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Sample records for acid dna ribonucleic

  1. Ribonucleic acid purification.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2014-08-15

    Research on RNA has led to many important biological discoveries and improvement of therapeutic technologies. From basic to applied research, many procedures employ pure and intact RNA molecules; however their isolation and purification are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality. The current techniques to isolate and purify RNA molecules still have several limitations and the requirement for new methods able to improve RNA quality to meet regulatory demands is growing. In fact, as basic research improves the understanding of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the purification of biological compounds, requiring high purity degree. In addition, its application in biopharmaceutical manufacturing is well established. This paper discusses the importance and the progress of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In particular and in view of the high specificity, affinity chromatography has been recently applied to RNA purification processes. Accordingly, recent chromatographic investigations based on biorecognition phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential contribution of these methods to overcome the challenges of RNA purification.

  2. Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis1

    PubMed Central

    Atherly, Alan G.

    1974-01-01

    A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis. PMID:4364330

  3. Control of dihydrofolate reductase messenger ribonucleic acid production

    SciTech Connect

    Leys, E.J.; Kellems, R.E.

    1981-11-01

    The authors used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

  4. Towards the elements of successful insect Ribonucleic acid interference (RNAi)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribonucleic acid interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that ...

  5. Saliva of Lygus lineolaris digests double stranded ribonucleic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The prospects for development of highly specific pesticides based on double stranded ribonucleic acid have been a recent focus of scientific research. Creative applications have been proposed and demonstrated. However, not all insects are sensitive to double stranded RNA (dsRNA) gene knockdown effec...

  6. Hybridizable ribonucleic acid of rat brain

    PubMed Central

    Bondy, S. C.; Roberts, Sidney

    1968-01-01

    1. Cerebral RNA of adult and newborn rats was labelled in vivo by intracervical injection of [5-3H]uridine or [32P]phosphate. Hepatic RNA of similar animals was labelled by intraperitoneal administration of [6-14C]orotic acid. Nuclear and cytoplasmic fractions were isolated and purified by procedures involving extraction with phenol and repeated precipitation with ethanol. 2. The fraction of pulse-labelled RNA from cerebral nuclei that hybridized to homologous DNA exhibited a wide range of turnover values and was heterogeneous in sucrose density gradients. 3. Base composition of the hybridizable RNA was similar to that of the total pulse-labelled material; both were DNA-like. 4. Pulse-labelled cerebral nuclear RNA hybridized to a greater extent than cytoplasmic RNA for at least a week after administration of labelled precursor. This finding suggested that cerebral nuclei contained a hybridizable component that was not transferred to cytoplasm. 5. The rates of decay of the hybridizable fractions of cerebral nuclei and cytoplasm were faster in the newborn animal than in the adult. Presumably a larger proportion of labile messenger RNA molecules was present in the immature brain. 6. Cerebral nuclear and cytoplasmic RNA fractions from newborn or adult rats, labelled either in vivo for periods varying from 4min. to 7 days or in vitro by exposure to [3H]-dimethyl sulphate, uniformly hybridized more effectively than the corresponding hepatic preparation. These data suggested that a larger proportion of RNA synthesis was oriented towards messenger RNA formation in brain than in liver. PMID:5683505

  7. Ribonucleic Acid Polymerase Activity in Sendai Virions and Nucleocapsid

    PubMed Central

    Robinson, William S.

    1971-01-01

    After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the 3H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA. PMID:4328418

  8. SYNTHESIS OF RIBONUCLEIC ACID BY X-IRRADIATED BACTERIA1

    PubMed Central

    Frampton, E. W.

    1964-01-01

    Frampton, E. W. (The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston). Synthesis of ribonucleic acid by X-irradiated bacteria. J. Bacteriol. 87:1369–1376. 1964.—Postirradiation synthesis of total ribonucleic acid (RNA) and of RNA components was measured after exposure of Escherichia coli B/r to X rays. Net synthesis of RNA measured by the orcinol reaction and by the incorporation of uridine-2-C14 was depressed in irradiated cells, but paralleled the period of postirradiation growth (30 to 40 min). Incorporation of uridine-2-C14, added after net synthesis of RNA had ceased, detected an apparent turnover in a portion of the RNA. Irradiated cells retained their ability to adjust RNA synthesis to growth rate. After a shift-down in growth rate, irradiated cells incorporated radioactive uridine, while the net synthesis of RNA ceased—presumptive evidence for a continued synthesis of messenger RNA. Chloramphenicol addition (100 μg/ml) did not influence the total amount of RNA synthesized. Synthesis of ribosomes and transfer RNA preceded by 0, 5, 10, and 15 min of postirradiation incubation was observed by the resolution of cell-free extracts on sucrose density gradients. Little immediate influence of irradiation could be detected on the synthesis of 50S and 30S ribosomes. A decline was observed in the synthesis of 50S ribosomes with continued postirradiation incubation; 30S ribosomes, ribosomal precursors, and 4S RNA continued to be synthesized. PMID:14188715

  9. Properties of Caulobacter Ribonucleic Acid Bacteriophage φCb5

    PubMed Central

    Bendis, Inakaren; Shapiro, Lucille

    1970-01-01

    The ribonucleic acid (RNA) bacteriophage φCb5, which specifically infects only one form of the dimorphic stalked bacterium Caulobacter crescentus, has been obtained in high yield. Since the phage is extremely salt-sensitive, a purification procedure was devised which avoided contact with solutions of high ionic strength. Phage φCb5 was studied with respect to the physical and chemical properties of both the phage and its RNA. In an electron microscope, the phage particles appear as small polyhedra, 23 nm in diameter. The phage is similar to the Escherichia coli RNA phages in that it (i) sediments at an S20, w of 70.6S, (ii) is composed of a single molecule of single-stranded RNA and a protein coat, (iii) contains two structural proteins, and (iv) apparently contains the genetic capacity to code for a coat protein subunit, a maturation-like protein, and an RNA polymerase. Phage φCb5 differs from the E. coli RNA phages in (i) host specificity, (ii) salt sensitivity, and (iii) the presence of histidine, but not methionine, in the coat protein. Images PMID:5495512

  10. Stimulation of Ribonucleic Acid Synthesis by Chloramphenicol in a rel+ Aminoacyl-Transfer Ribonucleic Acid Synthetase Mutant of Escherichia coli

    PubMed Central

    Yegian, Charles D.; Vanderslice, Rebecca W.

    1971-01-01

    Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel+ strain, it cannot carry out net RNA synthesis at high temperature. A 100-μg amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 μg of CAP or 100 μg of CAP plus 50 μg of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNAval at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases. PMID:4942766

  11. Deoxyribonucleic acid-ribonucleic acid hybridization studies on the L-Arabinose operon of Escherichia coli B-r.

    PubMed

    Wilcox, G; Singer, J; Heffernan, L

    1971-10-01

    An increase in the rate of synthesis of ara-specific messenger ribonucleic acid as measured by deoxyribonucleic acid-ribonucleic acid hybridization has been detected in the induced wild-type (ara(+)) strain of Escherichia coli B/r as compared with the uninduced control, thus providing evidence that regulation of the positively controlled l-arabinose operon is at the level of transcription.

  12. Comparison of methods of extracting messenger Ribonucleic Acid from ejaculated Porcine (Sus Scrofa) Spermatozoa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    H. D. Guthrie, G.R. Welch, and L. A. Blomberg. Comparison of Methods of Extracting Messenger Ribonucleic Acid from Ejaculated Porcine (Sus Scrofa) Spermatozoa. Biotechnology and Germplasm Laboratory, Agricultural Research Service U. S. Department of Agriculture, Beltsville, MD 20705 The purpos...

  13. Characterization of the Subunit Structure of the Ribonucleic Acid Genome of Influenza Virus

    PubMed Central

    Lewandowski, L. J.; Content, J.; Leppla, S. H.

    1971-01-01

    Ribonucleic acid extracted from influenza virus was labeled at the 3′ termini with 3H and analyzed by polyacrylamide gel electrophoresis. Influenza virus was found to contain a minimum of seven and possibly as many as 10 polynucleotide chains, most of which appear to terminate at the 3′ end in uridine. PMID:4332140

  14. Gene organization around the phenylalanyl-transfer ribonucleic acid synthetase locus in Escherichia coli.

    PubMed Central

    Comer, M M

    1981-01-01

    The organization of seven genes located at about 38 min on the genetic map of Escherichia coli was examined; these genes included pheS and pheT, which code for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid synthetase, and thrS, the structural gene for threonyl-transfer ribonucleic acid synthetase. Deletion mutants were isolated from an F-prime-containing merodiploid strain and were characterized genetically. Seventeen different kinds of deletions extending into pheS of pheT were identified. These deletions unambiguously defined the gene order as aroD pps himA pheT pheS thrS pfkB. Mutants with deletions covering either pheS or pheT, but not both, were analyzed further by assay of phenylalanyl-transfer ribonucleic acid synthetase. The phenotype of the mutants with a deletion from pfkB through pheS was anomalous; although the pheT gene was apparently still present, its product, the beta subunit, was much reduced in activity. PMID:7012115

  15. Transcription During the Development of Bacteriophage φ29: Production of Host-and φ29-Specific Ribonucleic Acid

    PubMed Central

    Schachtele, Charles F.; De Sain, Carol V.; Hawley, Louise A.; Anderson, Dwight L.

    1972-01-01

    The synthesis of ribonucleic acid (RNA) during development of the virulent Bacillus subtilis bacteriophage φ29 has been analyzed. Transcription of host deoxyribonucleic acid (DNA) continues at the preinfection rate throughout the latent period of viral growth. RNA-DNA hybridization was used to show that host messenger RNA synthesis continues late into the phage lytic cycle. Amino acid-labeling experiments show that this RNA is continuously used to produce protein. Ribosomal RNA production is not inhibited by phage infection. Small quantities of phage-specific RNA first appear between min 6 and 9 after infection. This RNA is made exclusively from one of the φ29 DNA strands. At 12 min postinfection, when phage DNA replication commences, large quantities of viral RNA start to be synthesized. This RNA appears to be transcribed from both strands of φ29 DNA. Studies with rifamycin and rifamycin-resistant host strains showed that the production of all phage φ29-specific RNA requires those components of the host RNA polymerase which are sensitive to this antibiotic. Thus, phage φ29 does not stop transcription of host DNA and may produce only one element for regulation of transcription of its own DNA. These findings may reflect the limited amount of genetic information carried by this phage. PMID:4630153

  16. Control of larval and egg development in Aedes aegypti with Ribonucleic acid interference (RNAi) against juvenile hormone acid methyl transferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribonucleic acid interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including mosquitoes and many other insects. Little has been done, however, to harness this approach in order to control adult and larval mosquitoes. Juvenile hormone (JH) plays a pi...

  17. Ribonucleic acid synthesis in yeast. The effect of cycloheximide on the synthesis of ribonucleic acid in Saccharomyces carlsbergensis

    PubMed Central

    de Kloet, S. R.

    1966-01-01

    1. Cycloheximide causes the release of the control amino acids have over RNA synthesis in Saccharomyces carlsbergensis N.C.T.C. 74. 2. The antibiotic causes a gradual deceleration of RNA formation. After incubation for 60min. at 30° RNA synthesis usually proceeds at a rate only a few per cent of that of the untreated control. 3. In the presence of cycloheximide two types of RNA accumulate in the cell: soluble RNA and a high-molecular-weight RNA. The latter has a base composition intermediate between those of yeast DNA and yeast ribosomal RNA, and sediments in a sucrose gradient at a rate faster than that of the 23s ribosomal RNA component. 4. Yeast ribosomal RNA contains methylated bases. Judged from the incorporation of [Me-14C]methionine, the extent of methylation of ribosomal RNA is about 20% of that of the `soluble' RNA fraction. The high-molecular-weight RNA formed in the presence of cycloheximide is less methylated than normal RNA. In this case the sucrose-density-gradient sedimentation patterns of newly methylated and newly synthesized RNA do not coincide. 5. In the presence of cycloheximide, polysomal material accumulates, indicating that messenger RNA is formed. 6. The effect of the antibiotic on protein and RNA synthesis can be abolished by washing of the cells. The RNA that has accumulated during incubation of the cells with the antibiotic is not stable on removal of cycloheximide. 7. The results presented in this study are discussed in relation to the regulation of RNA formation in yeast. PMID:5964958

  18. Correlation Between the Rate of Ribonucleic Acid Synthesis and the Level of Valyl Transfer Ribonucleic Acid in Mutants of Escherichia coli

    PubMed Central

    Kaplan, Sam

    1969-01-01

    By use of a mutant of Escherichia coli with a partially thermolabile transfer ribonucleic acid (tRNA) synthase, it was possible to regulate the rate of RNA synthesis over a 10-fold range. The addition of chloramphenicol to cultures kept at the nonpermissive temperature stimulated RNA synthesis. The longer the culture was kept at the nonpermissive temperature prior to addition of chloramphenicol, the lower was the resulting rate of RNA synthesis. The decrease in the rate of incorporation of labeled uracil into RNA was correlated with the decrease in the level of valyl tRNA. Additional experiments provided evidence which may be interpreted as indicating that valyl tRNA does not, by itself, react with the RNA-forming system. PMID:4891259

  19. Interaction of DAPI with double-stranded ribonucleic acids.

    PubMed Central

    Manzini, G; Xodo, L; Barcellona, M L; Quadrifoglio, F

    1985-01-01

    The interaction of DAPI with natural and synthetic double-stranded polyribonucleotides was studied with different optical and calorimetric methods. The results were similar to those obtained previously with double-stranded polydeoxynucleotides, i.e. two interaction modes, the first of which shows high affinity for AU clusters and consequent strong fluorescence enhancement. The results suggest caution in the use of DAPI as selective fluorescent staining agent for DNA in the presence of RNA. A narrow groove binding model with hydrogen bonds between DAPI and AU pairs is proposed. An intercalation mechanism can be excluded because of the non planarity of DAPI molecule. PMID:4080554

  20. Striking similarities are exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII

    SciTech Connect

    Rosa, M.D.; Gottlieb, E.; Lerner, M.R.; Steitz, J.A.

    1981-09-01

    The nucleotide sequence of the region of the Epstein-Barr virus genome that specified two small ribonucleic acids (RNAs), EBER 1 and EBER 2, has been determined. Both of these RNAs are encoded by the right-hand 1,000 base pairs of the EcoRI J fragment of EBV deoxyribonucleic acid. EBER 1 is 166 (167) nucleotides long and EBER 2 is 172 +- 1 nucleotides long; the heterogeneity resides at the 3' termini. The EBER genes are separated by 161 base pairs and are transcribed from the same deoxyribonucleic acid strand. In vitro, both EBER genes can be transcribed by RNA polymerase III; sequences homologous to previously identified RNA polymerase III intragenic transcription control regions are present. Striking similarities are therefore apparent both between the EBERs and the two adenovirus-associated RNAs, VAI and VAII, and between the regions of the two viral genomes that specify these small RNAs. We have shown that VAII RNA as well as VAI RNA and the EBERs exist in ribonucleoprotein complexes which are precipitable by anti-La antibodies associated with systemic lupus erythematosus. Finally the authors have demonstrated that the binding of protein(s) from uninfected cells confers antigenicity on each of the four virus-encoded small RNAs.

  1. Lack of protection afforded by ribonucleic acid preparations from Mycobacterium tuberculosis against Mycobacterium leprae infections in mice.

    PubMed Central

    Shepard, C C; Youmans, A Y; Youmans, G P

    1977-01-01

    Mycobacterial ribonucleic acid preparations from H37Ra, an attenuated strain of Mycobacterium tuberculosis, provide their usual marked protection against M. tuberculosis challenge; however, they provided no protection against Mycobacterium leprae challenge. Suspensions of intact H37Ra were not effective against M. leprae. Suspensions of BCG gave their usual distinct protection against M. leprae challenge. PMID:404242

  2. The role of renin-angiotensin aldosterone system related micro-ribonucleic acids in hypertension

    PubMed Central

    Wang, Hui-Bo; Yang, Jun

    2015-01-01

    Micro-ribonucleic acids (miRNAs) are small (21-25 nucleotide) single-stranded, evolutionarily conserved non-protein-coding RNAs, which control diverse cellular functions by interacting with the 3’ untranslated region of specific target messenger RNAs at the post-transcriptional level. Research shows that an aberrant expression profile of miRNAs has been linked to a series of diseases, including hypertension. In the past few decades, it has been demonstrated that excessive activation of the renin-angiotensin aldosterone system (RAAS) involves in the pathogenesis of hypertension. This article reviews the latest insights in the identification of RAAS-correlative miRNAs and the potential mechanisms for their roles in hypertension. PMID:26446323

  3. [Ubiquitous Ribonucleic Acid:miRNA is the Ubiquitin of RNA].

    PubMed

    Zheng, Xiao-fei

    2015-10-01

    Small RNAs, especially microRNAs (miRNAs),widely exist in eukaryotic cells, with their main functions being regulating gene expression and function of target molecules through the degradation of cellular target RNAs by the ribonuclease-based system. Ubiquitins and ubiquitin-like proteins are polypeptides that exist in most eukaryotic cells, and their main function is almost to regulate protein level through the degradation of cellular proteins by ubiquitin proteasome system. Small RNAs, including miRNAs,and ubiquitins or ubiquitin-like proteins have similarities in many aspects although small RNAs and ubiquitin or ubiquitin-like proteins interact different substrates respectively. Therefore, miRNAs can be defined as ubiquitra (ubiquitous ribonucleic acid, ubiquitra or uRNA), and the other small RNAs can be defined as ubiquitra-like RNA or uRNA-like RNA. The concept of ubiquitra may be applied for explaining the biological essence of small RNAs diversity.

  4. Protein and Ribonucleic Acid Synthesis During the Diploid Life Cycle of Allomyces arbuscula

    PubMed Central

    Burke, Daniel J.; Seale, Thomas W.; McCarthy, Brian J.

    1972-01-01

    The diploid life cycle of Allomyces arbuscula may be divided into four parts: spore induction, germination, vegetative growth, and mitosporangium formation. Spore induction, germination, and mitosporangium formation are insensitive to inhibition of actinomycin D, probably indicating that stable, pre-existing messenger ribonucleic acid (RNA) is responsible for these developmental events. Protein synthesis is necessary during the entire life cycle except for cyst formation. A system for obtaining synchronous germination of mitospores is described. During germination there is a characteristic increase in the rate of synthesis of RNA and protein although none of the other morphogenetic changes occurring during the life cycle are necessarily accompanied by an appreciable change in the rate of macromolecular synthesis. PMID:4113121

  5. Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor

    PubMed Central

    Rowe, Aaron A.; Miller, Erin A.; Plaxco, Kevin W.

    2011-01-01

    Biosensors built using ribonucleic acid (RNA) aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degradation when deployed in clinical samples. To explore methods for protecting RNA-based biosensors from such degradation we have constructed and characterized an electrochemical, aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2′-hydroxyl groups outside of the aptamer’s antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degradation-resistant deoxyribonucleic acid (DNA) equivalents. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-molecular-weight-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clinically relevant concentrations using the original RNA-based sensor. PMID:20687587

  6. Impact of size, secondary structure, and counterions on the binding of small ribonucleic acids to layered double hydroxide nanoparticles.

    PubMed

    Rodriguez, Blanca V; Pescador, Jorge; Pollok, Nicole; Beall, Gary W; Maeder, Corina; Lewis, L Kevin

    2015-12-30

    Use of ribonucleic acid (RNA) interference to regulate protein expression has become an important research topic and gene therapy tool, and therefore, finding suitable vehicles for delivery of small RNAs into cells is of crucial importance. Layered double metal hydroxides such as hydrotalcite (HT) have shown great promise as nonviral vectors for transport of deoxyribose nucleic acid (DNA), proteins, and drugs into cells, but the adsorption of RNAs to these materials has been little explored. In this study, the binding of small RNAs with different lengths and levels of secondary structure to HT nanoparticles has been analyzed and compared to results obtained with small DNAs in concurrent experiments. Initial experiments established the spectrophotometric properties of HT in aqueous solutions and determined that HT particles could be readily sedimented with near 100% efficiencies. Use of RNA+HT cosedimentation experiments as well as electrophoretic mobility shift assays demonstrated strong adsorption of RNA 25mers to HT, with twofold greater binding of single-stranded RNAs relative to double-stranded molecules. Strong affinities were also observed with ssRNA and dsRNA 54mers and with more complex transfer RNA molecules. Competition binding and RNA displacement experiments indicated that RNA-HT associations were strong and were only modestly affected by the presence of high concentrations of inorganic anions.

  7. The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli.

    PubMed

    Gray, W J; Midgley, J E

    1972-08-01

    The biosynthesis and stability of various RNA fractions was studied in RC(str) and RC(rel) multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RC(str) mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA-RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60-70% of the labelled RNA formed in RC(str) mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RC(rel) strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RC(rel) strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RC(str) or RC(rel) strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RC(str) auxotrophs was unchanged from that found in normally growing cells. In contrast, RC(rel) strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.

  8. Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

    PubMed Central

    Russell, Susan P.; Limbach, Patrick A.

    2013-01-01

    Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less. PMID:23500350

  9. Rates of synthesis and degradation of ribosomal ribonucleic acid during differentiation of Dictyostelium discoideum.

    PubMed Central

    Mangiarotti, G; Altruda, F; Lodish, H F

    1981-01-01

    Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells. PMID:6965093

  10. Incorporation of Precursors into Ribonucleic Acid, Protein, Glycoprotein, and Lipoprotein of Avian Myeloblastosis Virions

    PubMed Central

    Baluda, M. A.; Nayak, D. P.

    1969-01-01

    Freshly explanted leukemic myeloblasts produce avian myeloblastosis virus (AMV) at a constant rate without any obvious cytopathic effect; therefore, subviral components are continually synthesized at a steady rate. The incorporation of various radioactive precursors into virions was monitored by determination of radioactivity in purified virus after density equilibrium sedimentation in preformed sucrose gradients. The kinetics of incorporation of 3H-uridine have shown that there is an average time interval of 3 to 4 hr (half-life) between the time viral ribo-nucleic acid (RNA) is synthesized and the time it is released as a mature virus particle; this represents the average time interval spent by AMV-RNA in an intracellular pool. Studies with 14C-phenylalanine have revealed that some protein synthesis takes place at or near the cell surface immediately prior to maturation and release of virus. 14C-glucosamine also appears to be incorporated into the outer viral envelope shortly before maturation. On the other hand, there is an average lag of about 16 to 20 hr before 14C-ethanolamine incorporated into intracellular lipoprotein appears in free virions; this probably reflects the kinetics of replacement of cellular surface membrane. Actinomycin D inhibits AMV-RNA within 30 min but permits the maturation of AMV to continue for at least 2 hr. AMV released in the presence of actinomycin D contains AMV-RNA synthesized before the addition of the drug. PMID:4311791

  11. Rates of synthesis and degradation of ribosomal ribonucleic acid during differentiation of Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Altruda, F; Lodish, H F

    1981-01-01

    Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells.

  12. Human immunodeficiency virus trans-activator of transcription peptide detection via ribonucleic acid aptamer on aminated diamond biosensor

    NASA Astrophysics Data System (ADS)

    Rahim Ruslinda, A.; Wang, Xianfen; Ishii, Yoko; Ishiyama, Yuichiro; Tanabe, Kyosuke; Kawarada, Hiroshi

    2011-09-01

    The potential of ribonucleic acid (RNA) as both informational and ligand binding molecule have opened a scenario in the development of biosensors. An aminated diamond-based RNA aptasensor is presented for human immunodeficiency virus (HIV) trans-activator of transcription (Tat) peptide protein detection that not only gives a labeled or label-free detection method but also provides a reusable platform for a simple, sensitive, and selective detection of proteins. The immobilized procedure was based on the binding interaction between positively charged amine terminated diamond and the RNA aptamer probe molecules with the negatively charged surface carboxylic compound linker molecule such as terephthalic acid.

  13. RIBONUCLEIC ACID SYNTHESIS DURING MITOSIS AND MEIOSIS IN THE MOUSE TESTIS

    PubMed Central

    Monesi, Valerio

    1964-01-01

    The pattern of ribonucleic acid synthesis during germ cell development, from the stem cell to the mature spermatid, was studied in the mouse testis, by using uridine-H3 or cytidine-H3 labeling and autoradiography. Incorporation of tritiated precursors into the RNA occurs in spermatogonia, resting primary spermatocytes (RPS), throughout the second half of pachytene stage up to early diplotene, and in the Sertoli cells. Cells in leptotene, zygotene, and in the first half of pachytene stage do not synthesize RNA. No RNA synthesis was detected in meiotic stages later than diplotene, with the exception of a very low rate of incorporation in a fraction of secondary spermatocytes and very early spermatids. At long intervals after administration of the tracer, as labeled cells develop to more mature stages, late stages of spermatogenesis also become labeled. The last structures to become labeled are the residual bodies of Regaud. Thus, the RNA synthesized during the active meiotic stages is partially retained within the cell during further development. The rate of RNA synthesis declines gradually with the maturation from type A to intermediate to type B spermatogonia and to resting primary spermatocytes. "Dormant" type A spermatogonia synthesize little or no RNA. The incorporation of RNA precursors occurs exclusively within the nucleus: at later postinjection intervals the cytoplasm also becomes labeled. In spermatogonia all mitotic stages, except metaphase and anaphase, were shown to incorporate uridine-H3. RNA synthesis is then a continuous process throughout the cell division cycle in spermatogonia (generation time about 30 hours), and stops only for a very short interval (1 hour) during metaphase and anaphase. PMID:14206420

  14. Ribonucleic Acid and Protein Synthesis During Germination of Myxococcus xanthus Myxospores

    PubMed Central

    Juengst, Fredrick W.; Dworkin, Martin

    1973-01-01

    Ribonucleic acid (RNA) and protein synthesis during myxospore germination were examined. When RNA synthesis was inhibited more than 90% by either actinomycin D (Act D) or rifampin, germination was prevented. The data were consistent with the interpretation that rifampin did not interfere with protein synthesis in any way other than by inhibition of messenger RNA formation. Act D concentrations as high as 20 μg/ml did not totally inhibit RNA synthesis. In the presence of 8 μg of Act D/ml, germinating myxospores synthesized transfer RNA, 16S RNA, and 23S RNA. Evidence was presented which indicated that messenger RNA was also synthesized early in the germination period both in the presence and absence of 8 μg of Act D/ml. One explanation for the escape synthesis of RNA in germinating myxospores is that Act D exerts a differential effect on the transcription of larger versus smaller cistrons, the latter having a lower probability of binding Act D. We have found that in the presence of 8 μg of Act D/ml, escape RNA synthesis in myxospores was 25% for 23S RNA, 55% for 16S RNA, and more than 90% for 4S RNA. We have shown that germination of myxospores requires both RNA and protein synthesis during the first 25 to 35 min in germination medium. This finding does not support the earlier suggestion by Ramsey and Dworkin that a stable germination messenger RNA is required for germination of the myxospores of Myxococcus xanthus. PMID:4690965

  15. Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

    PubMed Central

    Melli, Marialuisa; Bishop, J. O.

    1970-01-01

    RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×105 the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro. PMID:5493851

  16. 5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

    NASA Technical Reports Server (NTRS)

    Van den Eynde, H.; De Baere, R.; Shah, H. N.; Gharbia, S. E.; Fox, G. E.; Michalik, J.; Van de Peer, Y.; De Wachter, R.

    1989-01-01

    The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

  17. Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis.

    PubMed Central

    Theil, K W; McCloskey, C M; Saif, L J; Redman, D R; Bohl, E H; Hancock, D D; Kohler, E M; Moorhead, P D

    1981-01-01

    A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays. Images PMID:6270190

  18. Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

    1994-01-01

    The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

  19. Selective precipitation of ribonucleic acid from a mixture of total cellular nucleic acids extracted from cultured mammalian cells

    PubMed Central

    Harrison, P. R.

    1971-01-01

    A simple and reproducible method is described for precipitating RNA selectively from total mammalian-cell nucleic acids extracted by the phenol–sodium dodecyl sulphate procedure at pH8.0. Under specified conditions bulk RNA is precipitated almost quantitatively whereas bulk DNA remains in solution. Minor components of RNA (detected by pulse-labelling and chromatography on methylated albumin–kieselguhr) and rapidly labelled components of DNA containing single-stranded regions are also precipitated. The usefulness of the method is discussed in the context of isolating separately both RNA and DNA from cultured cells that are difficult to obtain in quantity. PMID:5165620

  20. Prolactin messenger ribonucleic acid levels, prolactin synthesis, and radioimmunoassayable prolactin during the estrous cycle in the Golden Syrian hamster

    SciTech Connect

    Massa, J.S. ); Blask, D.E. )

    1990-01-01

    The purpose of this study was to observe the molecular dynamics of pituitary prolactin (PRL) gene expression during the estrous cycle of the Golden Syrian hamster. PRL messenger ribonucleic acid (mRNA) levels, PRL synthesis were measured in the morning on each day of the cycle. We observed that all of these PRL indices declined or did not change from Day 2 to Day 3 of the cycle. From Day 3 to Day 4 however, PRL mRNA levels increased 33-38% and media {sup 3}H-PRL increased 32-42%, while there were no significant changes in pituitary {sup 3}H-PRL, or RIA-PRL in the media or pituitary. From Day 4 to Day 1 (estrus) there was reciprocal change in the levels of {sup 3}H-PRL in the pituitary vs. the media, with the former increasing 37-50% and the latter decreasing 25-32%. Pituitary RIA-PRL did also increased 45-64% from Day 4 to Day 1 while media RIA-PRL did not change. These data are consistent with the following hypothesis: On the morning of proestrus(Day 4) in the hamster, PRL mRNA levels are elevated compared to those on Day 3, signaling an increase in PRL synthesis. This newly synthesized PRL is shunted into a readily releasable pool on the morning of Day 4 (contributing to the afternoon surge of serum PRL), and into a preferentially stored pool by the morning of Day 1.

  1. Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

    PubMed Central

    Whitfeld, P R; Leaver, C J; Bottomley, W; Atchison, B

    1978-01-01

    A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves. Images Fig. 8. PMID:743229

  2. Translation of satellite tobacco necrosis virus ribonucleic acid by an in vitro system from wheat germ.

    PubMed

    Leung, D W; Gilbert, C W; Smith, R E; Sasavage, N L; Clark, J M

    1976-11-02

    The RNA of satellite tobacco necrosis virus (STNV) is an effective messenger RNA when translated in an in vitro system from wheat germ. This RNA codes for only STNV coat protein, as indicated (1) by coincidence of the tryptic fingerprints of the translation product and of STNV coat protein, (2) by equivalent size of the translation product and STNV coat protein, and (3) by isolation of an initial peptide of the in vitro product containing the amino acid sequence of the N terminus of STNV coat protein. STNV RNA does not contain a 5'-terminal m7G(5')ppp(5')Np---group and translation of STNV RNA by the wheat germ system does not involve prior formation of 5'-terminal m7G(5')ppp(5') nP---groups on STNV RNA. STNV RNA and 125I-labeled STNV RNA form a specific initiation complex when incubated with initiator tRNA, GTP, initiation factors, and wheat germ ribosomes. Treatment of this specific initiation complex with ribonuclease A allows isolation of an 125I-labeled oligonucleotide protected from ribonuclease A by the initiation complex. This specific oligonucleotide contains approximately 38 nucleotides, including nucleotide sequences that coincide with the codons of the N-terminal amino acids of STNV coat proteins.

  3. Accumulation of methyl-deficient rat liver messenger ribonucleic acid on ethionine administration

    SciTech Connect

    Goswami, B.B.; Sharma, O.K.

    1980-01-01

    Highly purified poly(adenylic acid)-containing RNA isolated from livers of rats fed 0.25% DL-etionine in the diet for 7 days accepted methyl groups from S-adenosyl(methyl-/sup 3/H)methionine, when incubated in vitro with mRNA methyltransferases from vaccinia virus or Ehrlich ascites cells, whereas RNA from control rats had no such activity. Nuclease digestion followed by chromatographic analyses of mRNA methylated in vitro revealed that the methyl groups were incorporated at the 5' end into cap 1 structures (m/sup 7/GpppNmp...) by the viral enzyme, whereas both cap 0 (m/sup 7/GpppNp...) and cap 1 (m/sup 7/Gpppm/sup 6/Am...) structures were formed by the Ehrlich ascites cell enzymes. the methyl-deficient mRNA isolated from the liver of ethionine-fed rats differed in its translational properties from mRNA isolated from control animals in an in vitro protein synthesizing system from wheat germ.

  4. The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

    PubMed Central

    Knowler, J. T.; Smellie, R. M. S.

    1971-01-01

    Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17β action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3μg or more of oestradiol-17β. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2–2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17β is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA. PMID:5144761

  5. Formation of Viral Ribonucleic Acid and Virus in Cells that are Permissive or Nonpermissive for Murine Encephalomyelitis Virus (GDVII) 1

    PubMed Central

    Sturman, Lawrence S.; Tamm, Igor

    1969-01-01

    GDVII virus growth in BHK-21 cells, a permissive host for the virus, resembled productive infections with other picornaviruses. Virus yields ranged from 100 to 600 plaque-forming units (PFU)/cell. Virus replication in HeLa cells, a nonpermissive host for GDVII virus, was characterized by virus yields of only 0.1 to 5 PFU/cell. Similar low yields of virus have been obtained from HeLa cells at all multiplicities of input up to 6,000 per cell. The progeny particles from HeLa cells were, like the infecting particles, restricted in the HeLa cell host. Despite the great difference in final yields of virus from BHK-21 and HeLa cells, the times when maximal yields were reached were similar. GDVII virus stock grown in BHK-21 cells was designated HeLa-. A variant of GDVII virus which is capable of extensive growth in HeLa cells was obtained. This variant, designated HeLa+ GDVII virus, was passaged serially in HeLa cells. Virus yields of 50 to 150 infective virus particles per cell were obtained from infection of HeLa cells with HeLa+ GDVII virus. The major species of HeLa+ virus-specific ribonucleic acid (RNA) produced was single stranded and sedimented with an S value of 35S. The rate of accumulation of HeLa+ virus-specific RNA in HeLa cell cultures was about four times that of HeLa- RNA. The amount of virus-specific HeLa+ RNA formed in HeLa cells was several-fold greater than that of HeLa- RNA. With HeLa- parent GDVII virus undergoing productive replication in BHK-21 cells or abortive replication in HeLa cells, the major species of virus-specific RNA produced was single stranded and sedimented with an approximate S value of 35S. The amount of HeLa- virus-specific RNA extracted from BHK-21 cells was several-fold greater than the amount obtained from HeLa cells. PMID:4306304

  6. Double-Stranded Ribonucleic Acid from Mengo Virus: Production, Characterization, and Interferon-Inducing and Antiviral Activities in Comparison with Polyriboinosinic · Polyribocytidylic Acid

    PubMed Central

    Falcoff, E.; Falcoff, R.; Cherby, J.; Florent, J.; Lunel, J.; Ninet, L.; De Ratuld, Y.; Tissier, R.; Vuillemin, B.; Werner, G. H.

    1973-01-01

    Mengo virus double-stranded ribonucleic acid (dsRNA) was obtained on a semi-industrial scale from infected cultures of BHK-21 cells grown in suspension. Yield of the extraction and purification operations was small (about 22 mg from 1011 cells in a 100-liter culture). Physicochemical characterization of this dsRNA gave an estimated molecular weight close to 4 × 106, a density of 1.59 (similar to that of the poliovirus dsRNA), and a thermal transition midpoint of 94 C. This product was a little more toxic for the mouse, by the intravenous route, than polyriboinosinic · polyribocytidylic acid (poly I:C) and strictly comparable in this respect to poliovirus dsRNA. The interferon-inducing capacity in the mouse and the antiviral activities in the mouse (infected with encephalomyocarditis, Semliki Forest, influenza, foot-and-mouth disease, and murine hepatitis viruses) and in the rabbit (Shope fibroma virus) of the ultraviolet light-inactivated product were practically identical, on a quantitative basis, with those of poly I:C. In vitro and in vivo experiments showed the dsRNA from Mengo virus to be slightly but significantly more resistant than poly I:C to the inactivating effect of human serum. PMID:4364180

  7. The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase

    PubMed Central

    Henderson, A. R.

    1970-01-01

    1. The Widnell & Tata (1966) assay method for Mg2+-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [3H]GMP incorporation/min at 37°C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9μU/mg of DNA and 18h-starved rats had a mean activity of 53.2μU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15–120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg2+ ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg2+]– and pH–activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from

  8. Label-free impedimetric sensor for a ribonucleic acid oligomer specific to hepatitis C virus at a self-assembled monolayer-covered electrode.

    PubMed

    Park, Jin-Young; Lee, Yoon-suk; Chang, Byoung-Yong; Kim, Byeang Hyean; Jeon, Sangmin; Park, Su-Moon

    2010-10-01

    A ribonucleic acid (RNA) sensor based on hybridization of its peptide nucleic acid (PNA) molecule with a target RNA oligomer of the internal ribosome entry site sequence specific to the hepatitis C virus (HCV) and the electrochemical impedance detection is described. This RNA is one of the most conservative molecules of the whole HCV RNA genome. The ammonium ion terminated PNA molecule was immobilized via its host-guest interactions with the diaza crown ring of 3-thiophene-acetamide-diaza-18-crown-6 synthesized by a simple two-step method, which forms a well-defined self-assembled monolayer (SAM) on gold. Hybridization events of the probe PNA with the target RNA were monitored by measuring charge-transfer resistances for the Fe(CN)(6)(3-/4-) redox probe using Fourier transform electrochemical impedance spectroscopy. The ratio of the resistances of the SAM-covered electrode measured before and after hybridization increased linearly with log[RNA] in the rat liver lysate with a detection limit of about 23 pM.

  9. RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids.

    PubMed

    Sample, Paul J; Gaston, Kirk W; Alfonzo, Juan D; Limbach, Patrick A

    2015-05-26

    Ribosomal ribonucleic acid (RNA), transfer RNA and other biological or synthetic RNA polymers can contain nucleotides that have been modified by the addition of chemical groups. Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers. Mass spectrometry (MS) has become the conventional approach for determining the nucleotide composition, modification status and sequence of modified RNAs. Modified RNAs are analyzed by MS using collision-induced dissociation tandem mass spectrometry (CID MS/MS), which produces a complex dataset of oligomeric fragments that must be interpreted to identify and place modified nucleosides within the RNA sequence. Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers. There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined 'variable sequencing', which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing.

  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. Characterization of the viral ribonucleic acids and structural polypeptides of Anopheles A, Bunyamwera, Group C, California, Capim, Guama, Patois, and Simbu bunyaviruses.

    PubMed

    Ushijima, H; Klimas, R; Kim, S; Cash, P; Bishop, D H

    1980-11-01

    Analyses of the viral ribonucleic acids and structural polypeptides of 17-22 of the 119 accepted or proposed members of the Bunyavirus genus of arboviruses (family Bunyaviridae), have shown that from the standpoint of their structural components these viruses are highly comparable to each other. The average molecular weights for the three viral RNA species (L, large, M, medium, S, small) of 17 bunyaviruses were 2.93 X 10(6) (L, range 2.7-3.1 X 10(6)), 2.0 X 10(6) (M, range 1.8-2.3 X 10(6)), and 0.435 X 10(6) (Sm range 0.28-0.50 X 10(6)). The average molecular weights of the three major virion polypeptides (glycoproteins G1 and G2, and nucleocapsid protein, N) of 22 bunyaviruses were 115 X 10(3) (G1, range 108-120 X 10(3)), 37 X 10(3) (G2, range 20-41 X 10(3)) and 22 X 10(3) (N, range 19-25 X 10(3)). These results indicate that the structural components of bunyaviruses are different from those reported for Phlebotomus fever, Uukuniemi, and Crimean-Congo hemorrhagic fever, and other members of the Bunyaviridae family that are not currently assigned to a genus.

  13. Comparison of human rotaviruses isolated in Mexico City and in Santiago, Chile, by electrophoretic migration of their double-stranded ribonucleic acid genome segments.

    PubMed Central

    Espejo, R T; Avendaño, L F; Muñoz, O; Romero, P; Eternod, J G; Lopez, S; Moncaya, J

    1980-01-01

    During the period October to December 1979, rotaviruses were obtained from infants and young children hospitalized with acute gastroenteritis in Mexico City and were compared by analysis of the migration of their double-stranded ribonucleic acid (RNA) genome segments in gel electrophoresis. Comparison of the results of this analysis and of those of similar studies carried out in 1977 and 1978 showed that the two rotavirus electropherotypes designated 2s and 21 have been continuously present and that the proportion in which these two types have been found in hospitalized patients has varied greatly year to year. The RNAs from rotaviruses 2s and 21 differed in the electrophoretic migraton of at least eight genome segments. However, RNAs from virus assigned to the same electrophoreotypes were not necessarily identical: on the basis of small but significant differences in the migration of segment 7, 8, or 9, isolates of types 2s and 21 could be assigned to two and three different subtypes, respectively. Human rotaviruses obtained in a distant geographical region, Santiago, Chile, in July 1979 had RNA electrophoretic patterns similar to that of electropherotype 21 but different from it in the migration of one or two of the larger RNA segments. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:6254887

  14. Synaptic vesicles isolated from the electric organ of Torpedo californica and from the central nervous system of Mus musculus contain small ribonucleic acids (sRNAs).

    PubMed

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2017-06-01

    Synaptic vesicles (SVs) are presynaptic organelles that load and release small molecule neurotransmitters at chemical synapses. In addition to classic neurotransmitters, we have demonstrated that SVs isolated from the Peripheral Nervous Systems (PNS) of the electric organ of Torpedo californica, a model cholinergic synapse, and SVs isolated from the Central Nervous System (CNS) of Mus musculus (mouse) contain small ribonucleic acids (sRNAs; ≤ 50 nucleotides) (Scientific Reports, 5:1-14(14918) Li et al. (2015) [1]). Our previous publication provided the five most abundant sequences associated with the T. californica SVs, and the ten most abundant sequences associated with the mouse SVs, representing 59% and 39% of the total sRNA reads sequenced, respectively). We provide here a full repository of the SV sRNAs sequenced from T. californica and the mouse deposited in the NCBI as biosamples. Three data studies are included: SVs isolated from the electric organ of T. californica using standard techniques, SVs isolated from the electric organ of T. californica using standard techniques with an additional affinity purification step, and finally, SVs isolated from the CNS of mouse. The three biosamples are available at https://www.ncbi.nlm.nih.gov/biosample/ SRS1523467, SRS1523466, and SRS1523472 respectively.

  15. Thermal Death of Temperature-Sensitive Lysyl- and Tryptophanyl-Transfer Ribonucleic Acid Synthetase Mutants of Bacillus subtilis: Effect of Culture Medium and Developmental Stage

    PubMed Central

    Steinberg, William

    1974-01-01

    The growth of thermosensitive Bacillus subtilis lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants (lysS1 and trypS1) (l-lysine:transfer ribonucleic acid [tRNA] ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) was terminated when exponential phase cells were shifted from 30 to 43 C in a rich medium. Under these conditions, the temperature-inhibited cells undergo thermal death; they rapidly lose their ability to form colonies at 30 C. Another lysyl-tRNA synthetase mutant (lysS2) is refractory to thermal death even though its growth is inhibited at 43 C. The thermal death response of the lysS1 mutant is affected by the stage of cell development. At periods in spore outgrowth and sporogenesis these cells become refractory to thermal death. The refractory state does not result from the production of an inhibitor, or from the degradation of an activator of thermal death. However, culture medium composition does modify the thermal death response. Rich media enhance the effect, and no thermal death occurs in the lysS1 strain grown in a minimal medium. Temperature-sensitive cells can grow in a lysine- (0.25 mM) or tryptophan- (0.25 mM) supplemented minimal medium at 43 C, but amino acid concentrations of 25 mM only transiently protect trypS1 and lysS1 cells from thermal death in a rich medium. Osmotic agents such as sucrose (0.5 M) and NaCl (0.34 M) completely prevent thermal death in the lysS1 strain, although growth is still arrested. On solid media, sucrose stabilized lysS1 cells can form colonies at the restrictive temperature, but neither sucrose (0.5 M) nor NaCl (0.34 M) stabilized the lysS1 enzyme in vitro. Chloramiphenicol increased the rate of thermal death of the lysS1 strain but decreased the thermal death response of the trypS1 mutant. Considering the nature of the enzyme defect in the lysS1 strain, the common genetic origin of the spore and vegetative lysyl-tRNA synthetase, and the protective effects exerted by lysine

  16. Effects of estradiol-17beta administration on steady-state messenger ribonucleic acid (MRNA) encoding equine alpha and LH/CGbeta subunits in pituitaries of ovariectomized pony mares.

    PubMed

    Sharp, D C; Wolfe, M W; Cleaver, B D; Nilson, J

    2001-03-15

    The process of sexual recrudescence in the springtime in mares is characterized by renewal of follicular growth and acquisition of steroidogenic competence. Concomitant with renewal of follicular steroidogenesis is re-establishment of LH biosynthesis and secretion. Research results from our laboratory indicate that increased estradiol and LH secretion occur in close temporal association before the first ovulation of the year. Therefore, the hypothesis tested in this experiment was that estrogen administration to ovariectomized pony mares during the equivalent time of early vernal transition would enhance LH biosynthesis as monitored by messenger ribonucleic acid (mRNA) encoding for the pituitary subunits of LH (alpha and LH/CGbeta). Mares were administered either sesame oil vehicle control, or estradiol (5 mg i.m. twice daily in sesame oil) for 3, 6 or 9 days, beginning on February 2. The pituitary glands were harvested, and examined for LH subunit mRNA by Northern Blot and slot blot analysis. There was a significant increase in LH secretion after 6 days of estradiol secretion compared with control vehicle administration. Similarly, there was a significant increase in both alpha and LH/CGbeta subunit mRNA when estradiol was administered for 9 days. These data indicate that estrogen stimulates LH subunit formation in mares during early equivalent vernal transition. These data do not, however, discriminate between a direct pituitary effect of estrogen, and a hypothalamic effect. Whether the surge of estradiol just prior to the first ovulation of the year is essential for the renewed biosynthesis of LH subunits cannot be determined from these data. However an important role of estrogen in the final stages of sexual recrudescence is indicated.

  17. Elevation of growth hormone-releasing hormone receptor messenger ribonucleic acid expression in growth hormone-secreting pituitary adenoma with Gsalpha protein mutation.

    PubMed

    Sakai, Naoyuki; Kim, Kyongsong; Sanno, Naoko; Yoshida, Daizo; Teramoto, Akira; Shibasaki, Tamotsu

    2008-01-01

    Growth hormone-releasing hormone (GHRH) stimulates not only the synthesis and secretion of GH but also the proliferation of normal somatotrophs. The expression of GHRH receptor (GHRHR) is regulated by GHRH, both of which are known to be expressed in human GH-secreting pituitary adenoma cells. Somatic mutations in the subunit of Gsalpha protein (gsp), lead to the constitutive activation of adenylyl cyclase in pituitary adenomas that secrete GH. It has not been examined how gsp mutations influence GHRHR expression in GH-secreting adenomas. We therefore analyzed the expression levels of GHRHR messenger ribonucleic acid (mRNA) in GH-secreting pituitary adenomas focusing on a gsp mutation. Furthermore, we investigated the effect of GHRH on the expression of GHRHR mRNA in primary cultures of GH-secreting pituitary adenoma cells. GHRHR mRNA expression levels were significantly elevated in gsp mutation-positive GH-secreting adenomas compared with those in gsp mutation-negative ones. In primary-cultured GH-secreting adenoma cells, the increase of GH secretion in response to GHRH was shown in both gsp mutation-positive and -negative adenoma cells with a significantly higher response in the latter adenoma cells. GHRH increased GHRHR mRNA expression level in gsp mutation-negative adenoma cells while it was not influenced by GHRH in gsp mutation-positive adenoma cells. These results suggest that gsp mutations up-regulate GHRHR mRNA expression in GH-secreting pituitary adenoma cells, and that gsp mutations desensitize the adenoma cells to GHRH in terms of their GHRHR mRNA expression probably because of their saturation of GHRH signaling.

  18. Rift Valley Fever Virus: Molecular Biologic Studies of the M Segment RNA(Ribonucleic Acids) for Application in Disease Prevention

    DTIC Science & Technology

    1989-02-01

    report, the investigators have abided by the National Institutes Of Health Guidelines for Research Involving Recombinant DNA Molecules (April 1982) and...adhered to the "Guide for the Care and Use of Laboratory Arimals" of the Institute of Laboratory Animal Resources, National Research Council (DHEW...failed to suggest a precursor-product relationship between the 78kd and 14kd proteins (9) dowever, by combining site- directed mutagenesis techniques

  19. Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin–kieselguhr

    PubMed Central

    Johnson, Roger

    1970-01-01

    Chromatography on methylated albumin–kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The `chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source. PMID:4321853

  20. Prolactin messenger ribonucleic acid concentrations throughout the ovine estrous cycle: Assessment relative to prolactin serum and pituitary amounts

    SciTech Connect

    Landefeld, T.; Roulia, V.; Bagnell, T.; Ballard, T.; Levitan, I. )

    1991-01-01

    Prolactin (PRL) mRNA concentrations were assessed by nucleic acid hybridization assays in pituitaries of ewes representing the defined stages of the ovine estrous cycle. Concomitantly, pituitary and serum PRL concentrations were measured in these ewes using radioimmunoassays. It was observed that PRL serum, pituitary and mRNA concentrations tended to increase near the time of the gonadotropin preovulatory surge, particularly between 24 hrs before behavioral estrus to 5 hours after estrus. However, the changes in PRL mRNA, serum and pituitary concentrations were shown not to be statistically significant. These data suggest that PRL production during the sheep estrous cycle is maintained without dramatic changes in synthesis or secretion.

  1. Expression of luteinizing hormone and chorionic gonadotropin receptor messenger ribonucleic acid in human corpora lutea during menstrual cycle and pregnancy.

    PubMed

    Nishimori, K; Dunkel, L; Hsueh, A J; Yamoto, M; Nakano, R

    1995-04-01

    In the present study, we examined the expression of LH and CG receptor messenger RNA (mRNA) in human corpora lutea (CL) during the menstrual cycle and pregnancy. Poly(A)-enriched RNA was extracted from CL and analyzed by Northern and slot blots, using a radiolabeled complementary RNA probe derived from the human LH receptor complementary DNA. Northern blot analysis indicated the presence of multiple LH receptor mRNA transcripts with molecular sizes of 8.0, 7.0 and 4.5 kilobases in human CL during the menstrual cycle. The predominant transcript was 4.5 kilobases in size. However, no hybridization signals were observed in nongonadal tissues (heart, liver, and kidney). Densitometric analyses revealed that the levels of LH receptor mRNA increased from early luteal phase to midluteal phase and subsequently decreased during late luteal phase. After the onset of menstruation, the LH receptor mRNA level was undetectable in the regressing CL. Moreover, radioligand receptor assay (RRA) showed a close parallelism between LH receptor mRNA levels and LH receptor content in CL throughout the menstrual cycle. LH receptor mRNA expression was also found in CL during early pregnancy. The level of LH receptor mRNA was relatively high in early pregnancy CL, whereas LH receptor content was low. Using in situ hybridization, LH receptor mRNAs were uniformly expressed in both large and small luteal cells during early and midluteal phase and early pregnancy, but not in regressing CL. In conclusion, these data demonstrate that the regulation of LH receptor content in human CL during luteal phase is associated with similar changes in the receptor message levels, suggesting the physiological roles for LH receptor mRNA during the menstrual cycle in the human. In addition, the expression of LH receptor mRNA was demonstrated in human CL during early pregnancy.

  2. Changes in hepatic lipid parameters and hepatic messenger ribonucleic acid expression following estradiol administration in laying hens (Gallus domesticus).

    PubMed

    Lee, B K; Kim, J S; Ahn, H J; Hwang, J H; Kim, J M; Lee, H T; An, B K; Kang, C W

    2010-12-01

    Fatty liver hemorrhagic syndrome (FLHS) is characterized by increased hepatic triacylglycerol content associated with liver hemorrhages and results in a sudden decline in egg production. Genetic, environmental, nutritional, and hormonal factors have all been implicated in the etiology of FLHS, but the exact cause of FLHS is still unknown. Estrogens have been implicated in the development of excess fat content of the liver and in the etiology of FLHS. This study investigated estradiol (E(2)) administration in hens and its effect on lipid metabolism. Hy-Line Brown laying hens were intramuscularly injected with E(2) on a daily basis for 3 wk. The dosages were 0, 0.5, and 1.0 mg/kg of BW, with corn oil injections used as a control. Egg production and quality were measured among the groups, with no significant difference seen in egg production. Liver weights of hens treated with E(2) were greater than those of control hens, but the increase was not statistically significant. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities and E(2) plasma concentrations increased in a dose-dependent manner, with plasma concentration of E(2) increasing from 6,900 to 19,000 pg/mL. No significant differences in free cholesterol or phospholipids were observed, but there was a significant increase in hepatic triacylglycerol levels. Injection with E(2) showed an increased expression of mRNA for peroxisome proliferator-activated receptor γ (23-fold), but not for peroxisome proliferator-activated receptor α. A statistically significant increase was seen for fatty acid synthase, apolipoprotein B, and adenosine triphosphate citrate lyase, but not for acetyl coenzyme A carboxylase, apolipoprotein VLDL-II, microsomal triglyceride transport protein, or malic enzyme. For proteins involved in the oxidation of E(2), only cytochrome P450 3A37 showed a statistically significant increase. The present results suggest that E(2) upregulates the synthesis of fatty acids

  3. Effects of 17α-ethinylestradiol and bisphenol A on steroidogenic messenger ribonucleic acid levels in the rare minnow gonads.

    PubMed

    Liu, Shaozhen; Qin, Fang; Wang, Houpeng; Wu, Tingting; Zhang, Yingying; Zheng, Yao; Li, Meng; Wang, Zaizhao

    2012-10-15

    Previous studies showed that the endocrine disrupting chemicals (EDCs) affect reproductive physiology in teleosts. How the EDCs regulate gonadal steroidogenesis remains to be determined. The gonadal transcript changes of steroidogenic enzyme genes in adult rare minnow Gobiocypris rarus exposed to 17α-ethinylestradiol (EE2) and bisphenol A (BPA) were detected in the present study. The full-length cDNAs encoding steroidogenic enzymes, including steroidogenic acute regulatory protein (StAR), cytochrome P450-mediated side-chain cleavage enzyme (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 17 A1 (CYP17A1) were isolated and characterized by RT-PCR and RACE methods. The homology and phylogenetic analyses of the amino acid sequences confirmed that the nucleotide sequences of these steroidogenic genes were correct. The mRNA tissue distribution results indicated that StAR, cyp11a1, and cyp17a1 mRNAs were mainly expressed in the gonads and 3β-HSD was mainly expressed in both the gonads and the brains. The 233 dpf adult G. rarus were exposed to EE2 (25ng/L) and BPA (5, 15, and 50 μg/L) dissolved in dimethyl sulfoxide (DMSO) or control for 7 days. The gonadal mRNA levels of StAR, cyp11a1, 3β-HSD, cyp17a1 and ovarian cytochrome P450 aromatase (cyp19a1a) were quantified by qRT-PCR. Our data indicated that 25 ng/L EE2 had different degrees of inhibitory effects on the expression of steroidogenic genes in the gonads. BPA at different levels caused concentration-specific effects on the mRNA expression of the steroidogenic genes. The transcripts of several ovarian steroidogenic genes were more sensitive to 15 μg/L BPA than that at other two levels. These findings suggest that EE2 could impair gonadal steroidogenesis by suppressing mRNA expression of steroidogenic genes and BPA could cause variations in gonadal steroidogenesis modulation with a potential consequence of compensation for the disturbance.

  4. Molecular cloning of otoconin-22 complementary deoxyribonucleic acid in the bullfrog endolymphatic sac: effect of calcitonin on otoconin-22 messenger ribonucleic acid levels.

    PubMed

    Yaoi, Yuichi; Suzuki, Masakazu; Tomura, Hideaki; Sasayama, Yuichi; Kikuyama, Sakae; Tanaka, Shigeyasu

    2003-08-01

    Anuran amphibians have a special organ called the endolymphatic sac (ELS), containing many calcium carbonate crystals, which is believed to have a calcium storage function. The major protein of aragonitic otoconia, otoconin-22, which is considered to be involved in the formation of calcium carbonate crystals, has been purified from the saccule of the Xenopus inner ear. In this study, we cloned a cDNA encoding otoconin-22 from the cDNA library constructed for the paravertebral lime sac (PVLS) of the bullfrog, Rana catesbeiana, and sequenced it. The bullfrog otoconin-22 encoded a protein consisting of 147 amino acids, including a signal peptide of 20 amino acids. The protein had cysteine residues identical in a number and position to those conserved among the secretory phospholipase A(2) family. The mRNA of bullfrog otoconin-22 was expressed in the ELS, including the PVLS and inner ear. This study also revealed the presence of calcitonin receptor-like protein in the ELS, with the putative seven-transmembrane domains of the G protein-coupled receptors. The ultimobranchialectomy induced a prominent decrease in the otoconin-22 mRNA levels of the bullfrog PVLS. Supplementation of the ultimobranchialectomized bullfrogs with synthetic salmon calcitonin elicited a significant increase in the mRNA levels of the sac. These findings suggest that calcitonin secreted from the ultimobranchial gland, regulates expression of bullfrog otoconin-22 mRNA via calcitonin receptor-like protein on the ELS, thereby stimulating the formation of calcium carbonate crystals in the lumen of the ELS.

  5. Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

    PubMed Central

    Glasser, S. R.; Chytil, F.; Spelsberg, T. C.

    1972-01-01

    Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se. PMID:4656807

  6. Ribonucleic acid synthesis in chloroplasts

    PubMed Central

    Hartley, M. R.; Ellis, R. J.

    1973-01-01

    Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA. This incorporation shows an absolute requirement for light and does not occur in lysed chloroplasts. Fractionation by polyacrylamide-gel electrophoresis of the RNA synthesized in vitro reveals a major discrete product of molecular weight 2.7×106 and two minor products of molecular weight 1.2×106 and 0.47×106. These discrete products are super-imposed on a background of polydisperse RNA. The incorporation of 32Pi into chloroplast rRNA species (mol.wt. 1.05×106 and 0.56×106) in excised spinach leaves proceeds after a distinct lag period compared with the incorporation into cytoplasmic rRNA species (mol.wt. 1.34×106 and 0.7×106). Incorporation of 32Pi into chloroplast RNA species of molecular weight 2.7×106, 1.2×106, 0.65×106 and 0.47×106 proceeds without such a time-lag. The kinetics of labelling of the individual RNA components is consistent with the rapidly labelled RNA species of molecular weight 1.2×106 and 0.65×106 being precursors to the more slowly labelled rRNA species of molecular weight 1.05×106 and 0.56×106 respectively. PMID:4723226

  7. Synthesis of DNA mimics representing HypNA-pPNA hetero-oligomers.

    PubMed

    Efimov, Vladimir A; Chakhmakhcheva, Oksana G

    2005-01-01

    The methods for the synthesis and purification of negatively charged peptide nucleic acid (PNA)-relative deoxyribonucleic acid (DNA) mimics containing alternating residues of phosphono peptide nucleic acid (pPNA) monomers and PNA-like monomers on the base of trans-4-hydroxy-L-proline are described. Examples of the chimeric oligomers hybridization with complementary DNA and ribonucleic acid fragments are demonstrated.

  8. Congenital Visual Impairment and Progressive Microcephaly Due to Lysyl-Transfer Ribonucleic Acid (RNA) Synthetase (KARS) Mutations: The Expanding Phenotype of Aminoacyl-Transfer RNA Synthetase Mutations in Human Disease.

    PubMed

    McMillan, Hugh J; Humphreys, Peter; Smith, Amanda; Schwartzentruber, Jeremy; Chakraborty, Pranesh; Bulman, Dennis E; Beaulieu, Chandree L; Majewski, Jacek; Boycott, Kym M; Geraghty, Michael T

    2015-07-01

    Aminoacyl-transfer ribonucleic acid (RNA) synthetases (ARSs) are a group of enzymes required for the first step of protein translation. Each aminoacyl-transfer RNA synthetase links a specific amino acid to its corresponding transfer RNA component within the cytoplasm, mitochondria, or both. Mutations in ARSs have been linked to a growing number of diseases. Lysyl-transfer RNA synthetase (KARS) links the amino acid lysine to its cognate transfer RNA. We report 2 siblings with severe infantile visual loss, progressive microcephaly, developmental delay, seizures, and abnormal subcortical white matter. Exome sequencing identified mutations within the KARS gene (NM_005548.2):c.1312C>T; p.Arg438Trp and c.1573G>A; p.Glu525Lys occurring within a highly conserved region of the catalytic domain. Our patients' phenotype is remarkably similar to a phenotype recently reported in glutaminyl-transfer RNA synthetase (QARS), another bifunctional ARS gene. This finding expands the phenotypic spectrum associated with mutations in KARS and draws attention to aminoacyl-transfer RNA synthetase as a group of enzymes that are increasingly being implicated in human disease.

  9. Chronic improvement of amino acid nutrition stimulates initiation of global messenger ribonucleic acid translation in tissues of sheep without affecting protein elongation.

    PubMed

    Connors, M T; Poppi, D P; Cant, J P

    2010-02-01

    Initiation of mRNA translation and elongation of the polypeptide chain are 2 regulated processes responsible for the short-term postprandial acceleration of protein synthesis in animal tissues. It is known that a chronic increase in the absorptive supply of AA stimulates protein synthesis in ruminant animals, but effects on translation initiation and elongation are unknown. To determine whether initiation or elongation phases of global mRNA translation are affected by chronic elevation of AA supply, 24 ewe lambs of 25.9 +/- 2.5 kg of BW were randomly allocated to 4 treatment groups of 6 lambs each. All lambs received a basal diet of barley and hay at 1.2 times maintenance ME intake. Treatments were an intravenous (i.v.) saline infusion as a control, i.v. infusion of 6 essential AA (EAA; Arg, Lys, His, Thr, Met, Cys) for 10 d, i.v. infusion of the same EAA excluding Met and Cys (EAA-SAA) for 10 d, and an oral drench of fishmeal twice daily for 17 d. Fishmeal supplementation supplied an extra 719 mg of N x kg(-0.75) x d(-1) and N retention was increased 519 mg x kg(-0.75) x d(-1) over the control. The EAA treatment supplied an extra 343 mg of N x kg(-0.75) x d(-1) directly into the blood, and N balance was increased by 268 mg x kg(-0.75) x d(-1). Deletion of Met plus Cys from EAA had no effect on N balance. The results indicate that Met plus Cys did not limit body protein gain on the basal diet alone or the basal diet plus 6 AA. Protein fractional synthesis rates in liver, duodenum, skin, rumen, semimembranosus, and LM were measured by a flooding dose procedure using L-[ring-2,6-(3)H]-Phe. Ribosome transit times were estimated from the ratio of nascent to total protein-bound radioactivities. Fishmeal and EAA treatments had no effect on RNA, DNA, or protein contents of tissues, but fractional synthesis rate, translational efficiency, and concentrations of active ribosomes were consistently elevated. Ribosome transit time was not affected by long-term AA supply. We

  10. Dietary ribonucleic acid suppresses inflammation of adipose tissue and improves glucose intolerance that is mediated by immune cells in C57BL/6 mice fed a high-fat diet.

    PubMed

    Sakai, Tohru; Taki, Tomoyo; Nakamoto, Akiko; Tazaki, Shiho; Arakawa, Mai; Nakamoto, Mariko; Tsutsumi, Rie; Shuto, Emi

    2015-01-01

    Recent evidence suggests that immune cells play an important role in differentiation of inflammatory macrophages in adipose tissue, which contributes to systemic chronic inflammation. Dietary ribonucleic acid (RNA) has been shown to modulate immune function. We hypothesized that RNA affects immune cell function in adipose tissue and then improves inflammatory response in adipose tissue. C57/BL6 mice and recombination activating gene-1 (RAG-1) knockout mice on a C57BL/6 mice background were fed a high-fat diet containing 1% RNA for 12 wk. An oral glucose tolerance test was performed. Supplementation of dietary RNA in C57BL/6 mice fed a high-fat diet resulted in a smaller area under the curve (AUC) after oral glucose administration than that for control mice. The mRNA expression levels of inflammation-related cytokines in adipose tissue and serum interleukin-6 levels were reduced by dietary RNA supplementation. Interestingly, reduction of the AUC value by RNA supplementation was abolished in T and B cell-deficient RAG-1 knockout mice. These results indicate that RNA improves inflammation in adipose tissue and reduces the AUC value following oral glucose administration in a T and B cell-dependent manner.

  11. Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

    PubMed Central

    De Lucca, F. L.; Imaizumi, M. T.; Haddad, A.

    1974-01-01

    RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The `melting' temperature (Tm) of DNA in 0.15m-NaCl–0.015m-trisodium citrate, pH7.0, was 85°C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed. ImagesPLATE 1 PMID:4463939

  12. Characterization of the chicken follicle-stimulating hormone receptor (cFSH-R) complementary deoxyribonucleic acid, and expression of cFSH-R messenger ribonucleic acid in the ovary.

    PubMed

    You, S; Bridgham, J T; Foster, D N; Johnson, A L

    1996-11-01

    Studies were conducted to characterize the chicken (c) FSH receptor (R) cDNA, and to evaluate expression of cFSH-R mRNA in the hen ovary at known stages during follicle development. A total of 2.5 kb of nucleic acid sequence including the complete cFSH-R coding region was isolated by a combination of the reverse-transcription polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends techniques. Overall, the nucleic acid sequence homology of the cFSH-R cDNA coding region is 71.8% and 72.2% compared to the rat and bovine FSH-R, respectively, while the deduced amino acid sequence identity for the receptor protein (693 amino acids) is 71.9% and 72.4%, respectively. By comparison, the cFSH-R nucleic acid and amino acid sequences are 60.1% and 49.4% identical to the respective cLH-R sequences. Northern blot analysis detected a single 4.3-kb cFSH-R mRNA transcript, which was selectively expressed in ovarian (granulosa, theca, and stromal) tissues, but not the oviduct, adrenal, liver, muscle, or brain. As the follicle developed from the prehierarchical (6- to 8-mm diameter) to the largest preovulatory (F1 follicle) stage, cFSH-R mRNA levels progressively declined within both the granulosa and theca layers (p < 0.05). Moreover, cFSH-R mRNA levels were lower in whole atretic than in morphologically normal 3- to 5-mm follicles (p = 0.0015). The pattern of cFSH-R mRNA expression within the granulosa layer during follicle development was notably different from that of the recently reported cLH-R, in that cLH-R mRNA levels increase to become readily detectable coincident with dramatically increased steroidogenic capacity during the last few days before ovulation of the follicle. On the other hand, highest levels of cFSH-R mRNA in 6- to 8-mm (prehierarchical) follicles were consistent with a role for the cFSH-R in maintaining the viability of prehierarchical follicles and in initiating granulosa cell differentiation at the time when follicles are selected into the

  13. Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

    NASA Astrophysics Data System (ADS)

    Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

    2010-05-01

    Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

  14. Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes.

    PubMed

    Ma, Melissa; Crump, Doug; Farmahin, Reza; Kennedy, Sean W

    2015-02-01

    A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression. Bisphenol A (BPA) and 1 of its replacement alternatives, bisphenol S (BPS), were also screened for effects. Both TBBPA and BPA decreased CEH viability with calculated median lethal concentration (LC50) values of 40.6 μM and 61.7 μM, respectively. However, the replacement alternatives, TBBPA-DBPE and BPS, did not affect cell viability (up to 300 μM). Effects on mRNA expression were determined using an Avian ToxChip polymerse chain reaction (PCR) array and a real-time (RT)-PCR assay for the estrogen-responsive genes, apolipoproteinII (ApoII) and vitellogenin (Vtg). A luciferase reporter gene assay was used to assess dioxin-like effects. Tetrabromobisphenol-A altered mRNA levels of 4 genes from multiple toxicity pathways and increased luciferase activity in the luciferase reporter gene assay, whereas its alternative, TBBPA-DBPE, only altered 1 gene on the array, Cyp1a4, and increased luciferase activity. At 300 μM, a concentration that decreased cell viability for TBBPA and BPA, the BPA replacement, BPS, altered the greatest number of transcripts, including both ApoII and Vtg. Bisphenol A exposure did not alter any genes on the array but did up-regulate Vtg at 10 μM. Characterization of the potential toxicological and molecular-level effects of these compounds will ideally be useful to chemical regulators tasked with assessing the risk of new and existing chemicals.

  15. Studies on the Mechanism of Action of Hydrazine-Induced Methylation of DNA Guanne

    DTIC Science & Technology

    1984-10-03

    for aldehyde determinations measures both free aldehydes and those complexed with reduced glutathione (the major form of cellular aldehydes); it may...enzymes and naked DNA resulted in the expected formation of 7-methylguanine and 0 -methylguanine in the DNA. It may be that formaldehyde is complexed ...degradation products, other hydrazines, and isonicotinic acid hydrazide , J. Nat. Cancer Inst. 42:337-344. Kirby, K.S. (1962), Ribonucleic acids. IL Improved

  16. 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.

  17. 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.

  18. Role of polyadenylic acid in a deoxyribonucleic acid-membrane fraction extracted from pneumococci.

    PubMed Central

    Firshein, W; Meyer, B; Epner, E; Viggiani, J

    1976-01-01

    After the addition of radioactive polyadenylic acid to cell suspensions of pneumocci, part of the radioactivity becomes associated with a deoxyribonucleic acid (DNA)-membrane fraction extracted from the cells. A variety of techniques show that a portion of this associated radioactivity may represent oligoadenylates complexed to DNA, probaby as part of a ribonucleic acid (RNA) component. Polyadenylic acid, which had previously been shown to enhance DNA synthesis in cell suspensions (Firshein and Benson, 1968), also enhances the extent of DNA synthesis by the DNA-membrane fraction in vitro under specific conditions of concentration and conformation. The mechanism of action of this enhancement may be related to the ability of oligoadenylates to increase the number of initiation sites for DNA replication by stimulating the production of an RNA primer, thus providing additional 3'-OH groups with which DNA polymerase can react. PMID:6428

  19. Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2'-O-Ribonucleic Acid Modifications

    SciTech Connect

    Egli, Martin; Minasov, George; Tereshko, Valentina; Pallan, Pradeep S.; Teplova, Marianna; Inamati, Gopal B.; Lesnik, Elena A.; Owens, Steve R.; Ross, Bruce S.; Prakash, Thazha P.; Manoharan, Muthiah

    2010-03-05

    The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the

  20. 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

  1. 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.

  2. Simplified Identification of mRNA or DNA in Whole Cells

    NASA Technical Reports Server (NTRS)

    Almeida, Eduardo; Kadambi, Geeta

    2007-01-01

    A recently invented method of detecting a selected messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA) sequence offers two important advantages over prior such methods: it is simpler and can be implemented by means of compact equipment. The simplification and miniaturization achieved by this invention are such that this method is suitable for use outside laboratories, in field settings in which space and power supplies may be limited. The present method is based partly on hybridization of nucleic acid, which is a powerful technique for detection of specific complementary nucleic acid sequences and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes.

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

    PubMed

    Singh, Dinesh Pratap; Srivastava, Onkar Nath

    2007-06-01

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

  4. Body weight loss in beef cows: I. The effect of increased beta-oxidation on messenger ribonucleic acid levels of uncoupling proteins two and three and peroxisome proliferator-activated receptor in skeletal muscle.

    PubMed

    Brennan, K M; Michal, J J; Ramsey, J J; Johnson, K A

    2009-09-01

    Twenty-six Angus-cross cows were studied during BW loss (WL) and BW maintenance (WM) to examine the effects of elevated beta-oxidation on mRNA levels of NEFA-responsive signaling molecules in skeletal muscle. At the end of the WL and WM sampling periods, muscle biopsies were removed from the biceps femoris and mRNA levels were measured using real-time PCR. In comparison with WM, cows undergoing WL had elevated mRNA levels of carnitine palmitoyltransferase 1 (4.6-fold), fatty acid binding protein 3 (2.0-fold), and acyl-coenzyme A oxidase 1 (2.8-fold), all of which are indicators of beta-oxidation. Levels of mRNA of the NEFA-responsive signaling molecules PPAR alpha, delta, and gamma increased 2.0-fold, 2.2-fold, and 1.84-fold, respectively, during WL. Uncoupling proteins 2 and 3 also had increased mRNA (3.0-fold and 6.0-fold, respectively) during WL, but Western blot analysis found no changes in protein abundance of uncoupling protein 3. Uncoupling protein expression can be directly stimulated by elevated NEFA, potentially to protect cells from damage by lipid oxidation by-products. Thus, an increase in mRNA levels of genes involved in beta-oxidation of fatty acids and fatty acid by-products occurs during BW loss in beef cattle. These data support previous findings in nonruminants and suggest that these genes play a role in the same physiological processes in ruminants.

  5. 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

  6. 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.

  7. 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.

  8. Comparison of mammalian mitochondrial ribosomal ribonucleic acid from different species.

    PubMed

    Mitra, R S; Bartoov, B; Monahan, J; Freeman, K B

    1972-08-01

    Mitochondrial ribosomal RNA species from mouse L cells, rat liver, rat hepatoma, hamster BHK-21 cells and human KB cells were examined by electrophoresis on polyacrylamide-agarose gels and sedimentation in sucrose density gradients. The S(E) (electrophoretic mobility) and S values of mitochondrial rRNA of all species were highly dependent on temperature and ionic strength of the medium; the S(E) values increased and the S values decreased with an increase in temperature at a low ionic strength. At an ionic strength of 0.3 at 23-25 degrees C or an ionic strength of 0.01 at 3-4 degrees C the S and S(E) values were almost the same being about 16.2-18.0 and 12.3-13.6 for human and mouse mitochondrial rRNA. The molecular weights under these conditions were calculated to be 3.8x10(5)-4.3x10(5) and 5.9x10(5)-6.8x10(5), depending on the technique used. At 25 degrees C in buffers of low ionic strength mouse mitochondrial rRNA species had a lower electrophoretic mobility than those of human and hamster. Under these conditions the smaller mitochondrial rRNA species of hamster had a lower electrophoretic mobility than that of human but the larger component had an identical mobility. Mouse and rat mitochondrial rRNA species had identical electrophoretic mobilities. Complex differences between human and mouse mitochondrial rRNA species were observed on sedimentation in sucrose density gradients under various conditions of temperature and ionic strength. Mouse L-cell mitochondrial rRNA was eluted after cytoplasmic rRNA on a column of methylated albumin-kieselguhr.

  9. The molecular integrity of chloroplast ribosomal ribonucleic acid.

    PubMed

    Leaver, C J; Ingle, J

    1971-06-01

    Instability of chloroplast rRNA has been observed with essentially all chloroplast RNA preparations. This paper describes experiments that show that, under normal conditions of preparation and fractionation, only the heavy chloroplast component (mol.wt. 1.1x10(6)) is unstable, the light chloroplast rRNA (mol.wt. 0.56x10(6)) and the cytoplasmic rRNA species (mol.wt. 1.3x10(6) and 0.70x10(6)) being stable. The stability of the 1.1x10(6)-mol. wt. molecule varies with different plant species, as also does the size and the number of fragments produced. Cleavages in three particular regions of the molecule are very frequent within the range of tissues studied. The 1.1x10(6)-mol.wt. rRNA is, however, stabilized by the presence of Mg(2+) during the preparation and fractionation of the RNA.

  10. Messenger ribonucleic acid levels in disrupted human anterior cruciate ligaments.

    PubMed

    Lo, Ian K Y; Marchuk, Linda; Hart, David A; Frank, Cyril B

    2003-02-01

    Thirty patients had anterior cruciate ligament reconstruction for ongoing instability. Two groups were defined according to gross morphologic features identified during reconstruction: anterior cruciate ligament disruptions with scars attached to a structure in the joint and disruptions without reattachments. Reverse transcription polymerase chain reaction for a subset of extracellular matrix molecules, proteinases, and proteinase inhibitors was done on samples of scarred anterior cruciate ligament tissue removed during reconstructive surgery. Results of the nonattached scar group showed significantly increased mRNA levels for Type I collagen, and an increased Type I to Type III collagen ratio compared with that for the attached scar group. In the first year after injury, decorin mRNA levels in the nonattached scar group also were significantly higher than in the attached scar group. Biglycan mRNA levels in the nonattached scar group correlated closely with Type I collagen mRNA levels. These results suggest differences in cellular expression in torn anterior cruciate ligaments that attach to structures in the joint versus those which do not. Although the molecular mechanisms responsible for these differences have not been delineated, different molecular signals may influence the gross morphologic features of anterior cruciate ligament disruptions or alternatively, differing gross morphologic features may be subject to different mechanical loads leading to altered molecular expression. However, the finding of endogenous cellular activity in injured anterior cruciate ligaments raises the possibility that this activity may be enhanced to improve outcomes.

  11. Interfering ribonucleic acids that suppress expression of multiple unrelated genes

    PubMed Central

    Passioura, Toby; Gozar, Mary M; Goodchild, Amber; King, Andrew; Arndt, Greg M; Poidinger, Michael; Birkett, Donald J; Rivory, Laurent P

    2009-01-01

    Background Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of VEGF-A and ICAM-1; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy. Results Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated. Conclusion Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach. PMID:19531249

  12. 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.

  13. 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

  14. 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).

  15. 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.

  16. Deoxyribonucleic Acid Polymerase of Rous Sarcoma Virus: Reaction Conditions and Analysis of the Reaction Product Nucleic Acids

    PubMed Central

    Bishop, D. H. L.; Ruprecht, Ruth; Simpson, R. W.; Spiegelman, S.

    1971-01-01

    Reaction conditions for Rous sarcoma virus ribonucleic acid (RNA)-instructed deoxyribonucleic acid (DNA) polymerase activity are described whereby the viral RNA is relatively protected from endogenous or added nuclease activity. Three analyses of reaction product nucleic acids (3H-RNA, 32P-DNA) were compared, namely, gel electrophoresis, Cs2SO4 gradient centrifugation, and hydroxyapatite column chromatography. It was found that hydroxyapatite analysis could be misleading unless the state of the template RNA was monitored concomitantly with the DNA analysis. Gel electrophoresis and Cs2SO4 gradient centrifugation gave comparable results. It was concluded that analyses of the product of reverse transcriptase reactions should not only refer to the template RNA and product DNA species, but also be performed with virus or viral RNA which do not have or obtain nicks in the 60S RNA. Otherwise, interpretation of the results would have the ambiguity of potential artifacts caused by those degraded RNA molecules. PMID:4332143

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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)

  2. 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.

  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. 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

  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. Target Biological Structures: The Cell, Organelles, DNA and RNA

    NASA Astrophysics Data System (ADS)

    van Holst, Marcelis; Grant, Maxine P.; Aldrich-Wright, Janice

    Living organisms are self replicating molecular factories of staggering complexity [1]. As a result, we are often overwhelmed when trying to identify potential targets for therapeutics. Water, inorganic ions and a large array of relatively small organic molecules (e.g., sugars, vitamins and fatty acids) account for approximately 80% of living matter, with water being the most abundant. Macromolecules such as proteins, polysaccharides, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) constitute the rest. The majority of potential therapeutic targets are found within the cell. Small molecules which are vital for cellular function are imported into the cell by a variety of mechanisms but unlike smaller molecules, macromolecules are assembled within the cell itself. Drugs are usually designed to target cellular macromolecules, as they perform very specific roles in the metabolic processes.

  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. 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

  11. 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

  12. 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.

  13. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

    PubMed Central

    Jolly, Pawan; Estrela, Pedro

    2016-01-01

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Protein Synthesis and Deoxyribonucleic Acid-Membrane Attachment During Thymineless Death in Escherichia coli

    PubMed Central

    Dankberg, Frances; Cummings, Donald J.

    1973-01-01

    The proteins synthesized during thymineless death in Escherichia coli B and B/r were analyzed by polyacrylamide gel elctrophoresis. It was found that the amount of a protein of molecular weight 80,000 to 88,000 is greatly increased during thymineless death compared to the amounts of other cell proteins. A technique for the isolation of cell membrane-deoxyribonucleic acid (DNA)-nascent ribonucleic acid (RNA) complex on detergent crystals was used to determine whether DNA might be detached from the cell membrane as a result of thymineless death. It was found that under no conditions of thymineless death or immunity to thymineless death was there any change in the attachment of DNA or pulse-labeled RNA to cell membrane. Images PMID:4570604

  20. Computational Approaches to Nucleic Acid Origami.

    PubMed

    Jabbari, Hosna; Aminpour, Maral; Montemagno, Carlo

    2015-10-12

    Recent advances in experimental DNA origami have dramatically expanded the horizon of DNA nanotechnology. Complex 3D suprastructures have been designed and developed using DNA origami with applications in biomaterial science, nanomedicine, nanorobotics, and molecular computation. Ribonucleic acid (RNA) origami has recently been realized as a new approach. Similar to DNA, RNA molecules can be designed to form complex 3D structures through complementary base pairings. RNA origami structures are, however, more compact and more thermodynamically stable due to RNA's non-canonical base pairing and tertiary interactions. With all these advantages, the development of RNA origami lags behind DNA origami by a large gap. Furthermore, although computational methods have proven to be effective in designing DNA and RNA origami structures and in their evaluation, advances in computational nucleic acid origami is even more limited. In this paper, we review major milestones in experimental and computational DNA and RNA origami and present current challenges in these fields. We believe collaboration between experimental nanotechnologists and computer scientists are critical for advancing these new research paradigms.

  1. 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

  2. Sample to answer: a fully integrated nucleic acid identification system for bacteria monitoring

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Elsnab, John; Johnson, Michael; Gale, Bruce K.

    2010-02-01

    A fully integrated microfluidic system was developed and incorporates an EC-MWCNT (electrochemical multiwalled carbon nanotube) sensor for the detection of bacteria. Sample metering, reagent metering and delivery was implemented with microvalves and pumps embedded inside the microfluidic system. The nucleic acid extraction was performed using microchannels controlled using automated platforms and a disposable microfluidic silica cartridge. The target samples were flowed and hybridized with probe ssDNA (single strand DNA) across the MWCNT-EC sensor (built on a silicon chip), which was embedded in a microfluidic cell. The 9-pad sensor was scanned before and after hybridization to measure the quantity of RNA (Ribonucleic acid) bound to the array surface. A rapid and accurate sample-in answer-out nucleic acid system was realized with automated volume metering, microfluidic sample preparation, and integrated nano-biosensors.

  3. 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

  4. 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

  5. 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

  6. [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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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 ...

  13. 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

  14. 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

  15. [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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Improved Algorithm for Analysis of DNA Sequences Using Multiresolution Transformation

    PubMed Central

    Inbamalar, T. M.; Sivakumar, R.

    2015-01-01

    Bioinformatics and genomic signal processing use computational techniques to solve various biological problems. They aim to study the information allied with genetic materials such as the deoxyribonucleic acid (DNA), the ribonucleic acid (RNA), and the proteins. Fast and precise identification of the protein coding regions in DNA sequence is one of the most important tasks in analysis. Existing digital signal processing (DSP) methods provide less accurate and computationally complex solution with greater background noise. Hence, improvements in accuracy, computational complexity, and reduction in background noise are essential in identification of the protein coding regions in the DNA sequences. In this paper, a new DSP based method is introduced to detect the protein coding regions in DNA sequences. Here, the DNA sequences are converted into numeric sequences using electron ion interaction potential (EIIP) representation. Then discrete wavelet transformation is taken. Absolute value of the energy is found followed by proper threshold. The test is conducted using the data bases available in the National Centre for Biotechnology Information (NCBI) site. The comparative analysis is done and it ensures the efficiency of the proposed system. PMID:26000337

  3. 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

  4. 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.

  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. 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.

  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. Specificity of the photoreaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen with ribonucleic acid. Identificaton of reactive sites in Escherichia coli phenylalanine-accepting transfer ribonucleic acid

    SciTech Connect

    Bachellerie, J.P.; Hearst, J.E.

    1982-03-16

    In order to test the potential of psoralen photo-addition for the probing of RNA conformation at sequence resolution, the specificity of the reaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) with Escherichia coli tRNA/sup Phe/ was analyzed. The sites of HMT covalent addition have been identified by a combination of analytical techniques involving chemical cleavage of the tRNA/sup Phe/ molecule at the m/sup 7/G site and gel electrophoresis of RNase T/sub 1/ digests together with paper electrophoretic characterization of HMT-modified nucleotides and oligonucleotides. HMT photoaddition shows a very high preference for uracil residues. However, important differences in HMT photoreactivity are observed for various U sites of the tRNA/sup Phe/ molecule. Reactivity of specific bases has been correlated with partial melting of the molecule. Data available so far indicate a strong preference of the photo-reactive probe for a ''loose'' helical conformation as compared with a tight helix, whereas a random coil appears poorly reactive. (JMT)

  14. 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.

  15. 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 ...

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Spermidine-Deoxyribonucleic acid interaction in vitro and in Escherichia coli.

    PubMed Central

    Rubin, R L

    1977-01-01

    The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 10(5) M-1 in 1 mM sodium cacodylate, pH 7.5, to 3 x 10(2) M-1 in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered. PMID:320196

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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}.

  17. 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.

  18. ARRB1 enhances the chemosensitivity of lung cancer through the mediation of DNA damage response

    PubMed Central

    Shen, Hongchang; Wang, Liguang; Zhang, Jiangang; Dong, Wei; Zhang, Tiehong; Ni, Yang; Cao, Hongxin; Wang, Kai; Li, Yun; Wang, Yibing; Du, Jiajun

    2017-01-01

    ARRB1 (also known as β-arrestin-1) serves as a multifunctional adaptor contributing to the regulation of signaling pathways. ARRB1 may be involved in DNA damage accumulation; however the underlying mechanism involved is unclear. In the present study, non-small cell lung cancer (NSCLC) cell lines (H520 and SK-MES-1) were transfected with ARRB1 plasmids or small interfering ribonucleic acid (siRNA) and received treatment with DNA-damaging agents (cisplatin and etoposide). A mouse xenograft model was used to assess the impact of ARRB1 on the efficacy of cisplatin in vivo. A total of 30 surgically resected NSCLC patients were recruited for the present study and qRT-PCR was performed to determine the mRNA levels in cancer tissues compared with para-carcinoma tissues. Our data showed that DNA damage was abrogated in the ARRB1-knockdown cells and enhanced in the ARRB1-overexpressing cells. ATR and Chk1 were more activated in the ARRB1-overexpressing cells compared to the ARRB1-knockdown cells, followed by increased H2AX phosphorylation. DNA damage and apoptosis were increased in the ARRB1-overexpressing cells treated with cisplatin. These data provided strong evidence that ARRB1 contributes to the response of NSCLC to DNA-damaging agents and is essential for DNA damage response (DDR). ARRB1 may enhance the efficacy of DNA-damaging agents in NSCLC. PMID:28035404

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3 beta-hydroxysteroid dehydrogenase/delta (5-4)-isomerase and 17 alpha-hydroxylase/C17-20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line.

    PubMed

    Lefèvre, A; Rogier, E; Astraudo, C; Duquenne, C; Finaz, C

    1994-12-01

    Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17 alpha-hydroxylase/C17-20 lyase (P-450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3 beta HSD and P-450 17 alpha were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17 alpha and 3 beta HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17 alpha expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0 kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of primers, probes, enzymes, and controls for the amplification and detection of enterovirus ribonucleic...

  14. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of primers, probes, enzymes, and controls for the amplification and detection of enterovirus ribonucleic...

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Mesenchymal stem cells as novel micro-ribonucleic acid delivery vehicles in kidney disease.

    PubMed

    Yao, Kevin; Ricardo, Sharon D

    2016-05-01

    MicroRNAs (miRNAs) are short single strands of RNA responsible for post-transcriptional regulation of gene expression and have been implicated in the pathogenesis of chronic kidney disease (CKD). Emerging evidence reports that miRNAs can reduce kidney fibrosis through regulation of targets associated with collagen and extracellular matrix accumulation. However, the development of miRNA therapies has been hampered by the lack of targeted and sustainable methods of systemic miRNA delivery. Mesenchymal stem cells (MSCs) provide a promising miRNA delivery platform to overcome toxicity, the potential for insertional mutations and the low efficiency of previous methods. MSCs are endogenously immunoprivileged and home to sites of inflammation. They also release trophic growth factors to modulate the immune system, alter the polarization of macrophages and provide renal protection and repair. The potential to engineer MSCs to express or overexpress miRNAs, released by exosomes, may enhance their natural functions. Clinical studies are already being conducted individually for the use of miRNAs in cancer and MSCs in diseases associated with CKD. Hence, the combination of miRNAs and MSCs may provide an unparalleled cell-based therapy for treating CKD.

  2. Discovery of huanglongbing (HLB) pre-symptomatic Ribonucleic acid (RNA) biomarkers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Huanglongbing (HLB) is the most devastating citrus disease and is associated with vector-borne Liberibacter. Currently there is no cure for huanglongbing. Visual disease symptoms appear in only a few leaves months after initial Liberibacter exposure compromising disease management by tree removal. S...

  3. The selective reaction of methoxyamine with cytidine residues in mammalian initiator transfer ribonucleic acid

    PubMed Central

    Piper, Peter W.; Clark, Brian F.C.

    1974-01-01

    Methoxyamine reacts selectively with tRNA molecules at certain exposed cytosine residues usually located in non base-paired regions of the two dimensional clover leaf structure. Here methoxyamine is used for the first time in a study of a mammalian tRNA structure. One of the sequence abnormalities of myeloma initiator tRNA is a cytosine instead of the usual uracil immediately preceding the anticodon. A study of the reaction of the cytosine residues with methoxyamine indicates that the accessibility of bases to chemical reagents in the anticodon loop of this mammalian initiator tRNA is very similar to that observed for the bacterial initiator tRNA. Images PMID:10793658

  4. Factors Affecting the Extraction of Intact Ribonucleic Acid from Plant Tissues Containing Interfering Phenolic Compounds

    PubMed Central

    Newbury, H. John; Possingham, John V.

    1977-01-01

    Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions. PMID:16660134

  5. Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Clohisy, J. C.; Connolly, T. J.; Bergman, K. D.; Quinn, C. O.; Partridge, N. C.

    1994-01-01

    Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

  6. Nucleotide sequence of a lysine transfer ribonucleic Acid from bakers' yeast.

    PubMed

    Madison, J T; Boguslawski, S J; Teetor, G H

    1972-05-12

    The nucleotide sequence of one of the two major lysine transfer RNA's from bakers' yeast has been determined. Its structure is compared to that of a lysine tRNA from a haploid yeast. A total of 21 nucleotides differ in the two molecules. Only the T-psi-C-G (thymidine-pseudouridine-cytidine-guanosine) loop and its supporting stem are identical.

  7. Dependence of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen photoaddition on the conformation of ribonucleic acid

    SciTech Connect

    Thompson, J.F.; Bachellerie, J.P.; Hall, K.; Hearst, J.E.

    1982-03-16

    The photoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) to different conformational states of RNA was studied. Poly(U), poly(A,U) (random copolymer), poly(A-U) (alternating copolymer), poly(A)-poly(U) (double stranded), and poly(U)-poly(A)-poly(U) (triple stranded) were reacted with HMT at different temperatures and salt concentrations. The conformation of the polymers was monitored by UV absorption and circular dichroism. It was found that the rate of HMT photoaddition changed dramatically at structural transitions in the RNA. The alternating copolymer poly(A-U) was found to have the highest rate of addition. Low salt and temperature produced maximal incorporation.

  8. Structural proteins of ribonucleic acid tumor viruses. Purification of envelope, core, and internal components.

    PubMed

    Strand, M; August, J T

    1976-01-25

    Murine type C virus structural proteins, the envelope glycopeptides, 30,000 dalton major core protein, and 15,000 dalton internal protein have each been purified to near homogeneity and in high yield from the smae batch of virus by use of phosphocellulose column chromatography and gel filtration procedures. Evidence that these proteins are specified by the viral genome was obtained by competition radioimmunoassay analysis, comparing these polypeptides from Rauscher virus cultivated in a variety of mammalian cell lines; all of the reactive antigenic determinants of these proteins appeared to be virus-specific.

  9. Differentiation of human pituitary adenomas determines the pattern of chromogranin/secretogranin messenger ribonucleic acid expression.

    PubMed

    Jin, L; Chandler, W F; Smart, J B; England, B G; Lloyd, R V

    1993-03-01

    The distribution of chromogranin/secretogranin (Cg/Sg) mRNAs, determined by Northern and in situ hybridization, was analyzed in 14 cultured pituitary adenomas characterized by immunohistochemistry and hormone secretion in a defined medium in vitro. There were 5 functional GH adenomas, 1 silent GH adenoma, 7 null cell adenomas, and 1 oncocytoma. The null cell adenomas, oncocytoma, and silent GH adenomas were also analyzed by electron microscopy. Most null cell adenomas and the oncocytoma secreted FSH and LH into the culture medium. GH adenomas, which are examples of well differentiated tumors based on morphological examination, expressed significantly more SgIII mRNA compared to the null cell adenomas and oncocytoma (70 +/- 6% vs. 22 +/- 5%; P < 0.001). GH adenomas also expressed significantly less CgA mRNA compared to the less well differentiated null cell adenomas and oncocytoma (27 +/- 6% vs. 67 +/- 4%; P < 0.001), which could be considered less well differentiated based on ultrastructural morphological features. After treatment with phorbol 12-myristate 13-acetate (10(-7) M) for 7 days, there was an increase in the mRNA for CgB and SgII mRNAs in GH and null cell tumors, while dexamethasone treatment for 7 days increased CgA mRNA in GH and null cell adenomas. GnRH treatment for 7 days increased CgB mRNA in null cell adenomas. Phorbol 12-myristate 13-acetate also decreased the percentage of immunoreactive GH cells and GHm RNA, determined by in situ and Northern hybridization analyses. These results indicate that pituitary adenomas have a distinct pattern of Cg/Sg mRNA expression, which appears to be related to the degree of morphological differentiation of these neoplasms, and suggest that the effects of secretagogues on various Cg/Sg mRNA levels may be related to the stimulation of hormone secretion.

  10. Homologous down-regulation of growth hormone-releasing hormone receptor messenger ribonucleic acid levels.

    PubMed

    Aleppo, G; Moskal, S F; De Grandis, P A; Kineman, R D; Frohman, L A

    1997-03-01

    Repeated stimulation of pituitary cell cultures with GH-releasing hormone (GHRH) results in diminished responsiveness, a phenomenon referred to as homologous desensitization. One component of GHRH-induced desensitization is a reduction in GHRH-binding sites, which is reflected by the decreased ability of GHRH to stimulate a rise in intracellular cAMP. In the present study, we sought to determine if homologous down-regulation of GHRH receptor number is due to a decrease in GHRH receptor synthesis. To this end, we developed and validated a quantitative RT-PCR assay system that was capable of assessing differences in GHRH-R messenger RNA (mRNA) levels in total RNA samples obtained from rat pituitary cell cultures. Treatment of pituitary cells with GHRH, for as little as 4 h, resulted in a dose-dependent decrease in GHRH-R mRNA levels. The maximum effect was observed with 0.1 and 1 nM GHRH, which reduced GHRH-R mRNA levels to 49 +/- 4% (mean +/- SEM) and 54 +/- 11% of control values, respectively (n = three separate experiments; P < 0.05). Accompanying the decline in GHRH-R mRNA levels was a rise in GH release; reaching 320 +/- 31% of control values (P < 0.01). Because of the possibility that the rise in medium GH level is the primary regulator of GHRH-R mRNA, we pretreated pituitary cultures for 4 h with GH to achieve a concentration comparable with that induced by a maximal stimulation with GHRH (8 micrograms GH/ml medium). Following pretreatment, cultures were stimulated for 15 min with GHRH and intracellular cAMP accumulation was measured by RIA. GH pretreatment did not impair the ability of GHRH to induce a rise in cAMP concentrations. However, as anticipated, GHRH pretreatment (10 nM) significantly reduced subsequent GHRH-stimulated cAMP to 46% of untreated controls. These data suggest that GHRH, but not GH, directly reduces GHRH-R mRNA levels. To determine whether this effect was mediated through cAMP, cultures were treated with forskolin, a direct stimulator of adenylate cyclase. Forskolin (10 microM) significantly reduced GHRH-R mRNA concentrations (37 +/- 6% of control values) indicating that GHRH acts through the cAMP-second messenger system cascade to regulate GHRH-R mRNA. The somatostatin analogue, octreotide (10 nM), which has been previously reported to decrease adenylate cyclase activity, did not affect GHRH-R mRNA levels. Taken together, these results indicate that GHRH inhibits the production of its own receptor by a receptor-mediated, cAMP-dependent reduction of GHRH-R mRNA accumulation.

  11. Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization

    SciTech Connect

    Yang, G.; Matocha, M.F.; Rapoport, S.I.

    1988-08-01

    An in situ hybridization procedure was applied to quantify glucocorticoid receptor (GR) mRNAs in the hippocampus of rat brain. Hybridization was carried out using a radiolabeled antisense probe complementary to the rat liver GR gene. The specificity of the method was validated by showing: 1) a high cellular grain density in sections hybridized with an antisense but not a sense probe; 2) agreement between the experimental and theoretical temperature at which 50% of the hybrids melted, and 3) a high signal distribution of GR mRNA in the hippocampus, a region of brain known to preferentially concentrate steroid hormones. Within the hippocampus, however, subregional differences in hybridization densities were observed. Quantitative autoradiography indicated that the average neuronal silver grain number was highest in the pyramidal cell layers of CA2 and CA4 and lowest in those of CA1 and CA3. Also, there was a significant difference in the average grain number between all of the cell fields except for that between CA2 and CA4. These results show that contiguous but neuroanatomically distinct cell fields of the hippocampus express different levels of GR transcripts, and indicate that differential regulation of GR expression occurs in subpopulations of hippocampal neurons.

  12. THE NATURE AND PROCESSING OF RIBOSOMAL RIBONUCLEIC ACID IN A DINOFLAGELLATE

    PubMed Central

    Rae, Peter M. M.

    1970-01-01

    Certain features of the dinoflagellate nucleus suggest that it represents a primitive form of eukaryotic nucleus. For this reason, it was of interest to characterize dinoflagellate ribosomal RNA (rRNA) and its mode of synthesis to determine if it also deviated from typical eukaryotic patterns. Gyrodinium cohnii was chosen for this examination. Gyrodinium ribosomal RNA species are 16 and 25s as judged by their sedimentation velocities in isokinetic sucrose gradients. These values are typical of higher plants. In addition, the RNA cosedimented precisely with rRNA from the ciliate Tetrahymena. Nucleotide ratio analyses revealed a GMP + CMP content of 46% for both species of rRNA. The kinetics of incorporation of a radioactive precursor into ribosomal RNA have also been studied, and it seems likely that the maturation of rRNA starts with the synthesis of a 38s molecule. This serves as precursor to the 16s species, and, after a 27s intermediate, the 25s ribosomal component. The process is similar to that in other eukaryotes. The structure of the nucleolus has also been examined, and is seen to be typically eukaryotic. PMID:5459003

  13. Evolution of early life inferred from protein and ribonucleic acid sequences

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.; Schwartz, R. M.

    1978-01-01

    The chemical structures of ferredoxin, 5S ribosomal RNA, and c-type cytochrome sequences have been employed to construct a phylogenetic tree which connects all major photosynthesizing organisms: the three types of bacteria, blue-green algae, and chloroplasts. Anaerobic and aerobic bacteria, eukaryotic cytoplasmic components and mitochondria are also included in the phylogenetic tree. Anaerobic nonphotosynthesizing bacteria similar to Clostridium were the earliest organisms, arising more than 3.2 billion years ago. Bacterial photosynthesis evolved nearly 3.0 billion years ago, while oxygen-evolving photosynthesis, originating in the blue-green algal line, came into being about 2.0 billion years ago. The phylogenetic tree supports the symbiotic theory of the origin of eukaryotes.

  14. Normal level of thyroglobulin messenger ribonucleic acid in a human congenital goiter with thyroglobulin deficiency.

    PubMed

    Cabrer, B; Brocas, H; Perez-Castillo, A; Pohl, V; Navas, J J; Targovnik, H; Centenera, J A; Vassart, G

    1986-10-01

    Two siblings with congenital goiter were investigated from clinical, biochemical, and molecular biology standpoints. The association of clinical and biological hypothyroidism with undetectable levels of serum thyroglobulin (Tg) and the presence of iodohistidines in the urine suggested the diagnosis of defective Tg gene expression. This conclusion was confirmed by analysis of proteins present in goiter extracts. Only minute amounts of Tg-related material was detected by RIA (0.28 and 0.17 mg/g tissue compared to 80-100 mg/g in normal thyroid tissue), by Sepharose 6B chromatography, and by sucrose density gradient centrifugation. Surprisingly, the goiters contained normal amounts of Tg mRNA. The size of the mRNA and the sequence organization of its first five exons also were normal. We conclude that no gross alteration of structure or transcription of the Tg gene was present in these patients. The results are compatible with a lesion affecting the mRNA sequence (point mutation, splicing error etc.), leading to defective translation or abnormal routing of the translation product through the membrane system of the cell. This latter hypothesis is supported by the extreme distension of the goiter endoplasmic reticulum found on electron microscopy.

  15. Regulation of rat luteinizing hormone subunit messenger ribonucleic acids by gonadal steroid hormones.

    PubMed Central

    Gharib, S D; Bowers, S M; Need, L R; Chin, W W

    1986-01-01

    Little is known about the hormonal regulation of luteinizing hormone (LH) biosynthesis. We have studied the regulation of LH messenger RNA (mRNA) levels by gonadal-steroid hormones in the rat. In one set of experiments, male and female rats were surgically gonadectomized (GDX) and killed 1, 3, 7, 14, 22, and 31 d postoperatively. In another set of experiments, male and female rats were surgically GDX and were injected subcutaneously with testosterone propionate (500 micrograms/100 g body wt per d) or 17 beta-estradiol 3-benzoate (10 micrograms/100 g body wt per d), respectively, beginning 3 wk postoperatively. Levels of serum LH were determined by radioimmunoassay and levels of LH subunit mRNAs in single pituitary glands were determined by blot hybridization analysis using labeled synthetic oligodeoxyribonucleotide probes that correspond to portions of the coding regions of the rat alpha- and LH beta-subunit mRNAs. 4 wk after gonadectomy, serum LH levels rose nine- and 20-fold, while alpha-subunit mRNA levels rose six- and 10-fold, and LH beta-subunit levels rose seven- and 14-fold, compared with controls in males and females, respectively. In gonadal-steroid hormone-treated male and female GDX rats, serum LH levels fell to 8 and 36% of control values, while alpha-subunit mRNA levels declined to 22 and 19%, and LH beta-subunit mRNA levels declined to 6 and 10% of control values, 48 h after injections were initiated, in males and females, respectively. We conclude that gonadal-steroid hormones negatively regulate the levels of both subunit mRNAs in GDX rats in a pattern that parallels the changes in serum LH values. These data suggest that gonadal-steroid hormone regulation of LH biosynthesis occurs, at least in part, at the level of LH subunit mRNAs due to effects at the transcriptional and/or RNA stability levels. Images PMID:2418065

  16. 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.

  17. Transcription of exogenous and endogenous deoxyribonucleic acid templates in cold-shocked Bacillus subtilis.

    PubMed Central

    Kuhl, S J; Brown, L R

    1980-01-01

    Ribonucleic acid (RNA) synthesis was examined in cold-shocked Bacillus subtilis cells. The cells were grown to mid-log stage, harvested, and cold shocked. RNA synthesis was monitored by the incorporation of [3H]uridine triphosphate or [alpha 32P]adenosine triphosphate into trichloroacetic acid-precipitable material in the presence of all four nucleoside triphosphates. The inhibition of RNA synthesis in cold-shocked cells by lipiarmycin, ethidium bromide, rifampin. or streptolydigin was analyzed using mutant or wild-type cells. Also examined were the effects of temperature, salt concentration, and the addition of polyamines or highly phosphorylated nucleotides. In ultraviolet-irradiated and cold-shocked cells, RNA wynthesis decreased to low levels. The addition of exogenous phi 29 or TSP-1 template to these cells caused a 13- to 20-fold increase in RNA synthesis, as monitored by trichloroacetic acid-precipitable counts. RNA synthesized in the presence of phi 29 deoxyribonucleic acid (DNA) hybridizes mainly to EcoRI fragments A and C of phi 29 DBA, These two fragments direct transcription by purified RNA polymerase in vitro and hybridize to early phi 29 DNA produced in vivo. Our results with TSP-1 DNA in this system indicated that the RNA produced hybridizes to the same fragments as early RNA produced in vivo. Plasmic pUB110 DNA was not transcribed in this system. Images PMID:6157674

  18. Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ)

    PubMed Central

    Kim, Doyoun; Hur, Jeonghwan; Park, Kwangsoo; Bae, Sangsu; Shin, Donghyuk; Ha, Sung Chul; Hwang, Hye-Yeon; Hohng, Sungchul; Lee, Joon-Hwa; Lee, Sangho; Kim, Yang-Gyun; Kim, Kyeong Kyu

    2014-01-01

    Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate. PMID:24682817

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Effect of Nalidixic Acid and Hydroxyurea on Division Ability of Escherichia coli fil+ and lon− Strains

    PubMed Central

    Kantor, George J.; Deering, R. A.

    1968-01-01

    Short periods of incubation in medium containing nalidixic acid or hydroxyurea, followed by a return to normal growth conditions, induced filament formation in Escherichia coli B (fil+) and AB1899NM (lon−) but not in B/r (fil−) and AB1157 (lon+). These drugs reversibly stopped deoxyribonucleic acid (DNA) synthesis with little or no effect on ribonucleic acid (RNA) synthesis or mass increase. The initial imbalance caused by incubation in these drugs was the same for B and B/r as was macromolecular synthesis following a return to normal growth conditions. DNA degradation caused by nalidixic acid was measured and found to be the same for B and B/r. Hydroxyurea caused no DNA degradation in these two strains. Survival curves as determined under various conditions by colony formation suggested that the property of filament formation was responsible for the extrasensitivity of fil+ and lon− strains to either nalidixic acid or hydroxyurea. E. coli B was more sensitive to either drug than was B/r or Bs-1. Pantoyl lactone or liquid holding treatment aided division and colony formation of nalidixic acid-treated B but had no effect on B/r. Likewise, the filament-former AB1899NM was more sensitive to nalidixic acid than was the non-filament-former AB1157. The sensitivity of B/r and Bs-1 to nalidixic acid was nearly the same except at longer times in nalidixic acid, when Bs-1 appeared more resistant. Even though nalidixic acid, hydroxyurea, and ultraviolet light may produce quite different molecular alterations in E. coli, they all cause a metabolic imbalance resulting in a lowered ratio of DNA to RNA and protein. We propose that it is this imbalance per se rather than any specific primary chemical or photochemical alterations which leads to filament formation by some genetically susceptible bacterial strains such as lon− and fil+. PMID:4867744

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Synthesis of Nucleic Acid and Protein in L Cells Infected with the Agent of Meningopneumonitis

    PubMed Central

    Schechter, Esther M.

    1966-01-01

    Schechter, Esther M. (The University of Chicago, Chicago, Ill.). Synthesis of nucleic acid and protein in L cells infected with the agent of meningopneumonitis. J. Bacteriol. 91:2069–2080. 1966.—Synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in uninfected L cells and in L cells infected with the meningopneumonitis agent was compared by measuring rates of incorporation of H3-cytidine and C14-lysine into nuclear, cytoplasmic, and agent fractions in successive 5-hr periods during the meningopneumonitis growth cycle. Synthesis of meningopneumonitis DNA, RNA, and protein was first clearly evident in the labeling period 15 to 20 hr after infection, soon after initiation of agent multiplication. The rates of synthesis of agent DNA, RNA, and protein increased logarithmically for a brief period and then declined. However, rates of isotope incorporation into all three meningopneumonitis macromolecules were sustained at near maximal values throughout the remainder of the meningopneumonitis growth cycle. These data are most readily interpreted in terms of multiplication of the meningopneumonitis agent by binary fission. The L cell response to infection was a decreased rate of DNA and RNA synthesis and an accelerated rate of cell death. Host protein synthesis was unaffected. The inhibition of nucleic acid synthesis in infected L cells probably involved competition between host and parasite for nucleic acid precursors. Different sublines of L cells varied greatly in the degree to which their nucleic acid-synthesizing mechanisms were damaged by infection. The cytoplasm of infected L cells contained newly synthesized DNA and RNA that could not be accounted for as intact meningopneumonitis cells. This nucleic acid probably arose from disintegration of the fragile intracellular forms of the meningopneumonitis agent. Images PMID:5937251

  10. 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.

  11. A cDNA encoding a cold-induced glycine-rich RNA binding protein from Prunus avium expressed in embryonic axes.

    PubMed

    Stephen, John R; Dent, Katherine C; Finch-Savage, William E

    2003-11-27

    A cDNA clone encoding a presumed full-length glycine-rich ribonucleic acid (RNA) binding protein was isolated from a lambda-ZAP Express cDNA library generated from primarily nondormant Prunus avium (wild cherry) embryonic axes. The cDNA, designated Pa-RRM-GRP1 (Prunus avium RNA recognition motif glycine-rich protein 1), contains a single N-terminal RNA recognition motif (RRM) and single C-terminal glycine-rich domain. The glycine-rich domain is unusually long at 91 amino acids, 58 of which are glycines. The 534-base pair (bp) open reading frame (ORF) of this clone encodes a 178-amino-acid polypeptide with a predicted molecular weight of 17.33 kDa and pI of 7.84. Comparative sequence alignment of Pa-RRM-GRP1 reveals extensive homology to known and presumed glycine-rich RNA binding proteins from angiosperms and gymnosperms. Genomic Southern blot analysis suggests that this gene exists as a single copy in P. avium. Expression of this gene in P. avium embryonic axes during low-temperature dormancy-breaking treatments was studied and found to be induced by cold (3 degrees C) using real-time PCR of total cDNA supported by Northern blot analysis of total RNA. Expression dropped during prolonged storage at 3 degrees C and was reduced to control levels by interruption of cold treatment by warming to 20 degrees C.

  12. 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.

  13. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

    2012-01-01

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  14. 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.

  15. 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)

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Effects of trace elements and pesticides on dephosphorylation of RNA and DNA added to soils

    SciTech Connect

    Frankenberger, W.T. Jr.; Johanson, J.B.; Lund L.J.

    1986-01-01

    This study was carried out to assess the effects of 14 trace elements, 12 herbicides, and two fungicides on dephosphorylation of yeast ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) added to soils (Xerollic Calciorthids and Typic Haploxeralfs). The cumulative amount of ortho phosphate (Pi) released from nucleic acids increased linearly with time of incubation (up to 72 h), decreased with profile depth, and was highly influenced by soil pH. When trace elements were applied and compared by using 2.5 mmol kg/sup -1/ of soil, the average inhibition in dephosphorylation of RNA and DNA in two soils ranged from 17% with Co(II) to 52% with Cu(II). The most effective inhibitors of nucleic acid dephosphorylation were Ag(I), Cu(I), Cd(II), Cu(II), Mn(II), Ni(II), and Pb(II) (avg inhibition greater than or equal to 35%). Other elements that inhibited dephosphorylation of RNA and DNA added to soils included Ba(II), Co(II), Hg(II), Zn(II), Ti(IV), V(IV), and W(VI). When the pesticides were compared by using 5 mg of active ingredient kg/sup -1/ of soil, the average inhibition in nucleic acid dephosphorylation ranged from 14% with butylate to 39% with chloramben. The most effective inhibitors (> 25%) were atrazine, naptalam, chloramben, dicamba, trifluralin, and maneb. Other pesticides that inhibited RNA and DNA dephosphorylation in soils included cyanazine, 2,4-D, dinitroamine, EPTC plus R-25788, alachlor, paraquat, butylate, and captan.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  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. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. Cloning and characterization of cDNA for syndecan core protein in sea urchin embryos.

    PubMed

    Tomita, K; Yamasu, K; Suyemitsu, T

    2000-10-01

    The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Properties of the Deoxyribonucleic Acid Contained in the Defective Particle Coliphage 15 1

    PubMed Central

    Frampton, E. W.; Mandel, M.

    1970-01-01

    Escherichia coli strain 15 TAU, which requires thymine, arginine, and uracil for growth and harbors an apparently defective prophage, was induced by exposure to ultraviolet light (580 ergs/mm2) or to mitomycin C (5 μg/ml). Phage particles (coliphage 15) were recovered from the resulting lysate by treatment with deoxyribonuclease, filtration, and several cycles of differential centrifugation. Analysis of the phage particles obtained by using cesium chloride density gradient centrifugation in a preparative ultracentrifuge resulted in the resolution of three components. The major component had a peak density of 1.52 to 1.53 g/cm3 followed by components with densities of 1.5 and 1.49 g/cm3. The guanine plus cytosine content of coliphage 15 deoxyribonucleic acid (DNA) was determined by both analytical ultracentrifugation in cesium chloride and by thermal denaturation in standard saline citrate buffer. Respective values of 46.4 ± 1% and 46.6 ± 1% guanine plus cytosine content were obtained. Coliphage 15 DNA formed molecular hybrids with messenger ribonucleic acid (RNA) from both uninduced and ultraviolet-induced cultures of E. coli 15 TAU, but did not hybridize with E. coli ribosomal RNA. The molecular weight of coliphage 15 DNA was determined by constant velocity sucrose density gradient centrifugation to be about 33 × 106 daltons. PMID:4909911

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. [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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. "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

  16. 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

  17. 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.

  18. 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.

  19. Solution Preserves Nucleic Acids in Body-Fluid Specimens

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond P.

    2004-01-01

    A solution has been formulated to preserve deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in specimens of blood, saliva, and other bodily fluids. Specimens of this type are collected for diagnostic molecular pathology, which is becoming the method of choice for diagnosis of many diseases. The solution makes it possible to store such specimens at room temperature, without risk of decomposition, for subsequent analysis in a laboratory that could be remote from the sampling location. Thus, the solution could be a means to bring the benefits of diagnostic molecular pathology to geographic regions where refrigeration equipment and diagnostic laboratories are not available. The table lists the ingredients of the solution. The functions of the ingredients are the following: EDTA chelates divalent cations that are necessary cofactors for nuclease activity. In so doing, it functionally removes these cations and thereby retards the action of nucleases. EDTA also stabilizes the DNA helix. Tris serves as a buffering agent, which is needed because minor contaminants in an unbuffered solution can exert pronounced effects on pH and thereby cause spontaneous degradation of DNA. SDS is an ionic detergent that inhibits ribonuclease activity. SDS has been used in some lysis buffers and as a storage buffer for RNA after purification. The use of the solution is straightforward. For example, a sample of saliva is collected by placing a cotton roll around in the subject's mouth until it becomes saturated, then the cotton is placed in a collection tube. Next, 1.5 mL of the solution are injected directly into the cotton and the tube is capped for storage at room temperature. The effectiveness of the solution has been demonstrated in tests on specimens of saliva containing herpes simplex virus. In the tests, the viral DNA, as amplified by polymerase chain reaction, was detected even after storage for 120 days.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. [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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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/.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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...

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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…

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Streptozotocin-Induced Diabetes Decreases Mammary Gland Lipoprotein Lipase Activity and Messenger Ribonucleic Acid in Pregnant and Nonpregnant Rats

    PubMed Central

    Blanco-Dolado, Laura; Martín-Hidalgo, Antonia; Herrera, Emilio

    2002-01-01

    Diabetes mellitus is associated with a reduction of lipoprotein lipase (LPL) activity in adipose tissue and development of hypertriglyceridemia. To determine how a condition of severe insulin deficiency affects mammary gland LPL activity and mRNA expression during late pregnancy, streptozotocin (STZ) treated (40 mg/kg) and non-treated (control) virgin and 20 day pregnant rats were studied. In control rats, both LPL activity and mRNA were higher in pregnant than in virgin rats. When compared to control rats, STZ-treated rats, either pregnant or virgin, showed decreased LPL activity and mRNA content. Furthermore, mammary gland LPL activity was linearly correlated with mRNA content, and either variable was linearly correlated with plasma insulin levels. Thus, insulin deficiency impairs the expression of LPL in mammary glands, revealing the role of insulin as a modulator of the enzyme at the mRNA expression level. PMID:11900280

  9. Effects of irradiation and semistarvation on rat thyrotropin beta subunit messenger ribonucleic acid, pituitary thyrotropin content, and thyroid hormone levels

    SciTech Connect

    Litten, R.Z. ); Carr, F.E. ); Fein, H.G.; Smallridge, R.C. )

    1990-01-01

    The effect of radiation-induced anorexia on serum thyrotropin (TSH), pituitary TSH-{beta} mRNA, pituitary TSH content, serum thyroxine (T{sub 4}), and serum 3,5,3{prime}-triiodothyronine (T{sub 3}) was investigated using feed-matched controls. Rats received 10 Gy gamma whole-body irradiation and were examined 1-3 days postirradiation. Feed-matched and untreated controls were also studied. The average food intake of the irradiated and feed-matched groups was approximately 18% of the untreated controls. Over the three day period both the irradiated and feed-matched groups lost a significant amount of body weight. The serum T{sub 4} levels of both the irradiated and feed-matched groups were not significantly different from each other, but were significantly depressed when compared to the untreated control group. The serum TSH and T{sub 3} were, however, significantly greater in the irradiated than the feed-matched groups at day 3 posttreatment. To determine if the difference in the serum TSH level between the two groups was due to a pretranslational alteration in TSH production, we measured the TSH-{beta} mRNA using an RNA blot hybridization assay. We found that the TSH-{beta} mRNA level was the same in the irradiated and feed-matched groups, suggesting that the mechanism responsible for the radiation-induced increase in the serum TSH level is posttranscriptional. Pituitary TSH content in the irradiated rats was significantly less than in pair-fed controls, suggesting that irradiation may permit enhanced secretion of stored hormone.

  10. Alterations in polyribosome and messenger ribonucleic acid metabolism and messenger ribonucleoprotein utilization in osmotically stressed plant seedlings

    SciTech Connect

    Mason, H.S.

    1986-01-01

    Polyribosome aggregation state in growing tissues of barley and wheat leaf of stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf of stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of /sup 32/PO/sub 4//sup 3 -/ into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A)/sup +/RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A)/sup +/RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described.

  11. Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser-induced fluorescence.

    PubMed

    Chung, Yi-An; Chen, Yi-Hsin; Chang, Po-Ling

    2015-08-01

    In this work, five fluorescent dyes (SYTO-9, SYBR Green I, SYBR Green II, SYBR Safe, and SYBR Gold) were used as both on-column and precolumn stains for total RNA analysis by CE-LIF with Ar ion laser excitation. In the on-column RNA stain, the SYTO-9 provided the highest fluorescence intensity and the lowest detectable concentration, as low as 10 pg/μL, while the SYBR Green II and SYBR Gold were adsorbed on the poly(ethylene oxide) thus affected the separation efficiency. As a precolumn stain, SYBR Gold was the most sensitive among the five dyes due to the strong affinity between the dye and RNA molecules. As a result, a single-cell quantity of RNA (10-30 pg per cell) could be detected by CE-LIF with precolumn staining by SYBR Gold. Because of the great savings of fluorescent dye using precolumn stain (one button dye may use for one million stain), this method is the best strategy for RNA staining in terms of cost-effectiveness and sensitivity.

  12. Effect of ribonucleic acid (RNA) isolation methods on putative reference genes messenger RNA abundance in human spermatozoa.

    PubMed

    Barragán, M; Martínez, A; Llonch, S; Pujol, A; Vernaeve, V; Vassena, R

    2015-07-01

    Although the male gamete participates in a significant proportion of infertility cases, there are currently no proven molecular markers of sperm quality. The search for significant gene expression markers is partially hindered by the lack of a recognized set of reference genes (RGs) to normalize reverse transcription quantitative PCR (RT-qPCR) data across studies. The aim of this study is to define a set of RGs in assisted reproduction patients undergoing different sample collection and RNA isolation methods. Twenty-two normozoospermic men were included in the study. From each man, semen was either cryopreserved by slow freezing or analyzed fresh, and, for each, RNA was extracted with either phenol-free or phenol-based methods. In two cases, both methods were used to isolate RNA. Twenty putative RGs were analyzed and their mRNA abundance across samples was estimated by RT-qPCR. To determine the genes whose steady-state mRNA abundance remains unchanged, three different algorithms (geNorm, BestKeeper and NormFinder) were applied to the qPCR data. We found that RGs such as GAPDH or ACTB, useful in other biological contexts, cannot be used as reference for human spermatozoa. It is possible to compare gene expression from fresh and cryopreserved sperm samples using the same isolation method, while the mRNA abundance of expressed genes becomes different depending on the RNA isolation technique employed. In our conditions, the most appropriate RGs for RT-qPCR analysis were RPLP1, RPL13A, and RPLP2. Published discrepancies in gene expression studies in human spermatozoa may be due in part to inappropriate RGs selection, suggesting a possible different interpretation of PCR data in several reports, which were normalized using unstable RGs.

  13. Development of a Single-Step Subtraction Method for Eukaryotic 18S and 28S Ribonucleic Acids

    DTIC Science & Technology

    2011-01-01

    purification and probing of mRNA for neutral protease of Bacillus cereus ,” Journal of Microbiological Methods, vol. 37, no. 2, pp. 187–192, 1999. [17...of hepatitis C virus RNA by direct capture from blood ,” Journal of Medical Virology, vol. 42, no. 1, pp. 22–28, 1994. [13] T. C. H. Hsuih, Y. N. Park

  14. Glucocorticoid control of rat growth hormone gene expression: Effect on cytoplasmic messenger ribonucleic acid production and degradation

    SciTech Connect

    Gertz, B.J.; Gardner, D.G.; Baxter, J.D. )

    1987-12-01

    The effect of the glucocorticoid dexamethasone on the production and degradation of rat GH (rGH) cytoplasmic mRNA was studied in cultured rat pituitary tumor (GC) cells. The incorporation of (3H)uridine into both rGH cytoplasmic mRNA and the pyrimidine nucleotide precursor pool was determined in hormone-treated and control cells. From these measurements glucocorticoid effects on absolute production rates of rGH cytoplasmic mRNA were determined and compared to effects on rGH mRNA accumulation. Rat GH mRNA half-life was then calculated based on a first-order decay model. Rat GH mRNA half-life was also directly assayed by: (1) pulse-chase studies and (2) measuring the kinetics of decay of rGH mRNA in cells after transfer from serum-containing to hormone-deficient media. From these independent analyses rGH mRNA half-life estimates ranged from 28-55 h in different experiments. Within individual experiments there was little variability of rGH mRNA decay rates; glucocorticoids were found not to alter the stability of rGH cytoplasmic mRNA. Glucocorticoid induction of rGH cytoplasmic mRNA accumulation was accounted for solely on the basis of increased mRNA production.

  15. Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression.

    PubMed

    Sivitz, W I; Fink, B D; Donohoue, P A

    1999-04-01

    Leptin is believed to act through hypothalamic centers to decrease appetite and increase energy utilization, in part through enhanced thermogenesis. In this study, we examined the effects of fasting for 2 days and exogenous s.c. leptin, 200 microg every 8 h for 2 days, on the regulation of uncoupling protein (UCP) subtypes in brown adipose tissue (BAT) and gastrocnemius muscle. Northern blot analysis (UCP-1) and ribonuclease protection (UCP-2 and 3) were used for quantitative messenger RNA (mRNA) analysis, and specific antibodies were used to measure UCP-1 and UCP-3 total protein expression. Leptin, compared with vehicle, did not alter BAT UCP-1 or UCP-3 mRNA or protein expression when administered to normal ad libitum fed rats. Fasting significantly decreased BAT UCP-1 and UCP-3 mRNA expression, to 31% and 30% of ad libitum fed controls, respectively, effects which were prevented by administration of leptin to fasted rats. Fasting also significantly decreased BAT UCP-1 protein expression, to 67% of control; however, that effect was not prevented by leptin treatment. Fasting also decreased BAT UCP-3 protein, to 85% of control, an effect that was not statistically significant. Fasting, with or without leptin administration, did not affect BAT UCP-2 mRNA; however, leptin administration to ad libitum fed rats significantly increased BAT UCP-2 mRNA, to 138% of control. Fasting significantly enhanced gastrocnemius muscle UCP-3 mRNA (411% of control) and protein expression (168% of control), whereas leptin administration to fasted rats did not alter either of these effects. In summary, UCP subtype mRNA and protein are regulated in tissue- and subtype-specific fashion by leptin and food restriction. Under certain conditions, the effects of these perturbations on UCP mRNA and protein are discordant.

  16. Endothelin in human brain and pituitary gland: Presence of immunoreactive endothelin, endothelin messenger ribonucleic acid, and endothelin receptors

    SciTech Connect

    Takahashi, K.; Ghatei, M.A.; Jones, P.M.; Murphy, J.K.; Lam, H.C.; O'Halloran, D.J.; Bloom, S.R. )

    1991-03-01

    The presence of immunoreactive (IR) endothelin, endothelin mRNA, and endothelin receptors in human brain and pituitary gland has been studied by RIA, Northern blot hybridization, and receptor assay. IR endothelin was detected in all five brain regions examined (cerebral cortex, cerebellum, brain stem, basal ganglia, and hypothalamus) (6-10 fmol/g wet wt) and spinal cord (22 +/- 6 fmol/g wet wt, n = 7, mean +/- SEM). Higher concentrations of IR endothelin were found in the pituitary gland (147 +/- 30 fmol/g wet wt). Fast protein liquid chromatographic analysis of the IR endothelin in pituitary gland showed a large IR peak in the position of endothelin-3 and a smaller peak in the position of endothelin-1, whereas IR endothelin in the hypothalamus and brain stem was mainly endothelin-1. Endothelin messenger RNA was detected by Northern blot hybridization in the pituitary but not in hypothalamus. The receptor assay showed that 125I-endothelin-1 binding sites were present in large numbers in all five brain regions but were much less abundant in the pituitary gland. Binding capacity and dissociation constant were 5052 +/- 740 fmol/mg protein and 0.045 +/- 0.007 nM in brain stem and 963 +/- 181 fmol/mg protein and 0.034 +/- 0.009 nM in hypothalamus. In the pituitary gland, there were two classes of binding sites for endothelin with dissociation constants of 0.059 +/- 0.002 nM (binding capacity = 418 +/- 63 fmol/mg protein) and 0.652 +/- 0.103 nM (binding capacity = 1717 +/- 200 fmol/mg protein). Endothelin-1, -2 and -3 were almost equipotent in displacing the binding (IC50 approximately 0.04 nM). These findings are in accord with the possibility that endothelin acts as a neurotransmitter, neuromodulator or neurohormone in man.

  17. Heat shock of cultured GC cells enhances the level of triiodothyronine induced growth hormone (GH) and GH messenger ribonucleic acid.

    PubMed

    Shapiro, L E; Katz, C P; DeFesi, C R; Surks, M I

    1989-07-01

    We have previously proposed that the effects of heat shock on thyroid hormone-responsive rat pituitary tumor (GC) cells may be a model relevant to the in vivo effects of nonthyroidal disease on thyroid hormone action. To determine the effects of heat shock on thyroid hormone responses, GC cells (normally cultured at 37 C) were studied after incubation at 41 C. After 18 h at 41 C there was enhanced synthesis of proteins (mol wt, 70,000 and 90,000) considered to be universal markers of the cellular response to heat shock. Incubation at 41 C also resulted in a significant decrease in GC cell viability and (after 24 h) arrest of GC cell growth. However, the induction of GH synthesis by T3 was significantly enhanced in GC cells stressed by incubation at 41 C. The addition of 5 nM T3 to thyroid hormone-depeleted GC cells resulted in a significantly greater (P less than 0.001) accumulation of GH (2642 +/- 280 ng/18 h) during 41 C incubation than during 37 C incubation (1223 +/- 175 ng/18 h). The enhanced T3-induced production of GH was coincident with a proportional increase (P less than 0.05) in cellular GH mRNA determined by dot hybridization analysis. Thus, the stress of 41 C incubation elicits a heat shock response in GC cells characterized by decreased viability and growth arrest, but enhanced accumulation of GH mRNA in response to T3. Our recent report on the identical effects due to the stress of implantation of the Walker 256 carcinoma on T3-induced rat pituitary GH mRNA in vivo suggests that heat shock of cultured GC cells is a valid in vitro model of nonthyroidal disease.

  18. Antibodies to Yeast Phenylalanine Transfer Ribonucleic Acid Are Specific for the Odd Nucleoside Y in the Anticodon Loop

    PubMed Central

    Fuchs, Sara; Aharonov, Aharon; Sela, Michael; Von Der Haar, Friedrich; Cramer, Friedrich

    1974-01-01

    Antibodies with specificity to a single species of tRNA were elicited in a goat by immunization with a glutaraldehyde conjugate of yeast phenylalanine transfer RNA with bovine gamma globulin. The specificity of the antibodies was studied by a radioimmunoassay measuring the direct binding of [3H]tRNAPhe or the inhibition of the binding. The antibodies formed are predominantly directed towards the characteristic highly modified nucleoside Y, which is located right next to the anticodon. The antibodies bind specifically to tRNAPhe, to oligonucleotides derived by enzymatic digestion from the anticodon loop of tRNAPhe, and to the Y nucleoside itself. tRNA species which do not contain Y in their sequences, or tRNAPhe from which the Y base has been excised, do not bind to the antibodies. Yeast tRNAPhe can be separated from other tRNA species with an immunoadsorbent of antibodies to tRNAPhe. PMID:4527666

  19. Synthesis of ribonucleic acid in the venom gland of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom

    PubMed Central

    De Lucca, F. L.; Imaizumi, M. T.

    1972-01-01

    1. The incorporation of [5-3H]uridine into RNA of the venom gland of Crotalus durissus terrificus was studied after manual extraction (`milking') of the venom. The labelled precursor was injected immediately after milking. 2. The RNA was extracted 1, 2, 4, 6 and 8h after injection of the label and analysed by sucrose-density-gradient centrifugation. 3. The sedimentation analysis showed that 18S rRNA synthesis is higher than 28S rRNA at all time-intervals. The specific radioactivities of both ribosomal components did not reach a plateau even at 8h after injection. 4. An RNA fraction was detected sedimenting between 18S rRNA and 4S tRNA and was called the 10–14S fraction. The specific radioactivity was always higher than that of both classes of rRNA and reached the maximum value at 6h of labelling. 5. The incorporation of the precursor was also studied by radioautography, which helped to elucidate the intracellular origin of the RNA analysed by sucrose-density-gradient centrifugation. ImagesPLATE 1 PMID:4664568

  20. [Protein and ribonucleic acid metabolism in the central nervous system of rats during space flight in the "Kosmos-605" satellite].

    PubMed

    Gazenko, O G; Demin, N N; Panov, A N; Rubinskaia, N L; Tigranian, R A

    1976-01-01

    On the 2nd postflight day the activity of neutral protamine peptide hydrolase of different compartments of the rat brain did not differ from the control level. With respect to the protein and RNA content and concentration motoneurons of anterior horns of the spinal cord and their glial-cells-satellites of rats exposed to the ground-based synchronous experiment did not differ from those of vivarium controls, except cells of the supraoptic nucleus. That was found on the 2nd and 27th postflight days. On the 2nd postflight day the protein and RNA concentration in neurons decreased and the protein concentration and content in gliocytes lowered; the RNA concentration and content in Purkinje cells of the cerebellum became reduced. On the 27th postflight day the RNA concentration in neurons of the supraoptic nucleus remained diminished whereas the protein content increased; in spinal motoneurons the protein concentration decreased and in adjacent gliocytes the protein concentration and content lowered. All the above changes made no more than 15% of the control values.

  1. Hepatic messenger ribonucleic acid activity profiles in experimental azotemia in the rat. Relationship to food intake and thyroid function.

    PubMed Central

    Kinlaw, W B; Schwartz, H L; Mariash, C N; Bingham, C; Carr, F E; Oppenheimer, J H

    1984-01-01

    We have studied the hepatic messenger RNA (mRNA) activity profile in chronically azotemic rats and sought to determine whether the observed changes could be mediated either by reduced food intake or diminished thyroid function at the tissue level. mRNA activity profiles were produced by two-dimensional gel electrophoretic separation of radioactively labeled products of an in vitro reticulocyte lysate system which had been programmed by hepatic RNA. Of the approximately 240 translational products identified in this system, seven sequences were consistently altered in azotemia. In pair-fed animals six of these also decreased, but the alterations in three were depressed to a significantly lesser extent in the pair-fed group. Moreover, analysis of covariance suggested that food intake could account for the differences in only one sequence. The possibility that the mRNA activity profile in azotemia could represent the effects of diminished thyroid function was minimized by the finding that the reductions in plasma thyroxine (T4) and triiodothyronine (T3) levels observed were due largely to reduced plasma protein binding, with maintenance of the mean free T4 and free T3 concentrations within the normal range. The changes in only one mRNA sequence could be related to free T3 levels alone. Our findings, therefore, indicate that although diminished food intake and reduced thyroid function may contribute to some of the observed changes in the mRNA activity profiles, the bulk of alterations in azotemia appear to be mediated by other mechanisms. The striking overlap between the sequences affected by azotemia and pair-feeding raises the speculation that altered gene expression in azotemia may reflect an impaired hepatic response at the pretranslational level to metabolic signals associated with food intake. Images PMID:6511910

  2. Rift Valley Fever Virus: Molecular Biologic Studies of the M Segment RNA(Ribonucleic Acids) for Application in Disease Prevention

    DTIC Science & Technology

    1988-12-01

    investigators have abided by the National 7nstitutes Of Health Guidelines for Research Involving Recombinant -NA Molecules (April 1982) and the ...34Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, National Research Council (DHEW Publi lcat4on N...conditions (different pulse times; chase in the presence or absence of cycloheximide), failed to suggest a precursor-product relationship between the

  3. 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.

  4. 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

    acid (UA), which results in the metabolic adaptation of normal cells against UV-induced ROS, and the metabolic switch of tumor cells subject to UV-induced damage. The multifaceted natural compound, UA, specifically inhibits photo-oxidative DNA damage in retinal pigment epithelial cells while enhancing that in skin melanoma. Considering the UA-mediated differential effects on cell bioenergetics, this article reviews the disparities in glucose metabolism between tumor and normal cells, along with (peroxisome proliferator-activated receptor-γ coactivator 1α)-dependent mitochondrial metabolism and redox (reduction-oxidation) control to demonstrate UA-induced synthetic lethality in tumor cells. PMID:28250388

  5. 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.

  6. Identification of mitochondrial DNA substitutions related to meat quality in Japanese Black cattle.

    PubMed

    Mannen, H; Morimoto, M L; Oyamat, K; Mukai, F; Tsuji, S

    2003-01-01

    Complete sequences of mitochondrial (mt) genomes of eight Japanese Black cattle were determined to investigate the relationships between mt deoxyribonucleic acid (DNA) displacement loop (D-loop) types and other mtDNA regions and to identify the variation in the coding region that may influence the economic traits. The survey of mitochondrial sequences in the encoding region revealed 14 substitutions including six antonymous substitutions and one in 16S ribosomal ribonucleic acid (rRNA). Three methods of polymorphic DNA analyses (polymerase chain reaction [PCR]-restriction fragment length polymorphism [RFLP], mismatch PCR-RFLP, PCR-single-strand conformation polymorphism [SSCP]) were performed on these seven candidate substitutions (base pair [bp] 2,232, 12,158, 12,908, 13,310, 14,122, 14,140, and 14,565) for 202 Japanese Black cattle. The substitution of bp 13,310 was observed in all samples, but not in the reference sequence, indicating that this is a minor substitution or a sequencing mistake in the reference sequence. The substitutions at bp 14,122, 14,140, and 14,565 were observed in only a few samples, suggesting that these were also minor substitutions. The substitutions at bp 2,232 (16S rRNA), 12,158, and 12,908 (reduced nicotinamide adenine dinucleotide-ubiquinone oxidoreductase chain-5) were closely related to mitochondrial D-loop types that have previously been related to differences in the carcass traits of Japanese Black cattle. Evaluation of the effects on six carcass traits with mixed model procedures suggests that the bp 2,232 substitution affects longissimus muscle area and beef marbling score. The substitution at bp 2,232 is a strong candidate for the mitochondrial effect on meat quality.

  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. Effect of microwaves (2450-MHz) on the immune system in mice: studies of nucleic acid and protein synthesis

    SciTech Connect

    Wiktor-Jedrzejczak, W.; Ahmed, A.; Czerski, P.; Leach, W.M.; Sell, K.W.

    1980-01-01

    CBA/J adult male mice were given single or triple exposures to 2450-mHz microwaves in an environmentally controlled wave guide facility. The average absorbed dose rate for a single exposure varied from 12 to 15 mW/g. Sham-exposed mice served as controls. Lymphoid cells were collected and tested for metabolic activity on days 3, 6, and 9 following a single exposure, and on days 9, 12, and 16 following triple exposures on days 0, 3, and 6. Cells were cultured in vitro for four hours to seven days before their metabolic rates were assayed. Under these conditions, microwaves failed to produce any detectable change in deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis, as measured by the incorporation of methyl(3H)-thymidine (3H-TDR) (DNA substrate), 3H-uridine (3H-UR) (RNA substrate), and 3H-leucine (protein substrate) by spleen, bone marrow, and peripheral blood lymphocytes (PBL) in vitro. These data suggest that microwave-induced increases in the frequency of complement-receptor (CR)- or surface-immunoglobulin (sIg)-bearing cells were not associated with a concomitant increase in cell proliferation and/or protein synthesis, and favor the concept that microwaves under these conditions stimulate already existing B-cell precursors for maturation.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Fatty Acid Synthetase of Saccharomyces cerevisiae

    PubMed Central

    Klein, Harold P.; Volkmann, Carol M.; Chao, Fu-Chuan

    1967-01-01

    A light particle fraction of Saccharomyces cerevisiae, obtained from the crude ribosomal material, and containing the fatty acid synthetase, consisted primarily of 27S and 47S components. This fraction has a protein-ribonucleic acid ratio of about 13. Electron micrographs showed particles ranging in diameter between 100 and 300 A in this material. By use of density gradient analysis, the fatty acid synthetase was found in the 47S component. This component contained particles which were predominantly 300 A in diameter and which were considerably flatter than ribosomes, and it consisted almost entirely of protein. Images PMID:6025308

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. [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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

    PubMed

    Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

    2013-03-15

    Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.

  1. 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).

  2. Rapamycin‐induced autophagy sensitizes A549 cells to radiation associated with DNA damage repair inhibition

    PubMed Central

    Li, Yong; Liu, Fen; Wang, Yong; Li, Donghai; Guo, Fei; Xu, Liyao; Zeng, Zhengguo; Zhong, Xiaojun

    2016-01-01

    Abstract Background Autophagy has been reported to increase in cancer cells after radiation. However, it remains unknown whether increased autophagy as a result of radiation affects DNA damage repair and sensitizes cancer cells. In this study, the radiosensitization effect of rapamycin, a mammalian target of rapamycin inhibitor that induces autophagy, on human lung adenocarcinoma A549 cells was investigated. Methods A549 cells were treated with different concentrations of rapamycin. Cell viability was evaluated by methyl‐thiazolyl‐tetrazolium assay. Survival fraction values of A549 cells after radiotherapy were detected by colony formation assay. Autophagosome was observed by a transmission electron microscope. Furthermore, Western blot was employed to examine alterations in autophagy protein LC3 and p62, DNA damage protein γ–H2AX, and DNA damage repair proteins Rad51, Ku70, and Ku80. Rad51, Ku70, and Ku80 messenger ribonucleic acid (mRNA) expression levels were examined by real‐time polymerase chain reaction. Results Rapamycin suppressed A549 cell proliferation in dose and time‐dependent manners. An inhibitory concentration (IC) 10 dose of rapamycin could induce autophagy in A549 cells. Rapamycin combined with radiation significantly decreased the colony forming ability of cells, compared with rapamycin or radiation alone. Rapamycin and radiation combined increased γ–H2AX expression levels and decreased Rad51 and Ku80 expression levels, compared with single regimens. However, rapamycin treatment did not induce any change in Rad51, Ku70, and Ku80 mRNA levels, regardless of radiation. Conclusions These findings indicate that increasing autophagy sensitizes lung cancer cells to radiation. PMID:27385978

  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. 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 mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7568234

  6. Copper(II) interacting with the non-steroidal antiinflammatory drug flufenamic acid: structure, antioxidant activity and binding to DNA and albumins.

    PubMed

    Tolia, Charikleia; Papadopoulos, Athanassios N; Raptopoulou, Catherine P; Psycharis, Vassilis; Garino, Claudio; Salassa, Luca; Psomas, George

    2013-06-01

    Copper(II) complexes with the non-steroidal antiinflammatory drug flufenamic acid (Hfluf) in the presence of N,N-dimethylformamide (DMF) or nitrogen donor heterocyclic ligands (2,2'-bipyridylamine (bipyam), 1,10-phenanthroline (phen), 2,2'-bipyridine (bipy) or pyridine (py)) have been synthesized and characterized. The crystal structures of [Cu2(fluf)4(DMF)2], 1, and [Cu(fluf)(bipyam)Cl], 2, have been determined by X-ray crystallography. Density functional theory (DFT) (CAM-B3LYP/LANL2DZ/6-31G**) was employed to determine the structure of complex 2 and its analogues (complexes [Cu(fluf)(phen)Cl], 3, [Cu(fluf)(bipy)Cl], 4 and [Cu(fluf)2(py)2], 5). Time-dependent DFT calculations of doublet-doublet transitions show that the lowest-energy band in the absorption spectrum of 2-5 has a mixed d-d/LMCT character. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind to CT DNA with [Cu(fluf)(bipy)Cl] exhibiting the highest binding constant to CT DNA. The complexes can bind to CT DNA via intercalation as concluded by studying the cyclic voltammograms of the complexes in the presence of CT DNA solution and by DNA solution viscosity measurements. Competitive studies with ethidium bromide (EB) have shown that the complexes can displace the DNA-bound EB suggesting strong competition with EB. Flufenamic acid and its Cu(II) complexes exhibit good binding affinity to human or bovine serum albumin protein with high binding constant values. All compounds have been tested for their antioxidant and free radical scavenging activity as well as for their in vitro inhibitory activity against soybean lipoxygenase showing significant activity with [Cu(fluf)(phen)Cl] being the most active.

  7. Quantification of adducts formed in DNA treated with N-acetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene: comparison of trifluoroacetic acid and enzymatic degradation

    SciTech Connect

    Tang, M.; Lieberman, M.W.

    1983-01-01

    We have examined two methods of preparation of DNA adducts from phi X174 RF DNA modified by (/sup 3/H)N-acetoxy-2-acetylaminofluorene ((/sup 3/H)NA-AAF) or N-hydroxy-2-aminofluorene ((/sup 3/H)N-OH-AF). Hydrolysis by enzymes (DNase I, snake venom phosphodiesterase and alkaline or acid phosphatase) and subsequent reverse phase h.p.l.c. of phi X174 RF DNA treated with (/sup 3/H)NA-AAF yielded 73% N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), 7% 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF), and a peak of unidentified radioactivity (13%). When (/sup 3/H)N-OH-AF modified phi X174 DNA was analyzed, both N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and a large percentage of the imidazole ring-opened derivative and unidentified products were found. In contrast, when anhydrous trifluoroacetic acid (TFA) was used to degrade these DNAs, we found for the (/sup 3/H)NA-AAF modified DNA 86% N-(guanin-8-yl)-2-acetylaminofluorene (G-C8-AAF) and 6% 3-(guanin-N2-yl)-2-acetylaminofluorene (G-N2-AAF), while for (/sup 3/H)N-OH-AF modified DNA only the N-(guanin-8-yl)-2-aminofluorene (G-C8-AF) was found. When DNA was prepared from human fibroblasts treated with (/sup 3/H)NA-AAF, only the G-C8-AF product was obtained. Thus, anhydrous TFA solvolysis followed by reverse phase h.p.l.c. is a rapid and convenient method to obtain quantitative yields of DNA adducts formed with acetylaminofluorene and related compounds: quantification by this method prevents loss of G-N2-AAF adducts, the conversion of AAF adducts to AF adducts, and the production of ring opened products in guanine residue.

  8. 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

  9. Structure Effect of Some New Anticancer Pt(II) Complexes of Amino Acid Derivatives with Small Branched or Linear Hydrocarbon Chains on Their DNA Interaction.

    PubMed

    Kantoury, Mahshid; Eslami Moghadam, Mahboube; Tarlani, Ali Akbar; Divsalar, Adeleh

    2016-07-01

    The aim of this study was to investigate the structure effect and identify the modes of binding of amino acid-Pt complexes to DNA molecule for cancer treatment. Hence, three novel water soluble platinum complexes, [Pt(phen)(R-gly)]NO3 (where phen is 1,10-phenanthroline, R-gly is methyl, amyl, and isopentyl-glycine), have been synthesized and characterized by spectroscopic methods, conductivity measurements, and chemical analysis. The anticancer activities of synthesized complexes were investigated against human breast cancer cell line of MDA-MB 231. The 50% cytotoxic concentration values were determined to be 42.5, 58, and 70 μm for methyl-, amyl-, and isopentyl-gly complexes, respectively. These complexes were interacted with calf thymus DNA (ct-DNA) via positive cooperative interaction. The modes of binding of the complexes to DNA were investigated by fluorescence spectroscopy and circular dichroism in combination with a molecular docking study. The result indicates that complexes with small or branched hydrocarbon chains can intercalate with DNA. This is while amyl complexes with linear chains interacted additionally via groove binding. The results of the negative value of Gibbs energy for binding of isopentyl-platinum to DNA and those of the molecular docking were coherent. Furthermore, the docking results demonstrated that hydrophobic interaction plays an important role in the complex-DNA interaction.

  10. Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

    PubMed

    Wang, Amy; Wolf, Douglas C; Sen, Banalata; Knapp, Geremy W; Holladay, Steven D; Huckle, William R; Caceci, Thomas; Robertson, John L

    2009-06-01

    Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

  11. Nucleic acid sensing receptors in systemic lupus erythematosus: development of novel DNA- and/or RNA-like analogues for treating lupus

    PubMed Central

    Lenert, P

    2010-01-01

    Double-stranded (ds) DNA, DNA- or RNA-associated nucleoproteins are the primary autoimmune targets in SLE, yet their relative inability to trigger similar autoimmune responses in experimental animals has fascinated scientists for decades. While many cellular proteins bind non-specifically negatively charged nucleic acids, it was discovered only recently that several intracellular proteins are involved directly in innate recognition of exogenous DNA or RNA, or cytosol-residing DNA or RNA viruses. Thus, endosomal Toll-like receptors (TLR) mediate responses to double-stranded RNA (TLR-3), single-stranded RNA (TLR-7/8) or unmethylated bacterial cytosine (phosphodiester) guanine (CpG)-DNA (TLR-9), while DNA-dependent activator of IRFs/Z-DNA binding protein 1 (DAI/ZBP1), haematopoietic IFN-inducible nuclear protein-200 (p202), absent in melanoma 2 (AIM2), RNA polymerase III, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) mediate responses to cytosolic dsDNA or dsRNA, respectively. TLR-induced responses are more robust than those induced by cytosolic DNA- or RNA- sensors, the later usually being limited to interferon regulatory factor 3 (IRF3)-dependent type I interferon (IFN) induction and nuclear factor (NF)-κB activation. Interestingly, AIM2 is not capable of inducing type I IFN, but rather plays a role in caspase I activation. DNA- or RNA-like synthetic inhibitory oligonucleotides (INH-ODN) have been developed that antagonize TLR-7- and/or TLR-9-induced activation in autoimmune B cells and in type I IFN-producing dendritic cells at low nanomolar concentrations. It is not known whether these INH-ODNs have any agonistic or antagonistic effects on cytosolic DNA or RNA sensors. While this remains to be determined in the future, in vivo studies have already shown their potential for preventing spontaneous lupus in various animal models of lupus. Several groups are exploring the possibility of translating these INH-ODNs into

  12. 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.

  13. DNA Methylation of Cellular Retinoic Acid-Binding Proteins in Cervical Cancer.

    PubMed

    Arellano-Ortiz, Ana L; Salcedo-Vargas, Mauricio; Vargas-Requena, Claudia L; López-Díaz, José A; De la Mora-Covarrubias, Antonio; Silva-Espinoza, Juan C; Jiménez-Vega, Florinda

    2016-01-01

    This study determined the methylation status of cellular retinoic acid-binding protein (CRABP) gene promoters and associated them with demographic characteristics, habits, and the presence of human papilloma virus (HPV) in patients with cervical cancer (CC), low and high squamous intraepithelial lesions, and no intraepithelial lesion. Women (n = 158) were selected from the Colposcopy Clinic of Sanitary Jurisdiction II in Ciudad Juarez, Chihuahua, Mexico. Demographic characteristics and habit information were collected. Cervical biopsy and endocervical scraping were used to determine methylation in promoter regions by methylation-specific polymerase chain reaction technique. We found hemi-methylation patterns in the promoter regions of CRABP1 and CRABP2; there was 28.5% hemi-methylation in CRABP1 and 7.0% in that of CRABP2. Methylation in CRABP1 was associated with age (≥35 years, P = 0.002), family history of cancer (P = 0.032), the presence of HPV-16 (P = 0.013), and no alcohol intake (P = 0.035). These epigenetic changes could be involved in the CC process, and CRABP1 has the potential to be a predictive molecular marker of retinoid therapy response.

  14. Inhibitory Effects of Glycyrrhetinic Acid on DNA Polymerase and Inflammatory Activities

    PubMed Central

    Ishida, Tsukasa; Mizushina, Yoshiyuki; Yagi, Saori; Irino, Yasuhiro; Nishiumi, Shin; Miki, Ikuya; Kondo, Yasuyuki; Mizuno, Shigeto; Yoshida, Hiromi; Azuma, Takeshi; Yoshida, Masaru

    2012-01-01

    We investigated the inhibitory effect of three glycyrrhizin derivatives, such as Glycyrrhizin (compound 1), dipotassium glycyrrhizate (compound 2) and glycyrrhetinic acid (compound 3), on the activity of mammalian pols. Among these derivatives, compound 3 was the strongest inhibitor of mammalian pols α, β, κ, and λ, which belong to the B, A, Y, and X families of pols, respectively, whereas compounds 1 and 2 showed moderate inhibition. Among the these derivatives tested, compound 3 displayed strongest suppression of the production of tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in a cell-culture system using mouse macrophages RAW264.7 and peritoneal macrophages derived from mice. Moreover, compound 3 was found to inhibit the action of nuclear factor-κB (NF-κB) in engineered human embryonic kidney (HEK) 293 cells. In addition, compound 3 caused greater reduction of 12-O-tetradecanoylphorbol-13-acetate-(TPA-) induced acute inflammation in mouse ear than compounds 1 and 2. In conclusion, this study has identified compound 3, which is the aglycone of compounds 1 and 2, as a promising anti-inflammatory candidate based on mammalian pol inhibition. PMID:21785649

  15. DNA Methylation of Cellular Retinoic Acid-Binding Proteins in Cervical Cancer

    PubMed Central

    Arellano-Ortiz, Ana L.; Salcedo-Vargas, Mauricio; Vargas-Requena, Claudia L.; López-Díaz, José A.; De la Mora-Covarrubias, Antonio; Silva-Espinoza, Juan C.; Jiménez-Vega, Florinda

    2016-01-01

    This study determined the methylation status of cellular retinoic acid-binding protein (CRABP) gene promoters and associated them with demographic characteristics, habits, and the presence of human papilloma virus (HPV) in patients with cervical cancer (CC), low and high squamous intraepithelial lesions, and no intraepithelial lesion. Women (n = 158) were selected from the Colposcopy Clinic of Sanitary Jurisdiction II in Ciudad Juarez, Chihuahua, Mexico. Demographic characteristics and habit information were collected. Cervical biopsy and endocervical scraping were used to determine methylation in promoter regions by methylation-specific polymerase chain reaction technique. We found hemi-methylation patterns in the promoter regions of CRABP1 and CRABP2; there was 28.5% hemi-methylation in CRABP1 and 7.0% in that of CRABP2. Methylation in CRABP1 was associated with age (≥35 years, P = 0.002), family history of cancer (P = 0.032), the presence of HPV-16 (P = 0.013), and no alcohol intake (P = 0.035). These epigenetic changes could be involved in the CC process, and CRABP1 has the potential to be a predictive molecular marker of retinoid therapy response. PMID:27867303

  16. Multivalent ion-mediated nucleic acid helix-helix interactions: RNA versus DNA.

    PubMed

    Wu, Yuan-Yan; Zhang, Zhong-Liang; Zhang, Jin-Si; Zhu, Xiao-Long; Tan, Zhi-Jie

    2015-07-13

    Ion-mediated interaction is critical to the structure and stability of nucleic acids. Recent experiments suggest that the multivalent ion-induced aggregation of double-stranded (ds) RNAs and DNAs may strongly depend on the topological nature of helices, while there is still lack of an understanding on the relevant ion-mediated interactions at atomistic level. In this work, we have directly calculated the potentials of mean force (PMF) between two dsRNAs and between two dsDNAs in Co(NH3)6 (3+) (Co-Hex) solutions by the atomistic molecular dynamics simulations. Our calculations show that at low [Co-Hex], the PMFs between B-DNAs and between A-RNAs are both (strongly) repulsive. However, at high [Co-Hex], the PMF between B-DNAs is strongly attractive, while those between A-RNAs and between A-DNAs are still (weakly) repulsive. The microscopic analyses show that for A-form helices, Co-Hex would become 'internal binding' into the deep major groove and consequently cannot form the evident ion-bridge between adjacent helices, while for B-form helices without deep grooves, Co-Hex would exhibit 'external binding' to strongly bridge adjacent helices. In addition, our further calculations show that, the PMF between A-RNAs could become strongly attractive either at very high [Co-Hex] or when the bottom of deep major groove is fixed with a layer of water.

  17. Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells.

    PubMed

    Kumar, Devbrat; Basu, Soumya; Parija, Lucy; Rout, Deeptimayee; Manna, Sanjeet; Dandapat, Jagneshwar; Debata, Priya Ranjan

    2016-07-01

    Cervical cancer and precancerous lesions of the cervix continue to be a global health issue, and the medication for the treatment for chronic HPV infection so far has not been effective. Potential anticancer and anti HPV activities of two known phytochemicals, Curcumin and Ellagic acid were evaluated in HeLa cervical cancer cells. Curcumin is a natural compound found in the root of Curcuma longa plant and Ellagic acid a polyphenol found in fruits of strawberries, raspberries and walnuts. The combination of Curcumin and Ellagic acid at various concentrations showed better anticancer properties than either of the drug when used alone as evidenced by MTT assay. Besides this, Curcumin and Ellagic acid also restore p53, induce ROS formation and DNA damage. Mechanistic study further indicated that Curcumin and Ellagic acid show anti-HPV activity as evidenced by decrease in the HPV E6 oncoprotein on HeLa cells.

  18. Analysis of cloned cDNA and genomic sequences for phytochrome: complete amino acid sequences for two gene products expressed in etiolated Avena.

    PubMed Central

    Hershey, H P; Barker, R F; Idler, K B; Lissemore, J L; Quail, P H

    1985-01-01

    Cloned cDNA and genomic sequences have been analyzed to deduce the amino acid sequence of phytochrome from etiolated Avena. Restriction endonuclease site polymorphism between clones indicates that at least four phytochrome genes are expressed in this tissue. Sequence analysis of two complete and one partial coding region shows approximately 98% homology at both the nucleotide and amino acid levels, with the majority of amino acid changes being conservative. High sequence homology is also found in the 5'-untranslated region but significant divergence occurs in the 3'-untranslated region. The phytochrome polypeptides are 1128 amino acid residues long corresponding to a molecular mass of 125 kdaltons. The known protein sequence at the chromophore attachment site occurs only once in the polypeptide, establishing that phytochrome has a single chromophore per monomer covalently linked to Cys-321. Computer analyses of the amino acid sequences have provided predictions regarding a number of structural features of the phytochrome molecule. PMID:3001642

  19. Synthesis, spectroscopic characterization and in vitro antimicrobial, anticancer and antileishmanial activities as well interaction with Salmon sperm DNA of newly synthesized carboxylic acid derivative, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid.

    PubMed

    Sirajuddin, Muhammad; Ali, Saqib; McKee, Vickie; Ullah, Hameed

    2015-03-05

    This paper stresses on the synthesis, characterization of novel carboxylic acid derivative and its application in pharmaceutics. Carboxylic acid derivatives have a growing importance in medicine, particularly in oncology. A novel carboxylic acid, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid, was synthesized and characterized by elemental analysis, FT-IR, NMR ((1)H, and (13)C), mass spectrometry and single crystal X-ray structural analysis. The structure of the title compound, C11H12N2O6, shows the molecules dimerised by short intramolecular OH⋯O hydrogen bonds. The compound was screened for in vitro antimicrobial, anticancer, and antileishmanial activities as well as interaction with SS-DNA. The compound was also checked for in vitro anticancer activity against BHK-21, H-157 and HCEC cell lines, and showed significant anticancer activity. The compound was almost non-toxic towards human corneal epithelial cells (HCEC) and did not show more than 7.4% antiproliferative activity when used at the 2.0μg/mL end concentration. It was also tested for antileishmanial activity against the promastigote form of leishmania major and obtained attractive result. DNA interaction study exposes that the binding mode of the compound with SS-DNA is an intercalative as it results in hypochromism along with minor red shift. A new and efficient strategy to identify pharmacophores sites in carboxylic acid derivative for antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

  20. Synthesis, spectroscopic characterization and in vitro antimicrobial, anticancer and antileishmanial activities as well interaction with Salmon sperm DNA of newly synthesized carboxylic acid derivative, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid

    NASA Astrophysics Data System (ADS)

    Sirajuddin, Muhammad; Ali, Saqib; McKee, Vickie; Ullah, Hameed

    2015-03-01

    This paper stresses on the synthesis, characterization of novel carboxylic acid derivative and its application in pharmaceutics. Carboxylic acid derivatives have a growing importance in medicine, particularly in oncology. A novel carboxylic acid, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid, was synthesized and characterized by elemental analysis, FT-IR, NMR (1H, and 13C), mass spectrometry and single crystal X-ray structural analysis. The structure of the title compound, C11H12N2O6, shows the molecules dimerised by short intramolecular Osbnd H⋯O hydrogen bonds. The compound was screened for in vitro antimicrobial, anticancer, and antileishmanial activities as well as interaction with SS-DNA. The compound was also checked for in vitro anticancer activity against BHK-21, H-157 and HCEC cell lines, and showed significant anticancer activity. The compound was almost non-toxic towards human corneal epithelial cells (HCEC) and did not show more than 7.4% antiproliferative activity when used at the 2.0 μg/mL end concentration. It was also tested for antileishmanial activity against the promastigote form of leishmania major and obtained attractive result. DNA interaction study exposes that the binding mode of the compound with SS-DNA is an intercalative as it results in hypochromism along with minor red shift. A new and efficient strategy to identify pharmacophores sites in carboxylic acid derivative for antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.