species was amplified using PCR. This region included one ribosomal RNA gene, four transfer RNA genes, two protein-coding genes, the control region, and intergenic spacers. Sets of PCR primers for amplifying the same region from great auk were designed from sequences of the extant species. The authenticity of the great auk sequence was ascertained by alternative amplifications, cloning, and separate analyses in an independent laboratory. Phylogenetic analyses of the entire assemblage, made possible by the great auk sequence, fully resolved the phylogenetic relationships and split it into two primary lineages, Uria versus Alle, Alca, and Pinguinus. A sister group relationship was identified between Alca and Pinguinus to the exclusion of ALLE: Phylogenetically, the flightless great auk originated late relative to other divergences within the assemblage. This suggests that three highly divergent species in terms of adaptive specializations, Alca, Alle, and Pinguinus, evolved from a single lineage in the Atlantic Ocean, in a process similar to the initial adaptive radiation of alcids in the Pacific Ocean.

  14. Fluorescence methods to study DNA translocation and unwinding kinetics by nucleic acid motors.

    PubMed

    Fischer, Christopher J; Tomko, Eric J; Wu, Colin G; Lohman, Timothy M

    2012-01-01

    Translocation of nucleic acid motor proteins (translocases) along linear nucleic acids can be studied by monitoring either the time course of the arrival of the motor protein at one end of the nucleic acid or the kinetics of ATP hydrolysis by the motor protein during translocation using pre-steady state ensemble kinetic methods in a stopped-flow instrument. Similarly, the unwinding of double-stranded DNA or RNA by helicases can be studied in ensemble experiments by monitoring either the kinetics of the conversion of the double-stranded nucleic acid into its complementary single strands by the helicase or the kinetics of ATP hydrolysis by the helicase during unwinding. Such experiments monitor translocation of the enzyme along or unwinding of a series of nucleic acids labeled at one position (usually the end) with a fluorophore or a pair of fluorophores that undergo changes in fluorescence intensity or efficiency of fluorescence resonance energy transfer (FRET). We discuss how the pre-steady state kinetic data collected in these ensemble experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rates and processivities of translocation and/or unwinding, the rate-limiting step(s) in these mechanisms, the average "kinetic step-size," and the stoichiometry of coupling ATP binding and hydrolysis to movement along the nucleic acid.

  15. Cells Deficient in the Fanconi Anemia Protein FANCD2 are Hypersensitive to the Cytotoxicity and DNA Damage Induced by Coffee and Caffeic Acid.

    PubMed

    Burgos-Morón, Estefanía; Calderón-Montaño, José Manuel; Orta, Manuel Luis; Guillén-Mancina, Emilio; Mateos, Santiago; López-Lázaro, Miguel

    2016-07-08

    Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee.

  16. Cells Deficient in the Fanconi Anemia Protein FANCD2 are Hypersensitive to the Cytotoxicity and DNA Damage Induced by Coffee and Caffeic Acid

    PubMed Central

    Burgos-Morón, Estefanía; Calderón-Montaño, José Manuel; Orta, Manuel Luis; Guillén-Mancina, Emilio; Mateos, Santiago; López-Lázaro, Miguel

    2016-01-01

    Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee. PMID:27399778

  17. cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda.

    PubMed

    Du, Yu-Jie; Luo, Xiao-Yan; Hao, Yan-Zhe; Zhang, Tian; Hou, Wan-Ru

    2007-10-26

    RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.

  18. Development of artificial nucleic acid that recognizes a CG base pair in triplex DNA formation.

    PubMed

    Hari, Yoshiyuki

    2013-01-01

    An oligonucleotide that can form a triplex with double-stranded DNA is called a triplex-forming oligonucleotide (TFO). TFOs have gained considerable attention because of their potential as gene targeting tools. However, triplex DNA formation involves inherent problems for practical use. The most important problem is that natural nucleotides in TFO do not have sufficient affinity and base pair-selectivity to pyrimidine-purine base pair, like a CG or TA base pair, within dsDNA. This suggests that dsDNA region including a CG or TA base pair cannot be targeted. Therefore, artificial nucleotides, especially with non-natural nucleobases, capable of direct recognition of a CG or TA base pair via hydrogen bond formation have been developed; however, nucleotides with better selectivity and stronger affinity are necessary for implementing this dsDNA-targeting technology using TFOs. Under such a background, we considered that facile and efficient synthesis of various nucleobase derivatives in TFOs would be useful for finding an ideal nucleobase for recognition of a CG or TA base pair because detailed and rational exploration of nucleobase structures is facilitated. Recently, to develop a nucleobase recognizing a CG base pair, we have used post-elongation modification, i.e., modification after oligonucleotide synthesis, for the facile synthesis of nucleobase derivatives. This review mainly summarizes our recent findings on the development of artificial nucleobases and nucleotides for recognition of a CG base pair in triplexes formed between dsDNA and TFOs.

  19. Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA

    PubMed Central

    Edgcomb, Virginia P.; McDonald, John H.; Devereux, Richard; Smith, David W.

    1999-01-01

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 107 to 2.5 × 109 (average, 1.1 × 109 ± 5.2 × 108) cells g of sediment−1. In September, numbers of SRB detected ranged from 5.4 × 108 to 7.3 × 109 (average, 2.5 × 109 ± 1.5 × 109) cells g of sediment−1. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments. PMID:10103245

  20. Bioaugmentation of bromoamine acid degradation with Sphingomonas xenophaga QYY and DNA fingerprint analysis of augmented systems.

    PubMed

    Qu, Yuanyuan; Zhou, Jiti; Wang, Jing; Song, Zhiyong; Xing, Linlin; Fu, Xiang

    2006-02-01

    One high-effective bromoamine acid (1-amino-4-bromoanthraquinone-2-sulfonic acid, BAA) degrading strain was isolated previously with the ability to use BAA as sole source of carbon and nitrogen. It was identified as Sphingomonas xenophaga QYY by 16S rDNA sequence analysis and physio-biochemical tests. In this study, bioaugmentation of BAA degradation with suspended and immobilized cells of strain QYY was investigated. The optimal degradation conditions were as follows: temperature 30 degrees C, pH 6.0-7.0, 150 rev min(-1) and the immobilized cells maintained degradation activity to BAA after 60 days storage at 4 degrees C. The structure of BAA was evidently changed according to the analysis of total organic carbon removal of BAA (about 50%) and the UV-VIS spectra changes during the biodegradation. Bioaugmented systems exhibited stronger abilities degrading BAA than the non-bioaugmented control ones. And microbial community dynamics of augmented systems was revealed by amplified ribosomal DNA restriction analysis (ARDRA), a modern DNA fingerprint technique. The results indicated that the microbial community dynamics was substantially changed throughout the augmentation process. This study suggests that it is feasible and potentially useful to enhance BAA degradation using bioaugmentation with the immobilized cells of BAA-degrading bacterium.

  1. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  2. Bioaccumulation of perfluoroalkyl acids including the isomers of perfluorooctane sulfonate in carp (Cyprinus carpio) in a sediment/water microcosm.

    PubMed

    Fang, Shuhong; Zhang, Yifeng; Zhao, Shuyan; Qiang, Liwen; Chen, Meng; Zhu, Lingyan

    2016-12-01

    Carp (Cyprinus carpio) were exposed to perfluoroalkyl acids (PFAAs) including perfluorooctane sulfonate (PFOS) isomers in an artificially contaminated sediment/water microcosm. The uptake constant of PFAAs increased with increasing carbon chain length, whereas the elimination coefficient displayed the opposite trend, suggesting that carbon chain length plays an important role in the bioaccumulation of PFAAs. When the contribution of suspended particulate matter was taken into account, the bioaccumulation factors (BAFs) became lower (3.61-600 L/kg) compared with BAFs derived from only considering the absorption from free PFAAs in water (3.85-97000 L/kg). The results indicate that suspended particulate matter in water constitutes an important source of exposure for aquatic organisms to long-chain PFAAs. Linear (n-)PFOS was preferentially accumulated compared with branched isomers in carp. Among the branched isomers, 1m-PFOS displayed the greatest bioaccumulation, whereas m2 -PFOS had the lowest. Linear PFOS displayed greater partitioning ability from blood to other tissues over branched PFOS (br-PFOS) isomers, leading to a relatively lower n-PFOS proportion in blood. In summary, suspended particulate matter made a contribution to the accumulation of long-chain PFAAs in aquatic organisms, and n-PFOS was preferentially accumulated compared with br-PFOS isomers. Environ Toxicol Chem 2016;35:3005-3013. © 2016 SETAC.

  3. Chemical Cues which Include Amino Acids Mediate Species-Specific Feeding Behavior in Invasive Filter-Feeding Bigheaded Carps.

    PubMed

    Claus, Aaron W; Sorensen, Peter W

    2017-03-15

    This study tested whether and how dissolved chemicals might assist food recognition in two filter-feeding fishes, the silver (Hypophthalmichthys molitrix) and the bighead carp (H. nobilis). These species evolved in Asia, are now invasive in the Mississippi River, and feed voraciously on microparticles including plankton. The food habits and biology of these carps are broadly similar to many filter-feeding fish, none of whose chemical ecology has been examined. We conducted five experiments. First, we demonstrated that buccal-pharngeal pumping (BPP), a behavior in which fish pump water into their buccal cavities, is responsible for sampling food: BPP activity in both silver and bighead carps was low and increased nearly 25-fold after exposure to a filtrate of a planktonic food mixture (P < 0.01) and over 35-fold when planktonic food was added (P < 0.001). Next, we showed that of nine food filtrates, the one containing chemicals released by spirulina, a type of cyanobacterium, was the most potent planktonic component for both species. The potency of filtrates varied between species in ways that reflected their different chemical compositions. While L-amino acids could explain about half of the activity of food filtrate, other unknown chemical stimuli were also implicated. Finally, occlusion experiments showed the olfactory sense has a very important, but not exclusive, role in bigheaded carp feeding behaviors and this might be exploited in both their control and culture.

  4. Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA.

    PubMed Central

    Hübner, S; Michel, F; Rudloff, V; Appelhans, H

    1992-01-01

    In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5'-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3'-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins. PMID:1637296

  5. Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil.

    PubMed

    Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami

    2017-04-15

    The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil.

  6. Anionic magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid for DNA base discrimination

    NASA Astrophysics Data System (ADS)

    Khadsai, Sudarat; Rutnakornpituk, Boonjira; Vilaivan, Tirayut; Nakkuntod, Maliwan; Rutnakornpituk, Metha

    2016-09-01

    Magnetite nanoparticles (MNPs) were surface modified with anionic poly( N-acryloyl glycine) (PNAG) and streptavidin for specific interaction with biotin-conjugated pyrrolidinyl peptide nucleic acid (PNA). Hydrodynamic size ( D h) of PNAG-grafted MNPs varied from 334 to 496 nm depending on the loading ratio of the MNP to NAG in the reaction. UV-visible and fluorescence spectrophotometries were used to confirm the successful immobilization of streptavidin and PNA on the MNPs. About 291 pmol of the PNA/mg MNP was immobilized on the particle surface. The PNA-functionalized MNPs were effectively used as solid supports to differentiate between fully complementary and non-complementary/single-base mismatch DNA using the PNA probe. These novel anionic MNPs can be efficiently applicable for use as a magnetically guidable support for DNA base discrimination.

  7. Amino acid and DNA analyses in a family with ornithine transcarbamylase deficiency.

    PubMed

    Hou, J W; Wang, T R

    1996-02-01

    Ornithine transcarbamylase (OTC) is a hepatic mitochondrial enzyme involved in the detoxification of ammonia by the urea cycle. OTC deficiency is an X-linked genetic disorder, usually causing neonatal or infantile hyperammonemia, coma and death. We attended a male newborn who had poor feeding since 30 hours of age, at which time, he then rapidly progressed to a comatose state. Hyperammonemia and liver dysfunction were noted. Analysis of plasma amino acids showed elevated levels of glutamine and alanine, but a decreased level of arginine and no citrulline. OTC deficiency was diagnosed by family history of early death of newborn males on the maternal side and characteristic biochemical findings. In addition, it was proved by Southern blot analysis of genomic DNA. Although OTC deficiency has been described as the most common inborn error of ureagenesis in humans, to our knowledge, this is the first report in a Chinese family confirmed by biochemical and DNA analyses.

  8. Quantification of false positive reduction in nucleic acid purification on hemorrhagic fever DNA.

    SciTech Connect

    James, Conrad D.; Pohl, Kenneth Roy; Derzon, Mark Steven; McClain, Jaime; Achyuthan, Komandoor

    2006-11-01

    Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system.

  9. Chlorogenic acid prevents isoproterenol-induced DNA damage in vascular smooth muscle cells

    PubMed Central

    Wang, Jingshuai; Li, Jiyang; Liu, Jie; Xu, Mengjiao; Tong, Xiaowen; Wang, Jianjun

    2016-01-01

    Numerous clinical therapeutic agents have been identified as DNA damaging. The present study revealed that isoproterenol (Iso) resulted in DNA damage in vascular smooth muscle cells (VSMCs) and increased the levels of intracellular oxygen free radicals. Administration of chlorogenic acid (CGA) inhibited this effect. Pretreatment with CGA abrogated the increase in protein expression levels of γ-H2A histone family member X, phosphorylated ataxia telangiectasia mutated, phosphorylated Rad3-related protein, breast cancer 1 and C-terminal Src homologous kinase induced by Iso. In addition, the increase in levels of intracellular reactive oxygen species (ROS) induced by Iso was inhibited by CGA pretreatment in a dose-dependent manner. The results of the present study suggest that CGA may inhibit Iso-induced VSMC damage via the suppression of ROS generation. Therefore, CGA may be a novel agent for the treatment of vascular diseases. PMID:27634104

  10. Aristoxazole analogues. Conversion of 8-nitro-1-naphthoic acid to 2-methylnaphtho[1,2-d]oxazole-9-carboxylic acid: comments on the chemical mechanism of formation of DNA adducts by the aristolochic acids.

    PubMed

    Priestap, Horacio A; Barbieri, Manuel A; Johnson, Francis

    2012-07-27

    2-Methylnaphtho[1,2-d]oxazole-9-carboxylic acid was obtained by reduction of 8-nitro-1-naphthoic acid with zinc-acetic acid. This naphthoxazole is a condensation product between an 8-nitro-1-naphthoic acid reduction intermediate and acetic acid and is a lower homologue of aristoxazole, a similar condensation product of aristolochic acid I with acetic acid that was previously reported. Both oxazoles are believed to arise via a common nitrenium/carbocation ion mechanism that is likely related to that which leads to aristolochic acid-DNA-adducts.

  11. DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes

    PubMed Central

    Kleiner, Ralph E.; Brudno, Yevgeny; Birnbaum, Michael E.; Liu, David R.

    2009-01-01

    The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that the polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence-specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry, but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step towards the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each

  12. Selection and identification of DNA aptamers against okadaic acid for biosensing application.

    PubMed

    Eissa, Shimaa; Ng, Andy; Siaj, Mohamed; Tavares, Ana C; Zourob, Mohammed

    2013-12-17

    This work describes the selection and identification of DNA aptamers that bind with high affinity and specificity to okadaic acid (OA), a lipophilic marine biotoxin that accumulates in shellfish. The aptamers selected using systematic evolution of ligands by exponential enrichment (SELEX) exhibited dissociation constants in the nanomolar range. The aptamer with the highest affinity was then used for the fabrication of a label-free electrochemical biosensor for okadaic acid detection. The aptamer was first immobilized on the gold electrode by a self-assembly approach through Au-S interaction. The binding of okadaic acid to the aptamer immobilized on the electrode surface induces an alteration of the aptamer conformation causing a significant decrease in the electron-transfer resistance monitored by electrochemical impedance spectroscopy. The aptasensor showed a linear range for the concentrations of OA between 100 pg/mL and 60 ng/mL with a detection limit of 70 pg/mL. The dissociation constant of okadaic acid with the aptamer immobilized on the electrode surface showed good agreement with that determined using fluorescence assay in solution. Moreover, the aptasensor did not show cross-reactivity toward toxins with structures similar to okadaic acid such as dinophysis toxin-1 and 2 (DTX-1, DTX-2). Further biosensing applications of the selected aptamers are expected to offer promising alternatives to the traditional analytical and immunological methods for OA detection.

  13. A study of aneuploidy and DNA fragmentation in spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line.

    PubMed

    Nguyen, Minh Huong; Morel, Frederic; Bujan, Louis; May-Panloup, Pascale; De Braekeleer, Marc; Perrin, Aurore

    2015-06-01

    Meiotic segregation of mosaic males with a 45,X cell line has been little examined. In this study, we evaluated the risk of aneuploid gametes using fluorescence in situ hybridization (FISH) and DNA fragmentation in ejaculated spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line. Triple- and dual-color FISH were performed. Sperm DNA fragmentation was detected using the TUNEL assay. A significantly increased frequency of XY disomic spermatozoa was observed for patients (P)1 and P2. A significant increase in diploidy and autosomal aneuploidy was found in P2 and P3, respectively. The rate of DNA fragmentation was not different from that observed in a control group. Data from the literature are scarce (only 3 cases reported), making comparison of the present data difficult, especially as the frequencies of the cell lines comprising the mosaicism differed between patients. Furthermore, the proportion of the different cell lines can differ from one tissue to another in the same patient. Whether the relative levels of the several cell lines present in the mosaicism can influence the rate of aneuploid spermatozoa remains unknown.

  14. Suberoylanilide Hydroxyamic Acid Modification of Chromatin Architecture Affects DNA Break Formation and Repair

    SciTech Connect

    Singh, Sheetal; Le Hongan; Shih, S.-J.; Ho, Bay; Vaughan, Andrew T.

    2010-02-01

    Purpose: Chromatin-modifying compounds that inhibit the activity of histone deacetylases have shown potency as radiosensitizers, but the action of these drugs at a molecular level is not clear. Here we investigated the effect of suberoylanilide hydroxyamic acid (SAHA) on DNA breaks and their repair and induction of rearrangements. Methods and Materials: The effect of SAHA on both clonogenic survival and repair was assessed using cell lines SCC-25, MCF7, and TK6. In order to study unique DNA double-strand breaks, anti-CD95 antibody was employed to introduce a DNA double-strand break at a known location within the 11q23 region. The effects of SAHA on DNA cleavage and rearrangements were analyzed by ligation-mediated PCR and inverse PCR, respectively. Results: SAHA acts as radiosensitizer at 1 {mu}M, with dose enhancement factors (DEFs) at 10% survival of: SCC-25 - 1.24 +- 0.05; MCF7 - 1.16 +- 0.09 and TK6 - 1.17 +- 0.05, and it reduced the capacity of SCC-25 cells to repair radiation induced lesions. Additionally, SAHA treatment diffused site-specific fragmentation over at least 1 kbp in TK6 cells. Chromosomal rearrangements produced in TK6 cells exposed to SAHA showed a reduction in microhomology at the breakpoint between 11q23 and partner chromosomes. Conclusions: SAHA shows efficacy as a radiosensitizer at clinically obtainable levels. In its presence, targeted DNA strand breaks occur over an expanded region, indicating increased chromatin access. The rejoining of such breaks is degraded by SAHA when measured as rearrangements at the molecular level and rejoining that contributes to cell survival.

  15. Impact of boric acid exposure at different concentrations on testicular DNA and male rats fertility.

    PubMed

    El-Dakdoky, Mai H; Abd El-Wahab, Hanan M F

    2013-06-01

    The aim of this study was to investigate the consequences of exposure to three levels of boric acid (BA) on male rats reproduction, fertility and progeny outcome, with emphasis on testicular DNA level and quality. Adult male rats (12 weeks old) were treated orally with 125, 250 and 500 mg/kg bwt/d of BA for 60 d. The results indicated that BA administration at 125 mg/kg bwt had no adverse effects on fertility, sperm characteristics or prenatal development of the impregnated females. However, at dose 250 mg, BA treatment significantly increased serum nitric oxide, testosterone, estradiol levels and testicular boron and calcium levels and also significantly reduced serum arginase activity, sperm quality and testicular DNA content with minor DNA fragmentation. The impact of BA exposure at dose 250 mg on male rats fertility was translated into increases in pre-implantation loss with a resulting decrease in the number of live fetuses/litter. In addition to the significant alteration of biochemical measurements, observed at dose 250 mg, administration of BA at 500 mg caused testicular atrophy, severe damage of spermatogenesis, spermiation failure and significant reduction of Mg and Zn testicular levels. None of the male rats, treated with 500 mg/kg bwt, could impregnate untreated females, suggesting the occurrence of definitive loss of fertility. In conclusion, BA impaired fertility, in a dose-dependant manner, by targeting the highly proliferative cells, the germ cells, through decreasing DNA synthetic rate rather than the induction of DNA damage.

  16. Isolation and identification of microorganisms including lactic acid bacteria and their use in microbial deacidification of wines from domestic vineyards.

    PubMed

    Drozdz, Iwona; Makarewicz, Malgorzata; Tuszyński, Tadeusz

    2013-01-01

    The aim of this study was to identify various bacteria isolated from grapes and their wines. Additionally we investigated the capacity of lactic acid bacteria for microbiological deacidification of wines produced in Poland. We have identified Oenococcus oeni, Lactobacillus acidophilus and Lactobacillus delbrueckii. During the microbial deacidification process, we observed decreases of total acidity and increases of volatile acidity, with statistically significant changes noted for O. oeni in Marechal Foch and Seyval Blanc, and for Lb. acidophilus in Frontenac. On the other hand, a statistically significant increase in pH was observed in Marechal Foch and Seyval Blanc following deacidification by O. oeni.

  17. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results.

  18. Ursolic Acid-Regulated Energy Metabolism—Reliever or Propeller of Ultraviolet-Induced Oxidative Stress and DNA Damage?

    PubMed Central

    Lee, Yuan-Hao; Sun, Youping; Glickman, Randolph D.

    2014-01-01

    Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts (e.g., pyrimidine dimers) and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR (mammalian target of rapamycin), whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic

  19. Hyaluronic Acid--an "Old" Molecule with "New" Functions: Biosynthesis and Depolymerization of Hyaluronic Acid in Bacteria and Vertebrate Tissues Including during Carcinogenesis.

    PubMed

    Tsepilov, R N; Beloded, A V

    2015-09-01

    Hyaluronic acid is an evolutionarily ancient molecule commonly found in vertebrate tissues and capsules of some bacteria. Here we review modern data regarding structure, properties, and biological functions of hyaluronic acid in mammals and Streptococcus spp. bacteria. Various aspects of biogenesis and degradation of hyaluronic acid are discussed, biosynthesis and degradation metabolic pathways for glycosaminoglycan together with involved enzymes are described, and vertebrate and bacterial hyaluronan synthase genes are characterized. Special attention is given to the mechanisms underlying the biological action of hyaluronic acid as well as the interaction between polysaccharide and various proteins. In addition, all known signaling pathways involving hyaluronic acid are outlined. Impaired hyaluronic acid metabolism, changes in biopolymer molecular weight, hyaluronidase activity, and enzyme isoforms often accompany carcinogenesis. The interaction between cells and hyaluronic acid from extracellular matrix that may be important during malignant change is discussed. An expected role for high molecular weight hyaluronic acid in resistance of naked mole rat to oncologic diseases and the protective role of hyaluronic acid in bacteria are discussed.

  20. Porous hyaluronic acid hydrogels for localized nonviral DNA delivery in a diabetic wound healing model.

    PubMed

    Tokatlian, Talar; Cam, Cynthia; Segura, Tatiana

    2015-05-01

    The treatment of impaired wounds requires the use of biomaterials that can provide mechanical and biological queues to the surrounding environment to promote angiogenesis, granulation tissue formation, and wound closure. Porous hydrogels show promotion of angiogenesis, even in the absence of proangiogenic factors. It is hypothesized that the added delivery of nonviral DNA encoding for proangiogenic growth factors can further enhance this effect. Here, 100 and 60 μm porous and nonporous (n-pore) hyaluronic acid-MMP hydrogels with encapsulated reporter (pGFPluc) or proangiogenic (pVEGF) plasmids are used to investigate scaffold-mediated gene delivery for local gene therapy in a diabetic wound healing mouse model. Porous hydrogels allow for significantly faster wound closure compared with n-pore hydrogels, which do not degrade and essentially provide a mechanical barrier to closure. Interestingly, the delivery of pDNA/PEI polyplexes positively promotes granulation tissue formation even when the DNA does not encode for an angiogenic protein. And although transfected cells are present throughout the granulation tissue surrounding, all hydrogels at 2 weeks, pVEGF delivery does not further enhance the angiogenic response. Despite this, the presence of transfected cells shows promise for the use of polyplex-loaded porous hydrogels for local gene delivery in the treatment of diabetic wounds.

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

    PubMed

    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.

  2. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.; Selvaganapathy, M.; Mahalakshmi, R.

    2012-10-01

    Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen (1O2) and superoxide anion radical (O2rad -) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

  3. Dietary omega-3 polyunsaturated fatty acids induce plasminogen activator activity and DNA damage in rabbit spermatozoa.

    PubMed

    Kokoli, A N; Lavrentiadou, S N; Zervos, I A; Tsantarliotou, M P; Georgiadis, M P; Nikolaidis, E A; Botsoglou, N; Boscos, C M; Taitzoglou, I A

    2017-02-20

    The aim of this study was to determine the effect(s) of dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) on rabbit semen. Adult rabbit bucks were assigned to two groups that were given two diets, a standard diet (control) and a diet supplemented with ω-3 PUFA. Sperm samples were collected from all bucks with the use of an artificial vagina in 20-day intervals, for a total period of 120 days. The enrichment of membranes in ω-3 PUFA was manifested by the elevation of the 22:5 ω-3 (docosapentaenoic acid [DPA]) levels within 40 days. This increase in DPA content did not affect semen characteristics (i.e., concentration, motility and viability). However, it was associated with the induction of lipid peroxidation in spermatozoa, as determined on the basis of the malondialdehyde content. Lipid peroxidation was associated with DNA fragmentation in ω-3 PUFA-enriched spermatozoa and a concomitant increase in plasminogen activator (PA) activity. The effects of ω-3 PUFA on sperm cells were evident within 40 days of ω-3 PUFA dietary intake and exhibited peack values on day 120. Our findings suggest that an ω-3 PUFA-rich diet may not affect semen characteristics; however, it may have a negative impact on the oxidative status and DNA integrity of the spermatozoa, which was associated with an induction of PAs activity.

  4. DNA-PK assay

    DOEpatents

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  5. Rapid identification of triterpenoid sulfates and hydroxy fatty acids including two new constituents from Tydemania expeditionis by LC-MS

    PubMed Central

    Zhang, Jian-Long; Kubanek, Julia; Hay, Mark E.; Aalbersberg, William; Ye, Wen-Cai; Jiang, Ren-Wang

    2011-01-01

    Tydemania expeditionis Weber-van Bosse (Udoteaceae) is a weakly calcified green alga. In the present paper, liquid chromatography coupled with photodiode array detection and electrospray mass spectrometry was developed to identify the fingerprint components. A total of four triterpenoid sulfates and three hydroxy fatty acids in the ethyl acetate fraction of the crude extract were structurally characterized on the basis of retention time, online UV spectrum and mass fragmentation pattern. Furthermore, detailed LC-MS analysis revealed two new hydroxy fatty acids, which were then prepared and characterized by extensive NMR analyses. The proposed method provides a scientific and technical platform for the rapid identification of triterpenoid sulfates and hydroxy fatty acids in similar marine algae and terrestrial plants. PMID:21915955

  6. The DNA invertase Gin of phage Mu: formation of a covalent complex with DNA via a phosphoserine at amino acid position 9.

    PubMed Central

    Klippel, A; Mertens, G; Patschinsky, T; Kahmann, R

    1988-01-01

    The DNA invertase Gin encoded by bacteriophage Mu catalyses efficient site-specific recombination between inverted repeat sequences (IR) in vivo and in vitro in the presence of the host factor FIS and the recombinational enhancer. We demonstrate that Gin alone is able to introduce single strand breaks into duplex DNA fragments which contain the IR sequence. Strand cleavage is site-specific and can occur on either strand within the IR. Cleaved molecules contain Gin covalently attached to DNA. The covalent complex is formed through linkage of Gin to the 5' DNA phosphate at the site of the break via a phosphoserine. Extensive site-directed mutational analysis showed that all mutants altered at serine position 9 were completely recombination deficient in vivo and in vitro. The mutant proteins bind to DNA but lack topoisomerase activity and are unable to introduce nicks. This holds true even for a conservative amino acid substitution at position 9. We conclude that serine at position 9 is part of the catalytic domain of Gin. The intriguing finding that the DNA invertase Gin has the same catalytic center as the DNA resolvases that promote deletions without recombinational enhancer and host factor FIS is discussed. Images PMID:3042382

  7. Chemical kinetic behavior of chlorogenic acid in protecting erythrocyte and DNA against radical-induced oxidation.

    PubMed

    Tang, You-Zhi; Liu, Zai-Qun

    2008-11-26

    As an abundant ingredient in coffee, chlorogenic acid (CGA) is a well-known antioxidant. Although some works have dealt with its radical-scavenging property, the present work investigated the protective effects of CGA on the oxidation of DNA and on the hemolysis of human erythrocytes induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) by means of chemical kinetics. The inhibition period (t(inh)) derived from the protective effect of CGA on erythrocyte and DNA was proportional to its concentration, t(inh) = (n/R(i))[CGA], where R(i) refers to the radical-initiation rate, and n indicates the number of radical-propagation chains terminated by CGA. It was found that the n of CGA to protect erythrocytes was 0.77, lower than that of vitamin E (2.0), but higher than that of vitamin C (0.19). Furthermore, CGA facilitated a mutual protective effect with VE and VC on AAPH-induced hemolysis by increasing n of VE and VC. CGA was also found to be a membrane-stabilizer to protect erythrocytes against hemin-induced hemolysis. Moreover, the n of CGA was only 0.41 in the process of protecting DNA. This fact revealed that CGA served as an efficient antioxidant to protect erythrocytes more than to protect DNA. Finally, the reaction between CGA and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(+*)) or 2,2'-diphenyl-1-picrylhydrazyl (DPPH) revealed that CGA was able to trap radicals by reducing radicals more than by donating its hydrogen atoms to radicals.

  8. AN APPROACH TO REVEALING BLOOD FLUKE LIFE CYCLES, TAXONOMY, AND DIVERSITY: PROVISION OF KEY REFERENCE DATA INCLUDING DNA SEQUENCE FROM SINGLE LIFE CYCLE STAGES

    PubMed Central

    Brant, Sara V.; Morgan, Jess A. T.; Mkoji, Gerald M.; Snyder, Scott D.; Rajapakse, R. P. V. Jayanthe; Loker, Eric S.

    2008-01-01

    Revealing diversity among extant blood flukes, and the patterns of relationships among them, has been hindered by the difficulty of determining if specimens described from different life cycle stages, hosts, geographic localities, and times represent the same or different species. Persistent collection of all available life cycle stages and provision of exact collection localities, host identification, reference DNA sequences for the parasite, and voucher specimens eventually will provide the framework needed to piece together individual life cycles and facilitate reconciliation with classical taxonomic descriptions, including those based on single life cycle stages. It also provides a means to document unique or rare species that might only ever be recovered from a single life cycle stage. With an emphasis on the value of new information from field collections of any available life cycle stages, here we provide data for several blood fluke cercariae from freshwater snails from Kenya, Uganda, and Australia. Similar data are provided for adult worms of Macrobilharzia macrobilharzia and miracidia of Bivitellobilharzia nairi. Some schistosome and sanguinicolid cercariae that we recovered have peculiar morphological features, and our phylogenetic analyses (18S and 28S rDNA and mtDNA CO1) suggest that 2 of the new schistosome specimens likely represent previously unknown lineages. Our results also provide new insights into 2 of the 4 remaining schistosome genera yet to be extensively characterized with respect to their position in molecular phylogenies, Macrobilharzia and Bivitellobilharzia. The accessibility of each life cycle stage is likely to vary dramatically from one parasite species to the next, and our examples validate the potential usefulness of information gleaned from even one such stage, whatever it might be. PMID:16629320

  9. Multilocus, DNA-based phylogenetic analyses reveal three new species lineages in the Phellinus gabonensis-P. caribaeo-quercicola species complex, including P. amazonicus sp. nov.

    PubMed

    de Campos-Santana, Marisa; Amalfi, Mario; Castillo, Gabriel; Decock, Cony

    2016-09-01

    Species complexes in the poroid Hymenochaetaceae are well documented in the temperate areas. Potential species complexes are less known in tropical areas, however. In the last ten years, four phylogenetically and morphologically closely related species of Phellinus (Hymenochaetaceae) were described from various tropical/subtropical areas viz. P. caribaeo-quercicola, P. gabonensis, P. ellipsoideus, and P. castanopsidis They are characterized by cushion-shaped basidiomata, ventricose, commonly hamate hymenial setae, and broadly ellipsoid, thick-walled, pale yellowish basidiospores. Pursuing the studies of this complex, a phylogenetic approach based on DNA sequence data from the nuc rDNA regions ITS1-5.8S-ITS2 (ITS) and partial 28S (including the domains D1, D2, D3) and on part of the translation elongation factor 1-α (tef1, region between exons 4 and 8) revealed three new lineages or phylogenetic species. Two of these phylogenetic species are composed of exclusively on Neotropical specimens. One of them, described below as Phellinus amazonicus sp. nov., is represented by multiple collections originating from Neotropical, lowland, dense, moist forest at the western edge of the Amazon Basin in Ecuador, the Guiana Shield in French Guiana and (more likely) Trinidad. The second Neotropical phylogenetic species is represented in our phylogenetic analyses by a single collection from northeastern Argentina. It is also potentially known from two herbarium specimens originating from southern Brazil, for which no sequence data is available. It is left for now as Phellinus sp. 1, waiting to gather more specimens and DNA sequences data. The third new phylogenetic species is known by a single collection (pure culture) of uncertain origin. It is thought to represent Phellinus setulosus, a Southeast Asian taxa. From an evolutionary perspective, tree species occurring in the Neotropics (P. amazonicus, P. caribaeo-quercicola, and Phellinus sp. 1) have a closely related genetic

  10. An approach to revealing blood fluke life cycles, taxonomy, and diversity: provision of key reference data including DNA sequence from single life cycle stages.

    PubMed

    Brant, Sara V; Morgan, Jess A T; Mkoji, Gerald M; Snyder, Scott D; Rajapakse, R P V Jayanthe; Loker, Eric S

    2006-02-01

    Revealing diversity among extant blood flukes, and the patterns of relationships among them, has been hindered by the difficulty of determining if specimens described from different life cycle stages, hosts, geographic localities, and times represent the same or different species. Persistent collection of all available life cycle stages and provision of exact collection localities, host identification, reference DNA sequences for the parasite, and voucher specimens eventually will provide the framework needed to piece together individual life cycles and facilitate reconciliation with classical taxonomic descriptions, including those based on single life cycle stages. It also provides a means to document unique or rare species that might only ever be recovered from a single life cycle stage. With an emphasis on the value of new information from field collections of any available life cycle stages, here we provide data for several blood fluke cercariae from freshwater snails from Kenya, Uganda, and Australia. Similar data are provided for adult worms of Macrobilharzia macrobilharzia and miracidia of Bivitellobilharzia nairi. Some schistosome and sanguinicolid cercariae that we recovered have peculiar morphological features, and our phylogenetic analyses (18S and 28S rDNA and mtDNA CO1) suggest that 2 of the new schistosome specimens likely represent previously unknown lineages. Our results also provide new insights into 2 of the 4 remaining schistosome genera yet to be extensively characterized with respect to their position in molecular phylogenies, Macrobilharzia and Bivitellobilharzia. The accessibility of each life cycle stage is likely to vary dramatically from one parasite species to the next, and our examples validate the potential usefulness of information gleaned from even one such stage, whatever it might be.

  11. Biodegradable DNA-brush Block Copolymer Spherical Nucleic Acids Enable Transfection Agent-Free Intracellular Gene Regulation

    PubMed Central

    Zhang, Chuan; Hao, Liangliang; Calabrese, Colin M.; Zhou, Yu; Choi, Chung Hang J.; Xing, Hang; Mirkin, Chad A.

    2015-01-01

    A new strategy for synthesizing spherical nucleic acid (SNA) nanostructures from biodegradable DNA block copolymers is reported. Multiple DNA strands are grafted to one end of a polyester chain (poly-caprolactone) to generate an amphiphilic DNA brush block copolymer (DBBC) structure capable of assembling into spherical micelles in aqueous solution. These novel DBBC-based micelle-SNAs exhibit a higher surface density of nucleic acids compared to micelle structures assembled from an analogous linear DNA block copolymer (DBC), which endows them with the ability to more efficiently enter cells without the need for transfection agents. Importantly, the new SNAs show effective gene regulation without observable cellular toxicity in mammalian cell culture. PMID:26297167

  12. Health information impact on the relative importance of beef attributes including its enrichment with polyunsaturated fatty acids (omega-3 and conjugated linoleic acid).

    PubMed

    Kallas, Zein; Realini, Carolina E; Gil, José Maria

    2014-08-01

    This paper uses Choice Experiments (CE) to investigate Spanish consumers' preferences towards beef meat enriched with polyunsaturated fatty acids (omega-3 and conjugated linoleic acid). Data were gathered from self-completed questionnaires in a controlled environment with two different samples (320 and 322 consumers) differentiated by the information received. The surveys were carried out in three main Spanish cities (Barcelona, Zaragoza and Pamplona), representing the average consumer. A variation of the "Dual Response Choice Experiments" (DRCE) design was used due to its ability to emphasize the purchase context. Results showed that consumers who received information attach higher preference for enriched meat with polyunsaturated fatty acids. The utility associated with the higher content of fat increase for informed consumers, showing a substitute effect. Informed consumers are willing to accept meat with a higher amount of visible fat if it is enriched with beneficial fatty acids.

  13. Alkylation of nucleic acids by DNA-targeted 4-anilinoquinolinium aniline mustards: kinetic studies.

    PubMed

    O'Connor, C J; Denny, W A; Fan, J Y

    1991-01-01

    The rate of constant for hydrolysis of a series of 4-substituted aniline mustards Ar-X-pC6H4-N(CH2CH2Cl)2, where Ar is 4-anilinoquinolinium and X = O, CH2, CONH and CO, have been measured in water and 0.02 M imidazole buffer at 37 degrees C and in 50% aqueous acetone at 66 degrees C. The equilibrium binding constants of the compounds and their hydrolysis products to nucleic acids of differing base composition have been determined at varying ionic strengths, and the results are consistent with the compounds binding as expected in the DNA minor groove. The alkylating reactivity of the mustards towards these nucleic acids has been measured in water at 37 degrees C and in 0.01 M HEPES buffer over a range of temperatures from 25 degrees C to 60 degrees C. Evaluation of the thermodynamic parameters for these kinetic and equilibrium studies suggests that the interaction with nucleic acids is via an internal SN2 mechanism involving an aziridinium ion.

  14. Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid.

    PubMed

    Torabi, Forough; Binduraihem, Adel; Miller, David

    2017-03-01

    Mature spermatozoa bind hyaluronic acid in the extracellular matrix via hyaladherins. Immature spermatozoa may be unable to interact because they do not express the appropriate hyaladherins on their surface. Fresh human semen samples were fractionated using differential density gradient centrifugation (DDGC) and the ability of these fractions to bind hyaluronic acid was evaluated. The presence of sperm hyaladherins was also assessed. CD44 was located mainly on the acrosome and equatorial segment and became more restricted to the equatorial segment in capacitated spermatozoa. Hyaluronic acid-TRITC (hyaluronic acid conjugated with tetramethylrhodamine isothiocyanante), a generic hyaluronic-acid-binding reagent, labelled the membrane and the neck region, particularly after capacitation. Sperm populations obtained after DDGC or after interaction with hyaluronic acid were assessed for DNA fragmentation and chromatin maturity. Strong relationships between both measures and sperm sedimentation and hyaluronic-acid-binding profiles were revealed. Capacitation enhanced hyaluronic acid binding of both DDGC-pelleted sperm and sperm washed free of seminal fluid. In conclusion, hyaladherins were detected on human sperm and a higher capacity for sperm hyaluronic-acid-binding was shown to correspond with their DDGC sedimentation profiles and with lower levels of DNA fragmentation and better chromatin maturity. Capacitation induced changes in the distribution and presence of hyaladherins may enhance hyaluronic-acid-binding.

  15. Effect of metallic cations on the efficiency of DNA amplification. Implications for nucleic acid replication during early stages of life

    NASA Astrophysics Data System (ADS)

    Arribas, María; de Vicente, Aránzazu; Arias, Armando; Lázaro, Ester

    2005-04-01

    The process of catalysis of biochemical reactions has been essential since the first organic molecules appeared on Earth. As the complexity of the ensemble of primitive biomolecules was very low, primitive catalysts had necessarily to be very simple molecules or ions. The evolution of catalysts had to be in parallel with the evolution of the molecular species reacting. An example of this parallel evolution is nucleic acid polymerization. Synthesis of primitive short oligonucleotides could have been catalysed by metal ions either in solution or on the surface of minerals such as montmorillonite clays. Some oligonucleotides could start to function as templates for the synthesis of complementary copies and there is experimental evidence supporting the role also played by metal ions in this process. In later stages of evolution, a group of enzymatic proteins, nucleic acid polymerases, has been selected to catalyse nucleic acid replication. The presence of Mg2+ in the active centre of these enzymes suggests that evolution has preserved some of the primitive catalysts, including them as cofactors of more complex molecules. However, the reasons why Mg2+ was selected among other ions that possibly were present in primitive environments are unknown. In this paper we try to approach this question by analysing the amplification efficiency of the polymerase chain reaction of a DNA fragment in the presence of different metal ions. In some cases the conditions of the reaction have been displaced from optimum (by the presence of nucleotide imbalances and a suboptimal Mg2+concentration). The results obtained permit one to draw interesting conclusions about how some metallic cations can help replication to proceed in conditions of limited substrate availability, a circumstance that could have been frequent at prebiotic stages, when nucleic acid synthesis was dependent on the physico-chemical conditions of the environment.

  16. An Investigation into the Association between DNA Damage and Dietary Fatty Acid in Men with Prostate Cancer

    PubMed Central

    Bishop, Karen S.; Erdrich, Sharon; Karunasinghe, Nishi; Han, Dug Yeo; Zhu, Shuotun; Jesuthasan, Amalini; Ferguson, Lynnette R.

    2015-01-01

    Prostate cancer is a growing problem in New Zealand and worldwide, as populations adopt a Western style dietary pattern. In particular, dietary fat is believed to be associated with oxidative stress, which in turn may be associated with cancer risk and development. In addition, DNA damage is associated with the risk of various cancers, and is regarded as an ideal biomarker for the assessment of the influence of foods on cancer. In the study presented here, 20 men with prostate cancer adhered to a modified Mediterranean style diet for three months. Dietary records, blood fatty acid levels, prostate specific antigen, C-reactive protein and DNA damage were assessed pre- and post-intervention. DNA damage was inversely correlated with dietary adherence (p = 0.013) and whole blood monounsaturated fatty acids (p = 0.009) and oleic acid (p = 0.020). DNA damage was positively correlated with the intake of dairy products (p = 0.043), red meat (p = 0.007) and whole blood omega-6 polyunsaturated fatty acids (p = 0.015). Both the source and type of dietary fat changed significantly over the course of the dietary intervention. Levels of DNA damage were correlated with various dietary fat sources and types of dietary fat. PMID:25580814

  17. Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

    PubMed

    Tang, Sheau-Chung; Yeh, Jih-I; Hung, Sung-Jen; Hsiao, Yu-Ping; Liu, Fu-Tong; Yang, Jen-Hung

    2016-03-01

    AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

  18. Ability of hypochlorous acid and N-chloramines to chlorinate DNA and its constituents.

    PubMed

    Stanley, Naomi R; Pattison, David I; Hawkins, Clare L

    2010-07-19

    Myeloperoxidase is a heme enzyme released by activated phagocytes that is responsible for the generation of the strong oxidant hypochlorous acid (HOCl). Although HOCl has potent bactericidal properties and plays an important role in the human immune system, this oxidant also causes damage to tissues, particularly under inflammatory conditions. There is a strong link between chronic inflammation and the incidence of many cancers, which may be associated with the ability of HOCl and related oxidants such as N-chloramines to damage DNA. However, in contrast to HOCl, little is known about the reactivity of N-chloramines with DNA and its constituents. In this study, we examine the ability of HOCl and various N-chloramines to form chlorinated base products on nucleosides, nucleotides, DNA, and in cellular systems. Experiments were performed with N-chloramines formed on Nalpha-acetyl-histidine (His-C), Nalpha-acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-C), and ammonia (Mono-C). Treatment of DNA and related materials with HOCl and His-C resulted in the formation of 5-chloro-2'-deoxycytidine (5CldC), 8-chloro-2'-deoxyadenosine (8CldA) and 8-chloro-2'-deoxyguanosine (8CldG). With the nucleosides, 8CldG was the favored product in each case, and HOCl was the most efficient chlorinating agent. 5Cl(d)C was the most abundant product on exposure of the nucleotides and DNA to HOCl and His-C, with only low levels of chlorinated products observed with Lys-C, Gly-C, Tau-C, and Mono-C. 5CldC was also formed on exposure of smooth muscle cells to either HOCl or His-C. Cellular RNA was also a target for HOCl and His-C, with evidence for the formation of 5-chloro-cytidine (5ClC). This study shows that HOCl and the model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may play a role in the development of various inflammatory cancers.

  19. Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA

    PubMed Central

    Yamada, Tetsuji; Palm, Curtis J.; Brooks, Bob; Kosuge, Tsune

    1985-01-01

    We report the nucleotide sequences of iaaM and iaaH, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium Pseudomonas syringae pv. savastanoi. The sequence analysis indicates that the iaaM locus contains an open reading frame encoding 557 amino acids that would comprise a protein with a molecular weight of 61,783; the iaaH locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of P. savastanoi and the deduced product of the T-DNA tms-1 gene of the octopine-type plasmid pTiA6NC from Agrobacterium tumefaciens. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of iaaH and tms-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in P. savastanoi and in A. tumefaciens T-DNA. Images PMID:16593610

  20. SDR-ELISA: Ultrasensitive and high-throughput nucleic acid detection based on antibody-like DNA nanostructure.

    PubMed

    Wen, Junlin; Chen, Junhua; Zhuang, Li; Zhou, Shungui

    2017-04-15

    An ultrasensitive and high-throughput nucleic acid detection system, termed as strand displacement reaction-enzyme linked immunosorbent assay (SDR-ELISA), has been developed on the basis of antibody-like DNA nanostructures. Three digoxigenin or biotin modified hairpin probes are utilized to construct antibody-like DNA nanostructures that feature affinity toward streptavidin and anti-digoxigenin antibody via isothermal target-triggered SDR amplification. These antibody-like nanostructures have been employed to conjugate horseradish-peroxidase-labeled anti-digoxigenin antibody with streptavidin that is immobilized on microliter plate wells for enzyme-linked colorimetric assay. The resulting SDR-ELISA system is ultrasensitive for target DNA with a low detection limit of 5 fM. Moreover, the SDR-ELISA system is capable of discriminating DNA sequences with single base mutations, and do so in a high-throughput manner by detection and quantification of up to 96 or 384 DNA samples in a single shot. This detection system is further applied to detect other DNA targets such as Shewanella oneidensis specific DNA sequence, which indicates the generality of proposed SDR-ELISA system. The integration of SDR amplification and convenient ELISA technique advances an intelligent strategy for ultrasensitive and high-throughput nucleic acid detection, which may be amenable for direct visual detection and quantification using an accompanying quantitative color chart.

  1. Effect of pollution on DNA damage and essential fatty acid profile in Cirrhinus mrigala from River Chenab

    NASA Astrophysics Data System (ADS)

    Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, K. A.; Mahboob, Shahid

    2016-05-01

    The objective of this study was to evaluate the effect of anthropogenic pollution on DNA damage and the fatty acid profile of the bottom dweller fish (Cirrhinus mrigala), collected from the River Chenab, in order to assess the effect of the toxicants on the quality of the fish meat. The levels of Cd, Hg, Cu, Mn, Zn, Pb, Cr and Sn and of phenols from this river were significantly higher than the permissible limits set by the USEPA. Comet assays showed DNA damage in Cirrhinus mrigala collected from three different sampling sites in the polluted area of the river. Significant differences were observed for DNA damage through comet assay in fish collected from polluted compared to control sites. No significant differences were observed for DNA damage between farmed and fish collected from upstream. The micronucleus assay showed similar trends. Fish from the highly polluted sites showed less number of fatty acids and more saturated fatty acids in their meat compared to fish from less polluted areas. Several fatty acids were missing in fish with higher levels of DNA in comet tail and micronucleus induction. Long-chain polyunsaturated fatty acids, eicosapentaenoic acid (20:5n-3) was found missing in the fish from polluted environment while it was found in considerable amount in farmed fish 7.8±0.4%. Docosahexaenoic acid (22:6n-3) also showed significant differences as 0.1±0.0 and 7.0±0.1% respectively, in wild polluted and farmed fishes.

  2. Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

    PubMed

    Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

    2014-11-01

    A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity.

  3. Nucleic Acids Research Group (NRG): The Importance of DNA Extraction in Metagenomics: The Gatekeeper to Accurate Results!

    PubMed Central

    Carmical, R.; Nadella, V.; Herbert, Z.; Beckloff, N.; Chittur, S.; Rosato, C.; Perera, A.; Auer, H.; Robinson, M.; Tighe, S.; Holbrook, Jennifer

    2013-01-01

    It is well recognized that the field of metagenomics is becoming a critical tool for studying previously unobtainable population dynamics at both an identification of species level and a functional or transcriptional level. Because the power to resolve microbial information is so important for identifying the components in an mixed sample, metagenomics can be used to study nearly any possible environment or system including clinical, environmental, and industrial, to name a few. Clinically, it may be used to determine sub-populations colonizing regions of the body or determining a rare infection to assist in treatment strategies. Environmentally it may be used to identify microbial populations within a soil, water or air sample, or within a bioreactor to characterize a population- based functional process. The possibilities are endless. However, the accuracy of a metagenomics dataset relies on three important “gatekeepers” including 1) The ability to effectively extract all DNA or RNA from every cell within a sample, 2) The reliability of the methods used for deep or high-throughput sequencing, and 3) The software used to analyze the data. Since DNA extraction is the first step in the technical process of metagenomics, the Nucleic Acid Research Group (NARG) conducted a study to evaluate extraction methods using a synthetic microbial sample. The synthetic microbial sample was prepared from 10 known bacteria at specific concentrations and ranging in diversity. Samples were extracted in duplicate using various popular kit based methods as well as several homebrew protocols then analyzed by NextGen sequencing on an Illumina HiSeq. Results of the study include determining the percent recovery of those organisms by comparing to the known quantity in the original synthetic mix.

  4. Multiplex paper-based colorimetric DNA sensor using pyrrolidinyl peptide nucleic acid-induced AgNPs aggregation for detecting MERS-CoV, MTB and HPV oligonucleotides.

    PubMed

    Tee-Ngam, Prinjaporn; Siangproh, Weena; Tuantranont, Adisorn; Vilaivan, Tirayut; Chailapakul, Orawon; Henry, Charles S

    2017-04-10

    The development of simple fluorescent and colorimetric assays that enable point-of-care DNA and RNA detection has been a topic of significant research because of the utility of such assays in resource limited settings. The most common motifs utilize hybridization to a complementary detection strand coupled with a sensitive reporter molecule. Here, apaper-based colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid (acpcPNA)-induced nanoparticle aggregationis reported as an alternative to traditional colorimetric approaches. PNA probes are an attractive alternative to DNA and RNA probes because they are chemically and biologically stable, easily synthesized, and hybridize efficiently with the complementary DNA strands. The acpcPNA probe contains a single positive charge from the lysine at C-terminus and causes aggregation of citrate anion-stabilized silver nanoparticles (AgNPs) in the absence of complementary DNA. In the presence of target DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion of the AgNPs as a result of electrostatic repulsion, giving rise to a detectable color change. Factors affecting the sensitivity and selectivity of this assay were investigated, including ionic strength, AgNP concentration, PNA concentration, and DNA strand mismatches. The method was used for screening of synthetic Middle East respiratory syndrome coronavirus (MERS-CoV), mycobacterium tuberculosis (MTB) and human papillomavirus (HPV)DNA based on a colorimetric paper-based analytical device developed using the aforementioned principle. The oligonucleotide targets were detected by measuring the color change of AgNPs, giving detection limits of 1.53 nM (MERS-CoV), 1.27 nM (MTB) and 1.03 nM (HPV).The acpcPNA probe exhibited high selectivity for the complementary oligonucleotides over single-base-mismatch, two-base-mismatch and non-complementary DNA targets. The proposed paper-based colorimetric DNA sensor has potential to be an alternative

  5. Meat texture and antioxidant status are improved when carnosic acid is included in the diet of fattening lambs.

    PubMed

    Morán, Lara; Andrés, Sonia; Bodas, Raúl; Prieto, Nuria; Giráldez, F Javier

    2012-08-01

    Thirty-two Merino lambs fed barley straw and a concentrate alone (CONTROL group) or enriched with carnosic acid [0.6 g kg(-1) dry matter (DM), CARN006 group; 1.2 g kg(-1) DM, CARN012 group] or vitamin E (0.6 g kg(-1) DM, VITE006 group) were used to assess the effect of these antioxidant compounds on meat quality. After being fed the experimental diets for at least 5 weeks, the animals were slaughtered with the 25 kg intended body weight and the different muscles (longissimus lumborum; LL, gluteus medius; GM) were sliced and kept refrigerated under modified atmosphere packaging during 0, 7 and 14 days. The results indicate that carnosic acid seemed to be useful to delay lipid peroxidation in a medium colour-stable muscle such as GM, but this effect was lower than that observed when vitamin E was supplemented to fattening lambs. On the contrary, meat texture and protection against cholesterol oxidation were equally improved with both compounds.

  6. Ferulic acid inhibits gamma radiation-induced DNA strand breaks and enhances the survival of mice.

    PubMed

    Maurya, Dharmendra Kumar; Devasagayam, Thomas Paul Asir

    2013-02-01

    Ferulic acid (FA) is a monophenolic phenylpropanoid occurring in plant products such as rice bran, green tea, and coffee beans. It has been shown to have significant antioxidant effects in many studies. In the present study, we show that intraperitoneal administration of FA at a dose of 50 mg/kg body weight 1 hour prior to or immediately after whole-body γ-irradiation of mice with 4 Gy results in considerable reduction in the micronuclei formation in peripheral blood reticulocytes. Administration of the same amount of FA immediately after 4 Gy γ-irradiation showed significant decrease in the amount of DNA strand breaks in murine peripheral blood leukocytes and bone marrow cells as examined by comet assay. Further, immunostaining of mouse splenic lymphocytes for phspho-γH2AX was carried out, and it was observed that FA inhibits the γH2AX foci formation. Finally, the survival of mice upon 6, 8, and 10 Gy γ-ray exposure was monitored. FA enhances the survival of mice by a factor of 2.5 at a dose of 6 Gy γ-radiation but not at higher doses. In conclusion, FA has protective potential in both pre- and postirradiation exposure scenarios and enhances the survival of mice possibly by decreasing DNA damage as examined by γH2AX foci, micronuclei formation, and comet assay.

  7. The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology.

    PubMed

    Derkx, Patrick M F; Janzen, Thomas; Sørensen, Kim I; Christensen, Jeffrey E; Stuer-Lauridsen, Birgitte; Johansen, Eric

    2014-08-29

    The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.

  8. The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology

    PubMed Central

    2014-01-01

    The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes. PMID:25186244

  9. Deoxycholic acid causes DNA damage while inducing apoptotic resistance through NF-κB activation in benign Barrett's epithelial cells.

    PubMed

    Huo, Xiaofang; Juergens, Stefanie; Zhang, Xi; Rezaei, Davood; Yu, Chunhua; Strauch, Eric D; Wang, Jian-Ying; Cheng, Edaire; Meyer, Frank; Wang, David H; Zhang, Qiuyang; Spechler, Stuart J; Souza, Rhonda F

    2011-08-01

    Gastroesophageal reflux is associated with adenocarcinoma in Barrett's esophagus, but the incidence of this tumor is rising, despite widespread use of acid-suppressing medications. This suggests that refluxed material other than acid might contribute to carcinogenesis. We looked for potentially carcinogenetic effects of two bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on Barrett's epithelial cells in vitro and in vivo. We exposed Barrett's (BAR-T) cells to DCA or UDCA and studied the generation of reactive oxygen/nitrogen species (ROS/RNS); expression of phosphorylated H2AX (a marker of DNA damage), phosphorylated IkBα, and phosphorylated p65 (activated NF-κB pathway proteins); and apoptosis. During endoscopy in patients, we took biopsy specimens of Barrett's mucosa before and after esophageal perfusion with DCA or UDCA and assessed DNA damage and NF-κB activation. Exposure to DCA, but not UDCA, resulted in ROS/RNS production, DNA damage, and NF-κB activation but did not increase the rate of apoptosis in BAR-T cells. Pretreatment with N-acetyl-l-cysteine (a ROS scavenger) prevented DNA damage after DCA exposure, and DCA did induce apoptosis in cells treated with NF-κB inhibitors (BAY 11-7085 or AdIκB superrepressor). DNA damage and NF-κB activation were detected in biopsy specimens of Barrett's mucosa taken after esophageal perfusion with DCA, but not UDCA. These data show that, in Barrett's epithelial cells, DCA induces ROS/RNS production, which causes genotoxic injury, and simultaneously induces activation of the NF-κB pathway, which enables cells with DNA damage to resist apoptosis. We have demonstrated molecular mechanisms whereby bile reflux might contribute to carcinogenesis in Barrett's esophagus.

  10. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  11. Induction of CYP1A and cyp2-mediated arachidonic acid epoxygenation and suppression of 20-hydroxyeicosatetraenoic acid by imidazole derivatives including the aromatase inhibitor vorozole.

    PubMed

    Diani-Moore, Silvia; Papachristou, Fotini; Labitzke, Erin; Rifkind, Arleen B

    2006-08-01

    Cytochrome P450 (P450) enzymes metabolize the membrane lipid arachidonic acid to stable biologically active epoxides [eicosatrienoic acids (EETs)] and 20-hydroxyeicosatetraenoic acid (20-HETE). These products have cardiovascular activity, primarily acting as vasodilators and vasoconstrictors, respectively. EET formation can be increased by the prototype CYP1A or CYP2 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or phenobarbital (PB), respectively. We report here that imidazole derivative drugs: the anthelminthics, albendazole and thiabendazole; the proton pump inhibitor, omeprazole; the thromboxane synthase inhibitor, benzylimidazole; and the aromatase (CYP19) inhibitor vorozole (R76713, racemate; and R83842, (+) enantiomer) increased hepatic microsomal EET formation in a chick embryo model. Albendazole increased EETs by transcriptional induction of CYP1A5 and the others by combined induction of CYP1A5 and CYP2H, the avian orthologs of mammalian CYP1A2 and CYP2B, respectively. All inducers increased formation of the four EET regioisomers, but TCDD and albendazole had preference for 5,6-EET and PB and omeprazole for 14,15-EET. Vorozole, benzylimidazole, and TCDD also suppressed 20-HETE formation. Vorozole was a remarkably effective and potent inducer of multiple hepatic P450s at a dose range which overlapped its inhibition of ovarian aromatase. Increased CYP1A activity in mouse Hepa 1-6 and human HepG2 cells by vorozole and other imidazole derivatives demonstrated applicability of the findings to mammalian cells. The findings suggest that changes in P450-dependent arachidonic acid metabolism may be a new source of side effects for drugs that induce CYP1A or CYP2. They demonstrate further that in vivo induction of multiple hepatic P450s produces additive increases in arachidonic acid epoxygenase activity and can occur concurrently with inhibition of ovarian aromatase activity.

  12. Growth and development of the arborescent cactus Stenocereus queretaroensis in a subtropical semiarid environment, including effects of gibberellic acid.

    PubMed

    Pimienta, Eulogio; Hernandez, Gerardo; Domingues, Alejandro; Nobel, Park S.

    1998-01-01

    In Stenocereus queretaroensis (Weber) Buxbaum, an arborescent cactus cultivated in Jalisco, Mexico, for its fruits but studied here in wild populations, stem extension occurred in the autumn at the beginning of the dry season, flowering and fruiting occurred in the spring at the end of the dry season, and new roots grew in the summer during the wet season. The asynchrony of vegetative and reproductive growth reduces competitive sink effects, which may be advantageous for wild populations growing in infertile rocky soils. Seasonal patterns of sugars in the roots and especially the stems of S. queretaroensis were closely related to the main phenological stages, becoming lower in concentration during periods of major stem extension. Cessation of stem extension occurred in 100-year-old plants for which injection of GA(3) reinitiated such growth. Isolated chlorenchyma cylinders had maximum extension in a bathing solution containing 0.1 &mgr;M gibberellic acid.

  13. A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA.

    PubMed

    Shen, Li; Gao, Ge; Zhang, Ying; Zhang, He; Ye, Zhiqiang; Huang, Shichao; Huang, Jinyan; Kang, Jiuhong

    2010-10-01

    Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.

  14. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    PubMed

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring.

  15. A unified model of nucleic acid unwinding by the ribosome and the hexameric and monomeric DNA helicases.

    PubMed

    Xie, Ping

    2015-09-07

    DNA helicases are enzymes that use the chemical energy to separate DNA duplex into their single-stranded forms. The ribosome, which catalyzes the translation of messenger RNAs (mRNAs) into proteins, can also unwind mRNA duplex. According to their structures, the DNA helicases can fall broadly into hexameric and monomeric forms. A puzzling issue for the monomeric helicases is that although they have similar structures, in vitro biochemical data showed convincingly that in the monomeric forms some have very weak DNA unwinding activities, some have relatively high unwinding activities while others have high unwinding activities. However, in the dimeric or oligomeric forms all of them have high unwinding activities. In addition, in the monomeric forms all of them can translocate efficiently along the single-stranded DNA (ssDNA). Here, we propose a model of the translocation along the ssDNA and DNA unwinding by the monomeric helicases, providing a consistent explanation of these in vitro experimental data. Moreover, by comparing the present model for the monomeric helicases with the model for the hexameric helicases and that for the ribosome which were proposed before, a unified model of nucleic acid unwinding by the three enzymes is proposed.

  16. A Magnetic Nanoparticle Based Nucleic Acid Isolation and Purification Instrument for DNA Extraction of Escherichia Coli O157: H7.

    PubMed

    Chen, Yahui; Lin, Jianhan; Jiang, Qin; Chen, Qi; Zhang, Shengjun; Li, Li

    2016-03-01

    The objective of this study was to evaluate the performance of a nucleic acid isolation and purification instrument using Escherichia coli O157:H7 as the model. The instrument was developed with magnetic nanoparticles for efficiently capturing nucleic acids and an intelligent mechanical unit for automatically performing the whole nucleic acid extraction process. A commercial DNA extraction kit from Huier Nano Company was used as reference. Nucleic acids in 1 ml of E. coli O157: H7 at a concentration of 5 x 10(8) CFU/mL were extracted by using this instrument and the kit in parallel and then detected by an ultraviolet spectrophotometer to obtain A260 values and A260/A280 values for the determination of the extracted DNA's quantity and purity, respectively. The A260 values for the instrument and the kit were 0.78 and 0.61, respectively, and the A260/A280 values were 1.98 and 1.93. The coefficient of variations of these parallel tests ranged from 10.5% to 16.7%. The results indicated that this nucleic acid isolation and purification instrument could extract a comparable level of nucleic acid within 50 min compared to the commercial DNA extraction kit.

  17. Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid.

    PubMed

    Vermeersch, Jacqueline J; Christmann-Franck, Serge; Karabashyan, Leon V; Fermandjian, Serge; Mirambeau, Gilles; Der Garabedian, P Arsène

    2004-01-01

    The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the epsilon-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K(i) = 40 microM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR). Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505). In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K532-PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.

  18. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

    SciTech Connect

    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-05-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. (/sup 3/H)Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

  19. In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

    PubMed Central

    Fabbro, D; Tell, G; Leonardi, A; Pellizzari, L; Pucillo, C; Lonigro, R; Formisano, S; Damante, G

    1996-01-01

    The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases. PMID:8811078

  20. Invertrons, a class of structurally and functionally related genetic elements that includes linear DNA plasmids, transposable elements, and genomes of adeno-type viruses.

    PubMed Central

    Sakaguchi, K

    1990-01-01

    Invertrons are genetic elements composed of DNA with inverted terminal repeats at both ends, covalently bonded to terminal proteins involved in the initiation of DNA replication at both their 5' termini when they exist in the cytoplasm of their host in free form. They function as viruses, linear DNA plasmids, transposable elements, and sometimes combinations of two of these properties. They differ from retroviruses and related retro-type transposons which have direct repeats on both their genomic ends and exploit RNA intermediates for replication of their DNA. A model for replication and integration of invertrons is presented, as well as a model for transposition of transposable elements. PMID:2157134

  1. Multi-species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation.

    PubMed

    Park, Seongjun; Bae, Wookeun; Rittmann, Bruce E

    2010-04-15

    A multi-species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite-oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia-oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients.

  2. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE1[OPEN

    PubMed Central

    Van de Wouwer, Dorien; Decou, Raphaël; Audenaert, Dominique; Nguyen, Long

    2016-01-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  3. The Effects of Topically Applied Glycolic Acid and Salicylic Acid on Ultraviolet Radiation-Induced Erythema, DNA Damage and Sunburn Cell Formation in Human Skin

    PubMed Central

    Kornhauser, Andrija; Wei, Rong-Rong; Yamaguchi, Yuji; Coelho, Sergio G.; Kaidbey, Kays; Barton, Curtis; Takahashi, Kaoruko; Beer, Janusz Z.; Miller, Sharon A.; Hearing, Vincent J.

    2009-01-01

    Background α-Hydroxy acids (αHA) are reported to reduce signs of aging in the skin and are widely used cosmetic ingredients. Several studies suggest that αHA can increase the sensitivity of skin to ultraviolet radiation. More recently, β-hydroxy acids (βHA), or combinations of αHA and βHA have also been incorporated into antiaging skin care products. Concerns have also arisen about increased sensitivity to ultraviolet radiation following use of skin care products containing β-HA. Objective To determine whether topical treatment with glycolic acid, a representative αHA, or with salicylic acid, a βHA, modifies the short-term effects of solar simulated radiation (SSR) in human skin. Methods Fourteen subjects participated in this study. Three of the four test sites on the mid-back of each subject were treated daily Monday - Friday, for a total of 3.5 weeks, with glycolic acid (10%), salicylic acid (2%), or vehicle (control). The fourth site received no treatment. After the last treatment, each site was exposed to SSR, and shave biopsies from all 4 sites were obtained. The endpoints evaluated in this study were erythema (assessed visually and instrumentally), DNA damage and sunburn cell formation. Results Treatment with glycolic acid resulted in increased sensitivity of human skin to SSR, measured as an increase in erythema, DNA damage and sunburn cell formation. Salicylic acid did not produce significant changes in any of these biomarkers. Conclusions Short-term topical application of glycolic acid in a cosmetic formulation increased the sensitivity of human skin to SSR, while a comparable treatment with salicylic acid did not. PMID:19411163

  4. Noninvasive measurement of aristolochic acid-DNA adducts in urine samples from aristolochic acid-treated rats by liquid chromatography coupled tandem mass spectrometry: evidence for DNA repair by nucleotide-excision repair mechanisms.

    PubMed

    Leung, Elvis M K; Chan, Wan

    2014-01-01

    Nephrotoxic aristolochic acids (AAs) form covalently bonded DNA adducts upon metabolic activation. In this work, a non-invasive approach to detect AAs exposure by quantifying urinary excreted DNA-AA adducts is presented. The developed method entails solid-phase extraction (SPE) enrichment of the urine-excreted DNA-AAs adducts, addition of internal standard, and quantification by liquid chromatography coupled tandem mass spectrometric (LC-MS/MS) analysis. Quantitative analysis revealed 7-(deoxyadenosine-N(6)-yl)-aristolactam II and 7-(deoxyguanosine-N(2)-yl)-aristolactam I that were previously detected as major DNA-AA adducts in different organs of AA-dosed rats, were detected as the major urine excreted adducts. Lower levels of 7-(deoxyadenosine-N(6)-yl)-aristolactam I and 7-(deoxyguanosine-N(2)-yl)-aristolactam II were also detected in the collected urine samples. The identities of the detected urinary DNA-AA adducts were confirmed by comparing chromatographic retention time with synthetic standards, by high-accuracy MS, and MS/MS analyses. LC-MS/MS analysis of the urine samples collected from the AAs-dosed rats demonstrated a time-dependent decrease in the urinary adduct levels, indicating the urinary DNA-AA adduct levels were reflective of the tissue adduct levels. It is expected that the developed approach of detecting urinary DNA-AA adducts will facilitate further carcinogenesis investigations of AAs.

  5. Novel triterpenoids inhibit both DNA polymerase and DNA topoisomerase.

    PubMed Central

    Mizushina, Y; Iida, A; Ohta, K; Sugawara, F; Sakaguchi, K

    2000-01-01

    As described previously, we found that new triterpenoid compounds, designated fomitellic acids A and B, which selectively inhibit the activities of mammalian DNA polymerases alpha and beta [Mizushina, Tanaka, Kitamura, Tamai, Ikeda, Takemura, Sugawara, Arai, Matsukage, Yoshida and Sakaguchi (1998) Biochem. J. 330, 1325-1332; Tanaka, Kitamura, Mizushina, Sugawara and Sakaguchi (1998) J. Nat. Prod. 61, 193-197] and that a known triterpenoid, ursolic acid, is an inhibitor of human DNA topoisomerases I and II (A. Iida, Y. Mizushina and K. Sakaguchi, unpublished work). Here we report that all of these triterpenoids are potent inhibitors of calf DNA polymerase alpha, rat DNA polymerase beta and human DNA topoisomerases I and II, and show moderate inhibitory effects on plant DNA polymerase II and human immunodeficiency virus reverse transcriptase. However, these compounds did not influence the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes such as human telomerase, T7 RNA polymerase and bovine deoxyribonuclease I. These triterpenoids were not only mammalian DNA polymerase inhibitors but also inhibitors of DNA topoisomerases I and II even though the enzymic characteristics of DNA polymerases and DNA topoisomerases, including their modes of action, amino acid sequences and three-dimensional structures, differed markedly. These triterpenoids did not bind to DNA, suggesting that they act directly on these enzymes. Because the three-dimensional structures of fomitellic acids were shown by computer simulation to be very similar to that of ursolic acid, the DNA-binding sites of both enzymes, which compete for the inhibitors, might be very similar. Fomitellic acid A and ursolic acid prevented the growth of NUGC cancer cells, with LD(50) values of 38 and 30 microM respectively. PMID:10970789

  6. Breaking the dogma: PCB-derived semiquinone free radicals do not form covalent adducts with DNA, GSH, and amino acids

    PubMed Central

    Wangpradit, Orarat; Rahaman, Asif; Mariappan, S. V. Santhana; Buettner, Garry R.; Robertson, Larry W.; Luthe, Gregor

    2016-01-01

    Covalent bond formations of free radical metabolites with biomolecules like DNA and proteins are thought to constitute a major mechanism of toxicity and carcinogenesis. Glutathione (GSH) is generally accepted as a radical scavenger protecting the cell. In the present study, we investigated a semiquinone radical (SQ•-) metabolite of the semivolatile 4-chlorobiphenyl, using electron paramagnetic resonance spectroscopy, and oxygen consumption. Proton nuclear magnetic resonance (1H NMR) and liquid chromatography–mass spectrometry (LC-MS) were also employed to elucidate the radical interaction with DNA, amino acids, and GSH. We found that DNA and oligonucleotides stabilized SQ•- by electron delocalization in the π-stacking system, resulting in persistent radical intercalated, rather than forming a covalent bond with SQ•-. This finding was strongly supported by the semiempirical calculation of the semioccupied molecular orbital and the linear combination of the atomic orbitals, indicating 9.8 kcal mol−1 energy gain. The insertion of SQ•- into the DNA strand may result in DNA strand breaks and interruption of DNA replication process or even activate radical mediated secondary reactions. The presence of amino acids resulted in a decrease of the electron paramagnetic resonance (EPR) signal of SQ•- and correlated with their isoelectric points. The pH shifts the equilibrium of the dianions of hydroquinone and influenced indirectly the formation of SQ•-. Similar findings were observed with GSH and Cys. GSH and Cys functioned as indirect radical scavengers; their activities depend on their chemical equilibria with the corresponding quinones, and their further reaction via Michael addition. The generally accepted role of GSH as radical scavenger in biological systems should be reconsidered based upon these findings, questioning the generally accepted view of radical interaction of semiquinones with biologically active compounds, like DNA, amino acids, proteins

  7. A mushroom-derived amino acid, ergothioneine, is a potential inhibitor of inflammation-related DNA halogenation.

    PubMed

    Asahi, Takashi; Wu, Xiaohong; Shimoda, Hiroshi; Hisaka, Shinsuke; Harada, Etsuko; Kanno, Tomomi; Nakamura, Yoshimasa; Kato, Yoji; Osawa, Toshihiko

    2016-01-01

    Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous acid (HOBr), in inflammatory regions are postulated to contribute to disease progression. In this study, we showed that ergothioneine (EGT), derived from an edible mushroom, inhibited MPO activity as well as the formation of 8-bromo-2'-deoxyguanosine in vitro. The HOBr scavenging effect of EGT is higher than those of ascorbic acid and glutathione. We initially observed that the administration of Coprinus comatus, an edible mushroom containing a high amount of EGT, inhibited the UV-B-induced inflammatory responses and DNA halogenation, suggesting that EGT is a promising anti-inflammatory agent from mushrooms.

  8. Diversity of Dicotyledenous-Infecting Geminiviruses and Their Associated DNA Molecules in Southern Africa, Including the South-West Indian Ocean Islands

    PubMed Central

    Rey, Marie E. C.; Ndunguru, Joseph; Berrie, Leigh C.; Paximadis, Maria; Berry, Shaun; Cossa, Nurbibi; Nuaila, Valter N.; Mabasa, Ken G.; Abraham, Natasha; Rybicki, Edward P.; Martin, Darren; Pietersen, Gerhard; Esterhuizen, Lindy L.

    2012-01-01

    The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-βs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world. PMID:23170182

  9. Diversity of dicotyledenous-infecting geminiviruses and their associated DNA molecules in southern Africa, including the South-west Indian ocean islands.

    PubMed

    Rey, Marie E C; Ndunguru, Joseph; Berrie, Leigh C; Paximadis, Maria; Berry, Shaun; Cossa, Nurbibi; Nuaila, Valter N; Mabasa, Ken G; Abraham, Natasha; Rybicki, Edward P; Martin, Darren; Pietersen, Gerhard; Esterhuizen, Lindy L

    2012-09-01

    The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-βs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world.

  10. Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

    NASA Technical Reports Server (NTRS)

    Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

  11. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    PubMed

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection.

  12. Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with β-cyclodextrin as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Wang, Lun; Bian, Guirong; Wang, Leyu; Dong, Ling; Chen, Hongqi; Xia, Tingting

    2005-04-01

    A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/β-cyclodextrin(β-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/β-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 μg mL -1 for calf thymus DNA (ct-DNA) and 0.3-12 μg mL -1 for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 μg mL -1 for ct-DNA and 0.02 μg mL -1 for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 μg mL -1 ct-DNA and 1.4% for 2.0 μg mL -1 fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

  13. Topology of RNA-protein nucleobase-amino acid π-π interactions and comparison to analogous DNA-protein π-π contacts.

    PubMed

    Wilson, Katie A; Holland, Devany J; Wetmore, Stacey D

    2016-05-01

    The present work analyzed 120 high-resolution X-ray crystal structures and identified 335 RNA-protein π-interactions (154 nonredundant) between a nucleobase and aromatic (W, H, F, or Y) or acyclic (R, E, or D) π-containing amino acid. Each contact was critically analyzed (including using a visual inspection protocol) to determine the most prevalent composition, structure, and strength of π-interactions at RNA-protein interfaces. These contacts most commonly involve F and U, with U:F interactions comprising one-fifth of the total number of contacts found. Furthermore, the RNA and protein π-systems adopt many different relative orientations, although there is a preference for more parallel (stacked) arrangements. Due to the variation in structure, the strength of the intermolecular forces between the RNA and protein components (as determined from accurate quantum chemical calculations) exhibits a significant range, with most of the contacts providing significant stability to the associated RNA-protein complex (up to -65 kJ mol(-1)). Comparison to the analogous DNA-protein π-interactions emphasizes differences in RNA- and DNA-protein π-interactions at the molecular level, including the greater abundance of RNA contacts and the involvement of different nucleobase/amino acid residues. Overall, our results provide a clearer picture of the molecular basis of nucleic acid-protein binding and underscore the important role of these contacts in biology, including the significant contribution of π-π interactions to the stability of nucleic acid-protein complexes. Nevertheless, more work is still needed in this area in order to further appreciate the properties and roles of RNA nucleobase-amino acid π-interactions in nature.

  14. Target-catalyzed autonomous assembly of dendrimer-like DNA nanostructures for enzyme-free and signal amplified colorimetric nucleic acids detection.

    PubMed

    He, Hongfei; Dai, Jianyuan; Duan, Zhijuan; Meng, Yan; Zhou, Cuisong; Long, Yuyin; Zheng, Baozhan; Du, Juan; Guo, Yong; Xiao, Dan

    2016-12-15

    Self-assembly of DNA nanostructures is of great importance in nanomedicine, nanotechnology and biosensing. Herein, a novel target-catalyzed autonomous assembly pathway for the formation of dendrimer-like DNA nanostructures that only employing target DNA and three hairpin DNA probes was proposed. We use the sticky-ended Y shape DNA (Y-DNA) as the assembly monomer and it was synthesized by the catalyzed hairpin assembly (CHA) instead of the DNA strand annealing method. The formed Y-DNA was equipped with three ssDNA sticky ends and two of them were predesigned to be complementary to the third one, then the dendrimer-like DNA nanostructures can be obtained via an autonomous assembly among these sticky-ended Y-DNAs. The resulting nanostructure has been successfully applied to develop an enzyme-free and signal amplified gold nanoparticle (AuNP)-based colorimetric nucleic acids assay.

  15. Morphology and SSU rDNA sequence analysis of two hypotrichous ciliates (Protozoa, Ciliophora, Hypotrichia) including the new species Metaurostylopsis parastruederkypkeae n. sp.

    NASA Astrophysics Data System (ADS)

    Lu, Borong; Wang, Chundi; Huang, Jie; Shi, Yuhong; Chen, Xiangrui

    2016-10-01

    The morphology and phylogeny of two hypotrichous ciliates, Metaurostylopsis parastruederkypkeae n. sp. and Neourostylopsis flavicana (Wang et al., 2011) Chen et al., 2013 were investigated based on morphology, infraciliature and the small subunit (SSU) ribosomal RNA gene (rRNA) sequence. The new species, M. parastruederkypkeae n. sp. was identified according to its characteristics: body shape ellipsoidal, size about (165-200) × (45-60) μm in vivo, cell color reddish; two types of cortical granules including wheat grain-like and yellow-greenish larger ones along the marginal cirri rows and dorsal kineties and dot-like and reddish smaller ones, grouped around marginal cirri on ventral side and arranged in short lines on dorsal side; 26-41 adoral membranelles; three frontal and one parabuccal, five to seven frontoterminal, one buccal, and three to six transverse cirri; seven to thirteen midventral pairs; five to nine unpaired ventral cirri, five to seven left and three to five right marginal rows; and three complete dorsal kineties. Phylogenetic analysis based on SSU rDNA sequences showed that both Metaurostylopsis and Neourostylopsis are monophyletic. As the internal relationship between and within both genera are not clear, further studies on the species in these two genera are necessary. The key characteristics of all known twelve Metaurostylopsis-Apourostylopsis-Neourostylopsis species complex were updated.

  16. Evaluation of human brain damage in fire fatality by quantification of basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP) and single-stranded DNA (ssDNA) immunoreactivities.

    PubMed

    Wang, Qi; Ishikawa, Takaki; Michiue, Tomomi; Zhu, Bao-Li; Maeda, Hitoshi

    2011-09-10

    Burns and inhalation of toxic gases, including carbon monoxide (CO) and cyanide, which are produced by combustion, are major factors involved in fire death. The present study immunohistochemically investigated basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP) and single-stranded DNA (ssDNA) in the brains of fire fatalities (n=49) to examine the differences between fatal burns and CO intoxication, compared with those in cardiac deaths (n=24) and mechanical asphyxiation cases (n=23). In acute fire fatality, neuronal ssDNA immunopositivity in the cerebral cortex of the parietal lobe was high in both fatal burns and fatal CO intoxication, but that of the pallidum was higher for CO intoxication than for burns. The number of neurons was decreased in prolonged fire deaths, irrespective of the severity of burns or CO intoxication, but glias were increased in cases of fatal burns. Prolonged deaths due to burns had a higher glial bFGF immunopositivity in the cortex and white matter, higher and lower glial GFAP immunopositivity in the cortex and white matter, respectively, and a low neuronal ssDNA immunopositivity in the cerebral cortex and hippocampus. In prolonged deaths due to CO intoxication, however, glial bFGF and GFAP immunopositivities were low at each site, but neuronal ssDNA immunopositivity showed a higher value. These observations suggest increased cerebral neuronal ssDNA immunopositivity to be a finding of vitality in acute fire death, and a neuronal loss accompanied by active glial responses after severe burns, and a neuronal loss and progressive apoptosis without glial responses after CO intoxication to be characteristic in prolonged death.

  17. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

    1998-01-01

    The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

  18. Principles of DNA architectonics: design of DNA-based nanoobjects

    NASA Astrophysics Data System (ADS)

    Vinogradova, O. A.; Pyshnyi, D. V.

    2012-02-01

    The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

  19. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells.

    PubMed

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X; Huang, Guowei

    2015-10-20

    Alzheimer's disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid β-peptide (Aβ) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8-40 μmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production.

  20. DNA interaction with octahedral and square planar Ni(II) complexes of aspartic-acid Schiff-bases

    NASA Astrophysics Data System (ADS)

    Sallam, S. A.; Orabi, A. S.; Abbas, A. M.

    2011-12-01

    Ni(II) complexes of (S,E)-2-(2-OHbenzilydene)aspartic acid; (S,E)-2-(2,3-diOHbenzilydene)aspartic acid-; (S,E)-2-(2,4-diOH-benzilydene)aspartic acid; (S,E)-2-(2,5-diOHbenzilydene)aspartic acid and (S,E)-2-((2-OHnaphthalene-1-yl)methylene)aspartic acid Schiff-bases have been synthesized by template method in ethanol or ammonia media. They were characterized by elemental analyses, conductivity measurements, magnetic moment, UV, IR and 1H nmr spectra as well as thermal analysis (TG, DTG, DTA). The Schiff-bases are dibasic tridentate or tetradentate donors and the complexes have square planar and octahedral structures. The complexes decompose in two or three steps where kinetic and thermodynamic parameters of the decomposition steps were computed. The interactions of the formed complexes with FM-DNA were monitored by UV and fluorescence spectroscopy.

  1. Induction of DNA Damage Response by the Supravital Probes of Nucleic Acids

    PubMed Central

    Zhao, Hong; Traganos, Frank; Dobrucki, Jurek; Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2009-01-01

    The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV) and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981) and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2 or p53 phosphorylation. However, pronounced ATM, Chk2 and p53 phosphorylation and perturbed G2 progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2 and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2 and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G2M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5 and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis. PMID:19373929

  2. cDNA and derived amino acid sequence of ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC).

    PubMed Central

    Khani, S C; Zaphiropoulos, P G; Fujita, V S; Porter, T D; Koop, D R; Coon, M J

    1987-01-01

    Administration of ethanol to rabbits is known to induce a unique liver microsomal cytochrome P-450, termed isozyme 3a or P-450ALC, which is responsible for the increased oxidation of ethanol and other alcohols and the activation of toxic or carcinogenic compounds such as acetaminophen and N-nitrosodimethylamine. To further characterize this cytochrome P-450 we have identified cDNA clones to isozyme 3a by immunoscreening, DNA hybridization, and hybridization-selection. The cDNA sequence determined from two overlapping clones contains an open reading frame of 1416 nucleotides, and the first 25 amino acids of this reading frame correspond to residues 21-45 of cytochrome P-450 3a. The complete polypeptide, including residues 1 to 20, contains 492 amino acids and has a molecular weight of 56,820. Cytochrome P-450 3a is approximately 55% identical in sequence to P-450 isozymes 1 and 3b and 48% identical to isozyme 2. Hybridization of clone p3a-2 to electrophoretically fractionated rabbit liver poly(A)+ RNA revealed multiple bands, but, with a probe derived from the 3' nontranslated portion of this cDNA, only a 1.9-kilobase band was observed. Treatment of rabbits with imidazole, which increases the content of isozyme 3a, resulted in a transient increase in form 3a mRNA, but this was judged to be insufficient to account for the known 4.5-fold increase in form 3a protein. Genomic DNA analysis indicated that the cytochrome P-450 3a gene does not belong to a large subfamily. Images PMID:3027695

  3. Stimulation of Endomitotic DNA Synthesis and Cell Elongation by Gibberellic Acid in Epicotyls Grown from Gamma-irradiated Pea Seeds 1

    PubMed Central

    Callebaut, Alfons; Van Oostveldt, Patrick; Van Parijs, Roger

    1980-01-01

    Large doses of γ-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced. Application of gibberellic acid restores the endomitotic DNA synthesis and the cell elongation in epicotyls of irradiated seeds. The cortex cells reach again the 8 C DNA level in darkness. The results suggest that γ-irradiation blocks endomitotic DNA synthesis and cell elongation by lowering the concentration of endogenous gibberellins. PMID:16661127

  4. Synthesis, physicochemical studies, embryos toxicity and DNA interaction of some new Iron(II) Schiff base amino acid complexes

    NASA Astrophysics Data System (ADS)

    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.

  5. Possible role of mtDNA depletion and respiratory chain defects in aristolochic acid I-induced acute nephrotoxicity

    SciTech Connect

    Jiang, Zhenzhou Bao, Qingli Sun, Lixin Huang, Xin Wang, Tao Zhang, Shuang Li, Han Zhang, Luyong

    2013-01-15

    This report describes an investigation of the pathological mechanism of acute renal failure caused by toxic tubular necrosis after treatment with aristolochic acid I (AAI) in Sprague–Dawley (SD) rats. The rats were gavaged with AAI at 0, 5, 20, or 80 mg/kg/day for 7 days. The pathologic examination of the kidneys showed severe acute tubular degenerative changes primarily affecting the proximal tubules. Supporting these results, we detected significantly increased concentrations of blood urea nitrogen (BUN) and creatinine (Cr) in the rats treated with AAI, indicating damage to the kidneys. Ultrastructural examination showed that proximal tubular mitochondria were extremely enlarged and dysmorphic with loss and disorientation of their cristae. Mitochondrial function analysis revealed that the two indicators for mitochondrial energy metabolism, the respiratory control ratio (RCR) and ATP content, were reduced in a dose-dependent manner after AAI treatment. The RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. In additional experiments, the activity of respiratory complex I, which is partly encoded by mitochondrial DNA (mtDNA), was more significantly impaired than that of respiratory complex II, which is completely encoded by nuclear DNA (nDNA). A real-time PCR assay revealed a marked reduction of mtDNA in the kidneys treated with AAI. Taken together, these results suggested that mtDNA depletion and respiratory chain defects play critical roles in the pathogenesis of kidney injury induced by AAI, and that the same processes might contribute to aristolochic acid-induced nephrotoxicity in humans. -- Highlights: ► AAI-induced acute renal failure in rats and the proximal tubule was the target. ► Tubular mitochondria were morphologically aberrant in ultrastructural examination. ► AAI impair mitochondrial bioenergetic function and mtDNA replication.

  6. Microbiology of acidic, geothermal springs of Montserrat: environmental rDNA analysis.

    PubMed

    Burton, N P; Norris, P R

    2000-10-01

    DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Caribbean island of Montserrat. 16S rRNA genes were amplified by PCR, cloned, sequenced, and examined to indicate some of the organisms that might be significant components of the in situ microbiota. A clone bank representing the lowest temperature pool that was sampled (33 degrees C) was dominated by genes corresponding to two types of acidophiles: Acidiphilium-like mesophilic heterotrophs and thermotolerant Acidithiobacillus caldus. Three clone types with origins in low- and moderate- (48 degrees C) temperature pools corresponded to bacteria that could be involved in metabolism of sulfur compounds: the aerobic A. caldus and putative anaerobic, moderately thermophilic, sulfur-reducing bacteria (from an undescribed genus and from the Desulfurella group). A higher-temperature sample indicated the presence of a Ferroplasma-like organism, distinct from the other strains of these recently recognized acidophilic, iron-oxidizing members of the Euryarchaeota. Acidophilic Archaea from undescribed genera related to Sulfolobus and Acidianus were predicted to dominate the indigenous acidophilic archaeal population at the highest temperatures.

  7. Pseudogramma polyacantha complex (Serranidae, tribe Grammistini): DNA barcoding results lead to the discovery of three cryptic species, including two new species from French Polynesia.

    PubMed

    Williams, Jeffrey T; Viviani, Jeremie

    2016-05-16

    The Pseudogramma polyacantha species complex was found to harbor cryptic taxonomic diversity with three similar, but genetically divergent, species previously hidden in the complex. The true Pseudogramma polyacantha occurs from French Polynesia to South Africa and has modally 19 (many with 20) segmented dorsal-fin rays, modally 16 segmented anal-fin rays, a relatively short lateral line, no dermal flap or small tentacle dorsally on eye, and extensive scalation on the interorbital, suborbital and dentary. Pseudogramma brederi (previously synonymized with P. polyacantha) is recognized as a valid species occurring from Hawaii to Mauritius and having modally 21 segmented dorsal-fin rays, modally 17 segmented anal-fin rays, a relatively long lateral line, no dermal flap or small tentacle dorsally on eye, and relatively well-developed scalation on the interorbital, suborbital and dentary. Pseudogramma galzini n. sp. is described as a new species known only from French Polynesia and having modally 22 segmented dorsal-fin rays, modally 17 segmented anal-fin rays, a relatively long lateral line, no dermal flap or small tentacle dorsally on eye, and limited scalation on the interorbital, suborbital and dentary. Pseudogramma paucilepis n. sp. is described as a new species known only from French Polynesia and having 20 segmented dorsal-fin rays, modally 16 segmented anal-fin rays, a relatively long lateral line, no dermal flap or small tentacle dorsally on eye, and relatively reduced scalation on the interorbital, suborbital and dentary. A mtDNA COI analysis including all available Pseudogramma sequences shows well-supported genetic divergence between the two new species and among congeners.

  8. Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA

    PubMed Central

    Mangel, Walter F.; McGrath, William J.; Xiong, Kan; Graziano, Vito; Blainey, Paul C.

    2016-01-01

    Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a ‘molecular sled' named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 106 (bp)2 s−1. pVIc is a ‘molecular sled,' because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Characteristics of the ‘molecular sled' in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry. PMID:26831565

  9. Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA

    DOE PAGES

    Mangel, Walter F.; McGrath, William J.; Xiong, Kan; ...

    2016-02-02

    Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a ‘molecular sled’ named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 106 (bp)2 s−1. pVIc is a ‘molecular sled,’ because it can slide heterologous cargos along DNA, for example, amore » streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Finally, characteristics of the ‘molecular sled’ in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.« less

  10. Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

    NASA Astrophysics Data System (ADS)

    Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

    2014-06-01

    The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

  11. High-performance method for specific effect on nucleic acids in cells using TiO2~DNA nanocomposites

    NASA Astrophysics Data System (ADS)

    Levina, Asya S.; Repkova, Marina N.; Ismagilov, Zinfer R.; Shikina, Nadezhda V.; Malygin, Ernst G.; Mazurkova, Natalia A.; Zinov'ev, Victor V.; Evdokimov, Alexei A.; Baiborodin, Sergei I.; Zarytova, Valentina F.

    2012-10-01

    Nanoparticles are used to solve the current drug delivery problem. We present a high-performance method for efficient and selective action on nucleic acid target in cells using unique TiO2.PL-DNA nanocomposites (polylysine-containing DNA fragments noncovalently immobilized onto TiO2 nanoparticles capable of transferring DNA). These nanocomposites were used for inhibition of human influenza A (H3N2) virus replication in infected MDCK cells. They showed a low toxicity (TC50 ~ 1800 μg/ml) and a high antiviral activity (>99.9% inhibition of the virus replication). The specificity factor (antisense effect) appeared to depend on the delivery system of DNA fragments. This factor for nanocomposites is ten-times higher than for DNA in the presence of lipofectamine. IC50 for nanocomposites was estimated to be 1.5 μg/ml (30 nM for DNA), so its selectivity index was calculated as ~1200. Thus, the proposed nanocomposites are prospective for therapeutic application.

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

    PubMed Central

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

    2011-01-01

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

  13. Evaluation of DNA encoding acidic ribosomal protein P2 of Cryptosporidium parvum as a potential vaccine candidate for cryptosporidiosis.

    PubMed

    Benitez, Alvaro; Priest, Jeffrey W; Ehigiator, Humphrey N; McNair, Nina; Mead, Jan R

    2011-11-15

    The Cryptosporidium parvum acidic ribosomal protein P2 (CpP2) is an important immunodominant marker in C. parvum infection. In this study, the CpP2 antigen was evaluated as a vaccine candidate using a DNA vaccine model in adult C57BL/6 IL-12 knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding CpP2 (CpP2-DNA) cloned into the pUMVC4b vector induced a significant anti-CpP2 IgG antibody response that was predominantly of the IgG1 isotype. Compared to control KO mice immunized with plasmid alone, CpP2-immunized mice demonstrated specific in vitro spleen cell proliferation as well as enhanced IFN-γ production to recombinant CpP2. Further, parasite loads in CpP2 DNA-immunized mice were compared to control mice challenged with C. parvum oocysts. Although a trend in reduction of infection was observed in the CpP2 DNA-immunized mice, differences between groups were not statistically significant. These results suggest that a DNA vaccine encoding the C. parvum P2 antigen is able to provide an effective means of eliciting humoral and cellular responses and has the potential to generate protective immunity against C. parvum infection but may require using alternative vectors or adjuvant to generate a more potent and balanced response.

  14. Ultra-sensitive detection of zinc oxide nanowires using a quartz crystal microbalance and phosphoric acid DNA

    NASA Astrophysics Data System (ADS)

    Jang, Kuewhan; You, Juneseok; Park, Chanhoo; Park, Hyunjun; Choi, Jaeyeong; Choi, Chang-Hwan; Park, Jinsung; Lee, Howon; Na, Sungsoo

    2016-09-01

    Recent advancements of nanomaterials have inspired numerous scientific and industrial applications. Zinc oxide nanowires (ZnO NWs) is one of the most important nanomaterials due to their extraordinary properties. However, studies performed over the past decade have reported toxicity of ZnO NWs. Therefore, there has been increasing demand for effective detection of ZnO NWs. In this study, we propose a method for the detection of ZnO NW using a quartz crystal microbalance (QCM) and DNA probes. The detection method is based on the covalent interaction between ZnO NWs and the phosphoric acid group of single-stranded DNA (i.e., linker DNA), and DNA hybridization between the linker DNA and the probe DNA strand on the QCM electrode. Rapid, high sensitivity, in situ detection of ZnO NWs was demonstrated for the first time. The limit of detection was 10-4 μg ml-1 in deionized water, which represents a sensitivity that is 100000 times higher than the toxic ZnO NW concentration level. Moreover, the selectivity of the ZnO NW detection method was demonstrated by comparison with other types of nanowires and the method was able to detect ZnO NWs in tap water sensitively even after stored for 14 d in a refrigerator. The performance of our proposed method was sufficient to achieve detection of ZnO NW in the ‘real-world’ environment.

  15. Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA

    SciTech Connect

    Mangel, Walter F.; McGrath, William J.; Xiong, Kan; Graziano, Vito; Blainey, Paul C.

    2016-02-02

    Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a ‘molecular sled’ named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 106 (bp)2 s−1. pVIc is a ‘molecular sled,’ because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Finally, characteristics of the ‘molecular sled’ in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.

  16. Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells

    PubMed Central

    Angrisano, T.; Sacchetti, S.; Natale, F.; Cerrato, A.; Pero, R.; Keller, S.; Peluso, S.; Perillo, B.; Avvedimento, V. E.; Fusco, A.; Bruni, C. B.; Lembo, F.; Santoro, M.; Chiariotti, L.

    2011-01-01

    Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci. PMID:20952403

  17. Extension of a PBPK model for ethylene glycol and glycolic acid to include the competitive formation and clearance of metabolites associated with kidney toxicity in rats and humans

    SciTech Connect

    Corley, R.A.; Saghir, S.A.; Bartels, M.J.; Hansen, S.C.; Creim, J.; McMartin, K.E.; Snellings, W.M.

    2011-02-01

    A previously developed PBPK model for ethylene glycol and glycolic acid was extended to include glyoxylic acid, oxalic acid, and the precipitation of calcium oxalate that is associated with kidney toxicity in rats and humans. The development and evaluation of the PBPK model was based upon previously published pharmacokinetic studies coupled with measured blood and tissue partition coefficients and rates of in vitro metabolism of glyoxylic acid to oxalic acid, glycine and other metabolites using primary hepatocytes isolated from male Wistar rats and humans. Precipitation of oxalic acid with calcium in the kidneys was assumed to occur only at concentrations exceeding the thermodynamic solubility product for calcium oxalate. This solubility product can be affected by local concentrations of calcium and other ions that are expressed in the model using an ion activity product estimated from toxicity studies such that calcium oxalate precipitation would be minimal at dietary exposures below the NOAEL for kidney toxicity in the sensitive male Wistar rat. The resulting integrated PBPK predicts that bolus oral or dietary exposures to ethylene glycol would result in typically 1.4-1.6-fold higher peak oxalate levels and 1.6-2-fold higher AUC's for calcium oxalate in kidneys of humans as compared with comparably exposed male Wistar rats over a dose range of 1-1000 mg/kg. The converse (male Wistar rats predicted to have greater oxalate levels in the kidneys than humans) was found for inhalation exposures although no accumulation of calcium oxalate is predicted to occur until exposures are well in excess of the theoretical saturated vapor concentration of 200 mg/m{sup 3}. While the current model is capable of such cross-species, dose, and route-of-exposure comparisons, it also highlights several areas of potential research that will improve confidence in such predictions, especially at low doses relevant for most human exposures.

  18. Quantitative trait loci mapping for conjugated linoleic acid, vaccenic acid and ∆(9) -desaturase in Italian Brown Swiss dairy cattle using selective DNA pooling.

    PubMed

    Strillacci, M G; Frigo, E; Canavesi, F; Ungar, Y; Schiavini, F; Zaniboni, L; Reghenzani, L; Cozzi, M C; Samoré, A B; Kashi, Y; Shimoni, E; Tal-Stein, R; Soller, M; Lipkin, E; Bagnato, A

    2014-08-01

    A selective DNA pooling approach was applied to identify QTL for conjugated linoleic acid (CLA), vaccenic acid (VA) and Δ(9) -desaturase (D9D) milk content in Italian Brown Swiss dairy cattle. Milk samples from 60 animals with higher values (after correction for environmental factors) and 60 animals with lower values for each of these traits from each of five half-sib families were pooled separately. The pools were genotyped using the Illumina BovineSNP50 BeadChip. Sire allele frequencies were compared between high and low tails at the sire and marker level for SNPs for which the sires were heterozygous. An r procedure was implemented to perform data analysis in a selective DNA pooling design. A correction for multiple tests was applied using the proportion of false positives among all test results. BTA 19 showed the largest number of markers in association with CLA. Associations between SNPs and the VA and Δ(9) -desaturase traits were found on several chromosomes. A bioinformatics survey identified genes with an important role in pathways for milk fat and fatty acids metabolism within 1 Mb of SNP markers associated with fatty acids contents.

  19. A physiologically based in silico model for trans-2-hexenal detoxification and DNA adduct formation in human including interindividual variation indicates efficient detoxification and a negligible genotoxicity risk.

    PubMed

    Kiwamoto, R; Spenkelink, A; Rietjens, I M C M; Punt, A

    2013-09-01

    A number of α,β-unsaturated aldehydes are present in food both as natural constituents and as flavouring agents. Their reaction with DNA due to their electrophilic α,β-unsaturated aldehyde moiety may result in genotoxicity as observed in some in vitro models, thereby raising a safety concern. A question that remains is whether in vivo detoxification would be efficient enough to prevent DNA adduct formation and genotoxicity. In this study, a human physiologically based kinetic/dynamic (PBK/D) model of trans-2-hexenal (2-hexenal), a selected model α,β-unsaturated aldehyde, was developed to examine dose-dependent detoxification and DNA adduct formation in humans upon dietary exposure. The kinetic model parameters for detoxification were quantified using relevant pooled human tissue fractions as well as tissue fractions from 11 different individual subjects. In addition, a Monte Carlo simulation was performed so that the impact of interindividual variation in 2-hexenal detoxification on the DNA adduct formation in the population as a whole could be examined. The PBK/D model revealed that DNA adduct formation due to 2-hexenal exposure was 0.039 adducts/10⁸ nucleotides (nt) at the estimated average 2-hexenal dietary intake (0.04 mg 2-hexenal/kg bw) and 0.18 adducts/10⁸ nt at the 95th percentile of the dietary intake (0.178 mg 2-hexenal/kg bw) in the most sensitive people. These levels are three orders of magnitude lower than natural background DNA adduct levels that have been reported in disease-free humans (6.8-110 adducts/10⁸ nt), suggesting that the genotoxicity risk for the human population at realistic dietary daily intakes of 2-hexenal may be negligible.

  20. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed Central

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

  1. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  2. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

    PubMed Central

    Devault, A; Lazure, C; Nault, C; Le Moual, H; Seidah, N G; Chrétien, M; Kahn, P; Powell, J; Mallet, J; Beaumont, A

    1987-01-01

    Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase. Images Fig. 1. Fig. 3. PMID:2440677

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

    PubMed

    Gu, Bin; Smyth, Maeve; Kohanoff, Jorge

    2014-11-28

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

  4. Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

    PubMed Central

    Heston, Lee; El-Guindy, Ayman; Countryman, Jill; Dela Cruz, Charles; Delecluse, Henri-Jacques; Miller, George

    2006-01-01

    The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding

  5. Influence of amino acids Shiff bases on irradiated DNA stability in vivo.

    PubMed

    Karapetyan, N H; Malakyan, M H; Bajinyan, S A; Torosyan, A L; Grigoryan, I E; Haroutiunian, S G

    2013-01-01

    To reveal protective role of the new Mn(II) complexes with Nicotinyl-L-Tyrosinate and Nicotinyl-L-Tryptophanate Schiff Bases against ionizing radiation. The DNA of the rats liver was isolated on 7, 14, and 30 days after X-ray irradiation. The differences between the DNA of irradiated rats and rats pre-treated with Mn(II) complexes were studied using the melting, microcalorimetry, and electrophoresis methods. The melting parameters and the melting enthalpy of rats livers DNA were changed after the X-ray irradiation: melting temperature and melting enthalpy were decreased and melting interval was increased. These results can be explained by destruction of DNA molecules. It was shown that pre-treatment of rats with Mn(II) complexes approximates the melting parameters to norm. Agarose gel electrophoresis data confirmed the results of melting studies. The separate DNA fragments were revealed in DNA samples isolated from irradiated animals. The DNA isolated from animals pre-treated with the Mn(II) chelates had better electrophoretic characteristics, which correspond to healthy DNA. Pre-treatment of the irradiated rats with Mn(II)(Nicotinil-L-Tyrosinate) and Mn(II)(Nicotinil-L-Tryptophanate)2 improves the DNA characteristics.

  6. A modified acidic approach for DNA extraction from plant species containing high levels of secondary metabolites.

    PubMed

    Cavallari, M M; Siqueira, M V B M; Val, T M; Pavanelli, J C; Monteiro, M; Grando, C; Pinheiro, J B; Zucchi, M I; Gimenes, M A

    2014-08-25

    Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

  7. Could humic acid relieve the biochemical toxicities and DNA damage caused by nickel and deltamethrin in earthworms (Eisenia foetida)?

    PubMed

    Shen, Chen-Chao; Shen, Dong-Sheng; Shentu, Jia-Li; Wang, Mei-Zhen; Wan, Ming-Yang

    2015-12-01

    The aim of the study was to determine whether humic acid (HA) prevented gene and biochemical toxic effects in earthworms (Eisenia foetida) exposed to nickel and deltamethrin (at 100 and 1 mg kg(-1), respectively) in soil. Cellular- and molecular-level toxic effects of nickel and deltamethrin in earthworms were evaluated by measuring damage to lipid membranes and DNA and the production of protein carbonyls over 42 days of exposure. Nickel and deltamethrin induced significant levels of oxidative stress in earthworms, increasing the production of peroxidation products (malondialdehyde and protein carbonyls) and increasing the comet assay tail DNA% (determined by single-cell gel electrophoresis). DNA damage was the most sensitive of the three indices because it gave a higher sample/control ratio than did the other indices. The presence of HA alleviated (in decreasing order of effectiveness) damage to DNA, proteins, and lipid membranes caused by nickel and deltamethrin. A low HA dose (0.5-1% HA in soil) prevented a great deal of lipid membrane damage, but the highest HA dose (3% HA in soil) prevented still more DNA damage. However, the malondialdehyde concentrations in earthworms were higher at the highest HA dose than at the lower HA doses. The amounts of protein carbonyls produced at different HA doses were not significantly different. The toxic effects to earthworms caused by increased oxidizable nickel concentrations could be relieved by adding HA.

  8. Investigation of irradiated rats DNA in the presence of Cu(II) chelates of amino acids Schiff bases.

    PubMed

    Karapetyan, N H; Torosyan, A L; Malakyan, M; Bajinyan, S A; Haroutiunian, S G

    2016-01-01

    The new synthesized Cu(II) chelates of amino acids Schiff bases were studied as a potential radioprotectors. Male albino rats of Wistar strain were exposed to X-ray whole-body irradiation at 4.8 Gy. This dose caused 30% mortality of the animals (LD30). The survival of animals exposed to radiation after preliminary administration of 10 mg/kg Cu(II)(Nicotinyl-L-Tyrosinate)2 or Cu(II)(Nicotinyl-L-Tryptophanate)2 prior to irradiation was registered about 80 and 100% correspondingly. Using spectrophotometric melting and agarose gel electrophoresis methods, the differences between the DNA isolated from irradiated rats and rats pretreated with Cu(II) chelates were studied. The fragments of DNA with different breaks were revealed in DNA samples isolated from irradiated animals. While, the repair of the DNA structure was observed for animals pretreated with the Cu(II) chelates. The results suggested that pretreatment of the irradiated rats with Cu(II)(Nicotinyl-L-Tyrosinate)2 and Cu(II)(Nicotinyl-L-Tryptophanate)2 compounds improves the liver DNA characteristics.

  9. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    PubMed

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.

  10. Label-free DNA biosensor based on a peptide nucleic acid-functionalized microstructured optical fiber-Bragg grating

    NASA Astrophysics Data System (ADS)

    Candiani, Alessandro; Bertucci, Alessandro; Giannetti, Sara; Konstantaki, Maria; Manicardi, Alex; Pissadakis, Stavros; Cucinotta, Annamaria; Corradini, Roberto; Selleri, Stefano

    2013-05-01

    We describe a novel sensing approach based on a functionalized microstructured optical fiber-Bragg grating for specific DNA target sequences detection. The inner surface of a microstructured fiber, where a Bragg grating was previously inscribed, has been functionalized by covalent linking of a peptide nucleic acid probe targeting a DNA sequence bearing a single point mutation implicated in cystic fibrosis (CF) disease. A solution of an oligonucleotide (ON) corresponding to a tract of the CF gene containing the mutated DNA has been infiltrated inside the fiber capillaries and allowed to hybridize to the fiber surface according to the Watson-Crick pairing. In order to achieve signal amplification, ON-functionalized gold nanoparticles were then infiltrated and used in a sandwich-like assay. Experimental measurements show a clear shift of the reflected high order mode of a Bragg grating for a 100 nM DNA solution, and fluorescence measurements have confirmed the successful hybridization. Several experiments have been carried out on the same fiber using the identical concentration, showing the same modulation trend, suggesting the possibility of the reuse of the sensor. Measurements have also been made using a 100 nM mismatched DNA solution, containing a single nucleotide mutation and corresponding to the wild-type gene, and the results demonstrate the high selectivity of the sensor.

  11. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

    PubMed

    Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

    2010-01-01

    The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

  12. Targeted mutation of Δ12 and Δ15 desaturase genes in hemp produce major alterations in seed fatty acid composition including a high oleic hemp oil.

    PubMed

    Bielecka, Monika; Kaminski, Filip; Adams, Ian; Poulson, Helen; Sloan, Raymond; Li, Yi; Larson, Tony R; Winzer, Thilo; Graham, Ian A

    2014-06-01

    We used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic-based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven Δ12 desaturases and one of three Δ15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have Δx+3 activity, while CSFAD3A activity was exclusively at the Δ15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non-sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major Δ12 and Δ15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a-1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1(Δ9) ) oil at yields similar to wild type. Cold-pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2(Δ6,9) and 18:2(Δ9,15), were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2(Δ9,15) from endogenous 18:1(Δ9) when expressed in S. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop.

  13. Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean.

    PubMed Central

    Broun, P; Somerville, C

    1997-01-01

    A cDNA encoding the oleate 12-hydroxylase from castor bean (Ricinus communis L.) has previously been shown to direct the synthesis of small amounts of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in seeds of transgenic tobacco plants. Expression of the cDNA under control of the Brassica napus napin promoter in transgenic Arabidopsis thaliana plants resulted in the accumulation of up to 17% of seed fatty acids as ricinoleate and two novel fatty acids that have been identified by gas chromatography-mass spectrometry as lesquerolic (14-hydroxyeicos-cis-11-enoic acid) and densipolic (12-hydroxyoctadec-cis-9,15-dienoic acid) acids. Traces of auricolic acid were also observed. These results suggest that either the castor hydroxylase can utilize oleic acid and eicosenoic acid as substrates for ricinoleic and lesquerolic acid biosynthesis, respectively, or Arabidopsis contains an elongase that accepts ricinoleic acid as a substrate. These observations are also consistent with indirect biochemical evidence that an n-3 desaturase is capable of converting ricinoleic acid to densipolic acid. Expression of the castor hydroxylase also caused enhanced accumulation of oleic acid and a corresponding decrease in the levels of polyunsaturated fatty acids. Since the steady-state level of mRNA for the oleate-12 desaturase was not affected, it appears that the presence of the hydroxylase, directly or indirectly, causes posttranscriptional inhibition of desaturation. PMID:9085577

  14. Electron microscopic studies of the interaction between a Bacillus subtilis alpha/beta-type small, acid-soluble spore protein with DNA: protein binding is cooperative, stiffens the DNA, and induces negative supercoiling.

    PubMed Central

    Griffith, J; Makhov, A; Santiago-Lara, L; Setlow, P

    1994-01-01

    DNA within spores of Bacillus subtilis is complexed with a group of alpha/beta-type small acid-soluble spore proteins (alpha/beta-type SASPs), which have almost identical primary sequences and DNA binding properties. Here electron microscopic and cyclization studies were carried out on alpha/beta-type SASP-DNA complexes. When an alpha/beta-type SASP was incubated with linear DNA, the protein bound cooperatively, forming a helical coating 6.6 +/- 0.4 nm wide with a 2.9 +/- 0.3 nm periodicity. alpha/beta-Type SASP binding to an 890-bp DNA was weakest at an (A+T)-rich region that was highly bent, but binding eliminated the bending. alpha/beta-Type SASP binding did not alter the rise per bp in DNA but greatly increased the DNA stiffness as measured by both electron microscopic and cyclization assays. Addition of alpha/beta-type SASPs to negatively supertwisted DNA led to protein binding without significant alteration of the plectonemically interwound appearance of the DNA. Addition of alpha/beta-type SASPs to relaxed or nicked circular DNA led to molecules that by electron microscopy appeared similar to supertwisted DNA. The introduction of negative supertwists in nicked circular DNA by alpha/beta-type SASPs was confirmed by ligation of these molecules followed by topoisomer analyses using agarose gel electrophoresis. Images PMID:8058784

  15. Antileukemia component, dehydroeburicoic acid from Antrodia camphorata induces DNA damage and apoptosis in vitro and in vivo models.

    PubMed

    Du, Ying-Chi; Chang, Fang-Rong; Wu, Tung-Ying; Hsu, Yu-Ming; El-Shazly, Mohamed; Chen, Chieh-Fu; Sung, Ping-Jyun; Lin, Yan-Yu; Lin, Yi-Hsin; Wu, Yang-Chang; Lu, Mei-Chin

    2012-06-15

    Antrodia camphorata (AC) is a native Taiwanese mushroom which is used in Asian folk medicine as a chemopreventive agent. The triterpenoid-rich fraction (FEA) was obtained from the ethanolic extract of AC and characterized by high performance liquid chromatography (HPLC). FEA caused DNA damage in leukemia HL 60 cells which was characterized by phosphorylation of H2A.X and Chk2. It also exhibited apoptotic effect which was correlated to the enhancement of PARP cleavage and to the activation of caspase 3. Five major triterpenoids, antcin K (1), antcin C (2), zhankuic acid C (3), zhankuic acid A (4), and dehydroeburicoic acid (5) were isolated from FEA. The cytotoxicity of FEA major components (1-5) was investigated showing that dehydroeburicoic acid (DeEA) was the most potent cytotoxic component. DeEA activated DNA damage and apoptosis biomarkers similar to FEA and also inhibited topoisomerase II. In HL 60 cells xenograft animal model, DeEA treatment resulted in a marked decrease of tumor weight and size without any significant decrease in mice body weights. Taken together, our results provided the first evidence that pure AC component inhibited tumor growth in vivo model backing the traditional anticancer use of AC in Asian countries.

  16. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  17. Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase.

    PubMed

    Naggert, J; Witkowski, A; Mikkelsen, J; Smith, S

    1988-01-25

    A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.

  18. Diverse responses to UV light exposure in Acinetobacter include the capacity for DNA damage-induced mutagenesis in the opportunistic pathogens Acinetobacter baumannii and Acinetobacter ursingii.

    PubMed

    Hare, Janelle M; Bradley, James A; Lin, Ching-li; Elam, Tyler J

    2012-03-01

    Error-prone and error-free DNA damage repair responses that are induced in most bacteria after exposure to various chemicals, antibiotics or radiation sources were surveyed across the genus Acinetobacter. The error-prone SOS mutagenesis response occurs when DNA damage induces a cell's umuDC- or dinP-encoded error-prone polymerases. The model strain Acinetobacter baylyi ADP1 possesses an unusual, regulatory umuD allele (umuDAb) with an extended 5' region and only incomplete fragments of umuC. Diverse Acinetobacter species were investigated for the presence of umuDC and their ability to conduct UV-induced mutagenesis. Unlike ADP1, most Acinetobacter strains possessed multiple umuDC loci containing either umuDAb or a umuD allele resembling that of Escherichia coli. The nearly omnipresent umuDAb allele was the ancestral umuD in Acinetobacter, with horizontal gene transfer accounting for over half of the umuDC operons. Despite multiple umuD(Ab)C operons in many strains, only three species conducted UV-induced mutagenesis: Acinetobacter baumannii, Acinetobacter ursingii and Acinetobacter beijerinckii. The type of umuDC locus or mutagenesis phenotype a strain possessed was not correlated with its error-free response of survival after UV exposure, but similar diversity was apparent. The survival of 30 Acinetobacter strains after UV treatment ranged over five orders of magnitude, with the Acinetobacter calcoaceticus-A. baumannii (Acb) complex and haemolytic strains having lower survival than non-Acb or non-haemolytic strains. These observations demonstrate that a genus can possess a range of DNA damage response mechanisms, and suggest that DNA damage-induced mutation could be an important part of the evolution of the emerging pathogens A. baumannii and A. ursingii.

  19. Alpha-lipoic acid potently inhibits peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation: implications for the neuroprotective effects of alpha-lipoic acid.

    PubMed

    Jia, Zhenquan; Zhu, Hong; Vitto, Michael J; Misra, Bhaba R; Li, Yunbo; Misra, Hara P

    2009-03-01

    Alpha-lipoic acid (LA) has recently been reported to afford protection against neurodegenerative disorders in humans and experimental animals. However, the mechanisms underlying LA-mediated neuroprotection remain an enigma. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders, this study was undertaken to investigate the effects of LA in peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite-elicited cytotoxicity. Incubation of phi X-174 plasmid DNA with the 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time-dependent fashion. The presence of LA at 100-1,600 microM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by 250 microM SIN-1 was found to be decreased in the presence of high concentrations of LA (400-1,600 microM), indicating that LA at these concentrations may affect the generation of peroxynitrite from auto-oxidation of SIN-1. It is observed that incubation of the plasmid DNA with authentic peroxynitrite resulted in a significant formation of DNA strand breaks, which could also be dramatically inhibited by the presence of LA (100-1,600 microM). EPR spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap, resulted in the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite and LA at 50-1,600 microM inhibited the adduct signal. Taken together, these studies demonstrate for the first time that LA can potently inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. In view of the critical involvement of peroxynitrite in the pathogenesis of various neurodegenerative diseases, the inhibition of peroxynitrite-mediated DNA damage by LA may be responsible, at least

  20. Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.

    PubMed

    Jara, C; Mateo, E; Guillamón, J M; Torija, M J; Mas, A

    2008-12-10

    Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

  1. Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen.

    PubMed

    Yun, Byeong Hwa; Yao, Lihua; Jelaković, Bojan; Nikolić, Jovan; Dickman, Kathleen G; Grollman, Arthur P; Rosenquist, Thomas A; Turesky, Robert J

    2014-09-01

    DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected.

  2. Asymmetric Synthesis, Structure, and Reactivity of Unexpectedly Stable Spiroepoxy-β-Lactones Including Facile Conversion to Tetronic Acids: Application to (+)-Maculalactone A

    PubMed Central

    Duffy, Richard J.; Morris, Kay A.; Vallakati, Ravikrishna; Zhang, Wei; Romo, Daniel

    2009-01-01

    A novel class of small spirocyclic heterocycles, spiroepoxy-β-lactones (1,4-dioxaspiro[2.3]-hexan-5-ones), is described that exhibit a number of interesting reactivity patterns. These spiroheterocycles, including an optically active series, are readily synthesized by epoxidation of ketene dimers (4-alkylidene-2-oxetanones) available from homo- or heteroketene dimerization. An analysis of bond lengths in these systems by X-ray crystallography and comparison to data for known spirocycles and those determined computationally, suggest that anomeric effects in these systems may be more pronounced due to their rigidity and may contribute to their surprising stability. The synthetic utility of spiroepoxy-β-lactones was explored and one facile rearrangement identified under several conditions provides a 3-step route from acid chlorides to optically active tetronic acids, ubiquitous heterocycles in bioactive natural products. The addition of various nucleophiles to these spirocycles leads primarily to addition at C5 and C2. The utility of an optically active spiroepoxy-β-lactone was demonstrated in the concise, enantioselective synthesis of the anti-fouling agent, (+)-maculalactone A, which proceeds in 5 steps from hydrocinnamoyl chloride by way of a tetronic acid intermediate. PMID:19453152

  3. Method and apparatus for staining immobilized nucleic acids

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

    2000-01-01

    A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

  4. DNA cleavage in red light promoted by copper(II) complexes of alpha-amino acids and photoactive phenanthroline bases.

    PubMed

    Patra, Ashis K; Bhowmick, Tuhin; Ramakumar, Suryanarayanarao; Nethaji, Munirathinam; Chakravarty, Akhil R

    2008-12-28

    Ternary copper(II) complexes [Cu(L-trp)(B)(H(2)O)](NO(3)) (1-3) and [Cu(L-phe)(B)(H(2)O)](NO(3)) (4-6) of L-tryptophan (L-trp) and L-phenylalanine (L-phe) having phenanthroline bases (B), viz. 1,10-phenanthroline (phen, 1 and 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2 and 5) and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3 and 6), were prepared and characterized by physico-chemical techniques. Complexes 3 and 6 were structurally characterized by X-ray crystallography and show the presence of a square pyramidal (4 + 1) CuN(3)O(2) coordination geometry in which the N,O-donor amino acid (L-trp or L-phe) and N,N-donor phenanthroline base bind at the equatorial plane with an aqua ligand coordinated at the elongated axial site. Complex 3 shows significant distortion from the square pyramidal geometry and a strong intramolecular pi-pi stacking interaction between the pendant indole ring of L-trp and the planar dppz aromatic moiety. All the complexes display good binding propensity to the calf thymus DNA giving an order: 3,6 (dppz) > 2,5 (dpq) > 1,4 (phen). The binding constant (K(b)) values are in the range of 2.1 x 10(4)-1.1 x 10(6) mol(-1) with the binding site size (s) values of 0.17-0.63. The phen and dpq complexes are minor groove binders while the dppz analogues bind at the DNA major groove. Theoretical DNA docking studies on 2 and 3 show the close proximity of two photosensitizers, viz. the indole moiety of L-trp and the quinoxaline/phenazine of the dpq/dppz bases, to the complementary DNA strands. Complexes 2 and 3 show oxidative DNA double strand breaks (dsb) of supercoiled (SC) DNA forming a significant quantity of linear DNA along with the nicked circular (NC) form on photoexposure to UV-A light of 365 nm and red light of 647.1 nm (Ar-Kr laser). Complexes 1,5 and 6 show only single strand breaks (ssb) forming NC DNA. The red light induced DNA cleavage involves metal-assisted photosensitization of L-trp and dpq/dppz base resulting in the formation of a reactive

  5. 3DNA: a versatile, integrated software system for the analysis, rebuilding, and visualization of three-dimensional nucleic-acid structures

    PubMed Central

    Lu, Xiang-Jun; Olson, Wilma K.

    2010-01-01

    We present a set of protocols showing how to use the 3DNA suite of programs to analyze, rebuild, and visualize three-dimensional nucleic-acid structures. The software determines a wide range of conformational parameters, including the identities and rigid-body parameters of interacting bases and base-pair steps, the nucleotides comprising helical fragments, the area of overlap of stacked bases, etc. The reconstruction of three-dimensional structure takes advantage of rigorously defined rigid-body parameters, producing rectangular block representations of the nucleic-acid bases and base pairs and all-atom models with approximate sugar-phosphate backbones. The visualization components create vector-based drawings and scenes that can be rendered as raster-graphics images, allowing for easy generation of publication-quality figures. The utility programs use geometric variables to control the view and scale of an object, for comparison of related structures. The commands run in seconds even for large structures. The software and related information are available at http://3dna.rutgers.edu/. PMID:18600227

  6. Synthesis, characterization, quantum chemical calculations and evaluation of antioxidant properties of 1,3,4-thiadiazole derivatives including 2- and 3-methoxy cinnamic acids

    NASA Astrophysics Data System (ADS)

    Gür, Mahmut; Muğlu, Halit; Çavuş, M. Serdar; Güder, Aytaç; Sayıner, Hakan S.; Kandemirli, Fatma

    2017-04-01

    A series of 1,3,4-thiadiazole derivatives including 2- and 3-methoxy cinnamic acids were synthesized, and their structures were elucidated by the UV, IR, 1H NMR, 13C NMR spectroscopies and elemental analysis. The UV and IR calculations of the molecules were performed by using B3LYP, HF and MP2 methods with selected 6-311++G(2d,2p), 6-311++G(3df,3pd) and cc-pvtz basis sets. Dipole moment, polarizability, chemical hardness/softness and electronegativity were also calculated and analyzed. Experimental FT-IR spectra and UV-Vis spectrum of the compounds were compared with theoretical data. Furthermore, antioxidant activities of the compounds were practised via different test methods such as 2,2-diphenyl-1-picryl-hydrazyl (DPPHrad), N,N-dimethyl-p-phenylenediamine (DMPDrad +), and 2,2‧-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTSrad +) scavenging activity assays. When compared with standards (BHA-Butylated hydroxyanisole, RUT-Rutin, and TRO-Trolox), it was observed that especially XIII and XIV which include methoxy groups at the o- and m-positions, respectively, had effective activities.

  7. Double stranded nucleic acid biochips

    DOEpatents

    Chernov, Boris; Golova, Julia

    2006-05-23

    This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.

  8. New water-soluble copper (II) complexes including 4,7-dimethyl-1,10-phenanthroline and L-tyrosine: Synthesis, characterization, DNA interactions and cytotoxicities

    NASA Astrophysics Data System (ADS)

    İnci, Duygu; Aydın, Rahmiye; Yılmaz, Dilek; Gençkal, Hasene Mutlu; Vatan, Özgür; Çinkılıç, Nilüfer; Zorlu, Yunus

    2015-02-01

    Two new water-soluble copper(II) complexes, [Cu(dmphen)2(NO3)]NO3 (1), [Cu(dmphen)(tyr)(H2O)]NO3·H2O (2) and the diquarternary salt of dmphen (dmphen = 4,7-dimethyl-1,10-phenanthroline and tyr = L-tyrosine), have been synthesized and characterized by elemental analysis, 1H NMR, 13C NMR and IR spectroscopy, thermal analysis and single crystal X-ray diffraction techniques. The CT-DNA binding properties of these compounds have been investigated by absorption, emission spectroscopy and thermal denaturation measurements. The supercoiled pBR322 plasmid DNA cleavage activity of these compounds has been explored by agarose gel electrophoresis. The cytotoxicity of these compounds against MCF-7, Caco-2, A549 cancer cells and BEAS-2B healthy cells was also studied by the XTT method. Complexes 1 and 2 exhibit significant cytotoxicity, with lower IC50 values than those of cisplatin.

  9. Complete nucleotide sequences and construction of full-length infectious cDNA clones of cucumber green mottle mosaic virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters.

    PubMed

    Park, Chan-Hwan; Ju, Hye-Kyoung; Han, Jae-Yeong; Park, Jong-Seo; Kim, Ik-Hyun; Seo, Eun-Young; Kim, Jung-Kyu; Hammond, John; Lim, Hyoun-Sub

    2017-04-01

    Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus ribozyme sequence to precisely cleave RNA transcripts at the 3' end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to tobacco mosaic virus; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain, and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as 'Russian,' 'Israeli,' and 'Asian' groups. All of our new Korean isolates fell within the 'Asian' clade. Neither the 128 nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.

  10. Phenolic promiscuity in the cell nucleus--epigallocatechingallate (EGCG) and theaflavin-3,3'-digallate from green and black tea bind to model cell nuclear structures including histone proteins, double stranded DNA and telomeric quadruplex DNA.

    PubMed

    Mikutis, Gediminas; Karaköse, Hande; Jaiswal, Rakesh; LeGresley, Adam; Islam, Tuhidul; Fernandez-Lahore, Marcelo; Kuhnert, Nikolai

    2013-02-01

    Flavanols from tea have been reported to accumulate in the cell nucleus in considerable concentrations. The nature of this phenomenon, which could provide novel approaches in understanding the well-known beneficial health effects of tea phenols, is investigated in this contribution. The interaction between epigallocatechin gallate (EGCG) from green tea and a selection of theaflavins from black tea with selected cell nuclear structures such as model histone proteins, double stranded DNA and quadruplex DNA was investigated using mass spectrometry, Circular Dichroism spectroscopy and fluorescent assays. The selected polyphenols were shown to display affinity to all of the selected cell nuclear structures, thereby demonstrating a degree of unexpected molecular promiscuity. Most interestingly theaflavin-digallate was shown to display the highest affinity to quadruplex DNA reported for any naturally occurring molecule reported so far. This finding has immediate implications in rationalising the chemopreventive effect of the tea beverage against cancer and possibly the role of tea phenolics as "life span essentials".

  11. Size and distribution of polyadenylic acid sequences in Drosophila polytene DNA and RNA.

    PubMed

    Alonso, C; Pages, M; García, M L

    1977-12-02

    [3H]Poly(U) hybridizes very rapidly to polytene DNA from Drosophila hydei. When hybridization is performed at 30 degrees C in 2 X SSC to a large excess of DNA, 95% of the poly(U) becomes ribonuclease resistant. Also, complementary RNA transcribed in vitro from polytene DNA hybridizes to poly(U). 023--0.25% of the DNA is composed of (dA)-rich sequences and 0.23--0.31% of cRNA hybridizes to [3H]poly(U). The length of the (dA)-rich sequences on the DNA and cRNA is 40 nucleotides. The Tm values of these hybrids formed between DNA or cRNA-poly(U) is 45 degrees C. The poly(A) fragments from cytoplasmic RNA ranged from 80 to 170 nucleotides in lenght, and migrated in polyacrilamide gels as a broad peak. The average sizes of the poly(A) fragments from the poly(A)-containing RNA transcribed by nuclei isolated from salivary glands in vivo or in vitro were 40, 70, 170 and 70 nucleotides, respectively. Hybridization in situ of [3H]-poly(U) to chromosome squashes indicated that the (dA)-rich sequences are randomly distributed over the whole genome.

  12. Determination of mammalian deoxyribonucleic acid (DNA) in commercial vegetarian and vegan diets for dogs and cats.

    PubMed

    Kanakubo, K; Fascetti, A J; Larsen, J A

    2017-02-01

    The determination of undeclared ingredients in pet food using different analytical methods has been reported in recent years, raising concerns regarding adequate quality control, dietary efficacy and the potential for purposeful adulteration. The objective of this study was to determine the presence or absence of mammalian DNA using multiplex polymerase chain reaction (PCR) on diets marketed as vegetarian or vegan for dogs and cats. The diets were tested in duplicate; two samples were purchased approximately 3 to 4 months apart with different lot numbers. Multiplex PCR-targeted mitochondrial DNA with two species-specific primers was used to amplify and sequence two sections of the cytochrome b gene for each of the 11 mammalian species. Half of the diets assessed (7/14) were positive for one or more undeclared mammalian DNA source (bovine, porcine, or ovine), and the result was repeatable for one or more species in six diets. While most of the detected DNA was found at both time points, in some cases, the result was positive only at one time point, suggesting the presence may have been due to unintentional cross-contact with animal-sourced ingredients. DNA from feline, cervine, canine, caprine, equine, murine (mouse and rat) and leporine was not identified in any samples. However, evidence of mammalian DNA does not confirm adulteration by the manufacturer nor elucidate its clinical significance when consumed by animals that may benefit from a vegetarian or vegan diet.

  13. Functional characterization of an acidic SK(3) dehydrin isolated from an Opuntia streptacantha cDNA library.

    PubMed

    Ochoa-Alfaro, A E; Rodríguez-Kessler, M; Pérez-Morales, M B; Delgado-Sánchez, P; Cuevas-Velazquez, C L; Gómez-Anduro, G; Jiménez-Bremont, J F

    2012-03-01

    Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.

  14. Mining the bitter melon (momordica charantia l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

    PubMed Central

    2010-01-01

    Background Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids. Results Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis. Conclusions 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in developing bitter melon seeds

  15. Multi-Biomarkers for Early Detection of Type 2 Diabetes, Including 10- and 12-(Z,E)-Hydroxyoctadecadienoic Acids, Insulin, Leptin, and Adiponectin.

    PubMed

    Umeno, Aya; Yoshino, Kohzoh; Hashimoto, Yoshiko; Shichiri, Mototada; Kataoka, Masatoshi; Yoshida, Yasukazu

    2015-01-01

    We have previously found that fasting plasma levels of totally assessed 10- and 12-(Z,E)-hydroxyoctadecadienoic acid (HODE) correlated well with levels of glycated hemoglobin (HbA1c) and glucose during oral glucose tolerance tests (OGTT); these levels were determined via liquid chromatography-mass spectrometry after reduction and saponification. However, 10- and 12-(Z,E)-HODE alone cannot perfectly detect early impaired glucose tolerance (IGT) and/or insulin resistance, which ultimately lead to diabetes. In this study, we randomly recruited healthy volunteers (n = 57) who had no known history of any diseases, and who were evaluated using the OGTT, the HODE biomarkers, and several additional proposed biomarkers, including retinol binding protein 4 (RBP4), adiponectin, leptin, insulin, glycoalbumin, and high sensitivity-C-reactive protein. The OGTT revealed that our volunteers included normal individuals (n = 44; Group N), "high-normal" individuals (fasting plasma glucose 100-109 mg/dL) with IGT (n = 11; Group HN+IGT), and diabetic individuals (n = 2; Group D). We then used these groups to evaluate the potential biomarkers for the early detection of type 2 diabetes. Plasma levels of RBP4 and glycoalbumin were higher in Group HN+IGT, compared to those in Group N, and fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids were significantly correlated with levels of RBP4 (p = 0.003, r = 0.380) and glycoalbumin (p = 0.006, r = 0.316). Furthermore, we developed a stepwise multiple linear regression models to predict the individuals' insulin resistance index (the Matsuda Index 3). Fasting plasma levels of 10- and 12-(Z,E)-HODE/linoleic acids, glucose, insulin, and leptin/adiponectin were selected as the explanatory variables for the models. The risks of type 2 diabetes, early IGT, and insulin resistance were perfectly predicted by comparing fasting glucose levels to the estimated Matsuda Index 3 (fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids, insulin, and leptin/adiponectin).

  16. DAPI: a DNA-specific fluorescent probe.

    PubMed

    Kapuscinski, J

    1995-09-01

    DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

  17. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

    PubMed Central

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-01-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed). PMID:22638583

  18. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion.

    PubMed

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-07-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed).

  19. Nucleic acid binding properties of a helix stabilising nucleoid protein from the thermoacidophilic archaeon Sulfolobus acidocaldarius that condenses DNA into compact structures.

    PubMed

    Celestina, F; Suryanarayana, T

    1995-12-01

    Helix stabilising nucleoid protein (HSNP-C') from an acidothermophilic archaeon Sulfolobus acidocaldarius has been characterised with respect to interaction with nucleic acids by gel retardation assay, binding to nucleic acid columns, fluorescence titrations and electron microscopy. The protein exists in solution as very large multimeric aggregates as indicated by cross-linking studies. The protein binds strongly and co-operatively to double stranded DNA. Electron microscopy of the complexes of the protein with DNA shows compact structures suggesting that the protein condenses DNA.

  20. A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems

    PubMed Central

    Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.

    2011-01-01

    The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile

  1. The colorimetric determination of selectively cleaved adenosines and guanosines in DNA oligomers using bicinchoninic acid and copper.

    PubMed

    Thomas, Elizabeth M; Testa, Stephen M

    2017-01-01

    Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this proof-of-principle study, we demonstrate that bicinchoninic acid (BCA) and copper, when combined with purine-specific chemical cleavage reactions, can be a colorimetric probe for the identification and quantification of adenosines and/or guanosines in single-stranded DNA oligomers, even in the presence of pyrimidines. Furthermore, the reactions are stoichiometric, which allows for the quantification of the number of adenosines and/or guanosines in these oligomers. Because the BCA/copper reagent detects the reducing sugar, 2-deoxyribose, that results from the chemical cleavage of a given nucleotide's N-glycosidic bond, these colorimetric assays are effectively detecting apurinic sites in DNA oligomers, which are known to occur via DNA damage in biological systems. We demonstrate that simple digital analysis of the color-changing chromophore (BCA/copper) is all that is necessary to obtain quantifiable and reproducible data, which indicates that these assays should be broadly accessible.

  2. Fabrication, characterization, and biological assessment of multilayer DNA coatings on sandblasted-dual acid etched titanium surface.

    PubMed

    Liu, Li; Song, Li-Na; Yang, Guo-Li; Zhao, Shi-Fang; He, Fu-Ming

    2011-06-01

    As local gene therapy has received attention, immobilizing functional gene onto irregular oral implant surface has become an advanced challenge. Electrostatic layer-by-layer (LBL) assembly technique could achieve this goal and allow local and efficient administration of genes to the target cells. In this study, multilayers of cationic lipid/plasmid DNA (pEGFP-C1) complex (LDc) and anionic hyaluronic acid were assembled onto sandblasted-dual acid etched titanium disks by the LBL technique. Surface characteristics of the coatings were performed by x-ray photospectroscopy (XPS), contact angle measurements, and scanning electron microscopy (SEM). The cell biological characteristics of the coatings were evaluated by in vitro experiments. SEM results demonstrated that the porous titanium surface was gradually flattened with the increase of the multilayer. The XPS survey indicated that the N element was found from the coating. The coating degradation and pEGFP-C1 releasing kinetics showed that the more assembled layer numbers were, the larger the amount of DNA released in the first 30 h. MC3T3-E1 cells were cultured directly on the DNA-loaded surface. Higher enhanced green fluorescent protein (EGFP) expression efficiency was achieved by increasing the number of layers when cells were cultured after 24 or 72 h. The MC3T3-E1 cell viability on the surface of multilayer DNA coatings was significantly higher than that on control porous titanium surface. It was concluded that the approach established by the LBL technique had great potential in immobilizing gene coatings onto the porous titanium surface and subsequently influenced the function of the cultured cell.

  3. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  4. 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.

    PubMed

    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.

  5. Maternal folic acid supplementation modulates DNA methylation and gene expression in the rat offspring in a gestation period-dependent and organ-specific manner.

    PubMed

    Ly, Anna; Ishiguro, Lisa; Kim, Denise; Im, David; Kim, Sung-Eun; Sohn, Kyoung-Jin; Croxford, Ruth; Kim, Young-In

    2016-07-01

    Maternal folic acid supplementation can alter DNA methylation and gene expression in the developing fetus, which may confer disease susceptibility later in life. We determined which gestation period and organ were most sensitive to the modifying effect of folic acid supplementation during pregnancy on DNA methylation and gene expression in the offspring. Pregnant rats were randomized to a control diet throughout pregnancy; folic acid supplementation at 2.5× the control during the 1st, 2nd or 3rd week of gestation only; or folic acid supplementation throughout pregnancy. The brain, liver, kidney and colon from newborn pups were analyzed for folate concentrations, global DNA methylation and gene expression of the Igf2, Er-α, Gr, Ppar-α and Ppar-γ genes. Folic acid supplementation during the 2nd or 3rd week gestation or throughout pregnancy significantly increased brain folate concentrations (P<.001), while only folic acid supplementation throughout pregnancy significantly increased liver folate concentrations (P=.005), in newborn pups. Brain global DNA methylation incrementally decreased from early to late gestational folic acid supplementation and was the lowest with folic acid supplementation throughout pregnancy (P=.026). Folic acid supplementation in late gestation or throughout pregnancy significantly decreased Er-α, Gr and Ppar-α gene expression in the liver (P<.05). The kidney and colon were resistant to the effect of folic acid supplementation. Maternal folic acid supplementation affects tissue folate concentrations, DNA methylation and gene expression in the offspring in a gestation-period-dependent and organ-specific manner.

  6. [A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture].

    PubMed

    Vardevanian, P O; Davtian, A M; Tiratsuian, S G; Vardevanian, A O

    1990-01-01

    A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.

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

    PubMed

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

    2014-01-01

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

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

    SciTech Connect

    Naish, S.J.; Perreault, S.D.; Zirkin, B.R.

    1987-01-01

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

  9. Self-assembling DNA hydrogel-based delivery of immunoinhibitory nucleic acids to immune cells.

    PubMed

    Nishida, Yu; Ohtsuki, Shozo; Araie, Yuki; Umeki, Yuka; Endo, Masayuki; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi; Takahashi, Yuki; Takakura, Yoshinobu; Nishikawa, Makiya

    2016-01-01

    Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical Editor: Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.

  10. An unprecedented nucleic acid capture mechanism for excision of DNA damage

    SciTech Connect

    Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.; Gold, Barry; Eichmanbrand, Brandt F.

    2010-11-18

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

  11. Nucleic acid (DNA) immunization as a platform for dengue vaccine development.

    PubMed

    Porter, Kevin R; Raviprakash, Kanakatte

    2015-12-10

    Since the early 1990s, DNA immunization has been used as a platform for developing a tetravalent dengue vaccine in response to the high priority need for protecting military personnel deployed to dengue endemic regions of the world. Several approaches have been explored ranging from naked DNA immunization to the use of live virus vectors to deliver the targeted genes for expression. Pre-clinical animal studies were largely successful in generating anti-dengue cellular and humoral immune responses that were protective either completely or partially against challenge with live dengue virus. However, Phase 1 clinical evaluation of a prototype monovalent dengue 1 DNA vaccine expressing prM and E genes revealed anti-dengue T cell IFNγ responses, but poor neutralizing antibody responses. These less than optimal results are thought to be due to poor uptake and expression of the DNA vaccine plasmids. Because DNA immunization as a vaccine platform has the advantages of ease of manufacture, flexible genetic manipulation and enhanced stability, efforts continue to improve the immunogenicity of these vaccines using a variety of methods.

  12. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  13. The Phenazine 2-Hydroxy-Phenazine-1-Carboxylic Acid Promotes Extracellular DNA Release and Has Broad Transcriptomic Consequences in Pseudomonas chlororaphis 30–84

    PubMed Central

    Wang, Dongping; Yu, Jun Myoung; Dorosky, Robert J.; Pierson, Leland S.; Pierson, Elizabeth A.

    2016-01-01

    Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30–84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surface-attached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30–84 biofilm matrix is demonstrated. To better understand the role of different phenazines in biofilm matrix production and gene expression, transcriptomic analyses were conducted comparing gene expression patterns of populations of wild type, 30-84O* and a derivative of 30–84 producing only PCA (30-84PCA) to a phenazine defective mutant (30-84ZN) when grown in static cultures. RNA-Seq analyses identified a group of 802 genes that were differentially expressed by the phenazine producing derivatives compared to 30-84ZN, including 240 genes shared by the two 2-OH-PCA producing derivatives, the wild type and 30-84O*. A gene cluster encoding a bacteriophage-derived pyocin and its lysis cassette was upregulated in 2-OH-PCA producing derivatives. A holin encoded in this gene cluster was found to contribute to the release of eDNA in 30–84 biofilm matrices, demonstrating that the influence of 2-OH-PCA on eDNA production is due in part to cell autolysis as a result of pyocin production and release. The results expand the current understanding of the functions different phenazines play in the survival of bacteria in biofilm-forming communities. PMID:26812402

  14. Lysophosphatidic acid induces both EGFR-dependent and EGFR-independent effects on DNA synthesis and migration in pancreatic and colorectal carcinoma cells.

    PubMed

    Tveteraas, Ingun Heiene; Aasrum, Monica; Brusevold, Ingvild Johnsen; Ødegård, John; Christoffersen, Thoralf; Sandnes, Dagny

    2016-02-01

    Lysophosphatidic acid (LPA) is a small glycerophospholipid ubiquitously present in tissues and plasma. It acts through receptors belonging to the G-protein-coupled receptor (GPCR) family, is involved in several biological processes, and is strongly implicated in different cancers. In this paper, we have investigated the effects of LPA on DNA synthesis and migration in a panel of pancreatic and colon cancer cells, with particular focus on the involvement of the epidermal growth factor (EGF) receptor (EGFR) in LPA-induced signaling. LPA stimulated DNA synthesis and/or migration in all the cell lines included in this study. In five of the six cell lines, LPA induced phosphorylation of the EGFR, and the effects on EGFR and Akt, and in some of the cells also ERK, were sensitive to the EGFR tyrosine kinase inhibitor gefitinib, strongly suggesting LPA-induced EGFR transactivation in these cells. In contrast, in one of the pancreatic carcinoma cell lines (Panc-1), we found no evidence of transactivation of the EGFR. In the pancreatic carcinoma cell lines where transactivation took place (BxPC3, AsPC1, HPAFII), gefitinib reduced LPA-induced DNA synthesis and/or migration. However, we also found evidence of transactivation in the two colon carcinoma cell lines (HT29, HCT116) although gefitinib did not inhibit LPA-induced DNA synthesis or migration in these cells. Taken together, the data indicate that in many gastrointestinal carcinoma cells, LPA uses EGFR transactivation as a mechanism when exerting such effects as stimulation of cell proliferation and migration, but EGFR-independent pathways may be involved instead of, or in concerted action with, the EGFR transactivation.

  15. The Phenazine 2-Hydroxy-Phenazine-1-Carboxylic Acid Promotes Extracellular DNA Release and Has Broad Transcriptomic Consequences in Pseudomonas chlororaphis 30–84

    SciTech Connect

    Wang, Dongping; Yu, Jun Myoung; Dorosky, Robert J.; Pierson, Leland S.; Pierson, Elizabeth A.

    2016-01-26

    Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30–84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surfaceattached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30–84 biofilm matrix is demonstrated. To better understand the role of different phenazines in biofilm matrix production and gene expression, transcriptomic analyses were conducted comparing gene expression patterns of populations of wild type, 30-84O* and a derivative of 30–84 producing only PCA (30-84PCA) to a phenazine defective mutant (30-84ZN) when grown in static cultures. RNA-Seq analyses identified a group of 802 genes that were differentially expressed by the phenazine producing derivatives compared to 30-84ZN, including 240 genes shared by the two 2-OH-PCA producing derivatives, the wild type and 30-84O*. A gene cluster encoding a bacteriophage- derived pyocin and its lysis cassette was upregulated in 2-OH-PCA producing derivatives. A holin encoded in this gene cluster was found to contribute to the release of eDNA in 30–84 biofilm matrices, demonstrating that the influence of 2-OH-PCA on eDNA production is due in part to cell autolysis as a result of pyocin production and release. The results expand the current understanding of the functions different phenazines play in the survival of bacteria in biofilm-forming communities.

  16. The Phenazine 2-Hydroxy-Phenazine-1-Carboxylic Acid Promotes Extracellular DNA Release and Has Broad Transcriptomic Consequences in Pseudomonas chlororaphis 30–84

    DOE PAGES

    Wang, Dongping; Yu, Jun Myoung; Dorosky, Robert J.; ...

    2016-01-26

    Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30–84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surfaceattached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30–84 biofilm matrix is demonstrated. To better understand the role of different phenazines in biofilm matrix production and gene expression, transcriptomic analyses were conductedmore » comparing gene expression patterns of populations of wild type, 30-84O* and a derivative of 30–84 producing only PCA (30-84PCA) to a phenazine defective mutant (30-84ZN) when grown in static cultures. RNA-Seq analyses identified a group of 802 genes that were differentially expressed by the phenazine producing derivatives compared to 30-84ZN, including 240 genes shared by the two 2-OH-PCA producing derivatives, the wild type and 30-84O*. A gene cluster encoding a bacteriophage- derived pyocin and its lysis cassette was upregulated in 2-OH-PCA producing derivatives. A holin encoded in this gene cluster was found to contribute to the release of eDNA in 30–84 biofilm matrices, demonstrating that the influence of 2-OH-PCA on eDNA production is due in part to cell autolysis as a result of pyocin production and release. The results expand the current understanding of the functions different phenazines play in the survival of bacteria in biofilm-forming communities.« less

  17. Ferulic acid prevents methylglyoxal-induced protein glycation, DNA damage, and apoptosis in pancreatic β-cells.

    PubMed

    Sompong, Weerachat; Cheng, Henrique; Adisakwattana, Sirichai

    2017-02-01

    Methylglyoxal (MG) can react with amino acids of proteins to induce protein glycation and consequently the formation of advanced glycation end-products (AGEs). Previous studies reported that ferulic acid (FA) prevented glucose-, fructose-, and ribose-induced protein glycation. In this study, FA (0.1-1 mM) inhibited MG-induced protein glycation and oxidative protein damage in bovine serum albumin (BSA). Furthermore, FA (0.0125-0.2 mM) protected against lysine/MG-mediated oxidative DNA damage, thereby inhibiting superoxide anion and hydroxyl radical generation during lysine and MG reaction. In addition, FA did not have the ability to trap MG. Finally, FA (0.1 mM) pretreatment attenuated MG-induced decrease in cell viability and prevented MG-induced cell apoptosis in pancreatic β-cells. The results suggest that FA is capable of protecting β-cells from MG-induced cell damage during diabetes.

  18. Effect of the amino acid substitution in the DNA-binding domain of the Fur regulator on production of pyoverdine.

    PubMed

    Valešová, Renáta; Palyzová, Andrea; Marešová, Helena; Stěpánek, Václav; Babiak, Peter; Kyslík, Pavel

    2013-07-01

    The ferric uptake regulator gene (fur), its promoter region and Fur box of pvdS gene involved in siderophore-mediated iron uptake system were sequenced in the parent strain Pseudomonas aeruginosa PAO1 and in the fur mutant FPA121 derived from the strain PAO1. We identified the gene fur 179 bearing a novel, single-point mutation that changed the amino acid residue Gln60Pro in the DNA-binding domain of the Fur protein. The synthesis of pyoverdine was studied in cultures of the strains PAO1 and FPA121 grown in iron-deplete and iron-replete (60 μmol/L FeIII) medium. The amino acid replacement in the regulatory Fur protein is responsible for the overproduction of pyoverdine in iron-deplete and iron-replete medium. No mutation was identified in the Fur box of the gene pvdS.

  19. Dissociative Electron Attachment to Phosphoric Acid Esters: The Direct Mechanism for Single Strand Breaks in DNA

    SciTech Connect

    Koenig, Constanze; Kopyra, Janina; Bald, Ilko; Illenberger, Eugen

    2006-07-07

    We use dibutyl phosphate to simulate the behavior of the phosphate group in DNA towards the attack of low energy electrons. We find that the compound undergoes effective dissociative electron attachment within a low energy resonant feature at 1 eV and a further resonance peaking at 8 eV. The dissociative electron attachment (DEA) reactions are associated with the direct cleavage of the C-O and the P-O bond but also the excision of the PO{sup -}, PO{sub 3}{sup -}, H{sub 2}PO{sub 3}{sup -} units. For the phosphate group coupled in the DNA network these reactions represent single strand breaks. We hence propose that the most direct mechanism of single strand breaks occurring in DNA at subexcitation energies (<4 eV) is due to DEA directly to the phosphate group.

  20. Histidine-Based Lipopeptides Enhance Cleavage of Nucleic Acids: Interactions with DNA and Hydrolytic Properties.

    PubMed

    Bélières, M; Déjugnat, C; Chouini-Lalanne, N

    2015-12-16

    Interaction studies and cleavage activity experiments were carried out between plasmid DNA and a series of histidine-based lipopeptides. Specific fluorescent probes (ethidium bromide, Hoechst 33342, and pyrene) were used to monitor intercalation, minor groove binding, and self-assembly of lipopeptides, respectively. Association between DNA and lipopeptides was thus evidenced, highlighting the importance of both histidine and hydrophobic tail in the interaction process. DNA cleavage in the presence of lipopeptides was then detected by gel electrophoresis and quantified, showing the importance of histidine and the involvement of its side-chain imidazole in the hydrolysis mechanism. These systems could then be developed as synthetic nucleases while raising concern of introducing histidine in the design of lipopeptide-based transfection vectors.

  1. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis.

    PubMed

    Liu, Ying; Xu, Qiao-Xian; Xi, Pei-Yu; Chen, Hong-Hao; Liu, Chun-Sheng

    2013-05-01

    The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

  2. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  3. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

    PubMed

    Hong, S B; Li, C M; Rhee, H J; Park, J H; He, X; Levy, B; Yoo, O J; Schuchman, E H

    1999-12-01

    Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion

  4. Development of PCR-Based DNA markers flanking three low phytic acid mutant loci in barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytic acid (PA) is the most abundant form of phosphorus (P) in cereal grains. PA chelates mineral cations to form an indigestible salt, and is thus regarded as an antinutritional agent and a contributor to water pollution. Grain with low phytic acid (lpa) genotypes could aid in mitigating this prob...

  5. A new delimitation of the Afro-Eurasian plant genus Althenia to include its Australasian relative, Lepilaena (Potamogetonaceae) - Evidence from DNA and morphological data.

    PubMed

    Ito, Yu; Tanaka, Norio; García-Murillo, Pablo; Muasya, A Muthama

    2016-05-01

    Althenia (Potamogetonaceae) is an aquatic plant genus disjunctly distributed in the southern- (South Africa's Cape Floristic Region: CFR) and northern- (Mediterranean Eurasia) hemispheres. This genus and its Australasian relative, Lepilaena, share similar floral characters yet have been treated as different genera or sections of Althenia sensu lato (s.l.) due to the isolated geographic distribution as well as the differences in sex expression, stamen construction, and stigma morphology. The diagnostic characters, however, need reevaluation over the boundaries between the entities. Here we tested the taxonomic delimitation between the entities, assessed synapomorphies for evolutionary lineages, and inferred biogeographic history in a phylogenetic framework. Our results indicated that Lepilaena was resolved as non-monophyletic in both plastid DNA and nuclear PhyC trees and Althenia was nested within it. As Althenia has nomenclatural priority, we propose a new delimitation to recognize Althenia s.l., which can be diagnosed by the female flowers with 3-segmented perianths and male flowers with perianths. The previously used diagnostic characters are either autapomorphies or synapomorphies for small lineages within Althenia s.l., and evolutionary transitions to sessile female flowers and narrow leaves characterize larger clades. Biogeographic analyses suggested a Miocene origin of Althenia s.l. in Australasia and indicated at least one inter- and one intra-specific inter-continental dispersal events among Australasia, Mediterranean Eurasia, and CFR need to be hypothesized to explain the current distribution patterns.

  6. An update on potato crisps contents of moisture, fat, salt and fatty acids (including trans-fatty acids) with special emphasis on new oils/fats used for frying.

    PubMed

    Gonçalves Albuquerque, Tânia; Sanches-Silva, Ana; Santos, Lèlita; Costa, Helena S

    2012-09-01

    Eighteen brands of potato crisps, frequently consumed, were analyzed to establish their nutritional value in relation to salt, fat and fatty acid (FA) composition. The purpose of the present study was to determine moisture, total fat, salt contents and FA profiles (including trans-FAs), and to identify the oil/fat used for frying of the 18 brands of potato crisps. Our results show that salt content ranged from 0.127 to 2.77 g/100 g and total fat content of potato crisps varied between 20.0 and 42.8 g/100 g. With respect to FAs analysis, palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2) were the major FAs found in the analyzed potato crisps. It is clear from our work that nowadays most potato crisps are currently produced using oils with high contents in unsaturated FAs, which can be considered as healthier from a nutritional point of view. Nevertheless, some brands of potato crisps still use palm oil or a blend of palm oil and other fats/oils, which are very rich in saturated FAs.

  7. Genetic and epigenetic transgenerational implications related to omega-3 fatty acids. Part I: maternal FADS2 genotype and DNA methylation correlate with polyunsaturated fatty acid status in toddlers: an exploratory analysis.

    PubMed

    Lupu, Daniel S; Cheatham, Carol L; Corbin, Karen D; Niculescu, Mihai D

    2015-11-01

    Polyunsaturated fatty acid metabolism in toddlers is regulated by a complex network of interacting factors. The contribution of maternal genetic and epigenetic makeup to this milieu is not well understood. In a cohort of mothers and toddlers 16 months of age (n = 65 mother-child pairs), we investigated the association between maternal genetic and epigenetic fatty acid desaturase 2 (FADS2) profiles and toddlers' n-6 and n-3 fatty acid metabolism. FADS2 rs174575 variation and DNA methylation status were interrogated in mothers and toddlers, as well as food intake and plasma fatty acid concentrations in toddlers. A multivariate fit model indicated that maternal rs174575 genotype, combined with DNA methylation, can predict α-linolenic acid plasma concentration in all toddlers and arachidonic acid concentrations in boys. Arachidonic acid intake was predictive for its plasma concentration in girls, whereas intake of 3 major n-3 species (eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids) were predictive for their plasma concentrations in boys. FADS2 genotype and DNA methylation in toddlers were not related to plasma concentrations or food intakes, except for CpG8 methylation. Maternal FADS2 methylation was a predictor for the boys' α-linolenic acid intakes. This exploratory study suggests that maternal FADS2 genetic and epigenetic status could be related to toddlers' polyunsaturated fatty acid metabolism.

  8. Sequence of a cDNA clone encoding the polysialic acid-rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM.

    PubMed Central

    Hemperly, J J; Murray, B A; Edelman, G M; Cunningham, B A

    1986-01-01

    Purified fractions of the neural cell-adhesion molecule N-CAM from embryonic chicken brain contain two similar polypeptides (Mr, 160,000 and 130,000), each containing an amino-terminal external binding region, a carbohydrate-rich central region, and a carboxyl-terminal region that is associated with the cell. Previous studies indicate that the two polypeptides arise by alternative splicing of mRNAs transcribed from a single gene. We report here the 3556-nucleotide sequence of a cDNA clone (pEC208) that encodes 964 amino acids from the carbohydrate and cell-associated domains of the larger N-CAM polypeptide followed by 664 nucleotides of 3' untranslated sequence. The predicted protein sequence contains attachment sites for polysialic acid-containing oligosaccharides, four tandem homologous regions of polypeptide resembling those seen in the immunoglobulin superfamily, and a single hydrophobic sequence that appears to be the membrane-spanning segment. The cytoplasmic domain carboxyl terminal to this segment includes a block of approximately equal to 250 amino acids present in the larger but not in the smaller N-CAM polypeptide. We designate these the ld (large domain) polypeptide and the sd (small domain) polypeptide. The intracellular domains of the ld and sd polypeptides are likely to be critical for cell-surface modulation of N-CAM by interacting in a differential fashion with other intrinsic proteins or with the cytoskeleton. PMID:3458261

  9. Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis.

    PubMed

    Rossi, Stefano; Lesignoli, Francesca; Germini, Andrea; Faccini, Andrea; Sforza, Stefano; Corradini, Roberto; Marchelli, Rosangela

    2007-04-04

    PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.

  10. DNA recognition by peptide nucleic acid-modified PCFs: from models to real samples

    NASA Astrophysics Data System (ADS)

    Selleri, S.; Coscelli, E.; Poli, F.; Passaro, D.; Cucinotta, A.; Lantano, C.; Corradini, R.; Marchelli, R.

    2010-04-01

    The increased concern, emerged in the last few years, on food products safety has stimulated the research on new techniques for traceability of raw food materials. DNA analysis is one of the most powerful tools for the certification of food quality, and it is presently performed through the polymerase chain reaction technique. Photonic crystal fibers, due to the presence of an array of air holes running along their length, can be exploited for performing DNA recognition by derivatizing hole surfaces and checking hybridization of complementary nucledotide chains in the sample. In this paper the application of a suspended core photonic crystal fiber in the recognition of DNA sequences is discussed. The fiber is characterized in terms of electromagnetic properties by means of a full-vector modal solver based on the finite element method. Then, the performances of the fiber in the recognition of mall synthetic oligonucleotides are discussed, together with a test of the possibility to extend this recognition to samples of DNA of applicative interest, such as olive leaves.

  11. Effective DNA binding and cleaving tendencies of malonic acid coupled transition metal complexes

    NASA Astrophysics Data System (ADS)

    Pravin, Narayanaperumal; Utthra, Ponnukalai Ponya; Kumaravel, Ganesan; Raman, Natarajan

    2016-11-01

    Eight transition metal complexes were designed to achieve maximum biological efficacy. They were characterized by elemental analysis and various other spectroscopic techniques. The monomeric complexes were found to espouse octahedral geometry and non-electrolytic nature. The DNA interaction propensity of the complexes with calf thymus DNA (CT-DNA), studied at physiological pH by spectrophotometric, spectrofluorometric, cyclic voltammetry, and viscometric techniques revealed intercalation as the possible binding mode. Fascinatingly, the complexes were found to exhibit greater binding strength than that of the free ligands. A strong hypochromism and a slight red shift were exhibited by complex 5 among the other complexes. The intrinsic binding constant values of all the complexes compared to cisplatin reveal that they are excellent metallonucleases than that of cisplatin. The complexes were also shown to reveal displacement of the ethidium bromide, a strong intercalator using fluorescence titrations. Gel electrophoresis was used to divulge the competence of the complexes in cleaving the supercoiled pBR322 plasmid DNA. From the results, it is concluded that the complexes, especially 5, are excellent chemical nucleases in the presence of H2O2. Furthermore, the in vitro antimicrobial screening of the complexes exposes that these complexes are excellent antimicrobial agents. Overall the effect of coligands is evident from the results of all the investigations.

  12. Running DNA Mini-Gels in 20 Minutes or Less Using Sodium Boric Acid Buffer

    ERIC Educational Resources Information Center

    Jenkins, Kristin P.; Bielec, Barbara

    2006-01-01

    Providing a biotechnology experience for students can be challenging on several levels, and time is a real constraint for many experiments. Many DNA based methods require a gel electrophoresis step, and although some biotechnology procedures have convenient break points, gel electrophoresis does not. In addition to the time required for loading…

  13. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    SciTech Connect

    Tsujiuchi, Toshifumi . E-mail: ttujiuch@life.kindai.ac.jp; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

    2006-10-27

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.

  14. Poly(D,L-lactide-co-glycolide acid) nanoparticles for DNA delivery: waiving preparation complexity and increasing efficiency.

    PubMed

    Gvili, Koby; Benny, Ofra; Danino, Dganit; Machluf, Marcelle

    When designing a nonviral gene delivery system based on polymeric nanoparticles (NPs), it is important to keep in mind obstacles associated with future clinical applications. Simplifying the procedure of NPs production and taking toxicity into account are the most important issues that need to be addressed. Toxicity concerns in clinical trials may be raised when using additives such as cationic polymers/lipids, buffering reagents, and proteins. Therefore, the aim of this study was to simplify the formulation of poly (lactide-co-glycolide) acid NPs by shortening steps such as sonication time and by avoiding the use of additives while preserving its efficiency. NPs (300 nm) were formulated using a modified w/o/w technique with DNA entrapment efficiency of 80%. Once achieving such NPs, formulation parameters such as DNA loading, release kinetics, DNA integrity and bioactivity, uptake by cells, and toxicity were addressed. The NPs were readily taken by several cell lines and were localized mostly in their endo-lysosomal compartments. The NPs did not affect cells viability. Most importantly, transfection studies in COS-7 and Cf2th cells resulted with a 250-fold protein expression levels when compared with the control. These expression levels are higher than ones achieved with more complicated NPs systems, demonstrating the efficiency of our simplified NPs for gene delivery.

  15. Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues

    PubMed Central

    Waggoner, Jesse J.

    2014-01-01

    Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types. PMID:24765569

  16. Porous Hyaluronic Acid Hydrogels for Localized Non-Viral DNA Delivery in a Diabetic Wound Healing Model

    PubMed Central

    Tokatlian, Talar; Cam, Cynthia; Segura, Tatiana

    2015-01-01

    The treatment of impaired wounds requires the use of biomaterials that can provide mechanical and biological queues to the surrounding environment to promote angiogenesis, granulation tissue formation, and wound closure. Porous hydrogels have previously been shown to promote angiogenesis even in the absence of pro-angiogenic factors. We hypothesized that the added delivery of non-viral DNA encoding for pro-angiogenic growth factors could further enhance this effect. Here, 100 and 60 μm porous and non-porous (n-pore) hyaluronic acid-MMP hydrogels with encapsulated reporter (pGFPluc) or pro-angiogenic (pVEGF) plasmids were used to investigate scaffold-mediated gene delivery for local gene therapy in a diabetic wound healing mouse model. Porous hydrogels allowed for significantly faster wound closure compared to n-pore hydrogels, which did not degrade and essentially provided a mechanical barrier to closure. Interestingly, the delivery of pDNA/PEI polyplexes positively promoted granulation tissue formation even when the DNA did not encode for an angiogenic protein. And although transfected cells were present throughout the granulation tissue surrounding all hydrogels at 2 weeks, pVEGF delivery did not further enhance the angiogenic response. Despite this, the presence of transfected cells shows promise for the use of polyplex-loaded porous hydrogels for local gene delivery in the treatment of diabetic wounds. PMID:25694196

  17. Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids.

    PubMed Central

    Leonard, A E; Bobik, E G; Dorado, J; Kroeger, P E; Chuang, L T; Thurmond, J M; Parker-Barnes, J M; Das, T; Huang, Y S; Mukerji, P

    2000-01-01

    The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response. PMID:10970790

  18. The effects of dietary boric acid and borax supplementation on lipid peroxidation, antioxidant activity, and DNA damage in rats.

    PubMed

    Ince, Sinan; Kucukkurt, Ismail; Cigerci, Ibrahim Hakki; Fatih Fidan, A; Eryavuz, Abdullah

    2010-07-01

    The aims of this study were to clarify the effects of high dietary supplementation with boric acid and borax, called boron (B) compounds, on lipid peroxidation (LPO), antioxidant activity, some vitamin levels, and DNA damage in rats. Thirty Sprague Dawley male rats were divided into three equal groups: the animals in the first group (control) were fed with a standard rodent diet containing 6.4 mg B/kg, and the animals in the experimental group were fed with a standard rodent diet added with a supra-nutritional amount of boric acid and borax (100 mg B/kg) throughout the experimental period of 28 days. The B compounds decreased malondialdehyde (MDA), DNA damage, the protein carbonyl content (PCO) level in blood, and glutathione (GSH) concentration in the liver, Cu-Zn superoxide dismutase (SOD), and catalase (CAT) activity in the kidney. The B compounds increased GSH concentration in blood and the vitamin C level in plasma. Consequently, our results demonstrate that B supplementation (100 mg/kg) in diet decreases LPO, and enhances the antioxidant defense mechanism and vitamin status. There are no differences in oxidant/antioxidant balance and biochemical parameters except for serum vitamin A and liver GSH concentration, between the boron compounds used in this study.

  19. A novel cationic microbubble coated with stearic acid-modified polyethylenimine to enhance DNA loading and gene delivery by ultrasound.

    PubMed

    Jin, Qiaofeng; Wang, Zhiyong; Yan, Fei; Deng, Zhiting; Ni, Fei; Wu, Junru; Shandas, Robin; Liu, Xin; Zheng, Hairong

    2013-01-01

    A novel cationic microbubble (MB) for improvement of the DNA loading capacity and the ultrasound-mediated gene delivery efficiency has been developed; it has been prepared with commercial lipids and a stearic acid modified polyethylenimine 600 (Stearic-PEI600) polymer synthesized via acylation reaction of branched PEI600 and stearic acid mediated by N, N'-carbonyldiimidazole (CDI). The MBs' concentration, size distribution, stability and zeta potential (ζ-potential) were measured and the DNA loading capacity was examined as a function of the amount of Stearic-PEI600. The gene transfection efficiency and cytotoxicity were also examined using breast cancer MCF-7 cells via the reporter plasmid pCMV-Luc, encoding the firefly luciferase gene. The results showed that the Stearic-PEI600 polymer caused a significant increase in magnitude of ζ-potential of MBs. The addition of DNA into cationic MBs can shift ζ-potentials from positive to negative values. The DNA loading capacity of the MBs grew linearly from (5±0.2) ×10⁻³ pg/µm² to (20±1.8) ×10⁻³ pg/µm² when Stearic-PEI600 was increased from 5 mol% to 30 mol%. Transfection of MCF-7 cells using 5% PEI600 MBs plus ultrasound exposure yielded 5.76±2.58×10³ p/s/cm²/sr average radiance intensity, was 8.97- and 7.53-fold higher than those treated with plain MBs plus ultrasound (6.41±5.82) ×10² p/s/cm²/sr, (P<0.01) and PEI600 MBs without ultrasound (7.65±6.18) ×10² p/s/cm²/sr, (P<0.01), respectively. However, the PEI600 MBs showed slightly higher cytotoxicity than plain MBs. The cells treated with PEI600-MBs and plain MBs plus ultrasound showed 59.5±6.1% and 71.4±7.1% cell viability, respectively. In conclusion, our study demonstrated that the novel cationic MBs were able to increase DNA loading capacity and gene transfection efficiency and could be potentially applied in targeted gene delivery and therapy.

  20. Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

    PubMed Central

    Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

    2016-01-01

    Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

  1. An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability.

    PubMed Central

    Zelenaya-Troitskaya, O; Perlman, P S; Butow, R A

    1995-01-01

    The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA. Images PMID:7621838

  2. Hymenoic acid, a novel specific inhibitor of human DNA polymerase lambda from a fungus of Hymenochaetaceae sp.

    PubMed

    Nishida, Masayuki; Ida, Noriko; Horio, Mao; Takeuchi, Toshifumi; Kamisuki, Shinji; Murata, Hiroshi; Kuramochi, Kouji; Sugawara, Fumio; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2008-05-01

    Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(1'E,5'S)-5'-carboxy-1'-methyl-1'-hexenyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase lambda (pol lambda) in vitro, and 50% inhibition was observed at a concentration of 91.7microM. Compound 1 did not influence the activities of the other seven mammalian pols (i.e., pols alpha, gamma, delta, epsilon, eta, iota, and kappa), but also showed no effect even on the activity of pol beta, which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. This compound also did not inhibit the activities of prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of eukaryotic pol lambda. This compound had no inhibitory activities against two N-terminal truncated pol lambda, del-1 pol lambda (lacking nuclear localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133-575]), and del-2 pol lambda (lacking NLS, BRCT, domain and proline-rich region [residues 245-575]). The compound 1-induced inhibition of intact pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.

  3. Neurotransmitter transporter family including SLC6A6 and SLC6A13 contributes to the 5-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX and photodamage, through uptake of ALA by cancerous cells.

    PubMed

    Tran, Tai Tien; Mu, Anfeng; Adachi, Yuka; Adachi, Yasushi; Taketani, Shigeru

    2014-01-01

    δ-Aminolevulinic acid (ALA)-induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA-induced accumulation of protoporphyrin by the treatment of colon cancer DLD-1 and epithelial cancer HeLa cells with γ-aminobutyric acid (GABA)-related compounds. When the cells were treated with GABA, taurine and β-alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA- and ALA methylester-dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA-treated cells were exposed to white light, the extent of photodamage increased in SLC6A6- and SLC6A13-expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD-1 and HeLa cells decreased the ALA-induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA-induced accumulation of protoporphyrin in cancerous cells.

  4. DNA commission of the International Society of Forensic Genetics: Recommendations on the evaluation of STR typing results that may include drop-out and/or drop-in using probabilistic methods

    PubMed Central

    Gill, P.; Gusmão, L.; Haned, H.; Mayr, W.R.; Morling, N.; Parson, W.; Prieto, L.; Prinz, M.; Schneider, H.; Schneider, P.M.; Weir, B.S.

    2015-01-01

    DNA profiling of biological material from scenes of crimes is often complicated because the amount of DNA is limited and the quality of the DNA may be compromised. Furthermore, the sensitivity of STR typing kits has been continuously improved to detect low level DNA traces. This may lead to (1) partial DNA profiles and (2) detection of additional alleles. There are two key phenomena to consider: allelic or locus ‘drop-out’, i.e. ‘missing’ alleles at one or more genetic loci, while ‘drop-in’ may explain alleles in the DNA profile that are additional to the assumed main contributor(s). The drop-in phenomenon is restricted to 1 or 2 alleles per profile. If multiple alleles are observed at more than two loci then these are considered as alleles from an extra contributor and analysis can proceed as a mixture of two or more contributors. Here, we give recommendations on how to estimate probabilities considering drop-out, Pr(D), and drop-in, Pr(C). For reasons of clarity, we have deliberately restricted the current recommendations considering drop-out and/or drop-in at only one locus. Furthermore, we offer recommendations on how to use Pr(D) and Pr(C) with the likelihood ratio principles that are generally recommended by the International Society of Forensic Genetics (ISFG) as measure of the weight of the evidence in forensic genetics. Examples of calculations are included. An Excel spreadsheet is provided so that scientists and laboratories may explore the models and input their own data. PMID:22864188

  5. Detection of deoxyribonucleic acid (DNA) targets using polymerase chain reaction (PCR) and paper surface-enhanced Raman spectroscopy (SERS) chromatography.

    PubMed

    Hoppmann, Eric P; Yu, Wei W; White, Ian M

    2014-01-01

    Surface-enhanced Raman spectroscopy (SERS) enables multiplex detection of analytes using simple, portable equipment consisting of a single excitation source and detector. Thus, in theory, SERS is ideally suited to replace fluorescence in assays that screen for numerous deoxyribonucleic acid (DNA) targets, but in practice, SERS-based assays have suffered from complexity and elaborate processing steps. Here, we report an assay in which a simple inkjet-fabricated plasmonic paper device enables SERS-based detection of multiple DNA targets within a single polymerase chain reaction (PCR). In prior work, we demonstrated the principles of chromatographic separation and SERS-based detection on inkjet-fabricated plasmonic paper. The present work extends that capability for post-PCR gene sequence detection. In this design, hydrolysis DNA probes with 5' Raman labels are utilized; if the target is present, the probe is hydrolyzed during PCR, freeing the reporter. After applying the PCR sample to a paper SERS device, an on-device chromatographic separation and concentration is conducted to discriminate between hydrolyzed and intact probes. SERS is then used to detect the reporter released by the hydrolyzed probes. This simple separation and detection on paper eliminates the need for complex sample processing steps. In this work, we simultaneously detect the methicillin-resistant Staphylococcus aureus genes mecA and femB to illustrate the concept. We envision that this approach could contribute to the development of multiplex DNA diagnostic tests enabling screening for several target sequences within a single reaction, which is necessary for cases in which sample volume and resources are limited.

  6. Development of a small gantry robotic workcell for deoxyribonucleic acid (DNA) filter array construction

    SciTech Connect

    Beugelsdijk, T.J.; Hollen, R.M.; Snider, K.T.

    1990-01-01

    At Los Alamos National Laboratory, we have constructed a primary cosmid library of human chromosome 16. This library consists of an 11-fold representation of the chromosome and is arrayed in microtiter plate format. A need has arisen in the large scale physical mapping of this chromosome, to array spots of DNA from each of these colonies onto filter media for hybridization studies. We are currently developing a small gantry robot-based workcell to array small spots of DNA in an interleaved format. This allows for the construction of a high spot density format filter array. This paper will discuss the features incorporated into this workcell for the handling of thousands of colonies and their automatic tracking and positioning onto the filter. 7 refs., 3 figs., 1 tab.

  7. Analys. DNA: a computer program for nucleic acid sequence data processing.

    PubMed

    Amthauer, R; Araya, A

    1984-09-01

    A computer program written in BASIC language is described. The program allows processing and analysis of DNA data and has been designed to be used by persons with little or no computer experience. The operator using different options can search for direct homologies with varying degrees of matching, generate complementary strands, find restriction sites, invert the polarity of the sequence and edit a print-out.

  8. A highly acid-resistant novel strain of Lactobacillus johnsonii No. 1088 has antibacterial activity, including that against Helicobacter pylori, and inhibits gastrin-mediated acid production in mice

    PubMed Central

    Aiba, Yuji; Nakano, Yasuhiro; Koga, Yasuhiro; Takahashi, Kenji; Komatsu, Yasuhiko

    2015-01-01

    A novel strain of Lactobacillus johnsonii No. 1088 was isolated from the gastric juice of a healthy Japanese male volunteer, and characterized for its effectiveness in the stomach environment. Lactobacillus johnsonii No. 1088 was found to have the strongest acid resistance among several lactobacilli examined (>10% of cells survived at pH 1.0 after 2 h), and such a high acid resistance property was a specific characteristic of this strain of L. johnsonii. When cultured with various virulent bacteria, L. johnsonii No. 1088 inhibited the growth of Helicobacter pylori,Escherichia coli O-157, Salmonella Typhimurium, and Clostridium difficile, in which case its effectiveness was more potent than that of a type strain of L. johnsonii,JCM2012. In addition to its effect in vitro, L. johnsonii No. 1088 inhibited the growth of H. pylori in human intestinal microbiota-associated mice in both its live and lyophilized forms. Moreover, L. johnsonii No. 1088 suppressed gastric acid secretion in mice via decreasing the number of gastrin-positive cells in the stomach. These results taken together suggest that L. johnsonii No. 1088 is a unique lactobacillus having properties beneficial for supporting H. pylori eradication by triple therapy including the use of a proton pump inhibitor (PPI) and also for prophylaxis of gastroesophageal reflux disease possibly caused after H. pylori eradication as a side effect of PPI. PMID:25771812

  9. Changes in free amino acid content and activities of amination and transamination enzymes in yeasts grown on different inorganic nitrogen sources, including hydroxylamine.

    PubMed

    Norkrans, B; Tunblad-Johansson, I

    1981-01-01

    This study concerns inter- and intraspecific differences between yeasts at assimilation of different nitrogen sources. Alterations in the content of free amino acids in cells and media as well as in the related enzyme activities during growth were studied. The hydroxylamine (HA)-tolerant Endomycopsis lipolytica was examined and compared with the nitrate-reducing Cryptococcus albidus, and Saccharomyces cerevisiae, requiring fully reduced nitrogen for growth. Special attention was paid to alanine, aspartic acid, and glutamic acid, the amino acids closely related to the Krebs cycle keto acids. The amino acids were analyzed as their n-propyl N-acetyl esters by gas-liquid chromatography (GLC). The composition of the amino acid pool was similar for the three yeasts. Glutamic acid was predominant; in early log-phase cells of E. lipolytica contents of 200-234 micromol . g(-1) dry weight were found. A positive correlation between the specific growth rate and the size of the amino acid pool was observed. The assimilation of ammonia was mediated by glutamate dehydrogenase (GDH). The NADP-GDH was the dominating enzyme in all three yeasts showing the highest specific activity in Cr. albidus grown on nitrate (6980 nmol . (min(-1)).(mg protein(-1)). Glutamine synthetase (GS) displayed a high specific activity in S. cerevisiae, which also had a high amount of glutamine. The assimilation of HA did not differ greatly from the assimilation of ammonium in E. lipolytica. The existing differences could rather be explained as provoked by the concentration of available nitrogen.

  10. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

    PubMed

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2015-10-08

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

  11. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA)

    PubMed Central

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S.; Harlow, Mark L.

    2015-01-01

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission. PMID:26446566

  12. [Properties of glycyrrhizin in Kampo extracts including licorice root and changes in the blood concentration of glycyrrhetic acid after oral administration of Kampo extracts].

    PubMed

    Miyamura, M; Ono, M; Kyotani, S; Nishioka, Y

    1996-03-01

    We investigated in vitro the properties of glycyrrhizin (GL), such as dissolution, absorption and resolution, using a Sho-Seiryu-To extract, a Sho-Saiko-To extract, both including a licorice root, and licorice extract. The dissolution of GL differed with the pH of the solvent. The absorption (partition coefficient) of GL decreased with an increase in pH, and increased in the presence of other active constituents, such as baicalin, baicalein, and ephedrine. In the case of the Sho-Saiko-To extract, the conversion from GL to glycyrrhetic acid (GA) by beta-glucuronidase originated from E. coli occurred slowly. It was also suppressed by adding baicalin. We determined in vivo the pharmacokinetics of GA after oral administration of Kampo extracts in healthy volunteers. In each Kampo extract, the time of administration had no influence on the mean maximum blood concentration (Cmax) and the area under the blood concentration-time curve (AUC). Tmax was delayed in the case of the administration after meal (p < 0.05).

  13. Poly(lactic-co-glycolic acid) nanoparticles as candidate DNA vaccine carrier for oral immunization of Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus.

    PubMed

    Tian, Jiyuan; Yu, Juan

    2011-01-01

    In order to protect DNA vaccine against degradation in alimentary tract of fish, poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating vaccine were prepared using W/O/W emulsification combined with spray drying technique in our laboratory. The characteristics of PLGA nanoparticles were described as follows: (1) shape, spherical; (2) size, <500 nm; (3) yield, ∼96.2%; loading percentage, ∼0.5%; encapsulation efficiency, ∼63.7%; supercoiled conformation percentage, ∼65%; (4) release dynamics, gradual release. In vitro transfection in SISK cells showed that PLGA nanoparticles could be utilized to transfect eukaryotes. After oral administration, FITC-labeled PLGA nanoparticles were detected in blood of fish, and RNA containing major capsid protein (MCP) gene information existed in various tissues of fish 10-90 days. In addition, the analysis of immune parameters in sera of treatment fish showed that: (1) infection rate of LCDV post-challenge, ∼16.7%; (2) prophenoloxidase, superoxide dismutase, respiratory burst, lysozyme and antibody levels, increased significantly (p<0.05); (3) activities of serum complement, changed a little (p>0.05). Pearson's correlation displayed that correlation of immune factors mentioned above (not including serum complement) were all positive for fish vaccinated. The data in this study suggested that PLGA nanoparticles were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by oral approach.

  14. Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

    2006-10-03

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  15. Gibberellic Acid Activates Chromatin-bound DNA-dependent RNA Polymerase in Wounded Potato Tuber Tissue 1

    PubMed Central

    Wielgat, Bernard; Kahl, Günter

    1979-01-01

    Chromatin-bound DNA-dependent RNA polymerases react upon wounding of white potato tuber tissues with an increase in activity, which is additionally enhanced to 300% in the presence of 0.1 micromolar gibberellic acid (GA3). 2,4-Dichlorophenoxyacetic acid is only weakly effective and indoleacetic acid not at all. Wounding and treatment with GA3 affect template availability of chromatin only slightly. The hormone has no effect on chromatin-bound RNA polymerases, if added in vitro. The enzymes from intact, wounded, and hormone-treated tissues possess similar characteristics: their activity is dependent on the presence of all four ribonucleotides and a divalent cation such as Mg2+ or Mn2+. However, the sensitivity of the enzymes from different preparations toward α-amanitin differs. Total RNA polymerase activity of chromatin was inhibited by α-amanitin to about 44% in intact, to about 22% in wounded, and only 15% in GA3-treated tissues. The relative activities of polymerases I and II were estimated by varying the (NH4)2SO4 and α-amanitin concentrations in the assay system. It is evident that GA3 preferentially stimulates polymerase I and hence ribosomal RNA synthesis. RNA polymerase II is but slightly affected by GA3. Nearest neighbor frequency analysis revealed that the RNA synthesized by the enzymes from the intact tuber is different from that of wounded or GA3-treated tissues. PMID:16661071

  16. Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments.

    PubMed

    de Ruiter, Anita; Zagrovic, Bojan

    2015-01-01

    Despite the great importance of nucleic acid-protein interactions in the cell, our understanding of their physico-chemical basis remains incomplete. In order to address this challenge, we have for the first time determined potentials of mean force and the associated absolute binding free energies between all standard RNA/DNA nucleobases and amino-acid sidechain analogs in high- and low-dielectric environments using molecular dynamics simulations and umbrella sampling. A comparison against a limited set of available experimental values for analogous systems attests to the quality of the computational approach and the force field used. Overall, our analysis provides a microscopic picture behind nucleobase/sidechain interaction preferences and creates a unified framework for understanding and sculpting nucleic acid-protein interactions in different contexts. Here, we use this framework to demonstrate a strong relationship between nucleobase density profiles of mRNAs and nucleobase affinity profiles of their cognate proteins and critically analyze a recent hypothesis that the two may be capable of direct, complementary interactions.

  17. Focused upon hybridization: rapid and high sensitivity detection of DNA using isotachophoresis and peptide nucleic acid probes.

    PubMed

    Ostromohov, Nadya; Schwartz, Ortal; Bercovici, Moran

    2015-09-15

    We present a novel assay for rapid and high sensitivity detection of nucleic acids without amplification. Utilizing the neutral backbone of peptide nucleic acids (PNA), our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis (ITP) unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, significantly improving the signal-to-noise ratio while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model and experimentally demonstrate the ability to detect targets as short as 17 nucleotides (nt) and a limit of detection of 100 fM with a dynamic range of 5 decades. We also demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal. The assay requires 15 min to complete, and it could potentially be used in applications where rapid and highly sensitive amplification-free detection of nucleic acids is desired.

  18. cDNA cloning, localization, and candidate binding partners of acid-extractable vitelline-coat protein Ci-v-Themis-like in the ascidian Ciona intestinalis.

    PubMed

    Otsuka, Kei; Yamada, Lixy; Sawada, Hitoshi

    2013-10-01

    Ascidians are hermaphrodites, although several ascidian species show self-sterility because of the occurrence of a self/nonself-recognition system called the self-incompatibility system. We previously reported that two pairs of sperm polycystin 1-like receptors, s-Themis-A and s-Themis-B, and egg fibrinogen-like ligands, v-Themis-A and v-Themis-B, are responsible for self-incompatibility in the ascidian Ciona intestinalis. Our previous results showed that v-Themis-A and v-Themis-B were hardly extracted from the vitelline coat (VC) by acid treatment, which is not in accordance with a report that an acid-extractable VC factor has the ability to distinguish self- from nonself-sperm. These results led us to explore a novel factor from acid-extractable VC proteins that could be involved in self-incompatibility. Here, we report cDNA cloning, expression, and localization of Ci-v-Themis-like, a major acid-extractable VC protein. This protein has a fibrinogen-like domain, as do v-Themis-A and v-Themis-B, but it showed no polymorphisms. Phylogenic analysis suggested that Ci-v-Themis-like is an ancestral protein of v-Themis-A and v-Themis-B. Whole mount in situ hybridization revealed that Ci-v-Themis-like mRNA is expressed in the ovary and testis. Western blotting and immunocytochemistry showed the occurrence of Ci-v-Themis-like in developing oocytes and on the VC of mature eggs. Yeast two-hybrid screenings using testis and ovary libraries revealed candidate interacting proteins; among these candidates, we succeeded in identifying several testis-specific proteins, including sperm proteases and coiled-coil-domain-containing proteins. The results suggest that Ci-v-Themis-like and its binding partners are involved in sperm binding to the VC prior to the allorecognition process during C. intestinalis fertilization.

  19. Controlled gene-eluting metal stent fabricated by bio-inspired surface modification with hyaluronic acid and deposition of DNA/PEI polyplexes.

    PubMed

    Kim, Taek Gyoung; Lee, Yuhan; Park, Tae Gwan

    2010-01-15

    A metal stent that could elute plasmid DNA (pDNA) in a controlled manner for substrate-mediated gene transfection was fabricated by first coating with hyaluronic acid (HA) and subsequent deposition of pDNA. To create robust HA coating layer on stainless steel (SS316L) surface, HA was derivatized with dopamine which is a well-known adsorptive molecule involving mussel adhesion process. The HA-coated surface was verified by various analytical techniques and proved to be very hydrophilic and stable, also showing superior biocompatibility in terms of suppressed plasma protein adsorption. For surface loading of pDNA, cationic pDNA/polyethylenimine (PEI) polyplexes were prepared and ionically adsorbed onto the HA-coated SS316L surface. The adsorbed surface exhibited evenly distributed nano-granular topography while the polyplexes maintained the nano-particular morphology. The pDNA was released out in a controlled manner for a period of 10 days with maintaining structural integrity. The dual coated substrate with HA and pDNA/PEI polyplexes exhibited greatly enhanced gene transfection efficiency, when compared to both bare substrate adsorbed with the polyplexes and PEI/pDNA polyelectrolyte multilayers. Dually functionalized stent with HA and pDNA exhibited effective biocompatibility and gene transfection.

  20. Metabolic evidence of vitamin B-12 deficiency, including high homocysteine and methylmalonic acid and low holotranscobalamin, is more pronounced in older adults with elevated plasma folate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: An analysis of data from the National Health and Nutrition Examination Survey indicated that in older adults exposed to folic acid fortification, the combination of low serum vitamin B-12 and elevated folate is associated with higher concentrations of homocysteine and methylmalonic acid ...

  1. Prooxidant DNA breakage induced by caffeic acid in human peripheral lymphocytes: Involvement of endogenous copper and a putative mechanism for anticancer properties

    SciTech Connect

    Bhat, S.H.; Azmi, A.S.; Hadi, S.M. . E-mail: smhadi@vsnl.com

    2007-02-01

    Plant-derived dietary material contains several classes of polyphenols such as flavonoids, curcuminoids, stilbenes and hydroxycinnamic acids. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing cellular DNA degradation in the presence of transition metal ions such as copper. Earlier we have shown that the stilbene resveratrol is able to mobilize endogenous copper ions leading to oxidative breakage of cellular DNA. In this paper, we show that caffeic acid (a hydroxycinnamic acid), which is a major constituent of coffee, is also capable of DNA breakage in human peripheral lymphocytes. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that caffeic acid generates oxidative stress in lymphocytes, which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.

  2. DNA-damaging disinfection byproducts: alkylation mechanism of mutagenic mucohalic acids.

    PubMed

    Gómez-Bombarelli, Rafael; González-Pérez, Marina; Arenas-Valgañón, Jorge; Céspedes-Camacho, Isaac Fabián; Calle, Emilio; Casado, Julio

    2011-10-15

    Hydroxyhalofuranones form a group of genotoxic disinfection byproduct (DBP) of increasing interest. Among them, mucohalic acids (3,4-dihalo-5-hydroxyfuran-2(5H)-one, MXA) are known mutagens that react with nucleotides, affording etheno, oxaloetheno, and halopropenal derivatives. Mucohalic acids have also found use in organic synthesis due to their high functionalization. In this work, the alkylation kinetics of mucochloric and mucobromic acids with model nucleophiles aniline and NBP has been studied experimentally. Also, the alkylation mechanism of nucleosides by MXA has been studied in silico. The results described allow us to reach the following conclusions: (i) based on the kinetic and computational evidence obtained, a reaction mechanism was proposed, in which MXA react directly with amino groups in nucleotides, preferentially attacking the exocyclic amino groups over the endocyclic aromatic nitrogen atoms; (ii) the suggested mechanism is in agreement with both the product distribution observed experimentally and the mutational pattern of MXA; (iii) the limiting step in the alkylation reaction is addition to the carbonyl group, subsequent steps occurring rapidly; and (iv) mucoxyhalic acids, the hydrolysis products of MXA, play no role in the alkylation reaction by MXA.

  3. Hybridization-modulated ion fluxes through peptide-nucleic-acid- functionalized gold nanotubes. A new approach to quantitative label-free DNA analysis.

    PubMed

    Jágerszki, Gyula; Gyurcsányi, Róbert E; Höfler, Lajos; Pretsch, Ernö

    2007-06-01

    The inner walls of gold nanotubes, prepared by template synthesis in the nanopores of polycarbonate track etch membranes, have been chemically modified with peptide nucleic acid (PNA) and used for label-free quantification of complementary DNA sequences. Selective binding of DNA to the PNA-modified nanotubes is shown to decrease the flux of optically detected anionic markers through the nanotubes in a concentration-dependent manner. The strong dependence of the biorecognition-modulated ion transport through the nanopores on the ionic strength suggests a dominantly electrostatic exclusion mechanism of the ion flux decrease as a result of DNA binding to the PNA-modified nanopores.

  4. Effect of amino acids on X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine in DNA.

    PubMed

    Shtarkman, I N; Gudkov, S V; Chernikov, A V; Bruskov, V I

    2008-04-01

    Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.

  5. A label-free signal amplification assay for DNA detection based on exonuclease III and nucleic acid dye SYBR Green I.

    PubMed

    Zheng, Aihua; Luo, Ming; Xiang, Dongshan; Xiang, Xia; Ji, Xinghu; He, Zhike

    2013-09-30

    We have developed a new fluorescence method for specific single-stranded DNA sequences with exonuclease III (Exo III) and nucleic acid dye SYBR Green I. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 27 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of the target DNA, the hairpin-probe is in the stem-closed structure, the fluorescence of SYBR Green I is very strong. In the presence of the target DNA, the hairpin-probe hybridizes with the target DNA to form double-stranded structure with a blunt 3'-terminus. Thus, in the presence of Exo III, only the 3'-terminus of probe is subjected to digestion. Exo III catalyzes the stepwise removal of mononucleotides from this terminus, releasing the target DNA. The released target DNA then hybridizes with another probe, whence the cycle starts anew. The signal of SYBR Green I decreases greatly. This system provides a detection limit of 160 pM, which is comparable to the existing signal amplification methods that utilized Exo III as a signal amplification nuclease. Due to the unique property of Exo III, this method shows excellent detection selectivity for single-base discrimination. More importantly, superiors to other methods based on Exo III, these probes have the advantages of easier to design, synthesize, purify and thus are much cheaper and more applicable. This new approach could be widely applied to sensitive and selective nucleic acids detection.

  6. Comparison of Five Commercial Nucleic Acid Extraction Kits for the PCR-based Detection of Burkholderia Pseudomallei DNA in Formalin-Fixed, Paraffin-Embedded Tissues.

    PubMed

    Obersteller, Sonja; Neubauer, Heinrich; Hagen, Ralf Matthias; Frickmann, Hagen

    2016-09-29

    The extraction and further processing of nucleic acids (NA) from formalin-fixed paraffin-embedded (FFPE) tissues for microbiological diagnostic polymerase chain reaction (PCR) approaches is challenging. Here, we assessed the effects of five different commercially available nucleic acid extraction kits on the results of real-time PCR. FFPE samples from organs of Burkholderia pseudomallei-infected Swiss mice were subjected to processing with five different extraction kits from QIAGEN (FFPE DNA Tissue Kit, EZ1 DNA Tissue Kit, DNA Mini Kit, DNA Blood Mini Kit, and FlexiGene DNA Kit) in combination with three different real-time PCRs targeting B. pseudomallei-specific sequences of varying length after 16 years of storage. The EZ1 DNA Tissue Kit and the DNA Mini Kit scored best regarding the numbers of successful PCR reactions. In case of positive PCR, differences regarding the cycle-threshold (Ct) values were marginal. The impact of the applied extraction kits on the reliability of PCR from FFPE material seems to be low. Interfering factors like the quality of the dewaxing procedure or the sample age appear more important than the selection of specialized FFPE kits.

  7. Optimized catalytic DNA-cleaving ribozymes

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor)

    1996-01-01

    The present invention discloses nucleic acid enzymes capable of cleaving nucleic acid molecules, including single-stranded DNA, in a site-specific manner under physiologic conditions, as well as compositions including same. The present invention also discloses methods of making and using the disclosed enzymes and compositions.

  8. Selenium-Assisted Nucleic Acid Crystallography: Use of DNA Phosphoroselenoates for MAD Phasing

    SciTech Connect

    Wilds, C.J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M.

    2010-03-08

    The combination of synchrotron radiation and a variety of atoms or ions (either covalently attached to the biomolecule prior to crystallization or soaked into crystals) that serve as anomalous scatterers constitutes a powerful tool in the X-ray crystallographer's repertoire of structure determination techniques. Phosphoroselenoates in which one of the nonbridging phosphate oxygens in the backbone is replaced by selenium offer a simplified means for introducing an anomalous scatterer into oligonucleotides by conventional solid-phase synthesis. Unlike other methods that are used to derivatize DNA or RNA by covalent attachment of a heavy atom (i.e., bromine at the C5 position of pyrimidines), tedious synthesis of specialized nucleosides is not required. Introduction of selenium is readily accomplished in solid-phase oligonucleotide synthesis by replacing the standard oxidation agent with a solution of potassium selenocyanide. This results in a diastereomeric mixture of phosphoroselenoates that can be separated by strong anion-exchange HPLC. As a test case, all 10 DNA hexamers of the sequence CGCGCG containing a single phosphoroselenoate linkage (PSe) were prepared. Crystals were grown for a subset of them, and the structure of [d(C{sub PSe}GCGCG)]{sub 2} was determined by the multiwavelength anomalous dispersion technique and refined to 1.1 {angstrom} resolution.

  9. Contributions of the TEL-patch Amino Acid Cluster on TPP1 to Telomeric DNA Synthesis by Human Telomerase

    PubMed Central

    Dalby, Andrew B.; Hofr, Ctirad; Cech, Thomas R.

    2015-01-01

    Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer as well as telomere-shortening syndromes. Recent studies have shown that the TEL-patch – a cluster of amino acids on the surface of the shelterin component TPP1 – is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment – binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action. PMID:25623306

  10. Duplex DNA-Invading γ-Modified Peptide Nucleic Acids Enable Rapid Identification of Bloodstream Infections in Whole Blood

    PubMed Central

    Nölling, Jörk; Rapireddy, Srinivas; Amburg, Joel I.; Crawford, Elizabeth M.; Prakash, Ranjit A.; Rabson, Arthur R.

    2016-01-01

    ABSTRACT Bloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention. PMID:27094328

  11. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  12. Protective Effect of Borage Seed Oil and Gamma Linolenic Acid on DNA: In Vivo and In Vitro Studies

    PubMed Central

    Tasset-Cuevas, Inmaculada; Fernández-Bedmar, Zahira; Lozano-Baena, María Dolores; Campos-Sánchez, Juan; de Haro-Bailón, Antonio; Muñoz-Serrano, Andrés; Alonso-Moraga, Ángeles

    2013-01-01

    Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster. Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells. PMID:23460824

  13. Spectral characterization, cyclic voltammetry, morphology, biological activities and DNA cleaving studies of amino acid Schiff base metal(II) complexes.

    PubMed

    Neelakantan, M A; Rusalraj, F; Dharmaraja, J; Johnsonraja, S; Jeyakumar, T; Sankaranarayana Pillai, M

    2008-12-15

    Metal complexes are synthesized with Schiff bases derived from o-phthalaldehyde (opa) and amino acids viz., glycine (gly) l-alanine (ala), l-phenylalanine (pal). Metal ions coordinate in a tetradentate or hexadentate manner with these N(2)O(2) donor ligands, which are characterized by elemental analysis, molar conductance, magnetic moments, IR, electronic, (1)H NMR and EPR spectral studies. The elemental analysis suggests the stoichiometry to be 1:1 (metal:ligand). Based on EPR studies, spin-Hamiltonian and bonding parameters have been calculated. The g-values calculated for copper complexes at 300K and in frozen DMSO (77K) indicate the presence of the unpaired electron in the dx2-y2 orbital. The evaluated metal-ligand bonding parameters showed strong in-plane sigma- and pi-bonding. X-ray diffraction (XRD) and scanning electron micrography (SEM) analysis provide the crystalline nature and the morphology of the metal complexes. The cyclic voltammograms of the Cu(II)/Mn(II)/VO(II) complexes investigated in DMSO solution exhibit metal centered electroactivity in the potential range -1.5 to +1.5V. The electrochemical data obtained for Cu(II) complexes explains the change of structural arrangement of the ligand around Cu(II) ions. The biological activity of the complexes has been tested on eight bacteria and three fungi. Cu(II) and Ni(II) complexes show an increased activity in comparison to the controls. The metal complexes of opapal Schiff base were evaluated for their DNA cleaving activities with calf-thymus DNA (CT DNA) under aerobic conditions. Cu(II) and VO(II) complexes show more pronounced activity in presence of the oxidant.

  14. Spectral characterization, cyclic voltammetry, morphology, biological activities and DNA cleaving studies of amino acid Schiff base metal(II) complexes

    NASA Astrophysics Data System (ADS)

    Neelakantan, M. A.; Rusalraj, F.; Dharmaraja, J.; Johnsonraja, S.; Jeyakumar, T.; Sankaranarayana Pillai, M.

    2008-12-01

    Metal complexes are synthesized with Schiff bases derived from o-phthalaldehyde (opa) and amino acids viz., glycine (gly) L-alanine (ala), L-phenylalanine (pal). Metal ions coordinate in a tetradentate or hexadentate manner with these N 2O 2 donor ligands, which are characterized by elemental analysis, molar conductance, magnetic moments, IR, electronic, 1H NMR and EPR spectral studies. The elemental analysis suggests the stoichiometry to be 1:1 (metal:ligand). Based on EPR studies, spin-Hamiltonian and bonding parameters have been calculated. The g-values calculated for copper complexes at 300 K and in frozen DMSO (77 K) indicate the presence of the unpaired electron in the d orbital. The evaluated metal-ligand bonding parameters showed strong in-plane σ- and π-bonding. X-ray diffraction (XRD) and scanning electron micrography (SEM) analysis provide the crystalline nature and the morphology of the metal complexes. The cyclic voltammograms of the Cu(II)/Mn(II)/VO(II) complexes investigated in DMSO solution exhibit metal centered electroactivity in the potential range -1.5 to +1.5 V. The electrochemical data obtained for Cu(II) complexes explains the change of structural arrangement of the ligand around Cu(II) ions. The biological activity of the complexes has been tested on eight bacteria and three fungi. Cu(II) and Ni(II) complexes show an increased activity in comparison to the controls. The metal complexes of opapal Schiff base were evaluated for their DNA cleaving activities with calf-thymus DNA (CT DNA) under aerobic conditions. Cu(II) and VO(II) complexes show more pronounced activity in presence of the oxidant.

  15. Protective effect of borage seed oil and gamma linolenic acid on DNA: in vivo and in vitro studies.

    PubMed

    Tasset-Cuevas, Inmaculada; Fernández-Bedmar, Zahira; Lozano-Baena, María Dolores; Campos-Sánchez, Juan; de Haro-Bailón, Antonio; Muñoz-Serrano, Andrés; Alonso-Moraga, Angeles

    2013-01-01

    Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells.

  16. Naringin ameliorates acetic acid induced colitis through modulation of endogenous oxido-nitrosative balance and DNA damage in rats

    PubMed Central

    Kumar, Venkatashivam Shiva; Rajmane, Anuchandra Ramchandra; Adil, Mohammad; Kandhare, Amit Dattatraya; Ghosh, Pinaki; Bodhankar, Subhash Laxman

    2014-01-01

    The aim of this study was to evaluate the effect of naringin on experimentally induced inflammatory bowel disease in rats. Naringin (20, 40 and 80 mg/kg) was given orally for 7 days to Wistar rats before induction of colitis by intrarectal instillation of 2 mL of 4% (v/v) acetic acid solution. The degree of colonic mucosal damage was analyzed by examining mucosal damage, ulcer area, ulcer index and stool consistency. Intrarectal administration of 4% acetic acid resulted in significant modulation of serum alkaline phosphatase, lactate dehydrogenase, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and myeloperoxidase (MPO) content along with colonic nitric oxide (NO), xanthine oxidase (XO) level and protein carbonyl content in the colonic tissue as well as in blood. Naringin (40 and 80 mg/kg) exerted a dose dependent (P < 0.05) ameliorative effect, as it significantly increased hematological parameter as well as colonic SOD and GSH. There was a significant (P < 0.05) and dose dependant inhibition of macroscopical score, ulcer area along with colonic MDA, MPO activity by the 7 days of pretreatment of naringin (40 and 80 mg/kg). Biochemical studies revealed a significant (P < 0.05) dose dependant inhibition in serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) levels by pretreatment of naringin. Increased levels of colonic NO, XO, protein carbonyl content and DNA damage were also significantly decreased by naringin pretreatment. The findings of the present investigation propose that naringin has an anti-inflammatory, anti-oxidant and anti-apoptotic potential effect at colorectal sites as it modulates the production and expression of oxidative mediators such as MDA, MPO, NO and XO, thus reducing DNA damage. PMID:24683411

  17. Omega-3 Polyunsaturated Fatty Acids Inhibited Tumor Growth via Preventing the Decrease of Genomic DNA Methylation in Colorectal Cancer Rats.

    PubMed

    Huang, Qionglin; Wen, Juan; Chen, Guangzhao; Ge, Miaomiao; Gao, Yihua; Ye, Xiaoxia; Liu, Chunan; Cai, Chun

    2016-01-01

    Omge-3 polyunsaturated fatty acids (PUFAs) exhibited significant effect in inhibiting various tumors. However, the mechanisms of its anticancer role have not been fully demonstrated. The declination of 5-methylcytosine (5 mC) was closely associated with poor prognosis of tumors. To explore whether omega-3 PUFAs influences on DNA methylation level in tumors, colorectal cancer (CRC) rat model were constructed using N-methyl phosphite nitrourea and omega-3 PUFAs were fed to part of the rats during tumor induction. The PUFAs contents in the rats of 3 experimental groups were measured using gas chromatography and 5 mC level were detected by liquid chromatography tandem mass spectrometry. The results showed that tumor incidence in omega-3 treated rats was much lower than in CRC model rats, which confirmed significant antitumor role of omega-3 PUFAs. Six PUFA members categorized to omega-3 and omega-6 families were quantified and the ratio of omega-6/omega-3 PUFAs was remarkably lower in omega-3 PUFAs treatment group than in CRC model group. 5 mC content in omega-3 PUFAs treated rats was higher than in CRC model rats, suggesting omega-3 PUFAs promoted 5 mC synthesis. Therefore, omega-3 PUFAs probably inhibited tumor growth via regulating DNA methylation process, which provided a novel anticancer mechanism of omega-3 PUFAs from epigenetic view.

  18. Antibacterial effects of Brazilian and Bulgarian propolis and synergistic effects with antibiotics acting on the bacterial DNA and folic acid.

    PubMed

    Orsi, R O; Fernandes, A; Bankova, V; Sforcin, J M

    2012-01-01

    Propolis is a honeybee product that has been used since ancient times because of its therapeutic effects. It can be used in the development of alternative therapies for the treatment of many diseases, and because propolis shows antibacterial action, this work was carried out in order to investigate a possible synergism between propolis and antibiotics acting on DNA (ciprofloxacin and norfloxacin) and on the metabolism (cotrimoxazole) against Salmonella typhi. Propolis samples collected in Brazil and Bulgaria were compared in these assays, and the synergism was investigated by using ½ and ¼ of the minimal inhibitory concentration for propolis and antibiotics, evaluating the number of viable cells according to the incubation time. Brazilian and Bulgarian propolis showed antibacterial activity, but no synergistic effects with the three tested antibiotics were seen. Previous works by our laboratory have revealed that propolis has synergistic effects with antibiotics, acting on the bacterial wall and ribosome, but it does not seem to interact with antibiotics acting on DNA or folic acid, and only a bacteriostatic action was seen in these assay conditions.

  19. Cyclen Grafted with poly[(Aspartic acid)-co-Lysine]: Preparation, Assembly with Plasmid DNA, and in Vitro Transfection Studies.

    PubMed

    Ma, Chunying; Zhang, Jin; Guo, Liwen; Du, Changguo; Song, Ping; Zhao, Baojing; Li, Ling; Li, Chao; Qiao, Renzhong

    2016-01-04

    Development of safe and effective gene carriers is the key to the success of gene therapy. Nowadays, it is still required to develop new methods to improve nonviral gene delivery efficiency. Herein, copolymers of poly[(aspartic acid)-co-lysine] grafted with cyclen (cyclen-pAL) were designed and evaluated for efficient gene delivery. Two copolymers with different Asp/Lys block ratios were prepared and characterized by NMR and gel permeation chromatography analysis. Agarose gel retardation, circular dichroism, and fluorescent quenching assays showed the strong DNA-binding and protection ability for the title compounds. Atomic force microscopy studies clearly delineated uniform DNA globules with a diameter around 100 nm, induced by cyclen-pAL. By grafting cyclen on Asp, relatively high gene delivery efficiency and low cytotoxicity of the modified copolymers were achieved compared with their parent compounds. The present work might help to develop strategies for design and modification of polypeptide copolymers, which may also be applied to favorable gene expression and delivery.

  20. Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: A comparative approach

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sakthivel, A.; Pravin, N.

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 102 to 105 indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

  1. Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: a comparative approach.

    PubMed

    Raman, N; Sakthivel, A; Pravin, N

    2014-05-05

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 10(2) to 10(5) indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

  2. [Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of β-amyloid with DNA].

    PubMed

    Khmeleva, S A; Mezentsev, Y V; Kozin, S A; Mitkevich, V A; Medvedev, A E; Ivanov, A S; Bodoev, N V; Makarov, A A; Radko, S P

    2015-01-01

    Interaction of intranuclear β-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of β-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of β-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the β-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aβ facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

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

    PubMed

    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. Meiofaunal Richness in Highly Acidic Hot Springs in Unzen-Amakusa National Park, Japan, Including the First Rediscovery Attempt for Mesotardigrada.

    PubMed

    Suzuki, Atsushi C; Kagoshima, Hiroshi; Chilton, Glen; Grothman, Gary T; Johansson, Carl; Tsujimoto, Megumu

    2017-02-01

    Extreme environments sometimes support surprisingly high meiofaunal diversity. We sampled runoff from the acidic hot springs of Unzen, Japan. This is the type locality of Thermozodium esakii Rahm, 1937, the only tardigrade in the class Mesotardigrada, which remains contentious in the absence of corroboration or supporting specimens. Our sampling revealed at least three species of arthropods, four rotifers, and five nematodes living in the hot (ca. 40°C) and acidic (ca. pH 2.5) water, but no tardigrades.

  5. Ultraviolet irradiation of DNA complexed with. alpha. /. beta. -type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers

    SciTech Connect

    Nicholson, W.L.; Setlow, B.; Setlow, P. )

    1991-10-01

    UV irradiation of complexes of DNA and an {alpha}/{beta}-type small, acid-soluble protein (SASP) from Bacillus subtilis spores gave decreasing amounts of pyrimidine dimers and increasing amounts of spore photoproduct as the SASP/DNA ratio was increased. The yields of pyrimidine dimers and spore photoproduct were < 0.2% and 8% of total thymine, respectively, when DNA saturated with SASP was irradiated at 254 nm with 30 kJ/m{sup 2}; in the absence of SASP the yields were reversed - 4.5% and 0.3%, respectively. Complexes of DNA with {alpha}/{beta}-type SASP from Bacillus cereus, Bacillus megaterium, or Clostridium bifermentans spores also gave spore photoproduct upon UV irradiation. However, incubation of these SASPs with DNA under conditions preventing complex formation or use of mutant SASPs that do not form complexes did not affect the photoproducts formed in vitro. These results suggest that the UV photochemistry of bacterial spore DNA in vivo is due to the binding of {alpha}/{beta}-type SASP, a binding that is known to cause a change in DNA conformation in vitro from the B form to the A form. The yields of spore photoproduct in vitro were significantly lower than in vivo, perhaps because of the presence of substances other than SASP in spores. It is suggested that as these factors diffuse out in the first minutes of spore germination, spore photoproduct yields become similar to those observed for irradiation of SASP/DNA complexes in vitro.

  6. Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

    PubMed Central

    Muratovska, Aleksandra; Lightowlers, Robert N.; Taylor, Robert W.; Turnbull, Douglass M.; Smith, Robin A. J.; Wilce, Jacqueline A.; Martin, Stephen W.; Murphy, Michael P.

    2001-01-01

    The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA

  7. Structure, antimicrobial activity, DNA- and albumin-binding of manganese(II) complexes with the quinolone antimicrobial agents oxolinic acid and enrofloxacin.

    PubMed

    Zampakou, Marianthi; Akrivou, Melpomeni; Andreadou, Eleni G; Raptopoulou, Catherine P; Psycharis, Vassilis; Pantazaki, Anastasia A; Psomas, George

    2013-04-01

    The reaction of MnCl2 with the quinolone antibacterial drug oxolinic acid (Hoxo) results to the formation of [KMn(oxo)3(MeOH)3]. Interaction of MnCl2 with the quinolone Hoxo or enrofloxacin (Herx) and the N,N'-donor heterocyclic ligand 1,10-phenanthroline (phen) results in the formation of metal complexes with the general formula [Mn(quinolonato)2(phen)]. The crystal structures of [KMn(oxo)3(MeOH)3] and [Mn(erx)2(phen)], exhibiting a 1D polymeric and a mononuclear structure, respectively, have been determined by X-ray crystallography. In these complexes, the deprotonated bidentate quinolonato ligands are coordinated to manganese(II) ion through the pyridone oxygen and a carboxylato oxygen. All complexes can act as potential antibacterial agents with [Mn(erx)2(phen)] exhibiting the most pronounced antimicrobial activity against five different microorganisms. Interaction of the complexes with calf-thymus DNA (CT DNA), studied by UV spectroscopy, has shown that they bind to CT DNA. Competitive study with ethidium bromide (EB) has shown that all complexes can displace the DNA-bound EB indicating their binding to DNA in strong competition with EB. Intercalative binding mode is proposed for the interaction of the complexes with CT DNA and has also been verified by DNA solution viscosity measurements and cyclic voltammetry. DNA electrophoretic mobility experiments suggest that [Mn(erx)2(phen)] binds strongly to supercoiled pDNA and to linearized pDNA possibly by an intercalative manner provoking double-stranded cleavage reflecting in a nuclease-like activity. The complexes exhibit good binding propensity to human or bovine serum albumin protein showing relatively high binding constant values. The binding constants of the complexes towards CT DNA and albumins have been compared to their corresponding zinc(II) and nickel(II) complexes.

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

    PubMed Central

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

    2016-01-01

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

  9. Distribution and effects of amino acid changes in drug-resistant α and β herpesviruses DNA polymerase

    PubMed Central

    Topalis, D.; Gillemot, S.; Snoeck, R.; Andrei, G.

    2016-01-01

    Emergence of drug-resistance to all FDA-approved antiherpesvirus agents is an increasing concern in immunocompromised patients. Herpesvirus DNA polymerase (DNApol) is currently the target of nucleos(t)ide analogue-based therapy. Mutations in DNApol that confer resistance arose in immunocompromised patients infected with herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV), and to lesser extent in herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV) and human herpesvirus 6 (HHV-6). In this review, we present distinct drug-resistant mutational profiles of herpesvirus DNApol. The impact of specific DNApol amino acid changes on drug-resistance is discussed. The pattern of genetic variability related to drug-resistance differs among the herpesviruses. Two mutational profiles appeared: one favoring amino acid changes in the Palm and Finger domains of DNApol (in α-herpesviruses HSV-1, HSV-2 and VZV), and another with mutations preferentially in the 3′-5′ exonuclease domain (in β-herpesvirus HCMV and HHV-6). The mutational profile was also related to the class of compound to which drug-resistance emerged. PMID:27694307

  10. DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in "drug-type" and "fiber-type" Cannabis sativa L.

    PubMed

    Kojoma, Mareshige; Seki, Hikaru; Yoshida, Shigeo; Muranaka, Toshiya

    2006-06-02

    The cannabinoid content of 13 different strains of cannabis plant (Cannabis sativa L.) was analyzed. Six strains fell into the "drug-type" class, with high Delta-9-tetrahydrocannabinolic acid (THCA) content, and seven strains into the "fiber-type" class, with low THCA using HPLC analysis. Genomic DNA sequence polymorphisms in the THCA synthase gene from each strain were studied. A single PCR fragment of the THCA synthase gene was detected from six strains of "drug-type" plants. We could also detect the fragment from seven strains of "fiber-type" plants, although no or very low content of THCA were detected in these samples. These were 1638 bp from all 13 strains and no intron among the sequences obtained. There were two variants of the THCA synthase gene in the "drug-type" and "fiber-type" cannabis plants, respectively. Thirty-seven major substitutions were detected in the alignment of the deduced amino acid sequences from these variants. Furthermore, we identified a specific PCR marker for the THCA synthase gene for the "drug-type" strains. This PCR marker was not detected in the "fiber-type" strains.

  11. An amphipathic trans-acting phosphorothioate DNA element delivers uncharged PNA and PMO nucleic acid sequences in mammalian cells

    PubMed Central

    Jain, Harsh V.; Beaucage, Serge L.

    2016-01-01

    An innovative approach to the delivery of uncharged peptide nucleic acids (PNA) and phosphorodiamidate morpholino (PMO) oligomers in mammalian cells is described and consists of extending the sequence of those oligomers with a short PNA-polyA or PMO-polyA tail. Recognition of the polyA-tailed PNA or PMO oligomers by an amphipathic trans-acting polythymidylic thiophosphate triester element (dTtaPS) results in efficient internalization of those oligomers in several cell lines. Our findings indicate that cellular uptake of the oligomers occurs through an energy-dependent mechanism and macropinocytosis appears to be the predo-minant endocytic pathway used for internalization. The functionality of the internalized oligomers is demonstrated by alternate splicing of the pre-mRNA encoding luciferase in HeLa pLuc 705 cells. Amphipathic phosphorothioate DNA elements may represent a unique class of cellular transporters for robust delivery of uncharged nucleic acid sequences in live mammalian cells. PMID:27516815

  12. Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.

    PubMed

    Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke

    2012-08-21

    We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.

  13. Nuclear hormone receptors involved in neoplasia: erb A exhibits a novel DNA sequence specificity determined by amino acids outside of the zinc-finger domain.

    PubMed Central

    Chen, H; Smit-McBride, Z; Lewis, S; Sharif, M; Privalsky, M L

    1993-01-01

    The erb A oncogene is a dominant negative allele of a thyroid hormone receptor gene and acts in the cancer cell by encoding a transcriptional repressor. We demonstrate here that the DNA sequence recognition properties of the oncogenic form of the erb A protein are significantly altered from those of the normal thyroid hormone receptors and more closely resemble those of the retinoic acid receptors; this alteration appears to play an important role in defining the targets of erb A action in neoplasia. Unexpectedly, the novel DNA recognition properties of erb A are encoded by an N-terminal region not previously implicated as playing this function in current models of receptor-DNA interaction. Two N-terminal erb A amino acids in particular, histidine 12 and cysteine 32, contribute to this phenomenon, acting in conjunction with amino acids in the zinc finger domain. The effects of the N-terminal domain can be observed at the level of both DNA binding and transcriptional modulation. Our results indicate that unanticipated determinants within the nuclear hormone receptors participate in DNA sequence recognition and may contribute to the differential target gene specificity displayed by different receptor forms. Images PMID:8096060

  14. Biophysical characterization of DNA binding from single molecule force measurements

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-09-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

  15. Biophysical characterization of DNA binding from single molecule force measurements

    PubMed Central

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-01-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function. PMID:20576476

  16. Synthesis and DNA transfection properties of new head group modified malonic acid diamides.

    PubMed

    Wölk, Christian; Heinze, Martin; Kreideweiss, Patrick; Dittrich, Matthias; Brezesinski, Gerald; Langner, Andreas; Dobner, Bodo

    2011-05-16

    Malonic acid diamides with two long hydrophobic alkyl chains and a basic polar head group as a new class of non-viral gene transferring compounds have shown high transfection efficiency and moderate toxicity. Based on the results obtained with saturated and unsaturated alkyl residues new derivatives with a more complex head group structure have been synthesized. For this purpose, cationic respectively basic groups were introduced by one or two lysine residues bound via tris(aminoethyl)amine spacer to the malonic acid diamide backbone. By studying in vitro gene delivery an increase of transfection efficacy was observed when using lipids with at least one unsaturated alkyl chain. This leads to cationic lipids exhibiting comparable or even higher transfection efficacies compared to the commercially available transfection agents LipofectAmine™ and SuperFect™. Phase transitions and phase structures of selected compounds have been analyzed and discussed in terms of transfection abilities. Particle size and zeta potential of liposomes and lipoplexes were also determined.

  17. Chitosan-coated poly(lactic-co-glycolic) acid nanoparticles as an efficient delivery system for Newcastle disease virus DNA vaccine.

    PubMed

    Zhao, Kai; Zhang, Yang; Zhang, Xiaoyan; Shi, Ci; Wang, Xin; Wang, Xiaohua; Jin, Zheng; Cui, Shangjin

    2014-01-01

    We determined the efficacy and safety of chitosan (CS)-coated poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) as a delivery system for a vaccine to protect chickens against Newcastle disease virus (NDV). The newly constructed vaccine contained DNA (the F gene) of NDV. The Newcastle disease virus (NDV) F gene deoxyribonucleic acid (DNA) plasmid (pFDNA)-CS/PLGA-NPs were spherical (diameter =699.1 ± 5.21 nm [mean ± standard deviation]) and smooth, with an encapsulation efficiency of 98.1% and a Zeta potential of +6.35 mV. An in vitro release assay indicated that CS controlled the burst release of plasmid DNA, such that up to 67.4% of the entire quantity of plasmid DNA was steadily released from the pFDNA-CS/PLGA-NPs. An in vitro expression assay indicated that the expression of nanoparticles (NPs) was maintained in the NPs. In an immunization test with specific pathogen-free chickens, the pFDNA-CS/PLGA-NPs induced stronger cellular, humoral, and mucosal immune responses than the plasmid DNA vaccine alone. The pFDNA-CS/PLGA-NPs did not harm 293T cells in an in vitro assay and did not harm chickens in an in vivo assay. Overall, the results indicated that CS-coated PLGA NPs can serve as an efficient and safe mucosal immune delivery system for NDV DNA vaccine.

  18. Characterization of a DNA-damage-recognition protein from F9 teratocarcinoma cells, which is inducible by retinoic acid and cyclic AMP.

    PubMed

    Chao, C C; Sun, N K; Lin-Chao, S

    1993-02-15

    A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

  19. Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences.

    PubMed

    Suzuki, T; Inoue, N; Higashi, T; Mizobuchi, R; Sugimura, N; Yokouchi, K; Furukohri, T

    2000-12-01

    Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 micromole Pi min(-1) mg protein(-1) for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1,608 and 1,239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66-73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63-71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55-57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.

  20. Cloning of the. gamma. -aminobutyric acid (GABA). rho. sub 1 cDNA: A GABA receptor subunit highly expressed in the retina

    SciTech Connect

    Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr. ); O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi ); Uhl, G.R. Johns Hopkins Univ. School of Medicine, Baltimore, MD )

    1991-04-01

    Type A {gamma}-aminobutyric acid (GABA{sub A}) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA{sub A} subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA {rho}{sub 1}, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

  1. CDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  2. CD44-Targeted Hyaluronic Acid-Coated Redox-Responsive Hyperbranched Poly(amido amine)/Plasmid DNA Ternary Nanoassemblies for Efficient Gene Delivery.

    PubMed

    Gu, Jijin; Chen, Xinyi; Ren, Xiaoqing; Zhang, Xiulei; Fang, Xiaoling; Sha, Xianyi

    2016-07-20

    Hyaluronic acid (HA), which can specifically bind to CD44 receptor, is a specific ligand for targeting to CD44-overexpressing cancer cells. The current study aimed to develop ternary nanoassemblies based on HA-coating for targeted gene delivery to CD44-positive tumors. A novel reducible hyperbranched poly(amido amine) (RHB) was assembled with plasmid DNA (pDNA) to form RHB/pDNA nanoassemblies. HA/RHB/pDNA nanoassemblies were fabricated by coating HA on the surface of the RHB/pDNA nanoassembly core through electrostatic interaction. After optimization, HA/RHB/pDNA nanoassemblies were spherical, core-shell nanoparticles with nanosize (187.6 ± 11.4 nm) and negative charge (-9.1 ± 0.3 mV). The ternary nanoassemblies could efficiently protect the condensed pDNA from enzymatic degradation by DNase I, and HA could significantly improve the stability of nanoassemblies in the sodium heparin solution or serum in vitro. As expected, HA significantly decreased the cytotoxicity of RHB/pDNA nanoassemblies due to the negative surface charges. Moreover, it revealed that HA/RHB/pDNA nanoassemblies showed higher transfection activity than RHB/pDNA nanoassemblies in B16F10 cells, especially in the presence of serum in vitro. Because of the active recognition between HA and CD44 receptor, there was significantly different transfection efficiency between B16F10 (CD44+) and NIH3T3 (CD44-) cells after treatment with HA/RHB/pDNA nanoassemblies. In addition, the cellular targeting and transfection activity of HA/RHB/pDNA nanoassemblies were further evaluated in vivo. The results indicated that the interaction between HA and CD44 receptor dramatically improved the accumulation of HA/RHB/pDNA nanoassemblies in CD44-positive tumor, leading to higher gene expression than RHB/pDNA nanoassemblies. Therefore, HA/RHB/pDNA ternary nanoassemblies may be a potential gene vector for delivery of therapeutic genes to treat CD44-overexpressing tumors in vivo.

  3. Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes

    SciTech Connect

    Chang, L.W.; Daniel, F.B. ); DeAngelo, A.B. )

    1992-01-01

    An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri-(TCA), di-(CA), and mono-(MCA) chloroacetic acid and their corresponding aldehydes, tri-(chloralhydrate, CH), di(DCAA) and mono-(CAA) chloroacetaldehyde. None of the chloracetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strand breakage in mice (7% at 4hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other rodent tissues and cultured cell types. Two of the chloroacetaldehydes, DCAA and CAA, are direct acting DNA damaging agents in CCRF-CEM cells, but not in liver or splenocytes in vivo or in cultured hepatocytes. CH showed no activity in any system investigated. 58 refs., 6 figs., 2 tabs.

  4. Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

    PubMed Central

    Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

    1986-01-01

    A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

  5. Temporal trends of perfluoroalkane sulfonic acids and their sulfonamide-based precursors in herring from the Swedish west coast 1991-2011 including isomer-specific considerations.

    PubMed

    Ullah, Shahid; Huber, Sandra; Bignert, Anders; Berger, Urs

    2014-04-01

    A method was developed for simultaneous analysis of perfluoroalkane sulfonic acids (PFSAs) and their sulfonamide-based precursors (perfluoroalkane sulfonamidoacetic acids (FASAAs), sulfonamides (FASAs), and sulfonamidoethanols (FASEs)) in fish muscle. Extraction was performed with acetonitrile followed by a clean-up and fractionation step and instrumental analysis by UPLC/MS/MS and GC/MS. Time trends of PFSAs and their precursors in herring muscle samples originating from the Kattegat at the west coast of Sweden were investigated covering the years 1991-2011. The following analytes were detected, all with decreasing or unchanged trends between 1991 and 2011: Perfluorobutane sulfonic acid (PFBS, below the method detection limit (acid (PFHxS, 9-38pg/g), perfluorooctane sulfonic acid (PFOS, 240-930pg/g), perfluorodecane sulfonic acid (PFDS, acid (MeFOSAA and EtFOSAA, 2-39 and 2-31pg/g, respectively) and perfluorooctane sulfonamide (FOSA, 78-920pg/g). The highest concentrations were found for PFOS and FOSA around the turn of the century. Shorter disappearance half-lives were observed for precursors compared to PFSAs. Assuming that these trend differences are representative for fish consumed by the general Swedish population, this observation suggests that the relative contribution of precursors to total human exposure to PFOS via fish intake has decreased in Sweden over the study period. PFOS precursors in fish may have constituted a significant indirect exposure pathway for PFOS in the 1990s. Isomer-specific analysis of PFOS and FOSA revealed <10% relative contributions of branched isomers to total PFOS and total FOSA. Furthermore, the percentage branched isomers decreased over time for both compounds. These findings are contrary to patterns and temporal trends of PFOS isomers commonly found in human serum. In combination with literature data on isomer patterns

  6. Arsenic Metabolites, Including N-Acetyl-4-hydroxy-m-arsanilic Acid, in Chicken Litter from a Roxarsone-Feeding Study Involving 1600 Chickens.

    PubMed

    Yang, Zonglin; Peng, Hanyong; Lu, Xiufen; Liu, Qingqing; Huang, Rongfu; Hu, Bin; Kachanoski, Gary; Zuidhof, Martin J; Le, X Chris

    2016-07-05

    The poultry industry has used organoarsenicals, such as 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, ROX), to prevent disease and to promote growth. Although previous studies have analyzed arsenic species in chicken litter after composting or after application to agricultural lands, it is not clear what arsenic species were excreted by chickens before biotransformation of arsenic species during composting. We describe here the identification and quantitation of arsenic species in chicken litter repeatedly collected on days 14, 24, 28, 30, and 35 of a Roxarsone-feeding study involving 1600 chickens of two strains. High performance liquid chromatography separation with simultaneous detection by both inductively coupled plasma mass spectrometry and electrospray ionization tandem mass spectrometry provided complementary information necessary for the identification and quantitation of arsenic species. A new metabolite, N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA), was identified, and it accounted for 3-12% of total arsenic. Speciation analyses of litter samples collected from ROX-fed chickens on days 14, 24, 28, 30, and 35 showed the presence of N-AHAA, 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), inorganic arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), and ROX. 3-AHPAA accounted for 3-19% of the total arsenic. Inorganic arsenicals (the sum of As(III) and As(V)) comprised 2-6% (mean 3.5%) of total arsenic. Our results on the detection of inorganic arsenicals, methylarsenicals, 3-AHPAA, and N-AHAA in the chicken litter support recent findings that ROX is actually metabolized by the chicken or its gut microbiome. The presence of the toxic metabolites in chicken litter is environmentally relevant as chicken litter is commonly used as fertilizer.

  7. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

    PubMed Central

    Isola, L M; Zhou, S L; Kiang, C L; Stump, D D; Bradbury, M W; Berk, P D

    1995-01-01

    To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAs