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Sample records for heterocycle differences dna

  1. Caffeine and other xanthines as cytochemical blockers and removers of heterocyclic DNA intercalators from chromatin.

    PubMed

    Lyles, Mark B; Cameron, Ivan L

    2002-01-01

    Caffeine (CAF) and other xanthines non-covalently bind with the cationic fluorescent dye acridine orange (AO) and with other heterocyclic mutagens and carcinogens that are known to intercalate into double-stranded DNA (dsDNA). Fluorescence microscopy and spectrofluorometry studies were employed to test the ability of caffeine and certain other methyl substituted xanthines, with different binding affinities for AO, to inhibit and to reverse the intercalation of AO and other heterocyclic agents from intercalation with the DNA of nuclear chromatin of air-dried cells. Results indicated that xanthines with binding affinity for AO greater than 150 m(-1) block the AO molecule in a concentration dependent manner and comply with mass action kinetics. Thus CAF and other xanthines can be used to either inhibit intercalation of AO into nuclear DNA or to remove AO once intercalated into nuclear DNA. The interactions between other planar heterocyclics, xanthines, and nuclear chromatin dsDNA were also found to be non-covalent. Studies are needed to determine the ability of CAF and other xanthines to block and/or remove polyaromatic hydrocarbon (PAH) intercalators from the DNA of living cells.

  2. Complexation of heterocyclic ligands with DNA in aqueous solution

    NASA Astrophysics Data System (ADS)

    Baranovskii, S. F.; Bolotin, P. A.; Evstigneev, M. P.; Chernyshev, D. N.

    2008-03-01

    We have used spectrophotometry to study self-association and complexation with DNA by organic heterocyclic compounds in the acridine and phenothiazine series: proflavin, thionine, and methylene blue. Based on the experimental concentration dependences of the molar absorption coefficient of the molecules in an aqueous buffer solution (0.01 M NaCl, 0.01 M Na2EDTA, 0.01 M Tris, pH 7.4, T = 298 K), we have determined the equilibrium dimerization constants for the dyes and the DNA complexation parameters using the Scatchard and McGhee-von Hippel models. The observed increase in the cooperativity parameters as the dimerization constants of the ligands increase allowed us to hypothesize that the same interactions occur between dye molecules adsorbed on DNA as in their self-association. The equilibrium DNA-binding constants for the ligands, obtained using the McGhee-von Hippel cooperative model, are (20.9 ± 2.7)·103 M-1 for proflavin and (33.8 ± 4.1)·103 M-1 for thionine. Using the Scatchard model, taking into account intercalation and “external” binding of ligands with DNA, we determined the DNA complexation constants for methylene blue: (26.4 ± 4.6)·103 and (96 ± 17)·103 M-1 respectively. Based on analysis of the data obtained, we hypothesized that the predominant type of binding with DNA is intercalation binding in the case of proflavin and thionine, and “external” binding with the DNA surface in the case of methylene blue.

  3. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

    NASA Astrophysics Data System (ADS)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  4. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    PubMed

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-05

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes

    PubMed Central

    Nauwelaers, Gwendoline; Bessette, Erin E.; Gu, Dan; Tang, Yijin; Rageul, Julie; Fessard, Valérie; Yuan, Jian-Min; Yu, Mimi C.; Langouët, Sophie; Turesky, Robert J.

    2011-01-01

    DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 107 DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation. PMID:21456541

  6. 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.

  7. DNA sequence-selective C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates show anti-tubercular-specific activities.

    PubMed

    Brucoli, Federico; Guzman, Juan D; Basher, Mohammad A; Evangelopoulos, Dimitrios; McMahon, Eleanor; Munshi, Tulika; McHugh, Timothy D; Fox, Keith R; Bhakta, Sanjib

    2016-12-01

    New chemotherapeutic agents with novel mechanisms of action are in urgent need to combat the tuberculosis pandemic. A library of 12 C8-linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD)-heterocyclic polyamide conjugates (1-12) was evaluated for anti-tubercular activity and DNA sequence selectivity. The PBD conjugates were screened against slow-growing Mycobacterium bovis Bacillus Calmette-Guérin and M. tuberculosis H37Rv, and fast-growing Escherichia coli, Pseudomonas putida and Rhodococcus sp. RHA1 bacteria. DNase I footprinting and DNA thermal denaturation experiments were used to determine the molecules' DNA recognition properties. The PBD conjugates were highly selective for the mycobacterial strains and exhibited significant growth inhibitory activity against the pathogenic M. tuberculosis H37Rv, with compound 4 showing MIC values (MIC=0.08 mg l(-1)) similar to those of rifampin and isoniazid. DNase I footprinting results showed that the PBD conjugates with three heterocyclic moieties had enhanced sequence selectivity and produced larger footprints, with distinct cleavage patterns compared with the two-heterocyclic chain PBD conjugates. DNA melting experiments indicated a covalent binding of the PBD conjugates to two AT-rich DNA-duplexes containing either a central GGATCC or GTATAC sequence, and showed that the polyamide chains affect the interactions of the molecules with DNA. The PBD-C8 conjugates tested in this study have a remarkable anti-mycobacterial activity and can be further developed as DNA-targeted anti-tubercular drugs.

  8. Effects of (+)-catechin and (-)-epicatechin on heterocyclic amines-induced oxidative DNA damage.

    PubMed

    Haza, Ana Isabel; Morales, Paloma

    2011-01-01

    The aim of the present study was to evaluate the protective effect of (+)-catechin and (-)-epicatechin against 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline (4,8-diMeIQx) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Increasing concentrations of 8-MeIQx, 4,8-diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose-dependent manner. Among those, PhIP (300 µm) exerted the highest genotoxicity. (+)-Catechin exerted protection against oxidized purines induced by 8-MeIQx, 4,8-diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)-catechin. Otherwise, (-)-epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8-MeIQx and 4,8-diMeIQx. One feasible mechanism by which (+)-catechin and (-)-epicatechin exert their protective effect towards heterocyclic amines-induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O-deethylation (CYP1A1) activity was moderately inhibited by (+)-catechin, while little effect was observed by (-)-epicatechin. However, (+)-catechin showed the greatest increase in UDP-glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)-catechin was more efficient than (-)-epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8-MeIQx and 4,8-diMeIQx. Copyright © 2010 John Wiley & Sons, Ltd.

  9. Mechanism and nature of the different viscosity sensitivities of hemicyanine dyes with various heterocycles.

    PubMed

    Cao, Jianfang; Hu, Chong; Liu, Fei; Sun, Wen; Fan, Jiangli; Song, Fengling; Sun, Shiguo; Peng, Xiaojun

    2013-06-03

    A series of hemicyanine derivatives are excellent fluorescent viscosity sensors in live cells and in imaging of living tissues due to their low quantum yields in solution but large fluorescence enhancements in viscous environments. Herein, three carbazole-based hemicyanine dyes with different heterocycles are studied. They have different background quantum yields, and hence different sensitivities to viscosity detection, large Stokes shifts, and high sensitivity. Better understanding of the structure-property relationships for viscosity sensitivity could benefit the design of improved dyes. Computational studies on these dyes reveal the mechanism of viscosity sensitivity of fluorescent molecular rotors and the nature of the difference in viscosity sensitivity of the three dyes. The results show that the greatly raised HOMO and greatly lowered LUMO in the S1 state compared with the S0 state are responsible for the large Stokes shift of the three dyes. The heterocyclic moieties have the primary influence on the LUMO levels of the three hemicyanine dyes. Rotation about the C-C bond adjacent to the carbazole moiety of the three dyes drives the molecule toward a small energy gap between the ground state and the first excited state, which causes mainly nonradiative deactivation. The oscillator strengths in the lowest singlet excited state drop rapidly with increasing rotation between 0 and 95°, which leads to a dark state for these dyes when fully twisted at 95°. We draw a mechanistic picture at the molecular level to illustrate how these dyes work as viscosity-sensitive fluorescent probes. The activation barriers and energy gaps of C-C bond rotation strongly depend on the choice of heterocycle, which plays a major role in reducing fluorescence quantum yield in the free state and provides high sensitivity to viscosity detection in viscous environments for the carbazole-based hemicyanine dyes.

  10. Binding to the DNA Minor Groove by Heterocyclic Dications: From AT Specific Monomers to GC Recognition with Dimers

    PubMed Central

    Nanjunda, Rupesh; Wilson, W. David

    2012-01-01

    Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, they have found extensive uses in biotechnology and they are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds have shown us that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, and the compounds can form cooperative stacked dimers to recognize GC and mixed AT/GC base pair sequences. PMID:23255206

  11. Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

    PubMed Central

    Nhili, Raja; Peixoto, Paul; Depauw, Sabine; Flajollet, Sébastien; Dezitter, Xavier; Munde, Manoj M.; Ismail, Mohamed A.; Kumar, Arvind; Farahat, Abdelbasset A.; Stephens, Chad E.; Duterque-Coquillaud, Martine; David Wilson, W.; Boykin, David W.; David-Cordonnier, Marie-Hélène

    2013-01-01

    Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site. PMID:23093599

  12. Design and synthesis of heterocyclic cations for specific DNA recognition: from AT-rich to mixed-base-pair DNA sequences.

    PubMed

    Chai, Yun; Paul, Ananya; Rettig, Michael; Wilson, W David; Boykin, David W

    2014-02-07

    The compounds synthesized in this research were designed with the goal of establishing a new paradigm for mixed-base-pair DNA sequence-specific recognition. The design scheme starts with a cell-permeable heterocyclic cation that binds to AT base pair sites in the DNA minor groove. Modifications were introduced in the original compound to include an H-bond accepting group to specifically recognize the G-NH that projects into the minor groove. Therefore, a series of heterocyclic cations substituted with an azabenzimidazole ring has been designed and synthesized for mixed-base-pair DNA recognition. The most successful compound, 12a, had an azabenzimidazole to recognize G and additional modifications for general minor groove interactions. It binds to the DNA site -AAAGTTT- more strongly than the -AAATTT- site without GC and indicates the design success. Structural modifications of 12a generally weakened binding. The interactions of the new compound with a variety of DNA sequences with and without GC base pairs were evaluated by thermal melting analysis, circular dichroism, fluorescence emission spectroscopy, surface plasmon resonance, and molecular modeling.

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

    PubMed

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

    2016-10-07

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

  14. Trifluoromethylated heterocycles.

    PubMed

    Gakh, Andrei A; Shermolovich, Yuriy

    2014-01-01

    This review is a follow-up to the previous chapter, "Monofluorinated Heterocycles" (Topics in Heterocyclic Chemistry, 2012, 33-63), and presents an overview of synthetic chemistry of heterocycles with only one trifluoromethyl group directly attached to the ring (trifluoromethylated heterocycles). Particular attention is given to the modern direct trifluoromethylation methods, including catalytic reactions, organometallic reagents, carbene and hypervalent chemistry, utilization of ionic nucleophilic and electrophilic trifluoromethylating agents, and to other pertinent trends. One of the emphases of the review is compounds with biomedical potential.

  15. Vanadium(IV) and copper(II) complexes of salicylaldimines and aromatic heterocycles: Cytotoxicity, DNA binding and DNA cleavage properties.

    PubMed

    Correia, Isabel; Roy, Somnath; Matos, Cristina P; Borovic, Sladjana; Butenko, Nataliya; Cavaco, Isabel; Marques, Fernanda; Lorenzo, Julia; Rodríguez, Alejandra; Moreno, Virtudes; Pessoa, João Costa

    2015-06-01

    Five copper(II) complexes, [Cu(sal-Gly)(bipy)](1), [Cu(sal-Gly)(phen)] (2), [Cu(sal-l-Ala)(phen)] (3), [Cu(sal-D-Ala)(phen)] (4), [Cu(sal-l-Phe)(phen)] (5) and five oxidovanadium(IV) complexes, [V(IV)O(sal-Gly)(bipy)] (6), [V(IV)O(sal-Gly)(phen)] (7), [V(IV)O(sal-l-Phe)(H2O)] (8), [V(IV)O(sal-l-Phe)(bipy)] (9), [V(IV)O(sal-l-Phe)(phen)] (10) (sal=salicylaldehyde, bipy=2,2'-bipyridine, phen=1,10-phenanthroline) were synthesized and characterized, and their interaction with DNA was evaluated by different techniques: gel electrophoresis, fluorescence, UV-visible and circular dichroism spectroscopy. The complexes interact with calf-thymus DNA and efficiently cleave plasmid DNA in the absence (only 2 and 5) and/or presence of additives. The cleavage ability is concentration-dependent as well as metal and ligand-dependent. Moreover, DNA binding experiments show that the phen-containing Cu(II) and V(IV)O compounds display stronger DNA interaction ability than the corresponding bipy analogues. The complexes present cytotoxic activity against human ovarian (A2780) and breast (MCF7) carcinoma cells. Cell-growth inhibition (IC50) of compounds 1, 2 and 5 in human promyelocytic leukemia (HL60) and human cervical cancer (HeLa) cells were also determined. The copper complexes show much higher cytotoxic activity than the corresponding vanadium complexes and the reference drug cisplatin (except for the sal-Gly complexes); namely, the phenanthroline copper complexes 2-5 are ca. 10-fold more cytotoxic than cisplatin and more cytotoxic than their bipyridine analogues.

  16. Heterocyclic chemistry in crop protection.

    PubMed

    Lamberth, Clemens

    2013-10-01

    An overview is given of the significance of heterocycles in crop protection chemistry, which is enormous as more than two-thirds of all agrochemicals launched to the market within the last 20 years belong to this huge group of chemicals. This review focuses on two important aspects of heterocyclic agrochemistry: the different roles of heterocyclic scaffolds in crop protection agents and the major possibilities for their synthesis.

  17. DNA Cleavage, Cytotoxic Activities, and Antimicrobial Studies of Ternary Copper(II) Complexes of Isoxazole Schiff Base and Heterocyclic Compounds

    PubMed Central

    Chityala, Vijay Kumar; Sathish Kumar, K.; Macha, Ramesh; Tigulla, Parthasarathy; Shivaraj

    2014-01-01

    Novel mixed ligand bivalent copper complexes [Cu. L. A. ClO4] and [Cu. L. A] where “L” is Schiff bases, namely 2-((3,4-dimethylisoxazol-5-ylimino)methyl)-4-bromophenol (DMIIMBP)/2-((3,4-dimethylisoxazol-5-ylimino)methyl)-4-chlorophenol (DMIIMCP), and “A” is heterocyclic compound, such as 1,10-phenanthroline (phen)/2,21-bipyridyl (bipy)/8-hydroxyquinoline (oxine)/5-chloro-8-hydroxyquinoline (5-Cl-oxine), have been synthesized. These complexes have been characterized by IR, UV-Vis, ESR, elemental analysis, magnetic moments, TG, and DTA. On the basis of spectral studies and analytical data, five-coordinated square pyramidal/four-coordinated square planar geometry is assigned to all complexes. The ligands and their ternary complexes with Cu(II) have been screened for antimicrobial activity against bacteria and fungi by paper disc method. The antimicrobial studies of Schiff bases and their metal complexes showed significant activity and further it is observed that the metal complexes showed more activity than corresponding Schiff bases. In vitro antitumor activity of Cu(II) complexes was assayed against human cervical carcinoma (HeLa) cancer cells and it was observed that few complexes exhibit good antitumor activity on HeLa cell lines. The DNA cleavage studies have also been carried out on pBR 322 and it is observed that these Cu(II) complexes are capable of cleaving supercoiled plasmid DNA in the presence of H2O2 and UV light. PMID:24895493

  18. Synthesis, crystal structure, DNA binding and photo-induced DNA cleavage activity of (S-methyl-L-cysteine)copper(II) complexes of heterocyclic bases.

    PubMed

    Patra, Ashis K; Nethaji, Munirathinam; Chakravarty, Akhil R

    2007-02-01

    Ternary S-methyl-L-cysteine (SMe-l-cys) copper(II) complexes [Cu(SMe-L-cys)(B)(H(2)O)](X) (1-4), where the heterocyclic base B is 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyridoquinoxaline (dpq, 3) and dipyridophenazine (dppz, 4), and X is ClO(4)(-) (1-3) or NO(3)(-) (4), are prepared and their DNA binding and cleavage properties studied. Complexes 2 and 4 are structurally characterized by X-ray crystallography. Both the crystal structures show distorted square-pyramidal (4+1) CuN(3)O(2) coordination geometry of the complexes in which the N,O-donor S-methyl-L-cysteine and N,N-donor heterocyclic base bind at the basal plane with a water molecule as the axial ligand. In addition, the dppz structure shows the presence of a 1D-chain formed due to covalent linkage of the carboxylate oxygen atom belonging to another molecule at the elongated axial site. The crystal structures show chemically significant non-covalent interactions like hydrogen bonding involving the axial aqua ligand and pi-pi interactions between dppz ligands. The complexes display a d-d band in the range of 605-654 nm in aqueous dimethylformamide (DMF) solution (9:1 v/v). The redox active complexes show quasireversible cyclic voltammetric response near 0.1 V in DMF assignable to the Cu(II)/Cu(I) couple. The complexes show good binding affinity to calf thymus (CT) DNA giving the order: 4 (dppz)>3 (dpq)>2 (phen)>1 (bpy). The intrinsic binding constants, obtained from UV-visible spectroscopic studies, are 1.3x10(4) and 2.15 x 10(4) M(-1) for 3 and 4, respectively. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder distamycin suggest major groove binding propensity for the dppz complex, while the phen and dpq complexes bind at the minor groove of DNA. Complexes 2-4 show DNA cleavage activity in dark in the presence of a reducing agent 3-mercaptopropionic acid (MPA) via a mechanistic pathway involving formation of hydroxyl radical as the reactive

  19. Picolinic acid based Cu(II) complexes with heterocyclic bases--crystal structure, DNA binding and cleavage studies.

    PubMed

    Pulimamidi, Rabindra Reddy; Nomula, Raju; Pallepogu, Raghavaiah; Shaik, Hussain

    2014-05-22

    In view of the importance of picolinic acid (PA) in preventing cell growth and arresting cell cycle, new PA based metallonucleases were designed with a view to study their DNA binding and cleavage abilities. Three new Cu(II) complexes [Cu(II)(DPPA)].4H2O (1),[Cu(II)(DPPA)(bpy)].5H2O (2) and [Cu(II)(DPPA)(phen)].5H2O (3), were synthesized using a picolinic acid based bifunctional ligand (DPPA) and heterocyclic bases (where DPPA: Pyridine-2-carboxylic acid {2-phenyl-1-[(pyridin-2-ylmethyl)-carbonyl]-ethyl}-amide; bpy: 2, 2'-bipyridine and phen: 1, 10-phenanthroline). DPPA was obtained by coupling 2-picolinic acid and 2-picolyl amine with l-phenylalanine through amide bond‌‌. Complexes were structurally characterized by a single crystal X-ray crystallography. The molecular structure of 1 shows Cu(II) center essentially in a square planar coordination geometry, while complex 2 shows an approximate five coordinated square-pyramidal geometry. Eventhough we could not isolate single crystal for complex (3), its structure was established based on other techniques. The complex (3) also exhibits five coordinate square pyramidal geometry. The complexes show good binding affinity towards CT-DNA. The binding constants (Kb) decrease in the order 1.35 ± 0.01 × 10(5) (3) > 1.23 ± 0.01 × 10(5) (2) > 8.3 ± 0.01 × 10(4) (1) M(-1). They also exhibit efficient nuclease activity towards supercoiled pUC19 DNA both in the absence and presence of external agent (H2O2). The kinetic studies reveal that the hydrolytic cleavage reactions follow the pseudo first-order rate constant and the hydrolysis rates are in the range of (5.8-8.0) × 10(7) fold rate enhancement compared to non-catalyzed double stranded DNA (3.6 × 10(-8) h(-1)). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. Inhibition of DNA gyrase by levofloxacin and related fluorine-containing heterocyclic compounds.

    PubMed

    Tunitskaya, V L; Khomutov, A R; Kochetkov, S N; Kotovskaya, S K; Charushin, V N

    2011-10-01

    Fluoroquinolones are an important class of modern and efficient antibacterial drugs with a broad spectrum of activity. Levofloxacin (the optically active form of ofloxacin) is one of the most promising fluoroquinolone drugs, and its antibacterial activity is substantially higher than the activity of other drugs of the fluoroquinolone family. Earlier, in the Postovsky Institute of Organic Synthesis, UB RAS, an original method of levofloxacin synthesis was developed, and now the pilot batch of the drug is being prepared. Bacterial DNA gyrase is a specific target of fluoroquinolones; hence, the study of the enzyme-drug interaction is of theoretical and practical importance. Moreover, the parameters of DNA gyrase inhibition may serve as a criterion for drug quality. Here, we present the results of studying the interaction of DNA gyrase with a number of fluoroquinolones and their analogs: intermediates and semi-products of the levofloxacin synthesis, and also samples from the pilot batches of this drug. The importance of two structural elements of the levofloxacin molecule for the efficiency of the inhibition is revealed. The data obtained may be useful for the design of new drugs derived from levofloxacin.

  1. Striking Difference in Antiproliferative Activity of Ruthenium- and Osmium-Nitrosyl Complexes with Azole Heterocycles

    PubMed Central

    2013-01-01

    Ruthenium nitrosyl complexes of the general formulas (cation)+[cis-RuCl4(NO)(Hazole)]−, where (cation)+ = (H2ind)+, Hazole = 1H-indazole (Hind) (1c), (cation)+ = (H2pz)+, Hazole = 1H-pyrazole (Hpz) (2c), (cation)+ = (H2bzim)+, Hazole = 1H-benzimidazole (Hbzim) (3c), (cation)+ = (H2im)+, Hazole = 1H-imidazole (Him) (4c) and (cation)+[trans-RuCl4(NO)(Hazole)]−, where (cation)+ = (H2ind)+, Hazole = 1H-indazole (1t), (cation)+ = (H2pz)+, Hazole = 1H-pyrazole (2t), as well as osmium analogues of the general formulas (cation)+[cis-OsCl4(NO)(Hazole)]−, where (cation)+ = (n-Bu4N)+, Hazole =1H-indazole (5c), 1H-pyrazole (6c), 1H-benzimidazole (7c), 1H-imidazole (8c), (cation)+ = Na+; Hazole =1H-indazole (9c), 1H-benzimidazole (10c), (cation)+ = (H2ind)+, Hazole = 1H-indazole (11c), (cation)+ = H2pz+, Hazole = 1H-pyrazole (12c), (cation)+ = (H2im)+, Hazole = 1H-imidazole (13c), and (cation)+[trans-OsCl4(NO)(Hazole)]−, where (cation)+ = n-Bu4N+, Hazole = 1H-indazole (5t), 1H-pyrazole (6t), (cation)+ = Na+, Hazole = 1H-indazole (9t), (cation)+ = (H2ind)+, Hazole = 1H-indazole (11t), (cation)+ = (H2pz)+, Hazole = 1H-pyrazole (12t), have been synthesized. The compounds have been comprehensively characterized by elemental analysis, ESI mass spectrometry, spectroscopic techniques (IR, UV–vis, 1D and 2D NMR) and X-ray crystallography (1c·CHCl3, 1t·CHCl3, 2t, 3c, 6c, 6t, 8c). The antiproliferative activity of water-soluble compounds (1c, 1t, 3c, 4c and 9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c) in the human cancer cell lines A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon adenocarcinoma) has been assayed. The effects of metal (Ru vs Os), cis/trans isomerism, and azole heterocycle identity on cytotoxic potency and cell line selectivity have been elucidated. Ruthenium complexes (1c, 1t, 3c, and 4c) yielded IC50 values in the low micromolar concentration range. In contrast to most pairs of analogous ruthenium and osmium complexes known, they turned

  2. Striking difference in antiproliferative activity of ruthenium- and osmium-nitrosyl complexes with azole heterocycles.

    PubMed

    Büchel, Gabriel E; Gavriluta, Anatolie; Novak, Maria; Meier, Samuel M; Jakupec, Michael A; Cuzan, Olesea; Turta, Constantin; Tommasino, Jean-Bernard; Jeanneau, Erwann; Novitchi, Ghenadie; Luneau, Dominique; Arion, Vladimir B

    2013-06-03

    Ruthenium nitrosyl complexes of the general formulas (cation)(+)[cis-RuCl4(NO)(Hazole)](-), where (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (Hind) (1c), (cation)(+) = (H2pz)(+), Hazole = 1H-pyrazole (Hpz) (2c), (cation)(+) = (H2bzim)(+), Hazole = 1H-benzimidazole (Hbzim) (3c), (cation)(+) = (H2im)(+), Hazole = 1H-imidazole (Him) (4c) and (cation)(+)[trans-RuCl4(NO)(Hazole)](-), where (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (1t), (cation)(+) = (H2pz)(+), Hazole = 1H-pyrazole (2t), as well as osmium analogues of the general formulas (cation)(+)[cis-OsCl4(NO)(Hazole)](-), where (cation)(+) = (n-Bu4N)(+), Hazole =1H-indazole (5c), 1H-pyrazole (6c), 1H-benzimidazole (7c), 1H-imidazole (8c), (cation)(+) = Na(+); Hazole =1H-indazole (9c), 1H-benzimidazole (10c), (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (11c), (cation)(+) = H2pz(+), Hazole = 1H-pyrazole (12c), (cation)(+) = (H2im)(+), Hazole = 1H-imidazole (13c), and (cation)(+)[trans-OsCl4(NO)(Hazole)](-), where (cation)(+) = n-Bu4N(+), Hazole = 1H-indazole (5t), 1H-pyrazole (6t), (cation)(+) = Na(+), Hazole = 1H-indazole (9t), (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (11t), (cation)(+) = (H2pz)(+), Hazole = 1H-pyrazole (12t), have been synthesized. The compounds have been comprehensively characterized by elemental analysis, ESI mass spectrometry, spectroscopic techniques (IR, UV-vis, 1D and 2D NMR) and X-ray crystallography (1c·CHCl3, 1t·CHCl3, 2t, 3c, 6c, 6t, 8c). The antiproliferative activity of water-soluble compounds (1c, 1t, 3c, 4c and 9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c) in the human cancer cell lines A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon adenocarcinoma) has been assayed. The effects of metal (Ru vs Os), cis/trans isomerism, and azole heterocycle identity on cytotoxic potency and cell line selectivity have been elucidated. Ruthenium complexes (1c, 1t, 3c, and 4c) yielded IC50 values in the low micromolar concentration range. In contrast to most

  3. Inhibitory effects of pomegranate seed extract on the formation of heterocyclic aromatic amines in beef and chicken meatballs after cooking by four different methods.

    PubMed

    Keşkekoğlu, Hasan; Uren, Ali

    2014-04-01

    Beef and chicken meatballs with a 0.5% (w/w) pomegranate seed extract were cooked using four different cooking methods (oven roasting, pan cooking, charcoal-barbecue, and deep-fat frying) and six heterocyclic aromatic amines; IQ, MeIQx, 4,8-DiMeIQx, PhIP, norharman, and harman were observed. In the beef meatballs, the highest inhibitory effects of pomegranate seed extract on heterocyclic aromatic amines formation were 68% for PhIP, 24% for norharman, 18% for harman, 45% for IQ, and 57% for MeIQx. Total heterocyclic aromatic amine formation was reduced by 39% and 46% in beef meatballs cooked by charcoal-barbecue and deep-fat frying, respectively. In the chicken meatballs, the highest inhibitory effects were 75% for PhIP, 57% for norharman, 28% for harman, 46% for IQ, and 49% for MeIQx. When the pomegranate seed extract was added to the chicken meatballs cooked by deep-fat frying, the total heterocyclic aromatic amine formation was inhibited by 49%, in contrast the total heterocyclic aromatic amine contents after oven roasting increased by 70%.

  4. The Unusual Monomer Recognition of Guanine-Containing Mixed Sequence DNA by a Dithiophene Heterocyclic Diamidine

    PubMed Central

    2015-01-01

    DB1255 is a symmetrical diamidinophenyl-dithiophene that exhibits cellular activity by binding to DNA and inhibiting binding of ERG, an ETS family transcription factor that is commonly overexpressed or translocated in leukemia and prostate cancer [Nhili, R., Peixoto, P., Depauw, S., Flajollet, S., Dezitter, X., Munde, M. M., Ismail, M. A., Kumar, A., Farahat, A. A., Stephens, C. E., Duterque-Coquillaud, M., Wilson, W. D., Boykin, D. W., and David-Cordonnier, M. H. (2013) Nucleic Acids Res. 41, 125–138]. Because transcription factor inhibition is complex but is an attractive area for anticancer and antiparasitic drug development, we have evaluated the DNA interactions of additional derivatives of DB1255 to gain an improved understanding of the biophysical chemistry of complex function and inhibition. DNase I footprinting, biosensor surface plasmon resonance, and circular dichroism experiments show that DB1255 has an unusual and strong monomer binding mode in minor groove sites that contain a single GC base pair flanked by AT base pairs, for example, 5′-ATGAT-3′. Closely related derivatives, such as compounds with the thiophene replaced with furan or selenophane, bind very weakly to GC-containing sequences and do not have biological activity. DB1255 is selective for the ATGAT site; however, a similar sequence, 5′-ATGAC-3′, binds DB1255 more weakly and does not produce a footprint. Molecular docking studies show that the two thiophene sulfur atoms form strong, bifurcated hydrogen bond-type interactions with the G-N-H sequence that extends into the minor groove while the amidines form hydrogen bonds to the flanking AT base pairs. The central dithiophene unit of DB1255 thus forms an excellent, but unexpected, single-GC base pair recognition module in a monomer minor groove complex. PMID:24495039

  5. Influence of different oligosaccharides and inulin on heterocyclic aromatic amine formation and overall mutagenicity in fried ground beef patties.

    PubMed

    Shin, Han-Seung; Park, Heekyung; Park, Daewoo

    2003-11-05

    The effects of different oligosaccharides [fructooligosaccharide (FOS), galactooligosaccharide (GOS), and isomaltooligosaccharide (MOS)] and inulin on heterocyclic aromatic amine (HAA) formation and overall mutagenicity in fried ground patties were evaluated. Different oligosaccharides and inulin was added directly to ground beef. Patties (100 g) were fried at 225 degrees C (surface temperature) for 10 min per side. FOS added at levels of 0.5, 1.0, 1.5, 2.0, and 2.5 g to 100 g of ground beef inhibited total HAA formation by 19, 32, 45, 51, and 58%, respectively. The addition of 1.5 g of FOS, GOS, MOS, and inulin to ground beef patties inhibited total HAA formation by 50, 47, 46, and 54%, respectively. They also reduced overall mutagenicity by 52, 51, 48, and 59%, respectively. These studies confirm that oligosaccharides and inulin have the potential to reduce HAA formation in cooked beef patties.

  6. Design and Synthesis of Novel Pyrazole-Substituted Different Nitrogenous Heterocyclic Ring Systems as Potential Anti-Inflammatory Agents.

    PubMed

    Nossier, Eman S; Fahmy, Hoda H; Khalifa, Nagy M; El-Eraky, Wafaa I; Baset, Marawan A

    2017-03-24

    With the aim of developing novel anti-inflammatory scaffolds, a new series of pyrazole-substituted various nitrogenous heterocyclic ring systems at C-4 position were synthesized through different chemical reactions and validated by means of spectral and elemental data. The new obtained compounds were investigated for their anti-inflammatory activity using the carrageenan-induced paw edema standard technique and revealed that, compound 6b showed increased potency with % inhibition of edema 85.23 ± 1.92 and 85.78 ± 0.99, respectively, higher than the standard reference drugs indomethacin and celebrex (72.99% and 83.76%). Molecular modeling studies were initiated herein to validate the attained pharmacological data and provide understandable evidence for the observed anti-inflammatory behavior.

  7. Pyrido[1,2-a]pyrimidinium ions--a novel bridgehead nitrogen heterocycles: synthesis, characterisation, and elucidation of DNA binding and cell imaging properties.

    PubMed

    Manna, Susanta Kumar; Mandal, Arabinda; Mondal, Suresh Kumar; Adak, Arup Kr; Jana, Akash; Das, Somnath; Chattopadhyay, Sourav; Roy, Somenath; Ghorai, Shyamal Kr; Samanta, Shubhankar; Hossain, Maidul; Baidya, Mahiuddin

    2015-08-07

    A novel class of bridgehead nitrogen heterocycles, pyrido[1,2-a]pyrimidinium ions, has been readily synthesized by a two-step one-pot reaction in high yields (up to 93%). These ionic compounds are bench stable and moisture tolerant and have highly fluorescent properties (quantum yield up to 0.65). A characteristic bright bluish fluorescence was observed in polar solvents such as acetonitrile and fluorescent intensity gradually diminishes with decreasing the polarity of the medium, which becomes almost negligible in toluene. These compounds also show interesting bioactivity. DNA interaction, imaging, and viability experiments with human leukemic Jurkat and KG-1A cells revealed that they are potential candidates for cancer diagnosis.

  8. Synthesis and antitumor activity evaluation of new 2-(4-aminophenyl)benzothiazole derivatives bearing different heterocyclic rings.

    PubMed

    Yurttaş, Leyla; Tay, Funda; Demirayak, Şeref

    2015-06-01

    Twenty-five new N-[4-(benzothiazole-2-yl)phenyl]acetamide derivatives bearing different heterocyclic ring systems were synthesized using 2-(4-aminophenyl)benzothiazole structure as a pharmacophoric group. Final compounds were screened for their potential antitumor activity in vitro against approximately 60 human tumor cell lines derived from nine neoplastic diseases at National Cancer Institute, USA. 2-(4-Aminophenyl)benzothiazole structure was prepared by the reaction of 4-aminobenzoic acid and 2-aminothiophenol in polyphosphoric acid using microwave irradiation. After acetylation reaction, amide compounds 2a and 2b were obtained, which were then reacted with 2-mercapto(benz)imidazole/benzothiazole/benzoxazole derivatives in acetone with the presence of potassium carbonate to gain final compounds (3-27). Among all tested compounds, compound 10, namely N-[4-(benzothiazole-2-yl)-3-chlorophenyl]-2-[(benzimidazole-2-yl)thio]acetamide, and compound 16, namely N-[4-(benzothiazole-2-yl)phenyl]-2-[(1,5-diphenyl-1H-imidazole-2-yl)thio]acetamide, were found to be of considerable anticancer activity against some cancer cell lines.

  9. Impact of different pan-frying conditions on the formation of heterocyclic aromatic amines and sensory quality in fried bacon.

    PubMed

    Gibis, Monika; Kruwinnus, Miriam; Weiss, Jochen

    2015-02-01

    Heterocyclic aromatic amines (HAAs) are formed in the crust of cooked meat products. Most HAAs are carcinogenic in long-term animal studies. Besides precursors in raw materials, important factors are temperature and heating time. Bacon slices were investigated for concentrations of HAAs after pan-frying under different monitored heating conditions. Two HAAs, MeIQx (2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline) (1.5-5.6ng/g) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) (0.1-2.6ng/g), were found in pan-fried bacon slices. The bacon clearly contained higher concentrations of HAAs both with longer frying times and at temperatures of 200-220°C rather than 150-170°C, respectively. A similar continuous increase of the concentrations was observed for norharman (5.0-19.9ng/g) and harman (0.3-1.7ng/g). The sensory evaluation, using a hedonic test design for colour and flavour, of the pan-fried bacon slices resulted in a preferred frying time of 5min at 150-170°C. However, some testers clearly preferred crispy and darker bacon slices containing higher HAA concentrations.

  10. Difference Raman spectroscopy of DNA molecules

    NASA Astrophysics Data System (ADS)

    Anokhin, Andrey S.; Gorelik, Vladimir S.; Dovbeshko, Galina I.; Pyatyshev, Alexander Yu; Yuzyuk, Yury I.

    2015-01-01

    In this paper the micro-Raman spectra of calf DNA for different points of DNA sample have been recorded. The Raman spectra were made with help of difference Raman spectroscopy technique. Raman spectra were recorded with high spatial resolution from different points of the wet and dry samples in different spectral range (100÷4000cm-1) using two lasers: argon (514.5 nm) and helium -neon (632.8 nm). The significant differences in the Raman spectra for dry and wet DNA and for different points of DNA molecules were observed. The obtained data on difference Raman scattering spectra of DNA molecules may be used for identification of DNA types and for analysis of genetic information associated with the molecular structure of this molecule.

  11. [Synthesis of estrone derivatives containing different halogens and/or heterocyclic moieties].

    PubMed

    Frank, E; Wölfling, J; Schneider, G

    2001-12-01

    New compounds derived from the 3-methyl and 3-benzyl ethers of a D-secoestrone aldehyde were synthesized. In the presence of different Lewis acids D-homosteroids could form. These reactions which can be explained by an intramolecular Prinstype mechanism follow high stereoselectivity and reactivity and lead to compounds containing 16 beta-oriented halogens in the sterane skeleton. The formation and reaction of the imines derived from the aldehyde and aniline as well as substituted anilines provide a highly efficient access to steroid derivatives. The steroid alkaloids are of pharmacological interest.

  12. Heterocyclic Scaffolds: Centrality in Anticancer Drug Development.

    PubMed

    Ali, Imran; Lone, Mohammad Nadeem; Al-Othman, Zeid A; Al-Warthan, Abdulrahman; Sanagi, Mohd Marsin

    2015-01-01

    Cancer has been cursed for human beings for long time. Millions people lost their lives due to cancer. Despite of the several anticancer drugs available, cancer cannot be cured; especially at the late stages without showing any side effect. Heterocyclic compounds exhibit exciting medicinal properties including anticancer. Some market selling heterocyclic anticancer drugs include 5-flourouracil, methortrexate, doxorubicin, daunorubicin, etc. Besides, some natural products such as vinblastine and vincristine are also used as anticancer drugs. Overall, heterocyclic moeities have always been core parts in the expansion of anticancer drugs. This article describes the importance of heterocyclic nuclei in the development of anticancer drugs. Besides, the attempts have been made to discuss both naturally occurring and synthetic heterocyclic compounds as anticancer agents. In addition, some market selling anticancer heterocyclic compounds have been described. Moreover, the efforts have been made to discuss the mechanisms of actions and recent advances in heterocyclic compounds as anticancer agents. The current challenges and future prospectives of heterocyclic compounds have also been discussed. Finally, the suggestions for syntheses of effective, selective, fast and human friendly anticancer agents are discussed into the different sections.

  13. Switchable Access to Different Spirocyclopentane Oxindoles by N-Heterocyclic Carbene Catalyzed Reactions of Isatin-Derived Enals and N-Sulfonyl Ketimines.

    PubMed

    Wang, Lei; Li, Sun; Blümel, Marcus; Puttreddy, Rakesh; Peuronen, Anssi; Rissanen, Kari; Enders, Dieter

    2017-07-10

    A novel NHC-catalyzed annulation protocol for the asymmetric synthesis of biologically important β-lactam fused spirocyclopentane oxindoles with four contiguous stereocenters, including two quaternary carbon centers, was developed. Alternatively, spirocyclopentane oxindoles containing an enaminone moiety can be achieved using the same starting materials, isatin-derived enals, and N-sulfonyl ketimines, in the presence of a slightly different NHC catalytic system. This switchable annulation strategy enables the selective assembly of both heterocyclic scaffolds with good yields and excellent enantioselectivities for a broad range of substrates. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Effect of commercial marinades on the mutagenic activity, sensory quality and amount of heterocyclic amines in chicken grilled under different conditions.

    PubMed

    Tikkanen, L M; Latva-Kala, K J; Heiniö, R L

    1996-08-01

    The effect of processing conditions on the mutagenic activity and sensory quality of everyday food was studied by investigating grilled chicken samples seasoned with four different marinades and grilled at temperatures of about 110, 170 and 220 degrees C. The amounts of the heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5,-b]pyridine were determined only in samples grilled at 220 degrees C, using a gas chromatography-mass spectrometry technique with negative-ion chemical ionization. Sensory quality was determined using the extended ranking test method and the mutagenic activity using the Ames Salmonella assay. At 220 degrees C one of the marinades decreased the mutagenic activity in the chicken samples compared with the unseasoned control or samples treated with the other marinades. Great variations, without any clear correlation with mutagenicity, were observed in the amounts of heterocyclic amines between chicken samples treated with the same or different marinades. At the grilling temperatures of 170 degrees C and 110 degrees C the mutagenic activities of the chicken samples were lower or they were non-mutagenic. There was no correlation between mutagenic activity and sensory quality of the products. The samples with high mutagenic activity were ranked to be as good as the samples with lower or no mutagenicity. The results show that it is possible to prepare grilled products with reduced mutagenicity without compromising their sensory quality. It was also evident that marinades can have a reducing effect on the mutagenicity of grilled chicken. Variation observed in the amounts of heterocyclic amines between equivalent products makes it difficult to estimate their concentration in everyday foods.

  15. Tunable DNA cleavage activity promoted by copper(ii) ternary complexes with N-donor heterocyclic ligands.

    PubMed

    Bortolotto, T; Silva-Caldeira, P P; Pich, C T; Pereira-Maia, E C; Terenzi, H

    2016-06-04

    Several small molecules have the capacity to cleave DNA promptly at high yields, even under mild conditions. Usually, this activity has no constraints, occurring without external or user control. Here, we demonstrate that UV-light exposure can greatly enhance the DNA cleavage activity promoted by four ternary copper(ii) complexes. A remarkable photocontrolled activity was achieved, which may be interesting for chemical and biochemical applications.

  16. Genotoxicity and induction of DNA damage responsive genes by food-borne heterocyclic aromatic amines in human hepatoma HepG2 cells.

    PubMed

    Pezdirc, Marko; Žegura, Bojana; Filipič, Metka

    2013-09-01

    Heterocyclic aromatic amines (HAAs) are potential human carcinogens formed in well-done meats and fish. The most abundant are 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ). HAAs exert genotoxic activity after metabolic transformation by CYP1A enzymes, that is well characterized, however the genomic and intervening responses are not well explored. We have examined cellular and genomic responses of human hepatoma HepG2 cells after 24h exposure to HAAs. Comet assay revealed increase in formation of DNA strand breaks by PhIP, MeIQx and IQ but not 4,8-DiMeIQx, whereas increased formation of micronuclei was not observed. The four HAAs up-regulated expression of genes encoding metabolic enzymes CYP1A1, CYP1A2 and UGT1A1 and expression of TP53 and its downstream regulated genes CDKN1A, GADD45α and BAX. Consistent with the up-regulation of CDKN1A and GADD45α the cell-cycle analysis showed arrest in S-phase by PhIP and IQ, and in G1-phase by 4,8-DiMeIQx and MeIQx. The results indicate that upon exposure to HAAs the cells respond with the cell-cycle arrest, which enables cells to repair the damage or eliminate them by apoptosis. However, elevated expression of BCL2 and down-regulation of BAX may indicate that HAAs could suppress apoptosis meaning higher probability of damaged cells to survive and mutate. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Cytotoxic Action of Carboxyborane Heterocyclic Amine Adducts

    PubMed Central

    Miller, Merrill C.; Sood, Anup; Spielvogel, Bernard F.; Bastow, Ken

    1997-01-01

    The heterocyclic carboxyborane amines were found to be potent cytotoxic agents in the murine L1210 lymphoid leukemia and human HeLa suspended carcinoma cells. These agents were observed to inhibit HeLa DNA topoisomerase II activity ~ 200 μM and L1210 topoisomerase II activity ≥ 100 μM. These agents did not cause DNA protein linked breaks themselves, but upon incubation for 14-24 hr did enhance the ability of VP-16 to cause cleavable complexes. The heterocyclic amineboranes inhibited DNA synthesis and caused DNA strand scission. They were additive with VP-16 in affording these results as well as inhibiting colony growth of L1210 cells after co-incubation for 1 hr. The agents inhibited in vitro PKC phosphorylation of both L1210 lymphoid leukemia and human topoisomerase II enzyme. PMID:18475792

  18. Heterocycles from methylenecyclopropanes.

    PubMed

    Yu, Lei; Liu, Mingxuan; Chen, Fenglin; Xu, Qing

    2015-08-21

    The discovery of a series of novel organic reactions has made methylenecyclopropanes (MCPs) some of the most popular building blocks in synthetic organic chemistry during the past two decades. Among reported works, the construction of heterocycles from MCPs has highlighted new synthetic methodologies that afford more opportunities for the quick synthesis of elaborately substituted products, and this should draw a great deal of attention. However, reviews in this area are insufficient, and the latest monograph on heterocycle synthesis from MCPs was published 12 years ago. This review aims to summarize the novel organic reactions of MCPs to produce heterocycles published in recent years, which have provided specific and powerful tools for organic synthesis.

  19. DNA Minor Groove Induced Dimerization of Heterocyclic Cations: Compound Structure, Binding Affinity and Specificity for a TTAA Site

    PubMed Central

    Munde, Manoj; Kumar, Arvind; Nhili, Raja; Depauw, Sabine; David-Cordonnier, Marie-Hélène; Ismail, Mohamed A.; Stephens, Chad E.; Farahat, Abdelbasset A.; Batista-Parra, Adalgisa; Boykin, David W.; Wilson, W. David

    2010-01-01

    With the increasing number and variations of genome sequences available control of gene expression with synthetic, cell permeable molecules is within reach. The variety of sequence-specific-binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this paper is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole-diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, modifications were made to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine binds strongly to TTAA as a dimer and has selectivity (KTTAA/KAATT = 6). CD and DNAse I footprinting studies confirmed the preference of this compound for TTAA. In summary (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) –NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves. PMID:20713062

  20. Studies on DNA adduction with heterocyclic amines by accelerator mass spectrometry: a new technique for tracing isotope-labelled DNA adduction.

    PubMed

    Turteltaub, K W; Vogel, J S; Frantz, C E; Fultz, E

    1993-01-01

    DNA adduction in rodents at doses equivalent to human dietary exposure (10(4)-10(6)-fold lower than laboratory studies) is being studied using accelerator mass spectrometry (AMS). AMS is a nuclear physics technique for detection of cosmogenic isotopes and has been used for specifically selecting and counting 14C. Using AMS, DNA adducts are detectable at levels of 1-10 adducts/10(12) nucleotides following acute and chronic dosing regimes with 14C-labelled carcinogens. The adduct detection limit has been imposed by the natural abundance of 14C in the samples and animal-to-animal variation. AMS is also being coupled to HPLC, multidimensional TLC and radio-immunoassay. In addition, AMS's great sensitivity makes it useful for demonstrating that drugs and chemicals do not bind to DNA. The use of AMS, however, is limited to situations where radiolabelled agents can be used. The data suggest that AMS is extremely useful in obtaining quantitative data on the effects of carcinogens on DNA at the low doses common for actual human exposures and may be useful in human studies.

  1. Probing the difference in covalence by enthalpy measurements: a new heterocyclic N-donor ligand for actinide/lanthanide separation.

    PubMed

    Yang, Yanqiu; Liu, Jun; Yang, Liang; Li, Kun; Zhang, Huabei; Luo, Shunzhong; Rao, Linfeng

    2015-05-21

    Complexation of Am(III), Nd(III), and Eu(III) with a new heterocyclic nitrogen-donor ligand, 2,9-di(quinazolin-2-yl)-1,10-phenanthroline (denoted as BQPhen in this paper), was studied by thermodynamic measurements and theoretical computations. The stability constants of two successive complexes in dimethylformamide, ML(3+) and ML2(3+) where M stands for Nd, Eu, or Am while L stands for the BQPhen ligand, were determined by absorption spectrophotometry. The enthalpy of complexation was determined by microcalorimetry. Results show that BQPhen forms ten times stronger complexes with Am(III) than Eu(III) or Nd(III) under identical conditions, suggesting that BQPhen could be used as an efficient extractant for the separations of trivalent actinides from lanthanides. The higher binding strength of BQPhen towards Am(III) than Nd(III) or Eu(III) is mainly due to the more favourable enthalpy of complexation for Am(III)/BQPhen complexes, implying a higher degree of covalence in the Am(III)/BQPhen complexes than the lanthanide(III)/BQPhen complexes. The thermodynamic trend was corroborated with computational results and validated by solvent extraction experiments that demonstrated BQPhen preferably extracted Am(III) more than Eu(III), with a separation factor of about 10. Discussions have been made to compare BQPhen with other phenanthroline derivatives such as CyMe4-BTPhen, a bis-triazine-phenanthroline derivative that was reported in the literature. Data suggest that, under identical conditions, BQPhen would form stronger complexes with Am(III), Eu(III), and Nd(III) than CyMe4-BTPhen.

  2. Inhibitory Effect of Rosa rugosa Tea Extract on the Formation of Heterocyclic Amines in Meat Patties at Different Temperatures.

    PubMed

    Jamali, Muneer Ahmed; Zhang, Yawei; Teng, Hui; Li, Shun; Wang, Fulong; Peng, Zengqi

    2016-01-30

    In previous studies, heterocyclic amines (HCAs) have been identified as carcinogenic and a risk factor for human cancer. Therefore, the present study was designed to identify bioactive natural products capable of controlling the formation of HCAs during cooking. For this purpose we have evaluated the effect of Rosa rugosa tea extract (RTE) on the formation of HCAs in ground beef patties fried at 160 °C or 220 °C. RTE is rich in phenolic compounds and capable of inhibiting the formation of free radicals. The pyrido[3,4-b]indole (norharman) and 1-methyl-9H-pyrido[3,4-b]indole (harman) contents were significantly (p < 0.05) decreased in RTE-treated patties at 220 °C. 9H-3-Amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) and 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole acetate (Trp-P-1) were not detected at 160 °C and were statistically (p < 0.01) reduced at 220 °C compared to the control. RTE remarkably inhibited the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at 220 °C (p < 0.001) and at 160 °C (p < 0.05). 2-Amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-3-methyl-9H-pyrido[2,3-b]-indole (MeAαC) were only detected in the control group at 160 °C but were comparatively (p > 0.05) similar in the control and treated groups at 220 °C. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline (4,8-DiMeIQx) were not detected in any sample. Total HCAs were positively correlated with cooking loss. In the RTE-treated groups, 75% of the total HCAs were decreased at 160 °C and 46% at 220 °C, suggesting that RTE is effective at both temperatures and can be used during cooking at high temperatures to lessen the amount of HCAs formed.

  3. Formation of Heterocyclic Aromatic Amines and Migration Level of Bisphenol-A in Sous-Vide-Cooked Trout Fillets at Different Cooking Temperatures and Cooking Levels.

    PubMed

    Oz, Fatih; Seyyar, Esra

    2016-04-20

    The effects of different cooking temperatures (65, 75, and 85 °C) and cooking levels (medium and well) on some quality properties, the formation of heterocyclic aromatic amines (HCAs), and the migration level of bisphenol-A (BPA) in trout fillets cooked by sous-vide were investigated. As a result, as expected, cooking caused a reduction in water content of the samples, whereas pH, TBARS, L*, and b* values increased. Cooking loss values ranged between 14.78 and 20.51%. Whereas IQ, MeIQ, 7,8-DiMeIQx, 4,8-DiMeIQx, PhIP, AαC and MeAαC could not be detected in the analyzed samples, varying levels of IQx (up to 0.16 ng/g) and MeIQx (up to 5.66 ng/g) were detected. It was determined that total HCA amounts ranged between 1.28 and 5.75 ng/g, and all or a big part of the total HCAs belonged to MeIQx. In addition, the migration level of BPA in sous-vide-cooked samples ranged between 4.93 and 27.11 ng/g.

  4. A DNA Fingerprint Simulation: Different, Simple, Effective.

    ERIC Educational Resources Information Center

    Reed, Eileen

    2001-01-01

    Discusses the impact of biotechnology (i.e., the use of DNA profiling in the courtroom) on today's society. Presents a hands-on activity for DNA profiling simulation that actively involves students. (YDS)

  5. A DNA Fingerprint Simulation: Different, Simple, Effective.

    ERIC Educational Resources Information Center

    Reed, Eileen

    2001-01-01

    Discusses the impact of biotechnology (i.e., the use of DNA profiling in the courtroom) on today's society. Presents a hands-on activity for DNA profiling simulation that actively involves students. (YDS)

  6. Pd-Catalyzed Heterocycle Synthesis in Ionic Liquids

    NASA Astrophysics Data System (ADS)

    Li, Jianxiao; Jiang, Huanfeng

    Heterocyclic and fused heterocyclic compounds are ubiquitously found in natural products and biologically interesting molecules, and many currently marketed drugs hold heterocycles as their core structure. In this chapter, recent advances on Pd-catalyzed synthesis of heterocycles in ionic liquids (ILs) are reviewed. In palladium catalysis, ILs with different cations and anions are investigated as an alternative recyclable and environmentally benign reaction medium, and a variety of heterocyclic compounds including cyclic ketals, quinolones, quinolinones, isoindolinones, and lactones are conveniently constructed. Compared to the traditional methods, these new approaches have many advantages, such as environmentally friendly synthetic procedure, easy product and catalyst separation, recyclable medium, which make them have the potential applications in industry.

  7. Dissociative Ionization of Aromatic and Heterocyclic Molecules

    NASA Technical Reports Server (NTRS)

    Huo, Winifred M.

    2003-01-01

    Space radiation poses a major health hazard to humans in space flight. The high-energy charged particles in space radiation ranging from protons to high atomic number, high-energy (HZE) particles, and the secondary species they produce, attack DNA, cells, and tissues. Of the potential hazards, long-term health effects such as carcinogenesis are likely linked to the DNA lesions caused by secondary electrons in the 1 - 30 eV range. Dissociative ionization (DI) is one of the electron collision processes that can damage the DNA, either directly by causing a DNA lesion, or indirectly by producing radicals and cations that attack the DNA. To understand this process, we have developed a theoretical model for DI. Our model makes use of the fact that electron motion is much faster than nuclear motion and assumes DI proceeds through a two-step process. The first step is electron-impact ionization resulting in a particular state of the molecular ion in the geometry of the neutral molecule. In the second step the ion undergoes unimolecular dissociation. Thus the DI cross section sigma(sup DI)(sub a) for channel a is given by sigma(sup DI)(sub a) = sigma(sup I)(sub a) P(sub D) with sigma(sup I)(sub a) the ionization cross section of channel a and P(sub D) the dissociation probability. This model has been applied to study the DI of H2O, NH3, and CH4, with results in good agreement with experiment. The ionization cross section sigma(sup I)(sub a) was calculated using the improved binary encounter-dipole model and the unimolecular dissociation probability P(sub D) obtained by following the minimum energy path determined by the gradients and Hessians of the electronic energy with respect to the nuclear coordinates of the ion. This model is used to study the DI from the low-lying channels of benzene and pyridine to understand the different product formation in aromatic and heterocyclic molecules. DI study of the DNA base thymine is underway. Solvent effects will also be discussed.

  8. Dissociative Ionization of Aromatic and Heterocyclic Molecules

    NASA Technical Reports Server (NTRS)

    Huo, Winifred M.

    2003-01-01

    Space radiation poses a major health hazard to humans in space flight. The high-energy charged particles in space radiation ranging from protons to high atomic number, high-energy (HZE) particles, and the secondary species they produce, attack DNA, cells, and tissues. Of the potential hazards, long-term health effects such as carcinogenesis are likely linked to the DNA lesions caused by secondary electrons in the 1 - 30 eV range. Dissociative ionization (DI) is one of the electron collision processes that can damage the DNA, either directly by causing a DNA lesion, or indirectly by producing radicals and cations that attack the DNA. To understand this process, we have developed a theoretical model for DI. Our model makes use of the fact that electron motion is much faster than nuclear motion and assumes DI proceeds through a two-step process. The first step is electron-impact ionization resulting in a particular state of the molecular ion in the geometry of the neutral molecule. In the second step the ion undergoes unimolecular dissociation. Thus the DI cross section sigma(sup DI)(sub a) for channel a is given by sigma(sup DI)(sub a) = sigma(sup I)(sub a) P(sub D) with sigma(sup I)(sub a) the ionization cross section of channel a and P(sub D) the dissociation probability. This model has been applied to study the DI of H2O, NH3, and CH4, with results in good agreement with experiment. The ionization cross section sigma(sup I)(sub a) was calculated using the improved binary encounter-dipole model and the unimolecular dissociation probability P(sub D) obtained by following the minimum energy path determined by the gradients and Hessians of the electronic energy with respect to the nuclear coordinates of the ion. This model is used to study the DI from the low-lying channels of benzene and pyridine to understand the different product formation in aromatic and heterocyclic molecules. DI study of the DNA base thymine is underway. Solvent effects will also be discussed.

  9. Effects of heterocycles containing different atoms as π-bridges on the performance of dye-sensitized solar cells.

    PubMed

    Jia, Hailang; Ju, Xuehai; Zhang, Mingdao; Ju, Zemin; Zheng, Hegen

    2015-07-07

    Two new D-π-A zinc porphyrin dyes with thiophene and furan π-bridges have been synthesized and employed in dye-sensitized solar cells (DSSCs). Here, the triphenylamine (TPA) moiety was used as the electron donor, and the hexylthiophene chromophores were introduced onto the donor groups, which effectively extended the π-conjugation system. Although the two dyes had similar molecular structures, there was a significant difference between their optical and photoelectric properties. The EIS analysis suggested that the dye with the thiophene π-bridge had a lower charge recombination rate compared to the dye with the furan π-bridge. Based on their light-harvesting abilities, the power conversion efficiency (PCE) of dye JP-S was higher than that of dye JP-O. The JP-S-based DSSC showed a PCE of 5.84%, whereas the PCE of the JP-O-based DSSC was 4.68%. Moreover, using the dye TTR1 as a co-sensitizer made up for the poor absorption of porphyrin dyes in the 480-600 nm range and reduced the charge recombination. The JP-S + TTR1-based DSSCs showed a higher PCE of 6.71%, and the Jsc and Voc values of the device were both increased using this strategy.

  10. UV and ionizing radiations induced DNA damage, differences and similarities

    NASA Astrophysics Data System (ADS)

    Ravanat, Jean-Luc; Douki, Thierry

    2016-11-01

    Both UV and ionizing radiations damage DNA. Two main mechanisms, so-called direct and indirect pathways, are involved in the degradation of DNA induced by ionizing radiations. The direct effect of radiation corresponds to direct ionization of DNA (one electron ejection) whereas indirect effects are produced by reactive oxygen species generated through water radiolysis, including the highly reactive hydroxyl radicals, which damage DNA. UV (and visible) light damages DNA by again two distinct mechanisms. UVC and to a lesser extend UVB photons are directly absorbed by DNA bases, generating their excited states that are at the origin of the formation of pyrimidine dimers. UVA (and visible) light by interaction with endogenous or exogenous photosensitizers induce the formation of DNA damage through photosensitization reactions. The excited photosensitizer is able to induce either a one-electron oxidation of DNA (type I) or to produce singlet oxygen (type II) that reacts with DNA. In addition, through an energy transfer from the excited photosensitizer to DNA bases (sometime called type III mechanism) formation of pyrimidine dimers could be produced. Interestingly it has been shown recently that pyrimidine dimers are also produced by direct absorption of UVA light by DNA, even if absorption of DNA bases at these wavelengths is very low. It should be stressed that some excited photosensitizers (such as psoralens) could add directly to DNA bases to generate adducts. The review will described the differences and similarities in terms of damage formation (structure and mechanisms) between these two physical genotoxic agents.

  11. The Domino Way to Heterocycles

    PubMed Central

    Padwa, Albert; Bur, Scott K.

    2007-01-01

    Sequential transformations enable the facile synthesis of complex target molecules from simple building blocks in a single preparative step. Their value is amplified if they also create multiple stereogenic centers. In the ongoing search for new domino processes, emphasis is usually placed on sequential reactions which occur cleanly and without forming by-products. As a prerequisite for an ideally proceeding one-pot sequential transformation, the reactivity pattern of all participating components has to be such that each building block gets involved in a reaction only when it is supposed to do so. The development of sequences that combine transformations of fundamentally different mechanisms broadens the scope of such procedures in synthetic chemistry. This mini review contains a representative sampling from the last 15 years on the kinds of reactions that have been sequenced into cascades to produce heterocyclic molecules. PMID:17940591

  12. Synthesis and Antiproliferative Activity of Novel Heterocyclic Indole-Trimethoxyphenyl Conjugates

    PubMed Central

    Cahill, Michael M.; O’Shea, Kevin D.; Pierce, Larry T.; Winfield, Hannah J.; Eccles, Kevin S.; Lawrence, Simon E.

    2017-01-01

    The synthesis and biological evaluation of a series of novel heterocyclic indole derivatives is described. The consolidation of the combretastatin and bisindolylmaleimide templates towards the inclusion of a novel heterocyclic ring proffered a versatile pharmacophore with which to pursue chemical diversification. Given literature precedent, maleimide was initially investigated in this role and the bioactivity assessed by measurement of NCI-60 cell panel growth. Subsequently, a range of 5-aminopyrazoles was designed and developed to explore the specific effect of heterocycle hydrogen bonding on cell growth. The unique electronic nature of the 5-aminopyrazole moiety allowed for regiospecific monosubstitution on different sites of the ring, such as thiourea substitution at the N(1) position for derivative 45 or trifluoroacetylation on the 5-amino position for 43. Further derivatisation led to the ultimate development of bicyclic pyrazolotriazinedione 41 and pyrimidine 42 systems. The antiproliferative activities of these 3,4-diaryl-5-aminopyrazoles were assessed using the NCI-60 cell screen, disclosing the discovery of distinct selectivity profiles towards a number of cell lines, such as SNB-75 CNS cancer, UO-31 and CAKI-1 renal cancer cells. A series of DNA topological assays discounted the interaction with topoisomerase II as a putative mechanism of action. PMID:28678205

  13. Theoretical study on the catalytic reactivity of N-hydroxyphthalimide tuned by different heterocyclic substitutions on its phenyl ring for aerobic oxidation

    NASA Astrophysics Data System (ADS)

    Chen, Kexian; Xie, Haiying; Jiang, Kezhi; Mao, Jianyong

    2016-07-01

    The structure-reactivity relationship of new hydroxyimide organocatalysts based on the heterocyclic replacements of the phenyl ring of N-hydroxyphthalimide (NHPI) has been theoretically investigated to gain a mature understanding of this particular catalysis for aerobic oxidation. We find that the reactivity of catalysts with the common five-member aromatic rings is lower than that of NHPI. The catalyst with the recyclable structure of imidazolium ionic liquid may serve as a novel model catalyst for further improvements due to its reactivity comparable to that of NHPI. The catalytic reactivity of multi-nitroxyl catalysts is theoretically more fascinating than that of the highly efficient N,N-dihydroxypyromellitimide.

  14. Exposure to heterocyclic amines.

    PubMed Central

    Wakabayashi, K; Ushiyama, H; Takahashi, M; Nukaya, H; Kim, S B; Hirose, M; Ochiai, M; Sugimura, T; Nagao, M

    1993-01-01

    Many mutagenic heterocyclic amines (HAs) have been isolated from cooked foods and pyrolysates of amino acids and proteins, and the carcinogenicity of 10 of these HAs in rodents and of 1 in monkeys has been reported. Quantification of these carcinogenic HAs in various kinds of cooked foods indicated that the level of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was highest (0.56-69.2 ng/g), that of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was second highest (0.64-6.44 ng/g), and those of other HAs were 0.03-2.50 ng/g. Heterocyclic amines were found in urine samples of 10 healthy volunteers consuming a normal diet, but HAs were not detectable in urine samples of three patients receiving parenteral alimentation. These results strongly suggest that humans are continuously exposed to HAs derived from food in the normal diet. Based on quantitative data on the levels of HAs in cooked foods and urine samples, the daily exposures to PhIP and MeIQx were estimated to be 0.1-13.8 micrograms and 0.2-2.6 micrograms per person, respectively. These levels of carcinogenic HAs are in the same range as those of other carcinogens such as N-nitrosodimethylamine and benzo[a]pyrene to which humans are exposed. PMID:8319610

  15. Global methylation profiles in DNA from different blood cell types.

    PubMed

    Wu, Hui-Chen; Delgado-Cruzata, Lissette; Flom, Julie D; Kappil, Maya; Ferris, Jennifer S; Liao, Yuyan; Santella, Regina M; Terry, Mary Beth

    2011-01-01

    DNA methylation measured in white blood cell DNA is increasingly being used as in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran), and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources (WBC, Gran, mononuclear (MN), and lymphoblastoid cell lines (LCL)), we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA), and [(3)H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [(3)H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [(3)H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive correlations ranging from 0.3-0.7 for each cell type with one exception (Sat2 and Alu in MN). Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39-0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18-0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.

  16. DNA interaction, antimicrobial, electrochemical and spectroscopic studies of metal(II) complexes with tridentate heterocyclic Schiff base derived from 2‧-methylacetoacetanilide

    NASA Astrophysics Data System (ADS)

    Raman, Natarajan; Pothiraj, Krishnan; Baskaran, Thanasekaran

    2011-08-01

    A new Schiff base ligand (HL) was synthesized by the condensation reaction between 2'-methyleacetoacetanilide and 2-amino-3-hydroxypyridine. Its Co(II), Ni(II), Cu(II) and Zn(II) complexes were prepared by the interaction of the ligand with metal(II) chloride. They were characterized by elemental analysis, IR, 1H NMR, EPR, UV-Vis, magnetic susceptibility measurements, conductivity measurements and FAB-mass spectra. The interaction of the complexes with calf thymus DNA (CT-DNA) has been investigated by UV absorption, viscosity and cyclic voltammetry methods, and the mode of CT-DNA binding to the complexes has been explored. Furthermore, the DNA cleavage activity by the complexes was performed. It was found to be oxidative hydroxyl radical cleavage in the presence of 3-mercaptopropionic acid (MPA). The Schiff base and its complexes have been screened for their antibacterial ( Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa) and antifungal ( Aspergillus niger, Rhizopus stolonifer, Rhizoctonia bataicola and Candida albicans) activities and the data reveal that the complexes have higher activity than the free ligand.

  17. High resolution melting curve analysis of DNA samples isolated by different DNA extraction methods.

    PubMed

    Martín-Núñez, Gracia M; Gómez-Zumaquero, Juan M; Soriguer, Federico; Morcillo, Sonsoles

    2012-01-18

    High resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. Melting of double-stranded DNA molecules is influenced by several factors. We evaluated the influence of the DNA isolation method in the melting curve analysis to detect genetic variations. We isolated DNA from whole blood of 547 subjects by two different methods: Maxwell 16 Instrument and DNA FlexiGene Kit. A fragment of 159 bp was amplified and analyzed by high resolution melting. Those samples that showed a different melting curve pattern were sequenced. Of the samples extracted with the Maxwell 16 Instrument, 42% showed variation compared with 0.18% of the samples extracted with DNA FlexiGene Kit. After sequencing, we showed that all samples extracted with the Maxwell 16 Instrument were false positive except one, which coincided with the only sample that showed variation in those extracted with the DNA FlexiGene Kit. The method used to extract DNA is an important factor to consider in the analysis of melting curves obtained by high resolution melting, as it may influence the melting behaviour of the samples, giving false positive results in the detection of genetic variants. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Accelerator mass spectrometry in biomedical dosimetry: relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA.

    PubMed

    Turteltaub, K W; Felton, J S; Gledhill, B L; Vogel, J S; Southon, J R; Caffee, M W; Finkel, R C; Nelson, D E; Proctor, I D; Davis, J C

    1990-07-01

    Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a 14C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations (10Be, 14C, 26Al, 41Ca, and 129I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of the technology. In this study, the relationship between exposure to low levels of 2-amino-3,8-dimethyl[2-14C]imidazo[4,5-f]quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Instrument reproducibility in this study is 2%; sensitivity is 1 adduct per 10(11) nucleotides. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, we demonstrate at least 1 order of magnitude improvement over the best adduct detection sensitivity reported to date and 3-5 orders of magnitude improvement over other methods used for adduct measurement. An additional improvement of 2 orders of magnitude in sensitivity is suggested by preliminary experiments to develop bacterial hosts depleted in radiocarbon. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.

  19. Correlation between DNA interactions and cytotoxic activity of four new ternary compounds of copper(II) with N-donor heterocyclic ligands.

    PubMed

    Silva, Priscila P; Guerra, Wendell; Dos Santos, Geandson Coelho; Fernandes, Nelson G; Silveira, Josiane N; da Costa Ferreira, Ana M; Bortolotto, Tiago; Terenzi, Hernán; Bortoluzzi, Adailton João; Neves, Ademir; Pereira-Maia, Elene C

    2014-03-01

    Four new ternary complexes of copper(II) were synthesized and characterized: [Cu(hyd)(bpy)(acn)(ClO4)](ClO4)] (1), [Cu(hyd)(phen)(acn)(ClO4)](ClO4)] (2), [Cu(Shyd)(bpy)(acn)(ClO4)](ClO4)] (3) and [Cu(Shyd)(phen)(acn)(ClO4)](ClO4)] (4), in which acn=acetonitrile; hyd=2-furoic acid hydrazide, bpy=2,2-bipyridine; phen=1,10-phenanthroline and Shyd=2-thiophenecarboxylic acid hydrazide. The cytotoxic activity of the complexes in a chronic myelogenous leukemia cell line was investigated. All complexes are able to enter cells and inhibit cellular growth in a concentration-dependent manner, with an activity higher than that of the corresponding free ligands. The substitution of Shyd for hyd increases the activity, while the substitution of bpy for phen renders the complex less active. Therefore, the most potent complex is 4 with an IC50 value of 1.5±0.2μM. The intracellular copper concentration needed to inhibit 50% of cell growth is approximately 7×10(-15)mol/cell. It is worth notifying that a correlation between cytotoxic activity, DNA binding affinity and DNA cleavage was found: 1<3<2<4.

  20. DNA damage profiles induced by sunlight at different latitudes.

    PubMed

    Schuch, André Passaglia; Yagura, Teiti; Makita, Kazuo; Yamamoto, Hiromasa; Schuch, Nelson Jorge; Agnez-Lima, Lucymara Fassarella; MacMahon, Ricardo Monreal; Menck, Carlos Frederico Martins

    2012-04-01

    Despite growing knowledge on the biological effects of ultraviolet (UV) radiation on human health and ecosystems, it is still difficult to predict the negative impacts of the increasing incidence of solar UV radiation in a scenario of global warming and climate changes. Hence, the development and application of DNA-based biological sensors to monitor the solar UV radiation under different environmental conditions is of increasing importance. With a mind to rendering a molecular view-point of the genotoxic impact of sunlight, field experiments were undertaken with a DNA-dosimeter system in parallel with physical photometry of solar UVB/UVA radiation, at various latitudes in South America. On applying biochemical and immunological approaches based on specific DNA-repair enzymes and antibodies, for evaluating sunlight-induced DNA damage profiles, it became clear that the genotoxic potential of sunlight does indeed vary according to latitude. Notwithstanding, while induction of oxidized DNA bases is directly dependent on an increase in latitude, the generation of 6-4PPs is inversely so, whereby the latter can be regarded as a biomolecular marker of UVB incidence. This molecular DNA lesion-pattern largely reflects the relative incidence of UVA and UVB energy at any specific latitude. Hereby is demonstrated the applicability of this DNA-based biosensor for additional, continuous field experiments, as a means of registering variations in the genotoxic impact of solar UV radiation. © 2012 Wiley Periodicals, Inc.

  1. Photochemistry of hydrogen bonded heterocycles probed by photodissociation experiments and ab initio methods.

    PubMed

    Slavíček, Petr; Fárník, Michal

    2011-07-14

    In this perspective article, we focus on the photochemistry of five-membered nitrogen containing heterocycles (pyrrole, imidazole and pyrazole) in clusters. These heterocycles represent paradigmatic structures for larger biologically active heterocyclic molecules and complexes. The dimers of the three molecules are also archetypes of different bonding patterns: N-H···π interaction, N-H···N hydrogen bond and double hydrogen bond. We briefly review available data on photochemistry of the title molecules in the gas phase, but primarily we focus on the new reaction channels opened upon the complexation with other heterocycles or solvent molecules. Based on ab initio calculations we discuss various possible reactions in the excited states of the clusters: (1) hydrogen dissociation, (2) hydrogen transfer between the heterocyclic units, (3) molecular ring distortion, and (4) coupled electron-proton transfer. The increasing photostability with complexity of the system can be inferred from experiments with photodissociation in these clusters. A unified view on photoinduced processes in five-membered N-heterocycles is provided. We show that even though different deactivation channels are energetically possible for the complexed heterocycles, in most cases the major result is a fast reconstruction of the ground state. The complexed or solvated heterocycles are thus inherently photostable although the stability can in principle be achieved via different reaction routes.

  2. Comparison of DNA strand-break simulated with different DNA models

    NASA Astrophysics Data System (ADS)

    Xie, Wenzhang; Li, Junli; Li, Chunyan; Qiu, Rui; Yan, Congchong; Zeng, Zhi

    2014-06-01

    In Monte Carlo simulation of DNA damage, the geometric model of DNA is of great importance. To study the influence of DNA model on the simulation of DNA damage, three DNA models were created in this paper. They were a volume model and two atomic models with different parameters. Direct DNA strand-break induced by low-energy electrons were simulated respectively with the three models. The results show that most of the energy depositions in the DNA segments do not lead to strand-breaks. The simple single strand-break (SSB) tends to be the predominant damage type, and the contribution of complex double strand-break (DSB) to the total DSB cannot be neglected. Among the yields of all the three DNA target models applied here, the yields of the volume model are the highest, the yields of the atomic model with double van der Waals radii (r) take the second place, whereas the yields of the atomic model with single r come last. On average, the ratios of SSB yields are approximately equivalent to the corresponding ratios of the models' volume. However, there seems to be no clear relationship between the DSB yields and the models' volume.

  3. Comparative evaluation of different DNA extraction procedures from food samples.

    PubMed

    Di Bernardo, G; Del Gaudio, S; Galderisi, U; Cascino, A; Cipollaro, M

    2007-01-01

    Five methodologies for extracting DNA from food samples are described. The food products analyzed are from either soybean or maize. They were selected on the basis of the mechanical, thermal, and chemical treatments that they had been subjected to during industrial processing. DNA preparations were evaluated for purity, yield, and average fragment size. Two endogenous genes, soybean lectin gene and alcohol dehydrogenase gene (adh1), were used to assess the degree of DNA degradation at different stages of the transformation chain. The goal of this study was to determine the role that extraction methods play in DNA amplification in order to select the best protocol for a food sample. This comparative evaluation can be specifically useful for detection of genetically modified ingredients in a variety of food matrices.

  4. Detection of Different DNA Animal Species in Commercial Candy Products.

    PubMed

    Muñoz-Colmenero, Marta; Martínez, Jose Luis; Roca, Agustín; Garcia-Vazquez, Eva

    2016-03-01

    Candy products are consumed all across the world, but there is not much information about their composition. In this study we have used a DNA-based approach for determining the animal species occurring in 40 commercial candies of different types. We extracted DNA and performed PCR amplification, cloning and sequencing for obtaining species-informative DNA sequences. Eight species were identified including fish (hake and anchovy) in 22% of the products analyzed. Bovine and porcine were the most abundant appearing in 27 samples each one. Most products contained a mixture of species. Marshmallows (7), jelly-types, and gummies (20) contained a significantly higher number of species than hard candies (9). We demonstrated the presence of DNA animal species in candy product which allow consumers to make choices and prevent allergic reaction.

  5. RNA-DNA sequence differences spell genetic code ambiguities

    PubMed Central

    Nielsen, Michael L.

    2011-01-01

    A recent paper in Science by Li et al. 20111 reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized.  PMID:22567189

  6. Quantum Transport Through Heterocyclic Molecules

    NASA Astrophysics Data System (ADS)

    Maiti, Santanu K.; Karmakar, S. N.

    We explore electron transport properties in molecular wires made of heterocyclic molecules (pyrrole, furan and thiophene) by using the Green's function technique. Parametric calculations are given based on the tight-binding model to describe the electron transport in these wires. It is observed that the transport properties are significantly influenced by (a) the heteroatoms in the heterocyclic molecules and (b) the molecule-to-electrodes coupling strength. Conductance (g) shows sharp resonance peaks associated with the molecular energy levels in the limit of weak molecular coupling, while they get broadened in the strong molecular coupling limit. These resonances get shifted with the change of the heteroatoms in these heterocyclic molecules. All the essential features of the electron transfer through these molecular wires become much more clearly visible from the study of our current-voltage (I-V) characteristics, and they provide several key information in the study of molecular transport.

  7. The effects of cooking on wire and stone barbecue at different cooking levels on the formation of heterocyclic aromatic amines and polycyclic aromatic hydrocarbons in beef steak.

    PubMed

    Oz, Fatih; Yuzer, M Onur

    2016-07-15

    The effects of type of barbecue (wire and stone) and cooking levels (rare, medium, well-done and very well-done) on the formation of heterocyclic aromatic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) in beef steak were investigated. Varying levels of IQx (up to 0.29 ng/g), IQ (up to 0.93 ng/g), MeIQx (up to 0.08 ng/g), MeIQ (up to 0.75 ng/g), 7,8-DiMeIQx (up to 0.08 ng/g), 4,8-DiMeIQx (up to 4.95 ng/g), PhIP (up to 6.24 ng/g) and AαC (up to 0.20 ng/g) were determined, while MeAαC was not detected. The total HCA amounts in wire barbecued samples were higher than stone barbecued samples. Total HCA contents of the samples ranged between nd and 13.52 ng/g. In terms of PAHs, varying levels of BaA (up to 0.34 ng/g), Chry (up to 0.28 ng/g), BbF (up to 0.39 ng/g), BkF (up to 0.90 ng/g), BaP (up to 0.29 ng/g) and Bghip (up to 0.43 ng/g) were determined, while DahA and IncdP were not detected. The total PAH amounts in stone barbecued samples were higher than those of wire barbecued samples. Total PAH amounts of the samples ranged between nd and 2.63 ng/g.

  8. Mutagenic Potency of Food-Derived Heterocyclic Amines

    SciTech Connect

    Felton, J S; Knize, M G; Wu, R W; Colvin, M E; Hatch, F T; Malfatti, M A

    2006-10-26

    The understanding of mutagenic potency has been primarily approached using ''quantitative structure activity relationships'' (QSAR). Often this method allows the prediction of mutagenic potency of the compound based on its structure. But it does not give the underlying reason why the mutagenic activities differ. We have taken a set of heterocyclic amine structures and used molecular dynamic calculations to dock these molecules into the active site of a computational model of the cytochrome P-450 1A1 enzyme. The calculated binding strength using Boltzman distribution constants was then compared to the QSAR value (HF/6-31G* optimized structures) and the Ames/Salmonella mutagenic potency. Further understanding will only come from knowing the complete set of mutagenic determinants. These include the nitrenium ion half-life, DNA adduct half-life, efficiency of repair of the adduct, and ultimately fixation of the mutation through cellular processes. For two isomers, PhIP and 3-Me-PhIP, we showed that for the 100-fold difference in the mutagenic potency a 5-fold difference can be accounted for by differences in the P450 oxidation. The other factor of 20 is not clearly understood but is downstream from the oxidation step. The application of QSAR (chemical characteristics) to biological principles related to mutagenesis is explored in this report.

  9. RNA–DNA sequence differences in Saccharomyces cerevisiae

    PubMed Central

    Wang, Isabel X.; Grunseich, Christopher; Chung, Youree G.; Kwak, Hojoong; Ramrattan, Girish; Zhu, Zhengwei; Cheung, Vivian G.

    2016-01-01

    Alterations of RNA sequences and structures, such as those from editing and alternative splicing, result in two or more RNA transcripts from a DNA template. It was thought that in yeast, RNA editing only occurs in tRNAs. Here, we found that Saccharomyces cerevisiae have all 12 types of RNA–DNA sequence differences (RDDs) in the mRNA. We showed these sequence differences are propagated to proteins, as we identified peptides encoded by the RNA sequences in addition to those by the DNA sequences at RDD sites. RDDs are significantly enriched at regions with R-loops. A screen of yeast mutants showed that RDD formation is affected by mutations in genes regulating R-loops. Loss-of-function mutations in ribonuclease H, senataxin, and topoisomerase I that resolve RNA–DNA hybrids lead to increases in RDD frequency. Our results demonstrate that RDD is a conserved process that diversifies transcriptomes and proteomes and provide a mechanistic link between R-loops and RDDs. PMID:27638543

  10. Duplex structural differences and not 2'-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA.

    PubMed

    Olimpo, Jeffrey T; DeStefano, Jeffrey J

    2010-07-01

    Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA-DNA versus DNA-DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3' recessed DNA base) are required for stable binding to RNA-DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA-DNA. Replacing 2'- hydroxyls on pivotal RNA bases with 2'-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. RT's K(d) (k(off)/k(on)) for DNA-DNA and RNA-DNA were similar, although k(off) differed almost 40-fold, suggesting a faster k(on) for DNA-DNA. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, the ability of RT to form a more stable complex on RNA-DNA may aid in degradation of RNA fragments that remain after DNA synthesis.

  11. DNA methylation detection based on difference of base content

    NASA Astrophysics Data System (ADS)

    Sato, Shinobu; Ohtsuka, Keiichi; Honda, Satoshi; Sato, Yusuke; Takenaka, Shigeori

    2016-04-01

    Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

  12. Difference in DNA-binding abilities of Fur-homolog DNA binding protein from Neisseria gonorrhoeae.

    PubMed

    Bagchi, Angshuman

    2016-10-01

    Gonorrhea is a severe disease infecting both men and women worldwide. The causative agent of the disease is Neisseria gonorrhoeae. The organism mostly affects human beings in iron restricted environments. In such an environment the organism produces a set of proteins which are mostly absent in iron rich environments. The expressions of the genes for the proteins are regulated by the transcription factor (TF) belonging to the Fur family. Interestingly, the same TF acts as the activator and repressor of genes. In this present work, an attempt has been made to analyze the molecular details of the differential DNA-binding activities of the TF from Neisseria gonorrhoeae to come up with a plausible molecular reason behind the difference DNA binding activities of the same TF. Computational modelling technique was used to build the three dimensional structure of the TF. Molecular docking and molecular dynamics simulations were employed to determine the binding interactions between the TF and the promoter DNA. With the help of the computational techniques, the biochemical reason behind the different modes of DNA binding by the TF was analyzed. Results from this analysis may be useful to future drug development endeavours to curtail the spread of Gonorrhea.

  13. Molecular recognition of plant DNA: does it differ from conventional animal DNA?

    PubMed

    Maiti, Swati; Maiti, Protiti; Sinha, Sudarson Sekhar; Mitra, Rajib Kumar; Pal, Samir Kumar

    2009-03-01

    The recognition mechanism of DNA with small drugs/ligands is an important field of research from pharmacological point of view. Such studies are ample with DNAs extracted from animal cells, but are rare for those extracted from plant cells. However, such a study is strongly demanding for the formulation of pesticides and other agrochemicals. In this contribution, for the first time, we report the interaction of two well-known DNA binder ethidium bromide (EB) and Hoechst 33258 (H33258) with two genomic DNAs extracted from the leaves of Ricinus communis L. (castor bean) and Mangifera indica (mango) using steady-state and picosecond-resolved fluorescence spectroscopy. The purity of the extracted DNAs is confirmed from gel electrophoresis and optical absorption studies. As evidenced from the circular dichroism (CD) measurements the DNAs retain physiologically relevant B forms. The well-known DNA intercalator EB has been found to show an additional electrostatic mode of binding with the DNAs, which is not present in the conventional animal DNAs. The binding affinity of EB is found to be even weaker for the DNA extracted from M. indica compared to that in R. communis L. On the other hand, the binding affinity of H33258 with the plant DNAs is found to be comparable to that of animal DNAs. The difference in interaction could be rationalized from the possible differences in the base sequences.

  14. Viability and DNA fragmentation in differently sorted boar spermatozoa.

    PubMed

    De Ambrogi, M; Spinaci, M; Galeati, G; Tamanini, C

    2006-11-01

    Sperm cell defense against DNA damage relies on two factors: the tight packaging of chromatin, based on condensation and substitution of histones with protamines, and the antioxidant agents present in seminal plasma. These defenses are extremely important as mature sperm is unable to repair DNA damage and even if a successful fertilization occurs, embryo undergoes apoptosis at the time of genomic activation. Sex-sorting exposes spermatozoa to stress sources such as high pressure, laser beam and electrical charge. The aim of this work was to determine how sorting procedures affect viability and DNA integrity in boar spermatozoa, by using the newly developed Sperm-Sus-Halomax. Four sperm populations were considered: CONTROL (no treatment), REAL (sex-sorted semen), BULK (semen sorted without sex separation) and NO LASER (semen only exposed to the high pressure, but including also cells normally discarded from sex-sorting). A significantly (P=0.019) lower viability in NO LASER (64.71%) than in CONTROL (78.6%) and REAL (80.5%) groups was found; this was accompanied by a significantly (P=0.001) higher DNA fragmentation index (DFI) in NO LASER group (6.86%) respect to CONTROL (3.30%) and REAL (3.42%) groups. BULK group did not show any difference in viability or DFI as compared to the other groups. In conclusion, we may believe that sex-sorting procedure as a whole does not affect either viability or DFI and that shear mechanical forces are a relevant source of DNA damage for sorted semen.

  15. Exploring the Fate of Nitrogen Heterocycles in Complex Prebiotic Mixtures

    NASA Technical Reports Server (NTRS)

    Smith, Karen E.; Callahan, Michael P.; Cleaves, Henderson J.; Dworkin, Jason P.; House, Christopher H.

    2011-01-01

    A long standing question in the field of prebiotic chemistry is the origin of the genetic macromolecules DNA and RNA. DNA and RNA have very complex structures with repeating subunits of nucleotides, which are composed of nucleobases (nitrogen heterocycles) connected to sugar-phosphate. Due to the instability of some nucleobases (e.g. cytosine), difficulty of synthesis and instability of D-ribose, and the likely scarcity of polyphosphates necessary for the modern nucleotides, alternative nucleotides have been proposed for constructing the first genetic material. Thus, we have begun to investigate the chemistry of nitrogen heterocycles in plausible, complex prebiotic mixtures in an effort to identify robust reactions and potential alternative nucleotides. We have taken a complex prebiotic mixture produced by a spark discharge acting on a gas mixture of N2, CO2, CH4, and H2, and reacted it with four nitrogen heterocycles: uracil, 5-hydroxymethyluracil, guanine, and isoxanthopterin (2-amino-4,7-dihydroxypteridine). The products of the reaction between the spark mixture and each nitrogen heterocycle were characterized by liquid chromatography coupled to UV spectroscopy and Orbitrap mass spectrometry. We found that the reaction between the spark mixtUl'e and isoxanthopterin formed one major product, which was a cyanide adduct. 5-hydroxymethyluracil also reacted with the spark mixture to form a cyanide adduct, uracil-5-acetonitrile, which has been synthesized previously by reacting HCN with S-hydroxymethyluracil. Unlike isoxanthopterin, the chromatogram of the 5-hydroxymethyluracil reaction was much more complex with multiple products including spark-modified dimers. Additionally, we observed that HMU readily self-polymerizes in solution to a variety of oligomers consistent with those suggested by Cleaves. Guanine and uracil, the biological nucleobases, did not react with the spark mixture, even at high temperature (100 C). This suggests that there are alternative

  16. DNA differences found between Africanized and European honeybees.

    PubMed Central

    Hall, H G

    1986-01-01

    The harmful en masse introduction of Africanized honeybees into the United States will occur within 5 years. Possible means of control are dependent on a reliable way to distinguish the Africanized bees from the extant European bees. Current means of identification are inadequate. Reported here are the encouraging initial results to distinguish the bees by their nuclear DNA. With 9 restriction enzymes and 16 probes, six genetic differences have been found among three samples of European bees from California. Twelve additional differences were detected between the European samples and a sample of Africanized bees from Costa Rica. Images PMID:3014516

  17. Heterocycles from α-aminonitriles.

    PubMed

    Otto, Nicola; Opatz, Till

    2014-10-06

    Owing to their various modes of reactivity, α-aminonitriles represent versatile building blocks for the construction of a wide range of nitrogen heterocycles. The present Concept article focuses on synthetic methodologies using their bifunctional nature which is the basis of their reactivity as α-amino carbanions and as iminium ions. Reactions exclusively taking place on either the amine or on the nitrile moiety will not be considered.

  18. Recent Developments in the Chemistry of N-Heterocyclic Phosphines

    NASA Astrophysics Data System (ADS)

    Gudat, Dietrich

    This chapter gives a survey on five- and six-membered phosphorus-nitrogen heterocyclic compounds whose rings combine a phosphazene (>N-P = N-) or phosphazane (>N-P(X)-N<) unit with an unsaturated C2 or C3 building block. Representatives contain structurally diverse species like aromatic 1,3,2-diazaphosphinines and (benzo)-1,3,2-diazaphospholes, cationic counterparts of subvalent main-group carbene analogues like 1,3,2-diazaphospholenium ions and phosphenium-diketiminates, and neutral heterocycles like 1,3,2-diazaphospholenes featuring unusual structures and reactivities. The exploration of these species developed rapidly in the last two decades in the wake of cutting edge research on multiple bonding and low coordination in the chemistry of heavier main-group elements, and the discovery of stable carbenes. This review summarizes the elaboration of synthetic approaches for different types of N-heterocyclic phosphine derivatives, discusses their characterization by physical and computational methods which furnished a thorough understanding of structure and bonding, and finally highlights accomplishments in the exploration of the chemical properties at the border of classical organic heterocyclic chemistry and molecular inorganic chemistry.

  19. Modern advances in heterocyclic chemistry in drug discovery.

    PubMed

    Taylor, Alexandria P; Robinson, Ralph P; Fobian, Yvette M; Blakemore, David C; Jones, Lyn H; Fadeyi, Olugbeminiyi

    2016-07-12

    New advances in synthetic methodologies that allow rapid access to a wide variety of functionalized heterocyclic compounds are of critical importance to the medicinal chemist as it provides the ability to expand the available drug-like chemical space and drive more efficient delivery of drug discovery programs. Furthermore, the development of robust synthetic routes that can readily generate bulk quantities of a desired compound help to accelerate the drug development process. While established synthetic methodologies are commonly utilized during the course of a drug discovery program, the development of innovative heterocyclic syntheses that allow for different bond forming strategies are having a significant impact in the pharmaceutical industry. This review will focus on recent applications of new methodologies in C-H activation, photoredox chemistry, borrowing hydrogen catalysis, multicomponent reactions, regio- and stereoselective syntheses, as well as other new, innovative general syntheses for the formation and functionalization of heterocycles that have helped drive project delivery. Additionally, the importance and value of collaborations between industry and academia in shaping the development of innovative synthetic approaches to functionalized heterocycles that are of greatest interest to the pharmaceutical industry will be highlighted.

  20. Differences of DNA methylation profiles between monozygotic twins' blood samples.

    PubMed

    Li, Chengtao; Zhao, Shumin; Zhang, Na; Zhang, Suhua; Hou, Yiping

    2013-09-01

    Monozygotic twins (MZs) share an identical genomic sequence, which makes it impossible to discriminate one another with conventional genetic markers like STRs. On the other hand, phenotypic discordance between MZs implies the existence of different epigenetic characteristics. DNA methylation, an essential epigenetic modification, however, might be a potential biomarker to solve the forensic puzzle. In this study, we examined 22 pairs of MZs with a methylation BeadChip including 27,578 CpG sites. The results suggested that MZs exhibited remarkable differences of genome-wide 5-methylcytosine. According to a set of criteria of selection, 92 CpG sites with significant differences of methylation status within MZs were identified from the global epigenome. In conclusion, this pilot study suggested that CpG methylation profile could be a useful biomarker in individual identification of MZs.

  1. Duplex structural differences and not 2′-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA

    PubMed Central

    Olimpo, Jeffrey T.; DeStefano, Jeffrey J.

    2010-01-01

    Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA–DNA versus DNA–DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3′ recessed DNA base) are required for stable binding to RNA–DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA–DNA. Replacing 2′- hydroxyls on pivotal RNA bases with 2′-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. RT’s Kd (koff/kon) for DNA–DNA and RNA–DNA were similar, although koff differed almost 40-fold, suggesting a faster kon for DNA–DNA. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA–DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA–DNA and DNA–DNA, respectively, allow the former to conform more easily to HIV-RT’s binding cleft, leading to more stable binding. Biologically, the ability of RT to form a more stable complex on RNA–DNA may aid in degradation of RNA fragments that remain after DNA synthesis. PMID:20338878

  2. Identifying DNA Binding Motifs by Combining Data from Different Sources

    SciTech Connect

    Mao, Linyong; Resat, Haluk; Nagib Callaos; Katsuhisa Horimoto; Jake Chen; Amy Sze Chan

    2004-07-19

    A transcription factor regulates the expression of its target genes by binding to their operator regions. It functions by affecting the interactions between RNA polymerases and the gene's promoter. Many transcription factors bind to their targets by recognizing a specific DNA sequence pattern, which is referred to as a consensus sequence or a motif. Since it would remove the possible biases, combining biological data from different sources can be expected to improve the quality of the information extracted from the biological data. We analyzed the microarray gene expression data and the organism's genome sequence jointly to determine the transcription factor recognition sequences with more accuracy. Utilizing such a data integration approach, we have investigated the regulation of the photosynthesis genes of the purple non-sulphur photosynthetic bacterium Rhodobacter sphaeroides. The photosynthesis genes in this organism are tightly regulated as a function of environmental growth conditions by three major regulatory systems, PrrB/PrrA, AppA/PpsR and FnrL. In this study, we have detected a previously undefined PrrA consensus sequence, improved the previously known DNA-binding motif of PpsR, and confirmed the consensus sequence of the global regulator FnrL.

  3. Unreserved application of epigenetic methods to define differences of DNA methylation between urinary cellular and cell-free DNA.

    PubMed

    Ghanjati, Foued; Beermann, Agnes; Hermanns, Thomas; Poyet, Cedric; Araúzo-Bravo, Marcos J; Seifert, Hans-Helge; Schmidtpeter, Martin; Goering, Wolfgang; Sorg, Rüdiger; Wernet, Peter; Santourlidis, Simeon

    2014-01-01

    Urinary DNA is increasingly gaining importance in diagnosis of urological malignancies. Especially cell-free DNA originating from apoptotic and necrotic cells of the early tumor could become a key target for early stage tumor diagnosis. Aberrant DNA methylation forms tumor cell characteristic epigenetic profiles which are covalently established before any tumor related aberration at transcriptional or protein level has occurred. In addition, these epigenetic signatures are alterably adapted to and accompanying the individual stages of multistep, progressive tumorigenesis. Hence, they seem very promising for diagnosis as well as for monitoring the patient's follow-up care and even for decisions regarding personalized therapeutic options. The essential prerequisite at this approach will be a reliable methodological handling of the biological material of interest. In this study we present detailed analyses of LINE-1 DNA methylation profiles and demonstrate the sensitive detection of LINE-1 DNA methylation differences as well as between cancer patients and healthy individuals, between urinary cellular and cell-free DNA. In addition, we show methylome differences between both DNA fractions from a healthy individual and bladder cancer patients. In conclusion, we demonstrate here the unrestricted amenability of urinary cell-free DNA for both, a detailed characterization of a distinct DNA methylation alteration and its sensitive detection and a comprehensive global, array-based screening for DNA methylation differences.

  4. Sterically hindered complexes of platinum(II) with planar heterocyclic nitrogen donors. A novel complex with 1-methyl-cytosine has a spectrum of activity different from cisplatin and is able of overcoming acquired cisplatin resistance.

    PubMed

    Margiotta, Nicola; Natile, Giovanni; Capitelli, Francesco; Fanizzi, Francesco P; Boccarelli, Angelina; De Rinaldis, Pietro; Giordano, Domenico; Coluccia, Mauro

    2006-11-01

    A very interesting series of water soluble platinum compounds violating some of the classical structure-activity relationships, but still showing antitumor activity, was reported by Hollis and collaborators some 25 years ago [L.S. Hollis, A.R. Amundsenm, E.W. Stern. J. Med. Chem. 32 (1989) 128-136]. The compounds, having formula [PtClA(2)L](+) (A(2)=two monodentate or a bidentate amine, L=a secondary or tertiary amine or a N-donor heterocycle), were characterized by a positive charge and three non-labile N-donor ligands. We have extended the investigation to analogous compounds in which 2,9-dimethyl-1,10-phenanthroline has taken the place of the A(2) ligand(s) and L is 2-picoline (1), 6-amino-2-picoline (2), or 1-methyl-cytosine (3). The X-ray analysis of 2 has revealed a bow-like distortion of the phenanthroline plane, a sloping of the phenanthroline plane with respect to the coordination plane, and an overall shielding of the metallic core by the ortho substituents of the phenanthroline and pyridine ligands. In vitro grow inhibition assays have been performed on the most water soluble complex 3. The results indicate that this complex is characterized by a potent growth inhibitory activity with mean IC(50) value (in a panel of 11 human tumor cell lines) of 1.1 microM to be compared with a mean value of 3.8 microM for cisplatin. The same compound also appears to completely overcome the acquired cisplatin resistance stemming from reduced uptake or a multifocal mechanism, thus pointing to a mechanism of action distinctly different from that of cisplatin.

  5. [Features of binding of proflavine to DNA at different DNA-ligand concentration ratios].

    PubMed

    Berezniak, E G; gladkovskaia, N A; Khrebtova, A S; Dukhopel'nikov, E V; Zinchenko, A V

    2009-01-01

    The binding of proflavine to calf thymus DNA has been studied using the methods of differential scanning calorimetry and spectrophotometry. It was shown that proflavine can interact with DNA by at least 3 binding modes. At high DNA-ligand concentration ratios (P/D), proflavine intercalates into both GC- and AT-sites, with a preference to GC-rich sequences. At low P/D ratios proflavine interacts with DNA by the external binding mode. From spectrophotometric concentration dependences, the parameters of complexing of proflavine with DNA were calculated. Thermodynamic parameters of DNA melting were calculated from differential scanning calorimetry data.

  6. Photochromism of heterocyclic systems

    NASA Technical Reports Server (NTRS)

    Silversmith, E. F.

    1972-01-01

    The preparations of photochromic dimers of a 2-aryl-4,5-di-phenylimidazyl radical and of a 2,5-diphenyl-3,4-isobenzopyrrole with different substituents in the homocyclic ring are reported. Of the dienes investigated, only 1-4-diphenyl-1,3-butadiene and 2,3-dimethyl-1,3-butadiene gave isolable amounts of the Diels-Adler adducts.

  7. Potential antitumor agents. 29. Quantitative structure-activity relationships for the antileukemic bisquaternary ammonium heterocycles.

    PubMed

    Denny, W A; Atwell, G J; Baguley, B C; Cain, B F

    1979-02-01

    Quantitative relationships between physicochemical drug properties and antileukemic (L1210) efficacy have been examined for a series of bisquaternary ammonium heterocycles employing multiple variable regression analysis. Three measures of biologic response were examined: ILSmax, the percentage increase in mean life span of leukemic animals at the LD10 dose; D40, the drug dose necessary to provide 40% increase in life span; and CI (=LD 10/D40), the chemotherapeutic index. A cross correlation matrix between these three measures and the LD10 values demonstrates ILSmax and CI to be independent of toxicity. D40 is highly inversely correlated with LD10 and positively correlated with ILSmax, suggesting that this parameter measures a composite of both drug selectivity and toxicity. Superior regression equations resulted at all stages employing ILSmax as a measure of antitumor selectivity. Acceptable equations modeling LD10 could not be obtained. There was a parabolic relationship between agent lipophilic-hydrophilic balance, measured as chromatographic Rm values, and ILSmax. To reduce residual variance in the L1210 screening data, not accepted by this parabolic equation, measures of agent-DNA interaction were investigated as possible indices of site fit. Relative levels of drug-DNA interaction were obtained by spectrofluorimetric quantitation of drug displacement of DNA-bound ethidium. Addition to regression equations of agent C50 values for calf thymus DNA, those micromolar drug concentrations necessary to displace 50% of the ethidium bound to that DNA, provided a significant reduction in the screening data variance. C50 values for drug interactions with poly[d(A-T)] and poly[d(G-C)] were also investigated as possible indicators of drug selectivity towards different DNA sites. Marked differences were observed in the C50 values for the two synthetic nucleic acids, with those for calf thymus DNA and poly[d(G-C)] proving highly covariant. A regression equation containing a

  8. Nucleotidyl transferase assisted DNA labeling with different click chemistries.

    PubMed

    Winz, Marie-Luise; Linder, Eva Christina; André, Timon; Becker, Juliane; Jäschke, Andres

    2015-09-30

    Here, we present a simple, modular and efficient strategy that allows the 3'-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3'-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Nucleotidyl transferase assisted DNA labeling with different click chemistries

    PubMed Central

    Winz, Marie-Luise; Linder, Eva Christina; André, Timon; Becker, Juliane; Jäschke, Andres

    2015-01-01

    Here, we present a simple, modular and efficient strategy that allows the 3′-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3′-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation. PMID:26013812

  10. Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA.

    PubMed

    Worm Ørntoft, Mai-Britt; Jensen, Sarah Østrup; Hansen, Thomas Birkballe; Bramsen, Jesper Bertram; Andersen, Claus Lindbjerg

    2017-05-30

    Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. To compare cfDNA recovery of different BSC methods, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only 3 kits recovered DNA fragments of <100 bp well. In contrast, DNA input amount did not seem to affect DNA recovery: for quantities spanning between 820 and ∼25,000 genome equivalents per BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97-99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.

  11. On the regiochemical differences between Pd-catalyzed heterocyclization-allylation and -arylation reactions of alkynylbenzamides: preparation of 4-allyl-isochromen-1-imines and computational study.

    PubMed

    Álvarez, Rosana; Vilar, Unai; Madich, Youssef; Aurrecoechea, José M

    2017-10-05

    The Pd(0)-catalyzed cyclization-allylation reactions of 2-alkynylbenzamides proceed with high regioselectivity to afford the 6-endo-cyclization-derived products 4-allyl-isochromen-1-imines. DFT calculations have been performed on this and the related arylation reaction, that has been reported to afford the products corresponding to an exo-cyclization under similarly Pd(0)-catalyzed conditions. Under the reaction conditions, these cyclizations are presumed to be triggered by activation of the C-C triple bond with either an allyl- or an aryl palladium complex, generated by oxidative addition of an allyl- or aryl halide to the Pd(0) catalyst. For reactions promoted by allylpalladium species, calculations predict a reversible cyclization, followed by a regioselectivity-determining endo-selective reductive elimination. In contrast, according to calculations, the corresponding arylations would proceed through irreversible exo-selective cyclization and reductive elimination steps. These predictions are consistent with the experimental observations. The divergent regiochemical outcome appears to have its origin in the differences caused on the intermediate palladium complexes by the groups derived from the coupling agents (allyl or aryl) and by the reaction conditions (solvent and ligands) through a subtle interplay of polarity and coordinative effects.

  12. The Modern Face of Synthetic Heterocyclic Chemistry.

    PubMed

    Cabrele, Chiara; Reiser, Oliver

    2016-11-04

    The synthesis of heterocycles is arguably one of the oldest and at the same time one of the youngest disciplines of organic chemistry. Groundbreaking principles to form heterocycles, mainly by condensation reactions, were recognized in the beginning of the 19th century, and many of the classical reactions discovered at that time are still of great value today. In the 21st century, the wealth of synthetic methodology toward heterocycles is overwhelming, and catalysis, in particular, as one of the cornerstones of green and sustainable chemistry has contributed in a major way to these developments. This perspective tries the impossible by discussing some recent advances in the construction of heterocycles, focusing on catalytic methodology. We are aware that we do not come close to giving adequate credit to the great creativity of chemists in the field.

  13. Synthesis of saturated N-heterocycles.

    PubMed

    Vo, Cam-Van T; Bode, Jeffrey W

    2014-04-04

    Saturated N-heterocycles are prevalent in biologically active molecules and are increasingly attractive scaffolds in the development of new pharmaceuticals. Unlike their aromatic counterparts, there are limited strategies for facile construction of substituted saturated N-heterocycles by convergent, predictable methods. In this Synopsis, we discuss recent advances in the synthesis of these compounds, focusing on approaches that offer generality and convenience from widely available building blocks.

  14. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples.

    PubMed

    Claassen, Shantelle; du Toit, Elloise; Kaba, Mamadou; Moodley, Clinton; Zar, Heather J; Nicol, Mark P

    2013-08-01

    Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H'=2.30 and 1.27) and kit QS (H'=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed.

  15. Lactoperoxidase-catalyzed activation of carcinogenic aromatic and heterocyclic amines.

    PubMed

    Gorlewska-Roberts, Katarzyna M; Teitel, Candee H; Lay, Jackson O; Roberts, Dean W; Kadlubar, Fred F

    2004-12-01

    Lactoperoxidase, an enzyme secreted from the human mammary gland, plays a host defensive role through antimicrobial activity. It has been implicated in mutagenic and carcinogenic activation in the human mammary gland. The potential role of heterocyclic and aromatic amines in the etiology of breast cancer led us to examination of the lactoperoxidase-catalyzed activation of the most commonly studied arylamine carcinogens: 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), benzidine, 4-aminobiphenyl (ABP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). In vitro activation was performed with lactoperoxidase (partially purified from bovine milk or human milk) in the presence of hydrogen peroxide and calf thymus DNA. Products formed during enzymatic activation were monitored by HPLC with ultraviolet and radiometric detection. Two of these products were characterized as hydrazo and azo derivatives by means of mass spectrometry. The DNA binding level of 3H- and 14C-radiolabeled amines after peroxidase-catalyzed activation was dependent on the hydrogen peroxide concentration, and the highest levels of carcinogen binding to DNA were observed at 100 microM H2O2. Carcinogen activation and the level of binding to DNA were in the order of benzidine > ABP > IQ > MeIQx > PhIP. One of the ABP adducts was identified, and the level at which it is formed was estimated to be six adducts/10(5) nucleotides. The susceptibility of aromatic and heterocyclic amines for lactoperoxidase-catalyzed activation and the binding levels of activated products to DNA suggest a potential role of lactoperoxidase-catalyzed activation of carcinogens in the etiology of breast cancer.

  16. HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

    PubMed

    Nascimento-Brito, Sieberth; Paulo Zukurov, Jean; Maricato, Juliana T; Volpini, Angela C; Salim, Anna Christina M; Araújo, Flávio M G; Coimbra, Roney S; Oliveira, Guilherme C; Antoneli, Fernando; Janini, Luiz Mário R

    2015-01-01

    In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5) and C-X-C chemokine Receptor type 4 (CXCR4) Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

  17. γ irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells.

    PubMed

    Donà, Mattia; Ventura, Lorenzo; Macovei, Anca; Confalonieri, Massimo; Savio, Monica; Giovannini, Annalisa; Carbonera, Daniela; Balestrazzi, Alma

    2013-05-15

    In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50 Gy, delivered at 0.33 Gy min(-1)) and HDR (total doses 50 and 100 Gy, delivered at 5.15 Gy min(-1)) γ-ray in the 0-24h time period after treatments. Significant fluctuations of double strand breaks and different repair capacities were observed between treatments in the 0-4h time period following irradiation. Dose-rate-dependent changes in the expression of the PhMT2 and PhAPX genes encoding a type 2 metallothionein and the cytosolic isoform of ascorbate peroxidase, respectively, were detected by Quantitative RealTime-Polymerase Chain Reaction. The PhMT2 and PhAPX genes were significantly up-regulated (3.0- and 0.7-fold) in response to HDR. The results are discussed in light of the potential practical applications of LDR-based treatments in mutation breeding. Copyright © 2013 Elsevier GmbH. All rights reserved.

  18. DNA Repair and Ethnic Differences in Prostate Cancer Risk

    DTIC Science & Technology

    2007-03-01

    XRCC1 Polymorphisms: Effects on Aflatoxin B1 -DNA Adducts and Glycophorin A Variant Frequency. Cancer Res. 6-1-1999;59(11):2557-61. 33. Singh, N. P...Polymorphisms: Effects on Aflatoxin B1 -DNA Adducts. Cancer Res. 6-1-1999;59(11):2557-61. 20. Dogliotti, E., Hainaut, P., Hernandez, T., D’Errico, M...to ask you some general information about yourself. B1 . What is your marital status? ( )1 Widowed ( )2 Married or living as

  19. Comparison of different protocols for the extraction of microbial DNA from reef corals.

    PubMed

    Santos, H F; Carmo, F L; Leite, D C A; Jesus, H E; Maalouf, P De Carvalho; Almeida, C; Soriano, A U; Altomari, D; Suhett, L; Vólaro, V; Valoni, E; Francisco, M; Vieira, J; Rocha, R; Sardinha, B L; Mendes, L B; João, R R; Lacava, B; Jesus, R F; Sebastian, G V; Pessoa, A; van Elsas, J D; Rezende, R P; Pires, D O; Duarte, G; Castro, C B; Rosado, A S; Peixoto, R S

    2012-04-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

  20. Comparison of different protocols for the extraction of microbial DNA from reef corals

    PubMed Central

    Santos, H.F.; Carmo, F.L.; Leite, D.C.A.; Jesus, H.E.; Maalouf, P. De Carvalho; Almeida, C.; Soriano, A.U.; Altomari, D.; Suhett, L.; Vólaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B.L.; Mendes, L.B.; João, R.R.; Lacava, B.; Jesus, R.F.; Sebastian, G.V.; Pessoa, A.; van Elsas, J.D.; Rezende, R.P.; Pires, D.O.; Duarte, G.; Castro, C.B.; Rosado, A.S.; Peixoto, R.S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals. PMID:24031859

  1. [Heterocyclic aromatic amines, food-derived mutagens: metabolism and relevance to cancer susceptibility].

    PubMed

    Woziwodzka, Anna; Tarasewicz, Marta; Piosik, Jacek

    2010-01-01

    It is estimated that diet contributes to as much as one-third of cancer incidents. Heterocyclic aromatic amines (HCAs) are well-known mutagens/carcinogens found in thermal-processed meat and fish. HCAs require metabolic activation to exert their carcinogenic potential. First step in HCAs activation--the generation of N-hydroxy-HCA derivatives--is catalyzed by cytochrome P450, mainly isoenzyme CYP1A2. Further activation is carried out by N-acetyltransferases and sulfotransferases, which catalyze esterification of N-hydroxy-HCAs. The products of these reactions are highly genotoxic, capable of direct interaction with DNA by adduct formation. HCA-DNA adducts may cause errors in DNA replication and the generation of mutations, which, when not repaired, may contribute to cancer development. On the other hand, among enzymes involved in HCAs detoxication, UDP-glucuronosyltransferases and glutathione S-transferases can be mentioned. Balance between activation and detoxication processes of HCAs, together with genetically determined differences in HCA metabolism are crucial for the assessment of HCA-dependent cancer risk among individuals.

  2. Different Modes of Human Papillomavirus DNA Replication during Maintenance

    PubMed Central

    Hoffmann, Ralf; Hirt, Bernhard; Bechtold, Viviane; Beard, Peter; Raj, Kenneth

    2006-01-01

    Human papillomavirus (HPV) begins its life cycle by infecting the basal cells of the epithelium. Within these proliferating cells, the viral genomes are replicated, maintained, and passed on to the daughter cells. Using HPV episome-containing cell lines that were derived from naturally infected cervical tissues, we investigated the mode by which the viral DNAs replicate in these cells. We observed that, whereas HPV16 DNA replicated in an ordered once-per-S-phase manner in W12 cells, HPV31 DNA replicated via a random-choice mechanism in CIN612 cells. However, when HPV16 and HPV31 DNAs were separately introduced into an alternate keratinocyte cell line NIKS, they both replicated randomly. This indicates that HPV DNA is inherently capable of replicating by either random-choice or once-per-S-phase mechanisms and that the mode of HPV DNA replication is dependent on the cells that harbor the viral episome. High expression of the viral replication protein E1 in W12 cells converted HPV16 DNA replication to random-choice replication and, as such, it appears that the mode of HPV DNA replication in proliferating cells is dependent on the presence or the increased level of this protein in the host cell. The implications of these observations on maintenance, latency, and persistence are discussed. PMID:16611903

  3. Effect of intestinal microfloras from vegetarians and meat eaters on the genotoxicity of 2-amino-3-methylimidazo[4,5-f]quinoline, a carcinogenic heterocyclic amine.

    PubMed

    Kassie, F; Lhoste, E F; Bruneau, A; Zsivkovits, M; Ferk, F; Uhl, M; Zidek, T; Knasmüller, S

    2004-03-25

    Aim of this study was to investigate the impact of intestinal microfloras from vegetarians and non-vegetarians on the DNA-damaging activity of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), a carcinogenic heterocyclic amine that is found in fried meats. Floras from four vegetarians (Seventh Day Adventists) and from four individuals who consumed high amounts of meats were collected and inoculated into germfree F344 rats. The rats were kept on isocaloric diets that either contained animal derived protein and fat (meat consumers group) or proteins and fat of plant origin (vegetarian groups). IQ (90 mg/kg bw) was administered orally, after 4 h the extent of DNA-damage in colon and liver cells was determined in single cell gel electrophoresis assays. In all groups, the IQ induced DNA-migration was in the liver substantially higher than in the colon. In animals harbouring floras of vegetarians, the extent of damage was in both organs significantly (69.2% in the liver, P<0.016 and 64.7%, P<0.042 in the colon, respectively) lower than in the meat consumer groups. Our findings show that diet related differences in the microfloras have a strong impact on the genotoxic effects of IQ and suggest that heterocyclic amines are less genotoxic and carcinogenic in individuals that consume mainly plant derived foods.

  4. Ring cleavage of sulfur heterocycles: how does it happen?

    PubMed

    Bressler, D C; Norman, J A; Fedorak, P M

    Sulfur heterocycles are common constituents of petroleum and liquids derived from coal, and they are found in some secondary metabolites of microorganisms and plants. They exist primarily as saturated rings and thiophenes. There are two major objectives driving investigations of the microbial metabolism of organosulfur compounds. One is the quest to develop a process for biodesulfurization of fossil fuels, and the other is to understand the fates of organosulfur compounds in petroleum- or creosote-contaminated environments which is important in assessing bioremediation processes. For these processes to be successful, cleavage of different types of sulfur heterocyclic rings is paramount. This paper reviews the evidence for microbial ring cleavage of a variety of organosulfur compounds and discusses the few well-studied cases which have shown that the C-S bond is most susceptible to breakage leading to disruption of the ring. In most cases, the introduction of one or more oxygen atom(s) onto the adjacent C atom and/or onto the S atom weakens the C-S bond, facilitating its cleavage. Although much is known about the thiophene ring cleavage in dibenzothiophene, there is still a great deal to be learned about the cleavage of other sulfur heterocycles.

  5. Complexes of different nitrogen donor heterocyclic ligands with SbCl3 and PhSbCl2 as potential antileishmanial agents against Sb(III)-sensitive and -resistant parasites.

    PubMed

    Lizarazo-Jaimes, Edgar H; Reis, Priscila G; Bezerra, Filipe M; Rodrigues, Bernardo L; Monte-Neto, Rubens L; Melo, Maria N; Frézard, Frédéric; Demicheli, Cynthia

    2014-03-01

    Novel trivalent antimony complexes with the nitrogen donor heterocyclic ligand 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) or dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) have been synthesized by the reaction with SbCl3 or PhSbCl2. The crystal structures of [Sb(phen)Cl3] and [PhSb(phen)Cl2]CH3COOH were determined and shown to adopt a distorted square pyramid geometry with a five-coordinated Sb center. Surprisingly, all the complexes, the ligands and PhSbCl2 showed very high antileishmanial activities, with IC50 in the nanomolar range against Sb(III)-sensitive and -resistant Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis strains. These compounds were much more active against these Leishmania strains than the old trivalent drug potassium antimonyl tartrate. [PhSb(phen)Cl2]CH3COOH complex was found to be the most active compound and the lack of cross-resistance of PhSbCl2 suggests that the transport pathways of this compound across the cell membrane differ from those responsible for the resistance of Leishmania to Sb(OH)3. In the case of the complexes with PhSbCl2, our data supports the model that both ligand and metal contributed to the overall activity of the complex. Furthermore, among the complexes with SbCl3, only bipy showed an improved activity upon complexation. Cytotoxicity evaluations of these compounds against murine peritoneal macrophages showed high selective indexes in the range of 7-70 for [Sb(phen)Cl3], [Sb(bipy)Cl3] and [Sb(dpq)Cl3] complexes, being much more selective than potassium antimonyl tartrate. In conclusion, this study presents a set of new antileishmanial agents including one of the most active Sb-based compounds ever reported, which can contribute to the development of new chemotherapeutic strategies against leishmaniasis including Sb-resistant cases.

  6. Nickel-Catalyzed Reactions Directed toward the Formation of Heterocycles.

    PubMed

    Kurahashi, Takuya; Matsubara, Seijiro

    2015-06-16

    Heterocycles have garnered significant attention because they are important functional building blocks in various useful molecules, such as pharmaceuticals, agricultural chemicals, pesticides, and materials. Several studies have been conducted regarding the preparation of heterocyclic skeletons with an emphasis on selectivity and efficiency. Three strategies are typically employed to construct cyclic molecules, namely, cyclization, cycloaddition, and ring-size alterations. Although each method has certain advantages, cycloaddition may be superior from the viewpoint of divergence. Specifically, cycloadditions enable the construction of rings from several pieces. However, the construction of heterocycles via cycloadditions is more challenging than the construction of carbocycles. For heterocycle construction, simple pericyclic reactions rarely work smoothly because of the large HOMO-LUMO gap unless well-designed combinations, such as electron-rich dienes and aldehydes, are utilized. Thus, a different approach should be employed to prepare heterocycles via cycloadditions. To this end, the use of metallacycles containing heteroatoms is expected to serve as a promising solution. In this study, we focused on the preparation of heteroatom-containing nickelacycles. Because nickel possesses a relatively high redox potential and an affinity for heteroatoms, several methods were developed to synthesize heteronickelacycles from various starting materials. The prepared nickelacycles were demonstrated to be reasonable intermediates in cycloaddition reactions, which were used to prepare various heterocycles. In this Account, we introduce the following four methods to prepare heterocycles via heteronickelacycles. (1) Direct oxidative insertion of Ni(0) to α,β-unsaturated enone derivatives: treatment of 3-ethoxycarbonyl-4-phenyl-3-buten-2-one with Ni(0) afforded an oxa-nickelacycle, which reacted with alkynes to give pyrans. (2) Substitution of a part of a cyclic compound with

  7. The tert-amino effect in heterocyclic chemistry. Synthesis of spiro heterocycles.

    PubMed

    D'yachenko, Elisaveta V; Glukhareva, Tatiana V; Dyudya, Lyudmila V; Eltsov, Oleg V; Morzherin, Yury Yu

    2005-09-30

    The tert-amino reaction effect was examined. A new method to synthesize spiro heterocycles is presented. It was shown that the "tert-amino effect" could be applied to the formation of spiro-fused heterocycles. The formation of spiro compounds proceeds in most cases in good yields in a one-pot reaction.

  8. An efficient method for genomic DNA extraction from different molluscs species.

    PubMed

    Pereira, Jorge C; Chaves, Raquel; Bastos, Estela; Leitão, Alexandra; Guedes-Pinto, Henrique

    2011-01-01

    The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others.

  9. An Efficient Method for Genomic DNA Extraction from Different Molluscs Species

    PubMed Central

    Pereira, Jorge C.; Chaves, Raquel; Bastos, Estela; Leitão, Alexandra; Guedes-Pinto, Henrique

    2011-01-01

    The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others. PMID:22174651

  10. Immunization of mice against Salmonella typhimurium using different DNA preparations.

    PubMed

    Kalfayan, L H; Farhat, D; El-Nakat, H; Matar, G M; Abdelnoor, A M

    2001-11-01

    Groups of female BALB/c mice were given primary and booster injections of whole genomic DNA extracted from S. typhimurium, P. aeruginosa, or S. aureus. Other groups of mice were immunized in a similar manner with the 1.57kb fragment of the mouse virulence gene (mviA), pTargeT vector (plasmid DNA)/1.57kb construct, pTargeT vector, or saline. Mice in all groups were challenged intraperitoneally with 100 LD50 of S. typhimurium. The bacterial genomic DNA was extracted using the Pure Gene extraction kit. Specific primers were used to amplify the 1.57kb fragment by PCR. The pTargeT Mammalian Expression Vector System was used to prepare the plasmid/ 1.57kb construct. Bacterial genomic DNA extracted from P. aeruginosa and S. aureus appeared to induce non-specific resistance in mice. Specific, in addition to non-specific resistance appeared to be induced when genomic DNA from S. typhimurium was used. There was a prolongation of survival in the groups of mice that received either the 1.57kb fragment or the pTargeT vector/1.57kb construct and 16.67% and 33.34% respectively, of mice in each group survived at 40 days post challenge. None of the mice in the saline control group survived by day 7 post challenge. It is suggested that the non-specific resistance observed in this study might have been due to the adjuvant effect of the non-methylated CpG and other immunostimulatory motifs in bacterial DNA. Specific resistance obtained when genomic DNA from S. typhimurium was used might have been due to minute antigenic contamination, or virulence factor genes other than the mviA gene, might have been expressed in the host, which induced specific immunity.

  11. DNA Repair and Ethnic Differences in Prostate Cancer Risk

    DTIC Science & Technology

    2006-03-01

    2001;461(4):273-8. 32. Lunn, R. M., Langlois, R. G., Hsieh, L. L., Thompson, C. L., and Bell, D. A. XRCC1 Polymorphisms: Effects on Aflatoxin B1 -DNA...1992;18:1-29. 19. Lunn, R. M., Langlois, R. G., Hsieh, L. L., Thompson, C. L., and Bell, D. A. XRCC1 Polymorphisms: Effects on Aflatoxin B1 -DNA Adducts...about yourself. B1 . What is your marital status? ( )1 Widowed ( )2 Married or living as married ( )3 Divorced

  12. Cytotoxic, mutagenic, and carcinogenic effects of energy-related agents in diploid human cells which differ in DNA repair capacity. Progress report, 1980-1983

    SciTech Connect

    McCormick, J.J.; Maher, V.M.

    1983-01-01

    The mechanisms of action of a series of energy-related chemical carcinogens to bring about cell killing, the induction of mutations, and the transformation of diploid human fibroblasts in culture were examined. Some of these studies have employed direct-acting compounds (reactive derivatives or metabolites of parent compounds) such as (+-)-7..beta..,8..cap alpha..-dihydroxy9..cap alpha.., 10..cap alpha..-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti BPDE), the 4,5-epoxide of benzo(a)pyrene(B(a)P 4,5-oxide), and aflatoxin B/sub 1/-dichloride (AFB/sub 1/-Cl/sub 2/). In other studies, human epithelial cell lines have served as source of the various metabolizing enzymes needed to convert parent compounds into reactive intermediates. We screened a series of 15 epithelial cell lines with unlimited lifespan (derived from various human carcinomas) for the ability to activate representative polycyclic aromatic hydrocarbons, aromatic amides, nitrogen heterocyclics, nitro samines, and/or aflatoxins. Candidate metabolizing cells were then combined with diploid human fibroblasts as target cells in a cell-mediated assay of the mutagenic and/or cytotoxic effect of particular energy-related chemical carcinogens, such as B(a)P, benzofluoranthenes, and dibenzo(c,g)carbazole. The number and kind of major DNA adducts formed in human cells by these chemicals were determined. Comparative studies of anti BPDE and B(a)P 4,5-oxide showed that in diploid human fibroblasts, both normal and DNA repair deficient, these agents do not differ significantly in mutagenic efficiency (per mean lethal event) or mutagenic effectiveness (per DNA adduct). In diploid Chinese hamster embryo fibroblasts anti BPDE had approx. 4-fold higher mutagenic efficiency than B(a)P 4,5-oxide in the latter cells. FA cells were significantly more sensitive than normal to killing by anti BPDE.

  13. DNA Repair and Ethnic Differences in Prostate Cancer Risk

    DTIC Science & Technology

    2008-03-01

    Hsieh, L. L., Thompson, C. L., and Bell, D. A. XRCC1 Polymorphisms: Effects on Aflatoxin B1 -DNA Adducts. Cancer Res. 6-1-1999;59(11):2557-61. 20...3 B. DEMOGRAPHIC INFORMATION Now I would like to ask you some general information about yourself. B1 . What is your marital status

  14. Comprehensive database of Manufactured Gas Plant tars. Part C. Heterocyclic and hydroxylated polycyclic aromatic hydrocarbons.

    PubMed

    Gallacher, Christopher; Thomas, Russell; Lord, Richard; Kalin, Robert M; Taylor, Chris

    2017-08-15

    Coal tars are a mixture of organic and inorganic compounds that were by-products from the manufactured gas and coke making industries. The tar compositions varied depending on many factors such as the temperature of production and the type of retort used. For this reason a comprehensive database of the compounds found in different tar types is of value to understand both how their compositions differ and what potential chemical hazards are present. This study focuses on the heterocyclic and hydroxylated compounds present in a database produced from 16 different tars from five different production processes. Samples of coal tar were extracted using accelerated solvent extraction (ASE) and derivatized post-extraction using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS). The derivatized samples were analysed using two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC/TOFMS). A total of 865 heterocyclic compounds and 359 hydroxylated polycyclic aromatic hydrocarbons (PAHs) were detected in 16 tar samples produced by five different processes. The contents of both heterocyclic and hydroxylated PAHs varied greatly with the production process used, with the heterocyclic compounds giving information about the feedstock used. Of the 359 hydroxylated PAHs detected the majority would not have been be detected without the use of derivatization. Coal tars produced using different production processes and feedstocks yielded tars with significantly different heterocyclic and hydroxylated contents. The concentrations of the individual heterocyclic compounds varied greatly even within the different production processes and provided information about the feedstock used to produce the tars. The hydroxylated PAH content of the samples provided important analytical information that would otherwise not have been obtained without the use of derivatization and GCxGC/TOFMS. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Heterocyclic anions of astrobiological interest

    SciTech Connect

    Cole, Callie A.; Demarais, Nicholas J.; Bierbaum, Veronica M.; Yang, Zhibo; Snow, Theodore P. E-mail: Nicholas.Demarais@colorado.edu E-mail: Zhibo.Yang@ou.edu

    2013-12-20

    As more complex organic molecules are detected in the interstellar medium, the importance of heterocyclic molecules to astrobiology and the origin of life has become evident. 2-Aminothiazole and 2-aminooxazole have recently been suggested as important nucleotide precursors, highlighting azoles as potential prebiotic molecules. This study explores the gas-phase chemistry of three deprotonated azoles: oxazole, thiazole, and isothiazole. For the first time, their gas-phase acidities are experimentally determined with bracketing and H/D exchange techniques, and their reactivity is characterized with several detected interstellar neutral molecules (N{sub 2}O, O{sub 2}, CO, OCS, CO{sub 2}, and SO{sub 2}) and other reactive species (CS{sub 2}, CH{sub 3}Cl, (CH{sub 3}){sub 3}CCl, and (CH{sub 3}){sub 3}CBr). Rate constants and branching fractions for these reactions are experimentally measured using a modified commercial ion trap mass spectrometer whose kinetic data are in good accord with those of a flowing afterglow apparatus reported here. Last, we have examined the fragmentation patterns of these deprotonated azoles to elucidate their destruction mechanisms in high-energy environments. All experimental data are supported and complemented by electronic structure calculations at the B3LYP/6-311++G(d,p) and MP2(full)/aug-cc-pVDZ levels of theory.

  16. Heterocyclic Anions of Astrobiological Interest

    NASA Astrophysics Data System (ADS)

    Cole, Callie A.; Demarais, Nicholas J.; Yang, Zhibo; Snow, Theodore P.; Bierbaum, Veronica M.

    2013-12-01

    As more complex organic molecules are detected in the interstellar medium, the importance of heterocyclic molecules to astrobiology and the origin of life has become evident. 2-Aminothiazole and 2-aminooxazole have recently been suggested as important nucleotide precursors, highlighting azoles as potential prebiotic molecules. This study explores the gas-phase chemistry of three deprotonated azoles: oxazole, thiazole, and isothiazole. For the first time, their gas-phase acidities are experimentally determined with bracketing and H/D exchange techniques, and their reactivity is characterized with several detected interstellar neutral molecules (N2O, O2, CO, OCS, CO2, and SO2) and other reactive species (CS2, CH3Cl, (CH3)3CCl, and (CH3)3CBr). Rate constants and branching fractions for these reactions are experimentally measured using a modified commercial ion trap mass spectrometer whose kinetic data are in good accord with those of a flowing afterglow apparatus reported here. Last, we have examined the fragmentation patterns of these deprotonated azoles to elucidate their destruction mechanisms in high-energy environments. All experimental data are supported and complemented by electronic structure calculations at the B3LYP/6-311++G(d,p) and MP2(full)/aug-cc-pVDZ levels of theory.

  17. N-Heterocyclic molecule-capped gold nanoparticles as effective antibiotics against multi-drug resistant bacteria

    NASA Astrophysics Data System (ADS)

    Feng, Yan; Chen, Wenwen; Jia, Yuexiao; Tian, Yue; Zhao, Yuyun; Long, Fei; Rui, Yukui; Jiang, Xingyu

    2016-07-01

    We demonstrate that N-heterocyclic molecule-capped gold nanoparticles (Au NPs) have broad-spectrum antibacterial activity. Optimized antibacterial activity can be achieved by using different initial molar ratios (1 : 1 and 10 : 1) of N-heterocyclic prodrugs and the precursor of Au NPs (HAuCl4). This work opens up new avenues for antibiotics based on Au NPs.We demonstrate that N-heterocyclic molecule-capped gold nanoparticles (Au NPs) have broad-spectrum antibacterial activity. Optimized antibacterial activity can be achieved by using different initial molar ratios (1 : 1 and 10 : 1) of N-heterocyclic prodrugs and the precursor of Au NPs (HAuCl4). This work opens up new avenues for antibiotics based on Au NPs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr03317b

  18. Recent advances in rhodium-catalyzed asymmetric synthesis of heterocycles.

    PubMed

    Chen, Wen-Wen; Xu, Ming-Hua

    2017-02-01

    Heterocycles are crucial structural motifs that are ubiquitously present in biologically active natural products and pharmaceutically important compounds. Over the past few decades, great attention has been paid to develop efficient methodologies for the construction of diverse enantioenriched heterocyclic frameworks. This review focuses on the recent impressive progress and advances in the asymmetric synthesis of heterocycles under rhodium catalysis.

  19. 40 CFR 721.10003 - Manganese heterocyclic tetraamine complex (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Manganese heterocyclic tetraamine... Specific Chemical Substances § 721.10003 Manganese heterocyclic tetraamine complex (generic). (a) Chemical... as manganese heterocyclic tetraamine complex (PMNs P-98-625/626/627/628/629 and P-00-614/617) are...

  20. 40 CFR 721.10003 - Manganese heterocyclic tetraamine complex (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Manganese heterocyclic tetraamine... Specific Chemical Substances § 721.10003 Manganese heterocyclic tetraamine complex (generic). (a) Chemical... as manganese heterocyclic tetraamine complex (PMNs P-98-625/626/627/628/629 and P-00-614/617) are...

  1. 40 CFR 721.10003 - Manganese heterocyclic tetraamine complex (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Manganese heterocyclic tetraamine... Specific Chemical Substances § 721.10003 Manganese heterocyclic tetraamine complex (generic). (a) Chemical... as manganese heterocyclic tetraamine complex (PMNs P-98-625/626/627/628/629 and P-00-614/617) are...

  2. 40 CFR 721.10003 - Manganese heterocyclic tetraamine complex (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Manganese heterocyclic tetraamine... Specific Chemical Substances § 721.10003 Manganese heterocyclic tetraamine complex (generic). (a) Chemical... as manganese heterocyclic tetraamine complex (PMNs P-98-625/626/627/628/629 and P-00-614/617) are...

  3. 40 CFR 721.10003 - Manganese heterocyclic tetraamine complex (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Manganese heterocyclic tetraamine... Specific Chemical Substances § 721.10003 Manganese heterocyclic tetraamine complex (generic). (a) Chemical... as manganese heterocyclic tetraamine complex (PMNs P-98-625/626/627/628/629 and P-00-614/617)...

  4. Combinatorial Libraries of Bis-Heterocyclic Compounds with Skeletal Diversity

    PubMed Central

    Soural, Miroslav; Bouillon, Isabelle; Krchňák, Viktor

    2009-01-01

    Combinatorial solid-phase synthesis of bis-heterocyclic compounds, characterized by the presence of two heterocyclic cores connected by a spacer of variable length/structure, provided structurally heterogeneous libraries with skeletal diversity. Both heterocyclic rings were assembled on resin in a combinatorial fashion. PMID:18811208

  5. 40 CFR 721.5925 - Bis heterocyclic phenylene derivative (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Bis heterocyclic phenylene derivative... Specific Chemical Substances § 721.5925 Bis heterocyclic phenylene derivative (generic). (a) Chemical... as bis heterocyclic phenylene derivative (PMN P-01-0432) is subject to reporting under this...

  6. 40 CFR 721.5925 - Bis heterocyclic phenylene derivative (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Bis heterocyclic phenylene derivative... Specific Chemical Substances § 721.5925 Bis heterocyclic phenylene derivative (generic). (a) Chemical... as bis heterocyclic phenylene derivative (PMN P-01-0432) is subject to reporting under this...

  7. 40 CFR 721.5925 - Bis heterocyclic phenylene derivative (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Bis heterocyclic phenylene derivative... Specific Chemical Substances § 721.5925 Bis heterocyclic phenylene derivative (generic). (a) Chemical... as bis heterocyclic phenylene derivative (PMN P-01-0432) is subject to reporting under this...

  8. 40 CFR 721.5925 - Bis heterocyclic phenylene derivative (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Bis heterocyclic phenylene derivative... Specific Chemical Substances § 721.5925 Bis heterocyclic phenylene derivative (generic). (a) Chemical... as bis heterocyclic phenylene derivative (PMN P-01-0432) is subject to reporting under this...

  9. 40 CFR 721.5925 - Bis heterocyclic phenylene derivative (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Bis heterocyclic phenylene derivative... Specific Chemical Substances § 721.5925 Bis heterocyclic phenylene derivative (generic). (a) Chemical... as bis heterocyclic phenylene derivative (PMN P-01-0432) is subject to reporting under this...

  10. Excited state electronic structures and photochemistry of heterocyclic annulated perylene (HAP) materials tuned by heteroatoms: S, Se, N, O, C, Si, and B.

    PubMed

    Zhao, Guang-Jiu; Han, Ke-Li

    2009-04-23

    perylenes are significantly red-shifted in comparison with those of unsubstituted perylene. The differences of electronic structures and photochemistry of these heterocyclic annulated perylene materials can be ascribed to the electron delocalization of LUMO orbital from heteroatom into the perylene skeleton for Si- and B-heterocyclic annulated perylenes, because the electron of the LUMO orbital for S-, Se-, N-, O-, and C-heterocyclic annulated perylenes is localized on the heteroatoms.

  11. The Phosphinoboration of N-Heterocycles.

    PubMed

    Geier, Stephen J; Vogels, Christopher M; Mellonie, Niall R; Daley, Erika N; Decken, Andreas; Doherty, Simon; Westcott, Stephen A

    2017-08-11

    The addition of phosphinoboronate ester Ph2 PBpin (pin=1,2-O2 C2 Me4 ) (1) to a number of different N-heterocycles has been investigated. Reaction of 1 with pyridine resulted in highly selective formation of the corresponding 1,4-addition product, with addition of the electron-deficient Bpin group to the pyridine nitrogen atom and the phosphido group to the para carbon atom. Conversely, reactions of para-substituted pyridine derivatives occurred predominately to afford 1,2-addition products while quinoline reacted to afford the 1,2-adduct which ultimately isomerized to afford the corresponding 1,4-addition product. Preliminary computational studies have been undertaken to explore possible pathways for these transformations including transfer of the PPh2(-) anion from [B(PPh2 )2 pin](-) to the 4-position of a borenium/boronium activated pyridine and concerted pathways for 1,2-addition via intramolecular nucleophilic attack of PPh2 at C2 of a Ph2 PBpin-coordinated pyridine via a four-centered transition state and intramolecular transfer of PPh2 to the 2-position of a boron-activated pyridine in a phosphido-bridged dimer involving a six-centered transition state. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Marine Molecular Machines: Heterocyclization in Cyanobactin Biosynthesis

    PubMed Central

    McIntosh, John A.

    2012-01-01

    Natural products that contain amino acid-derived (Cys, Ser, Thr) heterocycles are ubiquitous in nature, yet key aspects of their biosynthesis remain undefined. Cyanobactins are heterocyclic ribosomal peptide natural products from cyanobacteria, including symbiotic bacteria living with marine ascidians. In contrast to other ribosomal peptide heterocyclases that have been studied, the cyanobactin heterocyclase is a single protein that does not require an oxidase enzyme. Using this simplifying condition, we provide new evidence to support the hypothesis that these enzymes are molecular machines, using ATP in a product binding or orientation cycle. Further, we show that both protease inhibitors and ATP analogues inhibit heterocyclization and define the order of biochemical steps in the cyanobactin biosynthetic pathway. The cyanobactin pathway enzymes, PatD and TruD, are thiazoline and oxazoline synthetases. PMID:20540059

  13. Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

    PubMed

    Jakubowska, Joanna; Maciejewska, Agnieszka; Pawłowski, Ryszard

    2012-01-01

    There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol-chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol-chloroform protocols are more efficient for successful DNA extraction.

  14. Multi-Component Reactions in Heterocyclic Chemistry

    NASA Astrophysics Data System (ADS)

    Müller, Thomas J. J.; Orru, Romano V. A.; Chebanov, Valentin A.; Sakhno, Yana I.; Saraev, Vyacheslav E.; Muravyova, Elena A.; Andrushchenko, Anastasia Yu.; Desenko, Sergey M.; Akhmetova, V. R.; Khabibullina, G. R.; Rakhimova, E. B.; Vagapov, R. A.; Khairullina, R. R.; Niatshina, Z. T.; Murzakova, N. N.; Maslivets, Andrey N.; Voskressensky, Leonid G.; Danagulyan, Gevorg G.; Murtchyan, Armen D.; Tumanyan, Araksya K.; Banfi, Luca; Basso, Andrea; de Moliner, Fabio; Guanti, Giuseppe; Petricci, Elena; Riva, Renata; Taddei, Maurizio; Naimi-Jamal, M. Reza; Mashkouri, Sara; Sharifi, Ali; Przhevalski, Nikolai M.; Rozhkova, Elena N.; Tokmakov, Gennadii P.; Magedov, Igor V.; Armisheva, M. N.; Rassudihina, N. A.; Vahrin, M. I.; Gein, V. L.; Shaabani, Ahmad; Rezayan, Ali Hossein; Sarvary, Afshin; Heidary, Marjan; Ng, Seik Weng; Beliaev, Nikolai A.; Mokrushin, Vladimir S.; Paramonov, Igor V.; Ilyin, Alexey; Garkushenko, Anna K.; Dushek, Maria A.; Sagitullina, Galina P.; Sagitullin, Reva S.; Kysil, Volodymyr; Khvat, Alexander; Tsirulnikov, Sergey; Tkachenko, Sergey; Ivachtchenko, Alexandre; Gein, Vladimir L.; Panova, Olga S.; Ilyn, Alexey P.; Kravchenko, Dmitri V.; Potapov, Victor V.; Ivachtchenko, Alexandre V.; Vichegjanina, V. N.; Levandovskaya, E. B.; Gein, V. L.; Vahrin, M. I.; Vladimirov, I. N.; Zorina, A. A.; Nosova, N. V.; Gein, V. L.; Fedorova, O. V.; Vahrin, M. I.

    Multi-component and domino reactions are efficient and effective methods in the sustainable and diversity-oriented synthesis of heterocycles. In particular, transition metal-catalyzed multi-component sequences have recently gained considerable interest. Based upon the Sonogashira entry to alkynones, alkenones, and intermediate allenes, we have opened new avenues to the one-pot synthesis of numerous classes of heterocyclic frameworks in an MCR fashion. This methodological approach has now found various applications in one-pot syntheses of functional chromophores, pharmaceutically active compounds, and marine alkaloids and derivatives.

  15. Syntheses of Novel Nitrogen and Phosphorus Heterocycles.

    DTIC Science & Technology

    2014-09-26

    Chemicals and Materials Research Department, Ultrasystems, Inc. under Contract F49620-82-C-0021, "Syntheses of Novel Nitrogen and Phosphorus Hetero- * cycles ...ADl-NISS9 449 SYNTHESES OF NOVEL NITROGEN AND PHOSPHORUS HETEROCYCLES In (U) ULTRRSYSTENS INC IRVINE CR K L PRCIOREK ET RL. 26 RPR 85 SN-209?-F RFOSR...MICROCOPY RESOLUTION TEST CHART NATIONAL BURE&U OF STAOACS-963-A SR-I"I" s, -Ŕ 500 4 SYNTHESES OF NOVEL NITROGEN AND PHOSPHORUS HETEROCYCLES Contract No

  16. Covalent Polymers Containing Discrete Heterocyclic Anion Receptors

    NASA Astrophysics Data System (ADS)

    Rambo, Brett M.; Silver, Eric S.; Bielawski, Christopher W.; Sessler, Jonathan L.

    This chapter covers recent advances in the development of polymeric materials containing discrete heterocyclic anion receptors, and focuses on advances in anion binding and chemosensor chemistry. The development of polymers specific for anionic species is a relatively new and flourishing area of materials chemistry. The incorporation of heterocyclic receptors capable of complexing anions through noncovalent interactions (e.g., hydrogen bonding and electrostatic interactions) provides a route to not only sensitive but also selective polymeric materials. Furthermore, these systems have been utilized in the development of polymers capable of extracting anionic species from aqueous media. These latter materials may lead to advances in water purification and treatment of diseases resulting from surplus ions.

  17. Different mechanisms for the photoinduced production of oxidative DNA damage by fluoroquinolones differing in photostability.

    PubMed

    Spratt, T E; Schultz, S S; Levy, D E; Chen, D; Schlüter, G; Williams, G M

    1999-09-01

    Several fluoroquinolone antibacterial agents exhibit an adverse phototoxic effect in humans and are photo-cocarcinogenic in mice. The UV-induced production of reactive oxygen species plays a role in the toxicity and may be involved in carcinogenicity. Four fluoroquinolones were examined for the ability to photochemically produce oxidative damage in naked DNA. The major structural difference in the fluoroquinolones that would have an effect on their photostability is the functionality at the 8-position. At this position, 1-cyclopropyl-7-(2,8-diazbicyclo[4.3.0]non-8-yl)-6, 8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (BAY y3118) contains a chlorine atom, lomefloxacin a fluorine atom, ciprofloxacin a proton, and moxifloxacin a methoxy group. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in calf thymus DNA was assessed by HPLC with electrochemical detection, and strand breaks were measured in pBR322 with agarose gel electrophoresis. The relative photolability of the fluoroquinolones correlated to the extent of production of 8-oxodGuo and strand breaks, with both UVA and UVB irradiation, in the following order: BAY y3118 approximately lomefloxacin > ciprofloxacin > moxifloxacin. Experiments were performed to determine whether the mechanism of damage was due to a type I (radical) or type II (singlet oxygen) pathway. Nitrogen depletion of oxygen resulted in a decrease in the extent of formation of 8-oxodGuo, suggesting that oxygen was involved. The use of selective radical or singlet oxygen inhibitors was inconclusive with respect to which pathway was involved. The use of D(2)O as a solvent, which would extend the lifetime of singlet oxygen, suggested that this species is involved in the formation of 8-oxodGuo by moxifloxacin and ciprofloxacin, but not by lomefloxacin and BAY y3118. Similarly, it was found that singlet oxygen was not involved in strand break formation. Thus, the evidence suggests that fluoroquinolones can photochemically

  18. Comparison of the binding of the therapeutically active nucleosides to DNA molecules with different level of lesions

    NASA Astrophysics Data System (ADS)

    Kruglova, E. B.; Gladkovskaya, N. A.

    2002-12-01

    Recently we have shown that DNA molecules extracted from epididymis of the Wistar male rats exposed to low doses of gamma radiation interact with some pyrimidine nucleosides. The bindign affinities of NUC to control DNA molecules are unessential. Comparing the UV melting curves for the various DNA sammples we show that observed differences are related to conformational chagnes in the DNA double helix. The samples of the damaged DNA have been obtained by partial denaturation of the calf thymus DNA in the salt-free aqueous solutions. The level of DNA damages in the model DNA smplase depends on the DNA concentration. It was shown that damages in the DNA molecules lead to changes of the melting curves of DNA-NUC mixtures that are similar to those for the DNA samples extracted from irradiated tissues. ALso it has been found that the binding mechanisms to cytosine arabinoside and 6-azacytosine to DNA molecuels having modifeid secondary structures are different.

  19. 2-Alkynylbenzaldoxime: a versatile building block for the generation of N-heterocycles.

    PubMed

    He, Linman; Nie, Hongming; Qiu, Guanyinsheng; Gao, Yueqiu; Wu, Jie

    2014-12-07

    2-Alkynylbenzaldoxime as a versatile building block has been applied widely for the construction of N-heterocycles in organic synthesis. Since it could be easily transferred to isoquinoline N-oxide via intramolecular 6-endo cyclization in the presence of metal catalysts or electrophiles, the subsequent [3 + 2] cycloaddition/nucleophilic addition and rearrangement could be expected. On the other hand, a Beckmann rearrangement could occur first since an oxime moiety is present in the molecule, which would then undergo an intramolecular cyclization to furnish nitrogen-containing heterocycles. This review reports the recent advancement in the generation of N-heterocycles starting from 2-alkynylbenzaldoximes via tandem reactions based on different reaction types.

  20. Hybrid Catalysis Enabling Room-Temperature Hydrogen Gas Release from N-Heterocycles and Tetrahydronaphthalenes.

    PubMed

    Kato, Shota; Saga, Yutaka; Kojima, Masahiro; Fuse, Hiromu; Matsunaga, Shigeki; Fukatsu, Arisa; Kondo, Mio; Masaoka, Shigeyuki; Kanai, Motomu

    2017-02-15

    Hybrid catalyst systems to achieve acceptorless dehydrogenation of N-heterocycles and tetrahydronaphthalenes-model substrates for liquid organic hydrogen carriers-were developed. A binary hybrid catalysis comprising an acridinium photoredox catalyst and a Pd metal catalyst was effective for the dehydrogenation of N-heterocycles, whereas a ternary hybrid catalysis comprising an acridinium photoredox catalyst, a Pd metal catalyst, and a thiophosphoric imide organocatalyst achieved dehydrogenation of tetrahydronaphthalenes. These hybrid catalyst systems allowed for 2 molar equiv of H2 gas release from six-membered N-heterocycles and tetrahydronaphthalenes under mild conditions, i.e., visible light irradiation at rt. The combined use of two or three different catalyst types was essential for the catalytic activity.

  1. Synthesis of Carbazoles and Carbazole-Containing Heterocycles via Rhodium-Catalyzed Tandem Carbonylative Benzannulations.

    PubMed

    Song, Wangze; Li, Xiaoxun; Yang, Ka; Zhao, Xian-liang; Glazier, Daniel A; Xi, Bao-min; Tang, Weiping

    2016-04-01

    Polycyclic aromatic compounds are important constituents of pharmaceuticals and other materials. We have developed a series of Rh-catalyzed tandem carbonylative benzannulations for the synthesis of tri-, tetra-, and pentacyclic heterocycles from different types of aryl propargylic alcohols. These tandem reactions provide efficient access to highly substituted carbazoles, furocarbazoles, pyrrolocarbazoles, thiophenocarbazoles, and indolocarbazoles. While tricyclic heterocycles could be derived from vinyl aryl propargylic alcohols, tetra- and pentacyclic heterocycles were synthesized from diaryl propargylic alcohols. The tandem carbonylative benzannulation is initiated by a π-acidic rhodium(I) catalyst-mediated nucleophilic addition to alkyne to generate a key metal-carbene intermediate, which is then trapped by carbon monoxide to form a ketene species for 6π electrocyclization. Overall, three bonds and two rings are formed in all of these tandem carbonylative benzannulation reactions.

  2. Structural and spectral comparisons between isomeric benzisothiazole and benzothiazole based aromatic heterocyclic dyes

    NASA Astrophysics Data System (ADS)

    Wang, Yin-Ge; Wang, Yue-Hua; Tao, Tao; Qian, Hui-Fen; Huang, Wei

    2015-09-01

    A pair of isomeric heterocyclic compounds, namely 3-amino-5-nitro-[2,1]-benzisothiazole and 2-amino-6-nitrobenzothiazole, are used as the diazonium components to couple with two N-substituted 4-aminobenzene derivatives. As a result, two pairs of isomeric aromatic heterocyclic azo dyes have been produced and they are structurally and spectrally characterized and compared including single-crystal structures, electronic spectra, solvatochromism and reversible acid-base discoloration, thermal stability and theoretically calculations. It is concluded that both benzisothiazole and benzothiazole based dyes show planar molecular structures and offset π-π stacking interactions, solvatochromism and reversible acid-base discoloration. Furthermore, benzisothiazole based aromatic heterocyclic dyes exhibit higher thermal stability, larger solvatochromic effects and maximum absorption wavelengths than corresponding benzothiazole based ones, which can be explained successfully by the differences of their calculated isomerization energy, dipole moment and molecular band gaps.

  3. Food heating and the formation of heterocyclic aromatic amine mutagens/carcinogens

    SciTech Connect

    Knize, M.G.; Salmon, C.P.; Felton, J.S.

    1997-12-31

    Several heterocyclic amines that are mutagenic and carcinogenic have been found as cooking products of muscle meats and some grain-based foods. Amounts in meats range from undetectable levels (less than 0.5 ppb) after boiling, microwave-cooking, and baking, to tens to hundreds of ppb for frying/grilling at high temperatures. A mutagenic response, believed to be caused by aromatic amines, was shown with some toasted foods, but the identity of the mutagenic chemicals are different from those found in meats. The airborne products from cooking also contain many of the same heterocyclic amines. Commercial cooking generally forms less of the heterocyclic amines than home cooking due to industry cooking practices.

  4. Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods.

    PubMed

    Gonzales, J L; Loza, A; Chacon, E

    2006-03-15

    There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.

  5. Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates.

    PubMed

    Oliveira, Caio Fernando de; Paim, Thiago Galvão da Silva; Reiter, Keli Cristine; Rieger, Alexandre; D'Azevedo, Pedro Alves

    2014-01-01

    Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

  6. Additive-Free Pd-Catalyzed α-Allylation of Imine-Containing Heterocycles.

    PubMed

    Kljajic, Marko; Puschnig, Johannes G; Weber, Hansjörg; Breinbauer, Rolf

    2017-01-06

    An additive-free Pd-catalyzed α-allylation of different imino-group-ontaining heterocycles is reported. The activation of α-CH pronucleophiles (pKa (DMSO) > 25) occurs without the addition of strong bases or Lewis acids using only the Pd/Xantphos catalyst system. The reaction scope has been studied for various 5- and 6-membered nitrogen-containing heterocycles (yields up to 96%). Mechanistic investigations suggest an initial allylation of the imine-N followed by a Pd-catalyzed formal aza-Claisen rearrangement.

  7. Synthesis of Five and Six-Membered Heterocycles Using Activated Nitriles for Industrial Applications.

    PubMed

    El-Sayed, Refat; Almalki, Meshal H K

    2017-01-01

    The aim of this study is a synthesis of new bioactive heterocyclic compounds incorporated fatty chain for use in different industrial applications. Cyanoacetamide derivative (2) was successfully transferred into five and six membered heterocyclic derivatives by the reaction with various chemical reagents. Addition number of moles of propylene oxide to these compounds gave nonionic surface-active agents having a good solubility, biodegradability and hence lowers the toxicity to human beings and becomes environmentally friendly. The antimicrobial and surface activities were investigated that showed the most of them have pronounced activity, which makes them suitable for diverse applications like the manufacturing of drugs, pesticides, emulsifiers, cosmetics, etc.

  8. Additive-Free Pd-Catalyzed α-Allylation of Imine-Containing Heterocycles

    PubMed Central

    2016-01-01

    An additive-free Pd-catalyzed α-allylation of different imino-group-ontaining heterocycles is reported. The activation of α-CH pronucleophiles (pKa (DMSO) > 25) occurs without the addition of strong bases or Lewis acids using only the Pd/Xantphos catalyst system. The reaction scope has been studied for various 5- and 6-membered nitrogen-containing heterocycles (yields up to 96%). Mechanistic investigations suggest an initial allylation of the imine-N followed by a Pd-catalyzed formal aza-Claisen rearrangement. PMID:27936786

  9. 1-Azadienes in cycloaddition and multicomponent reactions towards N-heterocycles.

    PubMed

    Groenendaal, Bas; Ruijter, Eelco; Orru, Romano V A

    2008-11-21

    1-Azadienes are versatile building blocks for the efficient construction of various N-heterocycles. Depending on the substitution pattern and reaction partner, they may participate in a range of different reactions. An overview of recent methods for the generation of 1-azadienes is presented, as well as their application in cycloaddition, electrocyclization, and multicomponent reactions. Considering the broad range of reactivities and resulting heterocyclic scaffold structures, 1-azadienes are very useful reactive intermediates for the development of modular reaction sequences in diversity-oriented synthesis.

  10. Applications of Multicomponent Reactions to the Synthesis of Diverse Heterocyclic Scaffolds

    PubMed Central

    Sunderhaus, James D.

    2009-01-01

    The sequencing of multicomponent reactions (MCRs) and subsequent cyclization reactions is a powerful stratagem for the rapid synthesis of diverse heterocyclic scaffolds. The optimal MCR is sufficiently flexible that it can be employed to generate adducts bearing a variety of functional groups that may then be selectively paired to enable different cyclization manifolds, thereby leading to a diverse collection of products. The growing interest in diversity-oriented synthesis has led to increased attention to this paradigm for library synthesis, which has inspired many advances in the design and implementation of MCRs for the construction of diverse heterocyclic scaffolds. PMID:19132705

  11. Multiplicity of Diverse Heterocycles from Polymer-Supported α-Acylamino Ketones

    PubMed Central

    Pudelová, Nadĕžda; Krchňák, Viktor

    2009-01-01

    Polymer-supported α-acylamino ketones were transformed to seven types of structurally unrelated heterocyclic compounds. Syntheses involved variety of chemical routes and comprised diverse chemistries (C-C, C=C, C-N, C=N, C-O bond formations). Different sizes of heterocycles (4-, 5-, 6-, and 7-membered rings) were prepared, including dihydro-pyrrol-2-ones, pyrazin-2-ones, dihydro-triazepin-6-ones, morpholin-3-ones, imidazoles, β-lactams, and isoquinolin-1-ones. Further elaboration to fused ring systems was also documented. PMID:19689103

  12. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk

    PubMed Central

    Leitner-Dagan, Yael; Sevilya, Ziv; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S.; Schechtman, Edna; Freedman, Laurence; Rennert, Gad; Paz-Elizur, Tamar

    2014-01-01

    DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case–control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/μg protein (95% CI 15.3–16.3), significantly higher than in control subjects—15.1 (14.6–15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1–2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6–4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT. PMID:25355292

  13. DNA extraction from plant food supplements: Influence of different pharmaceutical excipients.

    PubMed

    Costa, Joana; Amaral, Joana S; Fernandes, Telmo J R; Batista, Andreia; Oliveira, M Beatriz P P; Mafra, Isabel

    2015-12-01

    The consumption of plant food supplements (PFS) has been growing globally, with an increase of misleading labeling and fraudulent practices also being reported. Recently, the use of molecular biology techniques has been proposed to detect botanical adulterations, one of the possible frauds in PFS. However, difficulties in recovering DNA from some PFS samples have been described. Aiming at using DNA-based methods for the unequivocal identification of plant species in PFS, adequate DNA isolation is required. However, PFS often contain pharmaceutical excipients known to have adsorbent properties that might interfere with DNA extraction. Thus, the aim of this work was to assess the effect of different excipients (talc, silica, iron oxide and titanium dioxide) on the recovery/amplification of DNA. For that purpose, known amounts of template maize DNA were spiked either to PFS or to model mixtures of excipients and quantified by real-time PCR. The tested excipients evidenced clear adsorption phenomena that justify the hampering effect on DNA extraction from PFS. The use of either 10% talc or 0.5% dyes completely adsorbed DNA, resulting in negative PCR amplifications. For the first time, pharmaceutical excipients were shown to affect DNA extraction explaining the inability of recovering DNA from some PFS samples in previous studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Evaluating forensic DNA mixtures with contributors of different structured ethnic origins: a computer software.

    PubMed

    Hu, Yue-Qing; Fung, Wing K

    2003-08-01

    The effect of a structured population on the likelihood ratio of a DNA mixture has been studied by the current authors and others. In practice, contributors of a DNA mixture may belong to different ethnic/racial origins, a situation especially common in multi-racial countries such as the USA and Singapore. We have developed a computer software which is available on the web for evaluating DNA mixtures in multi-structured populations. The software can deal with various DNA mixture problems that cannot be handled by the methods given in a recent article of Fung and Hu.

  15. Discovery of novel heterocyclic factor VIIa inhibitors.

    PubMed

    Rai, Roopa; Kolesnikov, Aleksandr; Sprengeler, Paul A; Torkelson, Steven; Ton, Tony; Katz, Bradley A; Yu, Christine; Hendrix, John; Shrader, William D; Stephens, Robin; Cabuslay, Ronnell; Sanford, Ellen; Young, Wendy B

    2006-04-15

    Structure-activity relationships and binding mode of novel heterocyclic factor VIIa inhibitors will be described. In these inhibitors, a highly basic 5-amidinoindole moiety has been successfully replaced with a less basic 5-aminopyrrolo[3,2-b]pyridine scaffold.

  16. In vitro disposition profiling of heterocyclic compounds.

    PubMed

    Keemink, Janneke; Wuyts, Benjamin; Nicolaï, Johan; Jonghe, Steven De; Stella, Alessandro; Herdewijn, Piet; Augustijns, Patrick; Annaert, Pieter

    2015-08-01

    Compound libraries that are screened for biological activity commonly contain heterocycles. Besides potency, drug-like properties need to be evaluated to ensure in vivo efficacy of test compounds. In this context, we determined hepatic and intestinal disposition profiles for 17 heterocyclic compounds. All studied compounds showed rapid uptake in suspended rat hepatocytes, whereas metabolism was poor and the rate-limiting step in hepatic elimination. In vitro assays demonstrated a relatively low solubility and high intestinal permeability. Based on these in vitro data, heterocycles were categorized in the biopharmaceutics classification system (BCS) and the biopharmaceutics drug disposition classification system (BDDCS) to predict disposition characteristics before clinical data are available. Our findings emphasized the importance to use hepatocytes in addition to microsomes to study metabolism, since the latter lack non-microsomal enzymes and cellular context. Moreover, intracellular exposure should be considered to gain insight in the relevant fraction of the compound available at the enzymatic site. Finally, the study reveals discrepancies associated with the classification of heterocycles in BCS versus BDDCS. These probably originate from the binary character of both systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. International Society of Heterocyclic Chemistry--17th international congress. 1-6 August 1999, Vienna, Austria.

    PubMed

    Daneshtalab, M

    1999-10-01

    Approximately 1200 scientists attended this congress of heterocyclic chemistry, which focused on: New synthetic methods in heterocyclic chemistry; synthesis of bioactive heterocycles including natural products; heterocycles and asymmetric synthesis; heterocycles in bioorganic chemistry; new heterocyclic materials; structure and properties of heterocyclic compounds; solid-phase synthesis, combinatorial chemistry and heterocyclic scaffolds. These topics were covered in 600 posters and 100 plenary, invited and oral presentations. This report summarizes the highlights of the presentations related to the category of the synthesis of bioactive heterocycles including natural products.

  18. Perylene diimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase.

    PubMed

    Rossetti, Luigi; Franceschin, Marco; Bianco, Armandodoriano; Ortaggi, Giancarlo; Savino, Maria

    2002-09-16

    Four N,N'-disubstituted perylene diimides, having different side chains, have been studied for their ability in inducing G-quadruplex DNA structures. We found that electrostatic interactions between ligands side chains and DNA grooves play a main role not only in the amount of G-quadruplex formed, but also in selecting its topology. Moreover, such compounds show also a different ability to inhibit telomerase. The correlation of these findings suggests the intriguing possibility that different G-quadruplex structures could differently inhibit the enzyme.

  19. Centromeric and non-centromeric satellite DNA organisation differs in holocentric Rhynchospora species.

    PubMed

    Ribeiro, Tiago; Marques, André; Novák, Petr; Schubert, Veit; Vanzela, André L L; Macas, Jiri; Houben, Andreas; Pedrosa-Harand, Andrea

    2016-09-19

    Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.

  20. Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration.

    PubMed

    Pierrez, J; Guerci, A; Guerci, O

    1988-07-01

    The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes). In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.

  1. Comparison of DNA yield and STR success rates from different tissues in embalmed bodies.

    PubMed

    Wheeler, Amanda; Czado, Natalia; Gangitano, David; Turnbough, Meredith; Hughes-Stamm, Sheree

    2017-01-01

    Formalin fixation is commonly used to preserve tissue sections for pathological testing and embalming cadavers for medical dissection or burial. DNA extracted from formalin-fixed tissues may also provide an alternative source of genetic material for medical diagnosis and forensic casework, such as identifying unknown embalmed human remains. Formaldehyde causes DNA damage, chemical modifications, and degradation, thereby reducing the quantity and quality of DNA available for downstream genetic analyses. By comparing the DNA yield, level of DNA degradation, and short tandem repeat (STR) success of various tissue types, this study is the first of its kind to provide some guidance on which samples from embalmed bodies are likely to generate more complete STR profiles. Tissue samples were dissected from three male embalmed cadavers and included bone, cartilage, hair, muscle, internal organs, skin, teeth, and nail clippings. DNA was purified from all samples using the QIAamp® FFPE Tissue Kit (Qiagen), quantified using the QuantiFiler® Trio DNA Quantification kit (Life Technologies), and genotyped using the GlobalFiler® PCR Amplification Kit (Life Technologies). Results of this study showed variation in DNA quantity and STR success between different types of tissues and some variation between cadavers. Overall, bone marrow samples resulted in the highest DNA yields, the least DNA degradation, and greatest STR success. However, several muscle, hair, and nail samples generated higher STR success rates than traditionally harvested bone and tooth samples. A key advantage to preferentially using these tissue samples over bone (and marrow) and teeth is their comparative ease and speed of collection from the cadaver and processing during DNA extraction. Results also indicate that soft tissues affected by lividity (blood pooling) may experience greater exposure to formalin, resulting in more DNA damage and reduced downstream STR success than tissues under compression. Overall

  2. Differences in DNA Binding Specificity of Floral Homeotic Protein Complexes Predict Organ-Specific Target Genes.

    PubMed

    Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin

    2017-08-01

    Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.

  3. Super-resolution structure of DNA significantly differs in buccal cells of controls and Alzheimer's patients.

    PubMed

    Garcia, Angeles; Huang, David; Righolt, Amanda; Righolt, Christiaan; Kalaw, Maria Carmela; Mathur, Shubha; McAvoy, Elizabeth; Anderson, James; Luedke, Angela; Itorralba, Justine; Mai, Sabine

    2017-09-01

    The advent of super-resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super-resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) versus age- and gender-matched healthy, non-caregiver controls. In this super-resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D-SIM). Quantitation of the super-resolution DNA structure revealed that the nuclear super-resolution DNA structure of individuals with AD significantly differs from that of their controls (p < 0.05) with an overall increase in the measured DNA-free/poor spaces. This represents a significant increase in the interchromatin compartment. We also find that the DNA structure of AD significantly differs in mild, moderate, and severe disease with respect to the DNA-containing and DNA-free/poor spaces. We conclude that whole genome remodeling is a feature of buccal cells in AD. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  4. Controlled release of plasmid DNA from hydrogels prepared from gelatin cationized by different amine compounds.

    PubMed

    Kushibiki, Toshihiro; Tomoshige, Ryuji; Iwanaga, Kazunori; Kakemi, Masawo; Tabata, Yasuhiko

    2006-05-15

    This paper is an investigation to compare the in vivo controlled release of a plasmid DNA from biodegradable hydrogels prepared from gelatin cationized by different amine compounds, ethylenediamine, putrescine, spermidine, and spermine and the consequent profile of gene expression. Cationized gelatin prepared through the chemical introduction of each amine compound was crosslinked by various concentrations of glutaraldehyde to obtain cationized gelatin hydrogels for the carrier of plasmid DNA release. When the cationized gelatin hydrogels incorporating 125I-labeled plasmid DNA were implanted into the femoral muscle of mice, the radioactivity remaining decreased with time and the retention period of radioactivity prolonged with a decrease in the water content of hydrogels. When 125I-labeled cationized gelatin hydrogels with the higher water content was implanted, the radioactivity remaining was decreased faster with time. The remaining time profile of plasmid DNA radioactivity was in good accordance with that of hydrogel radioactivity, irrespective of the type of cationized gelatin. Following intramuscular implantation, any cationized gelatin hydrogel incorporating plasmid DNA enhanced the expression level of plasmid DNA to a significantly higher extent than the free plasmid DNA injection. In addition, prolonged time period of gene expression was observed although there was no significant difference in the expressed period between the cationized gelatin hydrogels. It was concluded that plasmid DNA of biological activity was released from every cationized gelatin hydrogel accompanied with the in vivo degradation, resulting in enhanced and prolonged gene expression.

  5. Biological properties of novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles.

    PubMed

    Novak, Maria S; Büchel, Gabriel E; Keppler, Bernhard K; Jakupec, Michael A

    2016-06-01

    Since the discovery that nitric oxide (NO) is a physiologically relevant molecule, there has been great interest in the use of metal nitrosyl compounds as antitumor pharmaceuticals. Particularly interesting are those complexes which can deliver NO to biological targets. Ruthenium- and osmium-based compounds offer lower toxicity compared to other metals and show different mechanisms of action as well as different spectra of activity compared to platinum-based drugs. Novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles were studied to elucidate their cytotoxicity and possible interactions with DNA. Apoptosis induction, changes of mitochondrial transmembrane potential and possible formation of reactive oxygen species were investigated as indicators of NO-mediated damage by flow cytometry. Results suggest that ruthenium- and osmium-nitrosyl complexes with the general formula (indazolium)[cis/trans-MCl4(NO)(1H-indazole)] have pronounced cytotoxic potency in cancer cell lines. Especially the more potent ruthenium complexes strongly induce apoptosis associated with depolarization of mitochondrial membranes, and elevated reactive oxygen species levels. Furthermore, a slight yet not unequivocal trend to accumulation of intracellular cyclic guanosine monophosphate attributable to NO-mediated effects was observed.

  6. Recurrent DNA virus domestication leading to different parasite virulence strategies

    PubMed Central

    Pichon, Apolline; Bézier, Annie; Urbach, Serge; Aury, Jean-Marc; Jouan, Véronique; Ravallec, Marc; Guy, Julie; Cousserans, François; Thézé, Julien; Gauthier, Jérémy; Demettre, Edith; Schmieder, Sandra; Wurmser, François; Sibut, Vonick; Poirié, Marylène; Colinet, Dominique; da Silva, Corinne; Couloux, Arnaud; Barbe, Valérie; Drezen, Jean-Michel; Volkoff, Anne-Nathalie

    2015-01-01

    Relics of ancient infections are abundant in eukaryote genomes, but little is known about how they evolve when they confer a functional benefit on their host. We show here, for the first time, that the virus-like particles shown to protect Venturia canescens eggs against host immunity are derived from a nudivirus genome incorporated by the parasitic wasp into its own genetic material. Nudivirus hijacking was also at the origin of protective particles from braconid wasps. However, we show here that the viral genes produce “liposomes” that wrap and deliver V. canescens virulence proteins, whereas the particles are used as gene transfer agents in braconid wasps. Our findings indicate that virus domestication has occurred repeatedly during parasitic wasp evolution but with different evolutionary trajectories after endogenization, resulting in different virulence molecule delivery strategies. PMID:26702449

  7. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

    PubMed Central

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  8. Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

    NASA Technical Reports Server (NTRS)

    Vercoutere, Wenonah; DeGuzman, Veronica

    2003-01-01

    The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

  9. Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

    NASA Technical Reports Server (NTRS)

    Vercoutere, Wenonah; DeGuzman, Veronica

    2003-01-01

    The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

  10. Effect of different concentration of HPV DNA probe immobilization for cervical cancer detection based IDE biosensor

    NASA Astrophysics Data System (ADS)

    Roshila, M. L.; Hashim, U.; Azizah, N.; Nadzirah, Sh.; Arshad, M. K. Md; Ruslinda, A. R.; Gopinath, Subash C. B.

    2017-03-01

    This paper principally delineates to the detection process of Human Papillomavirus (HPV) DNA test. HPV is an extremely common virus infection that infected to human by the progressions cell in the cervix cell. The types of HPV that give a most exceedingly awful infected with cervical cancer is 16 and 18 other than 31 and 45. The HPV DNA probe is immobilized with a different concentration to stabilize the sensitivity. A technique of rapid and sensitive for the HPV identification was proposed by coordinating basic DNA extraction with a quality of DNA. The extraction of the quality of DNA will make a proficiency of the discovery procedure. It will rely on the sequence of the capture probes and the way to support their attached. The fabrication, surface modification, immobilization and hybridization procedures are described by current-voltage (I-V) estimation by utilizing KEITHLEY 6487. This procedure will play out a decent affectability and selectivity of HPV discovery.

  11. Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency.

    PubMed

    Chang, Howard H Y; Watanabe, Go; Gerodimos, Christina A; Ochi, Takashi; Blundell, Tom L; Jackson, Stephen P; Lieber, Michael R

    2016-11-18

    The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency*

    PubMed Central

    Chang, Howard H. Y.; Watanabe, Go; Gerodimos, Christina A.; Ochi, Takashi; Blundell, Tom L.; Jackson, Stephen P.; Lieber, Michael R.

    2016-01-01

    The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5′ overhangs, and 3′ overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed. PMID:27703001

  13. [Species and tissue differences of reparative DNA synthesis in embryonic cell cultures treated with carcinogens].

    PubMed

    Budunova, I V; Belitskiĭ, G A

    1982-01-01

    DNA repair synthesis (RS) was studied in embryonic cell cultures exposed to different carcinogenic factors: UV-light, N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, aflatoxin BI and 7,12-dimethylbenz(a)anthracene. DNA RS level was shown to be higher in human liver cells than in murine ones. Tissue-dependent differences in DNA RS of cells damaged by carcinogens were found, too. RS-activity was higher in human, mouse and rat fibroblast cultures than in liver cultures of the same species. RS level in human kidney cultures was similar to that in human fibroblasts. The said differences should be taken into account in the evaluation of the results of testing of chemical agents for carcinogenicity, using their ability to cause DNA repair synthesis.

  14. Qualification study of two genomic DNA extraction methods in different clinical samples.

    PubMed

    Javadi, Alireza; Shamaei, Masoud; Mohammadi Ziazi, Leila; Pourabdollah, Mihan; Dorudinia, Atosa; Seyedmehdi, Seyed Mohammad; Karimi, Shirin

    2014-01-01

    The purity of genomic DNA (gDNA) extracted from different clinical specimens optimizes sensitivity of polymerase chain reaction (PCR) assays. This study attempted to compare two different DNA extraction techniques namely salting-out and classic phenol-chloroform. Qualification of two different DNA extraction techniques for 634 clinical specimens highly suspected of having mycobacterial infection was performed. Genomic DNA was extracted from 330 clinical samples using phenol-chloroform and 304 by non-toxic salting-out. Qualification of obtained gDNA was done through amplification of internal controls, β-actin and β-globin. β-actin-positive was detected in 279/330 (84%) and 272/304 (89%) samples by phenol-chloroform technique and salting-out, respectively. PCR inhibitor was found for the gDNA of 13/304 (4%) patient samples were negative by β-actin and β-globin tests via salting-out technique in comparison with gDNAs from 27/330 (8.5%) samples extracted by phenol-chloroform procedure. No statistically significant difference was found between phenol-chloroform technique and salting-out for 385 sputum, 29 bronchoalveolar lavage (BAL), 105 gastric washing, and 38 body fluid (P=0.04) samples. This illustrates that both techniques have the same quality for extracting gDNA. This study discloses salting-out as a non-toxic DNA extraction procedure with a superior time-efficiency and cost-effectiveness in comparison with phenol-chloroform and it can be routinely used in resource-limited laboratory settings.

  15. Ruthenium Catalysis in the Synthesis of Six-Membered Heterocycles

    NASA Astrophysics Data System (ADS)

    Zhang, Min

    Owing to the cost-effectiveness and versatility of ruthenium in catalysis, the utilization of ruthenium for developing atom and step-economic methodologies, allowing for the synthesis of six-membered heterocycles, is of particular importance in synthetic chemistry because of the important value of such compounds employed in discovering biologically and pharmacologically active compositions, the preparation of novel materials with specific functions, etc. This chapter highlights the recent 15 years' advances on ruthenium-catalyzed synthesis of six-membered heterocycles, with particular focus on the related approaches and mechanistic basis, which includes the synthesis of N-heterocycles, O-heterocycles, N,O-heterocycles, and other type of heteroatom-heterocycles.

  16. Comparison of Three Different DNA Extraction Methods for Linguatula serrata as a Food Born Pathogen

    PubMed Central

    ESLAMI, Gilda; KHALATBARI-LIMAKI, Sepideh; EHRAMPOUSH, Mohammad Hasan; GHOLAMREZAEI, Mostafa; HAJIMOHAMMADI, Bahador; ORYAN, Ahmad

    2017-01-01

    Background: One of the most important items in molecular characterization of food-borne pathogens is high quality genomic DNA. In this study, we investigated three protocols and compared their simplicity, duration and costs for extracting genomic DNA from Linguatula serrata. Methods: The larvae were collected from the sheep’s visceral organs from the Yazd Slaughterhouse during May 2013. DNA extraction was done in three different methods, including commercial DNA extraction kit, Phenol Chloroform Isoamylalcohol (PCI), and salting out. Extracted DNA in each method was assessed for quantity and quality using spectrophotometery and agarose gel electrophoresis, respectively. Results: The less duration was regarding to commercial DNA extraction kit and then salting out protocol. The cost benefit one was salting out and then PCI method. The best quantity was regarding to PCI with 72.20±29.20 ng/μl, and purity of OD260/OD280 in 1.76±0.947. Agarose gel electrophoresis for assessing the quality found all the same. Conclusion: Salting out is introduced as the best method for DNA extraction from L. seratta as a food-borne pathogen with the least costand appropriate purity. Although, the best purity was regarding to PCI but PCI is not safe as salting out. In addition, the duration of salting out was less than PCI. The least duration was seen in commercial DNA extraction kit, but it is expensive and therefore is not recommended for developing countries where consumption of offal is common. PMID:28761484

  17. Melting behavior and different bound states in three-stranded DNA models.

    PubMed

    Maji, Jaya; Bhattacharjee, Somendra M; Seno, Flavio; Trovato, Antonio

    2014-01-01

    Thermal denaturation of DNA is often studied with coarse-grained models in which native sequential base pairing is mimicked by the existence of attractive interactions only between monomers at the same position along strands (Poland and Scheraga models). Within this framework, the existence of a three-stranded DNA bound state in conditions where a duplex DNA would be in the denaturated state was recently predicted from a study of three directed polymer models on simplified hierarchical lattices (d>2) and in 1+1 dimensions. Such a phenomenon which is similar to the Efimov effect in nuclear physics was named Efimov-DNA. In this paper we study the melting of the three-stranded DNA on a Sierpinski gasket of dimensions d<2 by assigning extra weight factors to fork openings and closings, to induce a two-strand DNA melting. In such a context we can find again the existence of the Efimov-DNA-like state but quite surprisingly we discover also the presence of a different phase, to be called a mixed state, where the strands are pair-wise bound but without three chain contacts. Whereas the Efimov DNA turns out to be a crossover near melting, the mixed phase is a thermodynamic phase.

  18. Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia.

    PubMed

    Ribeiro, R A; Lovato, M B

    2007-03-29

    Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species.

  19. Comparison of Three Different DNA Extraction Methods for Linguatula serrata as a Food Born Pathogen.

    PubMed

    Eslami, Gilda; Khalatbari-Limaki, Sepideh; Ehrampoush, Mohammad Hasan; Gholamrezaei, Mostafa; Hajimohammadi, Bahador; Oryan, Ahmad

    2017-01-01

    One of the most important items in molecular characterization of food-borne pathogens is high quality genomic DNA. In this study, we investigated three protocols and compared their simplicity, duration and costs for extracting genomic DNA from Linguatula serrata. The larvae were collected from the sheep's visceral organs from the Yazd Slaughterhouse during May 2013. DNA extraction was done in three different methods, including commercial DNA extraction kit, Phenol Chloroform Isoamylalcohol (PCI), and salting out. Extracted DNA in each method was assessed for quantity and quality using spectrophotometery and agarose gel electrophoresis, respectively. The less duration was regarding to commercial DNA extraction kit and then salting out protocol. The cost benefit one was salting out and then PCI method. The best quantity was regarding to PCI with 72.20±29.20 ng/μl, and purity of OD260/OD280 in 1.76±0.947. Agarose gel electrophoresis for assessing the quality found all the same. Salting out is introduced as the best method for DNA extraction from L. seratta as a food-borne pathogen with the least costand appropriate purity. Although, the best purity was regarding to PCI but PCI is not safe as salting out. In addition, the duration of salting out was less than PCI. The least duration was seen in commercial DNA extraction kit, but it is expensive and therefore is not recommended for developing countries where consumption of offal is common.

  20. Melting behavior and different bound states in three-stranded DNA models

    NASA Astrophysics Data System (ADS)

    Maji, Jaya; Bhattacharjee, Somendra M.; Seno, Flavio; Trovato, Antonio

    2014-01-01

    Thermal denaturation of DNA is often studied with coarse-grained models in which native sequential base pairing is mimicked by the existence of attractive interactions only between monomers at the same position along strands (Poland and Scheraga models). Within this framework, the existence of a three-stranded DNA bound state in conditions where a duplex DNA would be in the denaturated state was recently predicted from a study of three directed polymer models on simplified hierarchical lattices (d >2) and in 1+1 dimensions. Such a phenomenon which is similar to the Efimov effect in nuclear physics was named Efimov-DNA. In this paper we study the melting of the three-stranded DNA on a Sierpinski gasket of dimensions d <2 by assigning extra weight factors to fork openings and closings, to induce a two-strand DNA melting. In such a context we can find again the existence of the Efimov-DNA-like state but quite surprisingly we discover also the presence of a different phase, to be called a mixed state, where the strands are pair-wise bound but without three chain contacts. Whereas the Efimov DNA turns out to be a crossover near melting, the mixed phase is a thermodynamic phase.

  1. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk.

    PubMed

    Leitner-Dagan, Yael; Sevilya, Ziv; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S; Schechtman, Edna; Freedman, Laurence; Rennert, Gad; Livneh, Zvi; Paz-Elizur, Tamar

    2014-12-01

    DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/μg protein (95% CI 15.3-16.3), significantly higher than in control subjects-15.1 (14.6-15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1-2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6-4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Facile synthesis of N-dialkylaminomethyl-substituted heterocycles.

    PubMed

    Love, Brian E

    2007-01-19

    Iminium ions are generated by treatment of aminals with succinic anhydride. These iminium ions are trapped by heterocycles, giving the corresponding N-dialkylamino-methyl-substituted heterocycles, which are easily separated from the succinic acid monoamide byproducts by means of an aqueous base wash. The heterocyclic products are obtained in good yield and in a high state of purity without need of recrystallization or distillation.

  3. Structural mechanisms of photoeffect in polyimide structures containing heterocyclic fragments

    SciTech Connect

    Aleksandrova, E. L.

    2006-11-15

    Trends in the variation in the quantum yields of charge-carrier photogeneration in polyimide structures containing heterocyclic fragments are studied. It is shown that the efficiency of sensitization of polyimides depends on the donor and acceptor properties of the fragments of monomeric units of the polyimide. It is established that the range of spectral sensitivity for heterocyclic fragments representing intramolecular complexes with charge transport is wider than that for heterocycles that do not represent such complexes.

  4. Thermally Stable Heterocyclic Imines as New Potential Nonlinear Optical Materials

    NASA Technical Reports Server (NTRS)

    Nesterov, Volodymyr V.; Antipin, Mikhail Y.; Nesterov, Vladimir N.; Moore, Craig E.; Cardelino, Beatriz H.; Timofeeva, Tatiana V.

    2004-01-01

    In the course of a search for new thermostable acentric nonlinear optical crystalline materials, several heterocyclic imine derivatives were designed, with the general structure D-pi-A(D'). Introduction of a donor amino group (D') into the acceptor moiety was expected to bring H-bonds into their crystal structures, and so to elevate their melting points and assist in an acentric molecular packing. Six heterocycle-containing compounds of this type were prepared, single crystals were grown for five of them, and these crystals were characterized by X-ray analysis. A significant melting temperature elevation was found for all of the synthesized compounds. Three of the compounds were also found to crystallize in acentric space groups. One of the acentric compounds is built as a three-dimensional H-bonded molecular network. In the other two compounds, with very similar molecular structure, the molecules form one-dimensional H-bonded head-to-head associates (chains). These chains are parallel in two different crystallographic directions and form very unusual interpenetrating chain patterns in an acentric crystal. Two of the compounds crystallized with centrosymmetric molecular packing.

  5. Thermally Stable Heterocyclic Imines as New Potential Nonlinear Optical Materials

    NASA Technical Reports Server (NTRS)

    Nesterov, Volodymyr V.; Antipin, Mikhail Y.; Nesterov, Vladimir N.; Moore, Craig E.; Cardelino, Beatriz H.; Timofeeva, Tatiana V.

    2004-01-01

    In the course of a search for new thermostable acentric nonlinear optical crystalline materials, several heterocyclic imine derivatives were designed, with the general structure D-pi-A(D'). Introduction of a donor amino group (D') into the acceptor moiety was expected to bring H-bonds into their crystal structures, and so to elevate their melting points and assist in an acentric molecular packing. Six heterocycle-containing compounds of this type were prepared, single crystals were grown for five of them, and these crystals were characterized by X-ray analysis. A significant melting temperature elevation was found for all of the synthesized compounds. Three of the compounds were also found to crystallize in acentric space groups. One of the acentric compounds is built as a three-dimensional H-bonded molecular network. In the other two compounds, with very similar molecular structure, the molecules form one-dimensional H-bonded head-to-head associates (chains). These chains are parallel in two different crystallographic directions and form very unusual interpenetrating chain patterns in an acentric crystal. Two of the compounds crystallized with centrosymmetric molecular packing.

  6. Upgrading carbon dioxide by incorporation into heterocycles.

    PubMed

    Yu, Bing; He, Liang-Nian

    2015-01-01

    Carbon dioxide is commonly regarded as the primary greenhouse gas, but from a synthetic standpoint can be utilized as an alternative and sustainable C1 synthon in organic synthesis rather than a waste. This results in the production of organic carbonates, carboxylic acids, and derivatives. Recently, CO2 has emerged as an appealing tool for heterocycle synthesis under mild conditions without using stoichiometric amounts of organometallic reducing reagents. This Minireview summarizes recent advances on methodologies for CO2 incorporation into N-, O-, and C-nucleophiles to provide various heterocycles, including cyclic carbamates, benzoxazine-2-one, 4-hydroxyquinolin-2-one, quinazoline-2,4(1 H,3 H)-diones, benzimidazolones, α-alkylidene cyclic carbonate.

  7. Heterocyclic Amaryllidaceae Alkaloids: Biosynthesis and Pharmacological Applications.

    PubMed

    Hotchandani, Tarun; Desgagne-Penix, Isabel

    2017-01-01

    Amaryllidaceae alkaloids (AAs), which are natural heterocyclic compounds, are isolated from Amaryllidaceae plants such as narcissus, snowdrop and spider lily. AAs have been extensively studied due to their multiple pharmacological properties. Nevertheless, knowledge of AA synthesis in plants is lacking and most genes encoding enzymes involved in their production remain unknown. AAs are structurally complex compounds which are challenging for total chemical synthesis that is economically viable. Therefore the understanding of AA biosynthesis could allow for the development of biotechnologies for the production of natural AAs or analogues, maintaining or improving their pharmacological properties. In this review, we describe the progress regarding the biosynthesis and pharmacological properties of AAs. The most recent developments in neurological, anti-cancer and anti-microbial bioactivities of heterocyclic AAs are covered.

  8. New heterocyclic compounds from Ranunculus ternatus Thunb.

    PubMed

    Feng, Zi-Ming; Zhan, Zhi-Lai; Yang, Ya-Nan; Jiang, Jian-Shuang; Zhang, Pei-Cheng

    2017-10-01

    Five new heterocyclic compounds, 5-α-d-fructofuranosylmethyl-furfural (1), 5-β-d-fructofuranosylmethyl-furfural (2), 5-β-d-fructopyranosylmethyl-furfural (3), 4-(2-((2S-2,3-dihydroxypropoxy)methyl)-5-formyl-1H-pyrrol-1-yl)butanoic acid (4), and 3S,4S-4,5,8-trihydroxy-3-(prop-1-en-2-yl)isochroman-1-one (5), were obtained from the root of Ranunculus ternatus Thunb., which is a traditional Chinese anti-tuberculosis medicine. Their structures were elucidated by UV, IR, HRESIMS, NMR data, and the comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Notably, compounds 1-3 are rarely occurring furfural fructosides in natural sources. These heterocyclic compounds could be further studied for the synthetic chemists and pharmacologists due to the source and structural properties. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Isolatable organophosphorus(III)-tellurium heterocycles.

    PubMed

    Nordheider, Andreas; Chivers, Tristram; Schön, Oliver; Karaghiosoff, Konstantin; Athukorala Arachchige, Kasun S; Slawin, Alexandra M Z; Woollins, J Derek

    2014-01-13

    A new structural arrangement Te3 (RP(III) )3 and the first crystal structures of organophosphorus(III)-tellurium heterocycles are presented. The heterocycles can be stabilized and structurally characterized by the appropriate choice of substituents in Tem (P(III) R)n (m=1: n=2, R=OMes* (Mes*=supermesityl or 2,4,6-tri-tert-butylphenyl); n=3, R=adamantyl (Ad); n=4, R=ferrocene (Fc); m=n=3: R=trityl (Trt), Mesor by the installation of a P(V) 2 N2 anchor in RP(III) [TeP(V) (tBuN)(μ-NtBu)]2 (R=Ad, tBu).

  10. Covalent Polymers Containing Discrete Heterocyclic Anion Receptors

    PubMed Central

    Rambo, Brett M.; Silver, Eric S.; Bielawski, Christopher W.; Sessler, Jonathan L.

    2010-01-01

    This chapter covers recent advances in the development of polymeric materials containing discrete heterocyclic anion receptors, and focuses on advances in anion binding and chemosensor chemistry. The development of polymers specific for anionic species is a relatively new and flourishing area of materials chemistry. The incorporation of heterocyclic receptors capable of complexing anions through non-covalent interactions (e.g., hydrogen bonding and electrostatic interactions) provides a route to not only sensitive but also selective polymer materials. Furthermore, these systems have been utilized in the development of polymers capable of extracting anionic species from aqueous environments. These latter materials may lead to advances in water purification and treatment of diseases resulting from surplus ions. PMID:20871791

  11. In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds.

    PubMed

    Kushibiki, Toshihiro; Tomoshige, Ryuji; Iwanaga, Kazunori; Kakemi, Masawo; Tabata, Yasuhiko

    2006-01-01

    The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P- mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin-plasmid DNA complexes depended on the N+/P- mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P- mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P- mixing ratio of 2. The present study indicates that there is an optimal N+/P- mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.

  12. DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection.

    PubMed

    Gorna, Alina E; Bowater, Richard P; Dziadek, Jaroslaw

    2010-05-25

    Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens.

  13. Synthesis of Pharmacological Heterocyclic Derivatives Based Surfactants.

    PubMed

    El-Sayed, Refat; Fadda, Ahmed A

    2016-01-01

    Synthesis of chromenopyrimidine derivatives and the related fused system carried out by the reaction of chromene derivative 1 with various reagents under suitable reaction conditions. Condensation of stearoyl chloride with these heterocycles, then, propoxylated the products using propylene oxide to produce surface active agents having a twofold capacity as surface and antimicrobial dynamic specialists which may be served in the production of medications, pesticides, beautifying agents or may be utilized as an antimicrobial. Some of the surface properties and antimicrobial activity were resolved.

  14. Novel pyrrolidine heterocycles as CCR1 antagonists.

    PubMed

    Merritt, J Robert; James, Ray; Paradkar, Vidyadhar M; Zhang, Chongwu; Liu, Ruiyan; Liu, Jinqi; Jacob, Biji; Chiriac, Camelia; Ohlmeyer, Michael J; Quadros, Elizabeth; Wines, Pamela; Postelnek, Jennifer; Hicks, Catherine M; Chen, Weiqing; Kimble, Earl F; O'Brien, Linda; White, Nicole; Desai, Hema; Appell, Kenneth C; Webb, Maria L

    2010-09-15

    A novel series of pyrrolidine heterocycles was prepared and found to show potent inhibitory activity of CCR1 binding and CCL3 mediated chemotaxis of a CCR1-expressing cell line. A potent, optimized triazole lead from this series was found to have acceptable pharmacokinetics and microsomal stability in rat and is suitable for further optimization and development. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  15. Sodium phosphaethynolate as a building block for heterocycles.

    PubMed

    Chen, Xiaodan; Alidori, Simone; Puschmann, Florian Frank; Santiso-Quinones, Gustavo; Benkő, Zoltán; Li, Zhongshu; Becker, Gerd; Grützmacher, Hans-Friedrich; Grützmacher, Hansjörg

    2014-02-03

    Phosphorus-containing heterocycles have evolved from laboratory curiosities to functional components, such as ligands in catalytically active metal complexes or molecular constituents in electronic devices. The straightforward synthesis of functionalized heterocycles on a larger scale remains a challenge. Herein, we report the use of the phosphaethynolate (OCP)(-) anion as a building block for various sterically unprotected and functionalized hydroxy substituted phosphorus heterocycles. Because the resulting heterocycles are themselves anions, they are building blocks in their own right and allow further facile functionalization. This property may be of interest in coordination chemistry and material science.

  16. Water purification by reverse osmosis using heterocyclic polymer membranes

    NASA Technical Reports Server (NTRS)

    Scott, H.

    1972-01-01

    Pyrrone (polyimidazopyrrolone) polymers are a new class of thermally stable, radiation and chemical resistant aromatic-heterocyclic polymers featuring a greater chemical and mechanical durability than cellulose acetate.

  17. Synthesis of functionalized tetrasubstituted pyrazolyl heterocycles--a review.

    PubMed

    Dadiboyena, Sureshbabu; Nefzi, Adel

    2011-11-01

    Heterocyclic chemistry constitutes an essential branch of organic chemistry and heterocycles are widely known to display an array of biological properties. Pyrazoles represent key structural motifs in heterocyclic chemistry and are present in a large number of biologically active molecules relevant to the pharmaceutical and agrochemical industries. Compounds incorporating the pyrazolyl structural unit are being developed in a wide variety of therapeutic areas including CNS, metabolic diseases, and oncology. The current review summarizes recent advances in the synthesis of tetrasubstituted pyrazoles. The contents are discussed in five sections: (a) 1,3-dipolar cycloadditions, (b) related 1,3-dipolar cycloadditions, (c) condensations, (d) allenylphosphonates, and (e) synthesis of fused pyrazole containing heterocycles.

  18. Isolation and taxonomic affiliation of N-heterocyclic aromatic hydrocarbon-transforming bacteria.

    PubMed

    Willumsen, Pia Arentsen; Johansen, Jens Efsen; Karlson, Ulrich; Hansen, Bjarne Munk

    2005-05-01

    The azaarenes (nitrogen-containing heterocyclic aromatic hydrocarbons) are products of incomplete combustion processes and thus are widely distributed with tar and oil products in the environment. Despite their adverse organoleptic, toxic, and carcinogenic characteristics, the biodegradability and fate of multi-ring azaarenes have received little attention. This work demonstrates the presence of genetically diverse azaarene-degrading bacteria in coal tar-contaminated soils. Thirty-eight bacterial strains able to transform the three-ring azaarenes, 5,6- and 7,8-benzoquinoline, phenanthridine, phenazine, or acridine, were isolated. Only seven of these strains grew in liquid medium on the specific azaarene compounds on which they were isolated using plates; and the rest transformed the azaarenes without growth. Taxonomic characterization by 16S ribosomal DNA sequencing revealed that our enrichment technique provided a diversity of 18 different azaarene-transforming bacterial species. Only a few strains were able to mineralize the homocyclic analogue, phenanthrene. Several of the isolates, e.g., Dyadobacter fermentans, Methylopila capsulata, and Agrobacterium tumefaciens, were related to genera relatively unknown with respect to the biodegradation of xenobiotic compounds. These strains can provide further information on the fate of azaarenes in the environment.

  19. Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.

    PubMed

    Anisenko, Andrey N; Knyazhanskaya, Ekaterina S; Zatsepin, Timofey S; Gottikh, Marina B

    2017-01-01

    Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  20. Circular dichroism anisotrophy of DNA with different modifications at N7 of guanine.

    PubMed

    Zavriev, S K; Minchenkova, L E; Vorlícková, M; Kolchinsky, A M; Volkenstein, M V; Ivanov, V I

    1979-09-27

    The complexex DNA-Ag1+, DNA-Cu1+, protonated DNA and DNA methylated at N7 of guanine were oriented by pumping the solutions through a multicapillary cell in the direction of a light beam. The CD components along the DNA axis, delta epsilon parallel, and normal to it, 2 delta epsilon perpendicular, were calculated from the CD spectra of the oriented samples by the method of Chung and Holzwarth, (1975) J. Mol. Biol. 92, 449--466. It was shown that in most cases, except that of the protonated DNA, the degree of orientation was only slightly less than that for pure DNA. This demonstrated the absence of aggregation and of appreciable denaturation. In all cases the modifications of DNA give rise to a negative component 2 delta epsilon perpendicular, whose magnitude increased as the extent of modification increased. From both the CD spectra of non-oriented samples and the absorption spectra, an inference is drawn that Ag1+ and Cu1+ are attached to the same site as CH3 groups i.e., to the N7 atom of guanine. Proton transfer along the H-bond from the N1 atom of G to the N3 atom of the complementary cytosine is suggested to be a result of the modifications, although the case of H+-DNA may differ from the others. Based on the CD spectra for the anisotropic components, delta epsilon parallel and 2 delta epsilon perpendicular, it is proposed that ligand binding is accompanied by winding of the DNA helix.

  1. Extracellular histones, cell-free DNA, or nucleosomes: differences in immunostimulation

    PubMed Central

    Marsman, Gerben; Zeerleder, Sacha; Luken, Brenda M

    2016-01-01

    In inflammation, extensive cell death may occur, which results in the release of chromatin components into the extracellular environment. Individually, the purified chromatin components double stranded (ds)DNA and histones have been demonstrated, both in vitro and in vivo, to display various immunostimulatory effects, for example, histones induce cytotoxicity and proinflammatory signaling through toll-like receptor (TLR)2 and 4, while DNA induces signaling through TLR9 and intracellular nucleic acid sensing mechanisms. However, DNA and histones are organized in nucleosomes in the nucleus, and evidence suggests that nucleosomes are released as such in inflammation. The cytotoxicity and proinflammatory signaling induced by nucleosomes have not been studied as extensively as the separate effects brought about by histones and dsDNA, and there appear to be some marked differences. Remarkably, little distinction between the different forms in which histones circulate has been made throughout literature. This is partly due to the limitations of existing techniques to differentiate between histones in their free or DNA-bound form. Here we review the current understanding of immunostimulation induced by extracellular histones, dsDNA and nucleosomes, and discuss the importance of techniques that in their detection differentiate between these different chromatin components. PMID:27929534

  2. Comparison of three different techniques of human sperm DNA isolation for methylation assay.

    PubMed

    Yuan, Hong-fang; Kuete, Martin; Su, Li; Yang, Fan; Hu, Zhi-yong; Tian, Bo-zhen; Zhang, Hui-ping; Zhao, Kai

    2015-12-01

    Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (P<0.01). PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 (DDX4) and copy number variations (CNVs) than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.

  3. A comparative evaluation of different DNA vaccine candidates against experimental murine leishmaniasis due to L. major.

    PubMed

    Ahmed, Sami Ben Hadj; Bahloul, Chokri; Robbana, Cyrine; Askri, Souhir; Dellagi, Koussay

    2004-04-16

    Over the past few years, several reports of DNA vaccines against murine cutaneous experimental leishmaniasis came out with promising but sometimes discordant results. The present studies were designed to compare, under similar conditions, the protective effects in the highly susceptible BALB/c mice of DNA vaccine candidates encoding to various Leishmania major antigens. The candidate DNA vaccines encode to the following antigens: LACK, PSA2, Gp63, LeIF and two newly identified p20 and Ribosomal like protein, in addition to different truncated portions of the LACK antigen. The most promising gene was LACK and it is more protective when it is used as a p24 truncated form. Furthermore, the presence of a tandem repeats of immunostimulating sequences (ISS) in the plasmid backbone played an important adjuvant effect in the observed protective effect induced by the DNA vaccine encoding to the LACKp24. Nevertheless, neither of the DNA vaccine candidates was able to mount a full protection in BALB/c mice challenged with a highly virulent L. major strain. Further improvements of the DNA vaccination approach are still needed to design a fully protective vaccine against leishmaniasis. Three directions of investigations are currently explored: DNA vaccines using a cocktail of antigens; Prime/Boost approach; and association of immune modulators with the candidate antigens.

  4. Nanomechanical properties of protein-DNA layers with different oligonucleotide tethers.

    PubMed

    Sánchez, Cristina Gutiérrez; Su, Qiang; Wenderhold-Reeb, Sabine; Nöll, Gilbert

    2016-06-24

    The multi-ligand binding flavoprotein dodecin is reconstituted on top of flavin-terminated oligonucleotide monolayers. A detailed quartz crystal microbalance with a dissipation monitoring (QCM-D) study showing how the length and flexibility of the oligonucleotide tethers influence the stability and the viscoelastic properties of the resulting DNA-protein layers is presented. Relatively dense protein layers can be obtained, if the length of the tethers is in the same range as the diameter of dodecin. When significantly longer tethers are used, less dense layers are formed. When rather short tethers are used, the reaching area of individual tethers is too low to capture single apododecin molecules cooperatively, and the formation of stable and dense protein layers is not possible. On top of the DNA-dodecin layers additional flavin-DNA ligands may be captured to form sandwich-type DNA-protein-DNA layers. Differences in the binding and unbinding behavior of flavin-dsDNA and flavin-ssDNA ligands are measured by QCM-D and surface plasmon fluorescence spectroscopy (SPFS). Both type of ligands show relatively low kon values, which might be explained by the structural rigidity of the binding pockets allowing a ligand to enter only when it approaches precisely in the right orientation. Apparently apododecin-flavin binding follows Fischer's classic lock-and-key binding model.

  5. Structural basis for the Smad5 MH1 domain to recognize different DNA sequences

    PubMed Central

    Chai, Nan; Li, Wan-Xin; Wang, Jue; Wang, Zhi-Xin; Yang, Shi-Ming; Wu, Jia-Wei

    2015-01-01

    Smad proteins are important intracellular mediators of TGF-β signalling, which transmit signals directly from cell surface receptors to the nucleus. The MH1 domain of Smad plays a key role in DNA recognition. Two types of DNA sequence were identified as Smad binding motifs: the Smad binding element (SBE) and the GC-rich sequence. Here we report the first crystal structure of the Smad5 MH1 domain in complex with the GC-rich sequence. Compared with the Smad5-MH1/SBE complex structure, the Smad5 MH1 domain contacts the GC-rich site with the same β-hairpin, but the detailed interaction modes are different. Conserved β-hairpin residues make base specific contacts with the minimal GC-rich site, 5′-GGC-3′. The assembly of Smad5-MH1 on the GC-rich DNA also results in distinct DNA conformational changes. Moreover, the crystal structure of Smad5-MH1 in complex with a composite DNA sequence demonstrates that the MH1 domain is targeted to each binding site (GC-rich or SBE) with modular binding modes, and the length of the DNA spacer affects the MH1 assembly. In conclusion, our work provides the structural basis for the recognition and binding specificity of the Smad MH1 domain with the DNA targets. PMID:26304548

  6. Aspects of Ancient Mitochondrial DNA Analysis in Different Populations for Understanding Human Evolution

    PubMed Central

    Nesheva, DV

    2014-01-01

    The evolution of modern humans is a long and difficult process which started from their first appearance and continues to the present day. The study of the genetic origin of populations can help to determine population kinship and to better understand the gradual changes of the gene pool in space and time. Mitochondrial DNA (mtDNA) is a proper tool for the determination of the origin of populations due to its high evolutionary importance. Ancient mitochondrial DNA retrieved from museum specimens, archaeological finds and fossil remains can provide direct evidence for population origins and migration processes. Despite the problems with contaminations and authenticity of ancient mitochondrial DNA, there is a developed set of criteria and platforms for obtaining authentic ancient DNA. During the last two decades, the application of different methods and techniques for analysis of ancient mitochondrial DNA gave promising results. Still, the literature is relatively poor with information for the origin of human populations. Using comprehensive phylogeographic and population analyses we can observe the development and formation of the contemporary populations. The aim of this study was to shed light on human migratory processes and the formation of populations based on available ancient mtDNA data. PMID:25741209

  7. Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification.

    PubMed

    Calacal, Gayvelline C; Apaga, Dame Loveliness T; Salvador, Jazelyn M; Jimenez, Joseph Andrew D; Lagat, Ludivino J; Villacorta, Renato Pio F; Lim, Maria Cecilia F; Fortun, Raquel D R; Datar, Francisco A; De Ungria, Maria Corazon A

    2015-11-01

    The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store. We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼ 1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA(®) cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor(®)HY. All samples were amplified using

  8. DNA methylation profiling in different phases of temporomandibular joint osteoarthritis in rats.

    PubMed

    Xiao, Jia-Ling; Meng, Juan-Hong; Gan, Ye-Hua; Li, Ya-Li; Zhou, Chun-Yan; Ma, Xu-Chen

    2016-08-01

    Temporomandibular joint osteoarthritis (TMJOA) is a complex disease with strong genetic and epigenetic components in its pathogenesis. The aim of this study was to evaluate DNA methylation in mandibular head cartilage in different phases of experimentally-induced TMJOA in rats. DNA methylation was evaluated using microarrays in the mandibular head cartilage of early, intermediate and late stage experimentally-induced TMJOA, and of the normal age-matched control groups. Genes with differentially methylated CpG sites were analyzed to reveal the over-represented gene ontologies and pathways at different stages, and were compared with published expression profiles to assess their overlappings. The DNA methylation patterns of the target genes were validated by methylated DNA immunoprecipitation qPCR in additional independent cartilage samples and mRNA levels were analyzed by real-time PCR. We observed 9489 differentially methylated regions between the TMJOA and controls. A total of 440 consistently altered genes were revealed in all three stages; most (80%) were hypomethylated and many were associated with cell cycle regulation. We also detected different DNA methylation changes in early and late stage TMJOA (Rearly=0.68, Rlate=0.47), while the differences between age-matched healthy cartilage were subtle. Strong inverse changes between methylation status and mRNA levels were confirmed in Adamts5, Chad, Cldn11 and Tnf. Our data reveals dynamic DNA methylation patterns during the progression of TMJOA, with a different host of genes and pathways. The changes of cartilage DNA methylation patterns might contribute to understand the etiologic mechanisms of TMJOA epigenetically. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Evaluation of different buffers on plasmid DNA encapsulation into PLGA microparticles.

    PubMed

    Tse, Man Tsuey; Blatchford, Chris; Oya Alpar, H

    2009-03-31

    Double emulsion solvent evaporation is a widely used method to prepare poly(dl-lactide-co-glycolide) (PLGA) microparticles encapsulating plasmid DNA. There are inherent problems associated with preparing plasmid DNA in this form, in particular the DNA is liable to degrade during manufacture and the resulting powder has low encapsulation efficiencies. This study compares the use of two buffers, 0.1M NaHCO(3) and 0.07M Na(2)HPO(4) and the effect these have on the encapsulation efficiency and other critical parameters associated with these encapsulated DNA materials. Both buffers preserved the conformation of the original plasmid DNA during the homogenization process, but those made with 0.07M Na(2)HPO(4) had higher encapsulation efficiencies, as well as smaller diameters, compared with those made with 0.1M NaHCO(3) (encapsulation efficiencies of 40.72-45.65%, and mean volume diameters of 2.96-4.45microm). Buffers with a range of pH from 5 to 12 were investigated, and it was demonstrated that pH 9 was the point at which the highest amount of supercoiled DNA was balanced with the highest encapsulation efficiency. To simulate in vitro release, it was shown that microparticles made with 0.07M Na(2)HPO(4) had lower DNA release rates than those made with 0.1M NaHCO(3). These results demonstrate that the use of different buffers can aid in retaining the conformation of plasmid DNA, and can also modulate the amount of DNA encapsulated and the release profiles of microparticles.

  10. Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters.

    PubMed

    Jathoul, Amit P; Holley, Jane L; Garmory, Helen S

    2004-09-28

    DNA vaccines which expressed the Hc fragment of the Clostridium botulinum type F neurotoxin (BoNT/F Hc) fused to a signal peptide downstream of four different eukaryotic promoters were prepared. Subsequently, the immunogenicity of the DNA vaccines and protection afforded in mice against challenge with 10(4) MLD of type F botulinum toxin was evaluated. The DNA vaccine containing the human ubiquitin gene (UbC) promoter induced the highest BoNT/F Hc-specific antibody concentration following two intramuscular immunisations and afforded 90% protection against challenge. The results from this study indicate that the selection of promoter used in DNA vaccination studies may be of importance in designing optimised vaccines.

  11. A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes.

    PubMed

    Wyroba, E; Satir, B H

    2000-01-01

    Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phosphoglycoprotein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3--I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.

  12. Sex differences of leukocytes DNA methylation adjusted for estimated cellular proportions.

    PubMed

    Inoshita, Masatoshi; Numata, Shusuke; Tajima, Atsushi; Kinoshita, Makoto; Umehara, Hidehiro; Yamamori, Hidenaga; Hashimoto, Ryota; Imoto, Issei; Ohmori, Tetsuro

    2015-01-01

    DNA methylation, which is most frequently the transference of a methyl group to the 5-carbon position of the cytosine in a CpG dinucleotide, plays an important role in both normal development and diseases. To date, several genome-wide methylome studies have revealed sex-biased DNA methylation, yet no studies have investigated sex differences in DNA methylation by taking into account cellular heterogeneity. The aim of the present study was to investigate sex-biased DNA methylation on the autosomes in human blood by adjusting for estimated cellular proportions because cell-type proportions may vary by sex. We performed a genome-wide DNA methylation profiling of the peripheral leukocytes in two sets of samples, a discovery set (49 males and 44 females) and a replication set (14 males and 10 females) using Infinium HumanMethylation450 BeadChips for 485,764 CpG dinucleotides and then examined the effect of sex on DNA methylation with a multiple linear regression analysis after adjusting for age, the estimated 6 cell-type proportions, and the covariates identified in a surrogate variable analysis. We identified differential DNA methylation between males and females at 292 autosomal CpG site loci in the discovery set (Bonferroni-adjusted p < 0.05). Of these 292 CpG sites, significant sex differences were also observed at 98 sites in the replication set (p < 0.05). These findings provided further evidence that DNA methylation may play a role in the differentiation or maintenance of sexual dimorphisms. Our methylome mapping of the effects of sex may be useful to understanding the molecular mechanism involved in both normal development and diseases.

  13. Different Effects of CSA and CSB Deficiency on Sensitivity to Oxidative DNA Damage

    PubMed Central

    de Waard, Harm; de Wit, Jan; Andressoo, Jaan-Olle; van Oostrom, Conny T. M.; Riis, Bente; Weimann, Allan; Poulsen, Henrik E.; van Steeg, Harry; Hoeijmakers, Jan H. J.; van der Horst, Gijsbertus T. J.

    2004-01-01

    Mutations in the CSA and CSB genes cause Cockayne syndrome, a rare inherited disorder characterized by UV sensitivity, severe neurological abnormalities, and progeriod symptoms. Both gene products function in the transcription-coupled repair (TCR) subpathway of nucleotide excision repair (NER), providing the cell with a mechanism to remove transcription-blocking lesions from the transcribed strands of actively transcribed genes. Besides a function in TCR of NER lesions, a role of CSB in (transcription-coupled) repair of oxidative DNA damage has been suggested. In this study we used mouse models to compare the effect of a CSA or a CSB defect on oxidative DNA damage sensitivity at the levels of the cell and the intact organism. In contrast to CSB−/− mouse embryonic fibroblasts (MEFs), CSA−/− MEFs are not hypersensitive to gamma-ray or paraquat treatment. Similar results were obtained for keratinocytes. In contrast, both CSB−/− and CSA−/− embryonic stem cells show slight gamma-ray sensitivity. Finally, CSB−/− but not CSA−/− mice fed with food containing di(2-ethylhexyl)phthalate (causing elevated levels of oxidative DNA damage in the liver) show weight reduction. These findings not only uncover a clear difference in oxidative DNA damage sensitivity between CSA- and CSB-deficient cell lines and mice but also show that sensitivity to oxidative DNA damage is not a uniform characteristic of Cockayne syndrome. This difference in the DNA damage response between CSA- and CSB-deficient cells is unexpected, since until now no consistent differences between CSA and CSB patients have been reported. We suggest that the CSA and CSB proteins in part perform separate roles in different DNA damage response pathways. PMID:15340056

  14. Cloning of apoptosis-related genes by representational difference analysis of cDNA.

    PubMed

    Hubank, Michael; Bryntesson, Fredrik; Regan, Jennifer; Schatz, David G

    2004-01-01

    Apoptosis is frequently triggered by events that alter the expression of key target genes. Under these circumstances, the genes involved can be identified by techniques that analyze gene expression. Researchers now have a choice of reliable and effective methods for differential gene expression analysis. Comparative approaches, including gene microarray analysis, serial analysis of gene expression, and differential display provide global information about expression levels. Subtractive approaches like complementary DNA representational difference analysis (cDNA RDA) and suppression subtraction polymerase chain reaction identify a focused set of differentially expressed genes. The most suitable technique to apply depends on individual circumstances. cDNA RDA is particularly useful in nonstandard model organisms for which comprehensive gene microarrays are not available and is best used for the identification of genes with a large difference in expression levels between two populations. The technique involves the generation of amplified mixtures of cDNA fragments that are typically smaller than 1000 base pairs and represent >86% of mRNA species from each starting population. Transcriptional differences between two populations can then be identified by subtraction of cDNA amplicons followed by further polymerase chain reaction amplification. The technique is capable of detecting differences for genes expressed at less than one copy per cell and is achievable using standard laboratory apparatus. cDNA RDA can identify genes not previously described in the database, can detect low abundance transcripts (e.g., from mixed cell populations), and is best applied in experiments where relatively few differentially expressed genes are expected. Here, we describe the application of cDNA RDA to the identification of apoptosis-related genes.

  15. Innovative methods for soil DNA purification tested in soils with widely differing characteristics.

    PubMed

    Sagova-Mareckova, Marketa; Cermak, Ladislav; Novotna, Jitka; Plhackova, Kamila; Forstova, Jana; Kopecky, Jan

    2008-05-01

    Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO(3) or purification of extracted DNA by CaCl(2). The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl(2) purification and in 93% of cases by the method based on CaCO(3) pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

  16. 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)

  17. 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)

  18. Carotenoids play a positive role in the degradation of heterocycles by Sphingobium yanoikuyae.

    PubMed

    Liu, Xiaorui; Gai, Zhonghui; Tao, Fei; Tang, Hongzhi; Xu, Ping

    2012-01-01

    Microbial oxidative degradation is a potential way of removing pollutants such as heterocycles from the environment. During this process, reactive oxygen species or other oxidants are inevitably produced, and may cause damage to DNA, proteins, and membranes, thereby decreasing the degradation rate. Carotenoids can serve as membrane-integrated antioxidants, protecting cells from oxidative stress. Several genes involved in the carotenoid biosynthetic pathway were cloned and characterized from a carbazole-degrading bacterium Sphingobium yanoikuyae XLDN2-5. In addition, a yellow-pigmented carotenoid synthesized by strain XLDN2-5 was identified as zeaxanthin that was synthesized from β-carotene through β-cryptoxanthin. The amounts of zeaxanthin and hydrogen peroxide produced were significantly and simultaneously enhanced during the biodegradation of heterocycles (carbazole < carbazole + benzothiophene < carbazole + dibenzothiophene). These higher production levels were consistent with the transcriptional increase of the gene encoding phytoene desaturase, one of the key enzymes for carotenoid biosynthesis. Sphingobium yanoikuyae XLDN2-5 can enhance the synthesis of zeaxanthin, one of the carotenoids, which may modulate membrane fluidity and defense against intracellular oxidative stress. To our knowledge, this is the first report on the positive role of carotenoids in the biodegradation of heterocycles, while elucidating the carotenoid biosynthetic pathway in the Sphingobium genus.

  19. Pentannulation of Heterocycles by Virtue of Precious Metal Catalysis.

    PubMed

    Petrović, Martina; Occhiato, Ernesto G

    2016-03-04

    Pentannulated heterocycles are the key structural subunit of many natural and biologically active compounds. Over the last decades, many precious metal-assisted pentannulations have been described as a consequence of an extensive research. This Focus Review gives an overview of precious metal-catalyzed reactions applied to the synthesis of cyclopenta-fused heterocycles in the last five years.

  20. Alkynylation of heterocyclic compounds using hypervalent iodine reagent.

    PubMed

    Kamlar, M; Císařová, I; Veselý, J

    2015-03-14

    The alkynylation of various nitrogen- and/or sulphur-containing heterocyclic compounds using hypervalent iodine TMS-EBX by utilization of tertiary amines under mild conditions is described. The developed metal-free methodology furnishes the corresponding alkynylated heterocycles bearing quaternary carbon in high yields.

  1. Iron-Catalyzed Synthesis of Sulfur-Containing Heterocycles.

    PubMed

    Bosset, Cyril; Lefebvre, Gauthier; Angibaud, Patrick; Stansfield, Ian; Meerpoel, Lieven; Berthelot, Didier; Guérinot, Amandine; Cossy, Janine

    2016-10-13

    An iron-catalyzed synthesis of sulfur- and sulfone-containing heterocycles is reported. The method is based on the cyclization of readily available substrates and proceeded with high efficiency and diastereoselectivity. A variety of sulfur-containing heterocycles bearing moieties suitable for subsequent functionalization are prepared. Illustrative examples of such postcyclization modifications are also presented.

  2. Application of denaturing gradient gel electrophoresis to detect DNA sequence differences encoding apolipoprotein E isoforms

    SciTech Connect

    Parker, S.; Angelico, M.C.; Laffel, L.; Krolewski, A.S. Harvard Medical School, Boston, MA )

    1993-04-01

    Apolipoprotein E (apoE) plays an important role in plasma lipid metabolism. Three common isoforms of this protein have been identified by the isoelectric focusing method. In this report the authors describe a new method for distinguishing these isoforms. Their method employs PCR amplification of the DNA sequence of exon 4 in the apoE gene followed by denaturing gradient gel electrophoresis (DGGE) to distinguish its different melting characteristics. Identification of the ApoE isoforms through DNA melting behavior rather than protein charge differences eliminates the problems associated with isoelectric focusing and facilitates screening for additional mutations at the apoE locus. 12 refs., 2 figs.

  3. The same, only different - DNA damage checkpoints and their reversal throughout the cell cycle.

    PubMed

    Shaltiel, Indra A; Krenning, Lenno; Bruinsma, Wytse; Medema, René H

    2015-02-15

    Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made. © 2015. Published by The Company of Biologists Ltd.

  4. Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy

    PubMed Central

    Cassuto, Nino Guy; Montjean, Debbie; Siffroi, Jean-Pierre; Bouret, Dominique; Marzouk, Flora; Copin, Henri; Benkhalifa, Moncef

    2016-01-01

    Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM) at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p < 10−6), OR = 2.4; and p < 0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI. PMID:27148551

  5. Distinct circular single-stranded DNA viruses exist in different soil types.

    PubMed

    Reavy, Brian; Swanson, Maud M; Cock, Peter J A; Dawson, Lorna; Freitag, Thomas E; Singh, Brajesh K; Torrance, Lesley; Mushegian, Arcady R; Taliansky, Michael

    2015-06-15

    The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

  6. One-Step Synthetic Access to Isosteric and Potent Anticancer Nitrogen Heterocycles with the Benzo[c]phenanthridine Scaffold.

    PubMed

    Steinhauer, Tamara N; Girreser, Ulrich; Meier, Christopher; Cushman, Mark; Clement, Bernd

    2016-06-06

    A versatile one-step two-component cyclization to build new tetracyclic nitrogen heterocycles is described. Ortho-methylhetarenecarbonitrile components were condensed with aldehydes to access a large library of differently substituted ring systems. The heterocyclic core can be easily modified by variation of the position of the endocyclic nitrogen atom in the o-methylhetarenecarbonitrile substrate. The manner of the nucleophilic attack that leads to the condensation can be triggered by different electron-density distribution in the molecule induced by the position of the nitrogen atom. Taking this into account, there is an electronic preference that leads to either pyridophenanthrolines or the corresponding pyridoazacarbazoles as the main products. We demonstrate the high antitumor potential of some of our synthesized heterocycles, which is strongly dependent on the substitution pattern introduced through the aldehyde component. The position and number of endocyclic nitrogen atoms play an important role regarding cytotoxicity of the studied compounds.

  7. Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

    PubMed

    Grasso, Chiara; Trevisan, Morena; Fiano, Valentina; Tarallo, Valentina; De Marco, Laura; Sacerdote, Carlotta; Richiardi, Lorenzo; Merletti, Franco; Gillio-Tos, Anna

    2016-01-01

    Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

  8. Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing

    PubMed Central

    Grasso, Chiara; Trevisan, Morena; Fiano, Valentina; Tarallo, Valentina; De Marco, Laura; Sacerdote, Carlotta; Richiardi, Lorenzo; Merletti, Franco; Gillio-Tos, Anna

    2016-01-01

    Background Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Objective Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. Methods We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. Results We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. Conclusion The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing. PMID

  9. ortho-Quinone methides as key intermediates in cascade heterocyclizations

    NASA Astrophysics Data System (ADS)

    Osipov, D. V.; Osyanin, V. A.; Klimochkin, Yu N.

    2017-07-01

    Development of new methods of heterocyclic synthesis is still a topical issue. In this connection, the trend related to the use of highly reactive o-quinone methides for the synthesis and functionalization of heterocycles appears rather promising. Since most of o-quinone methides are unstable, the choice of precursors and generation conditions is highly important for subsequent transformations involving them. Various methods of generation of o-quinone methides and cascade heterocyclizations in which the formation of these compounds is a key step are surveyed in the review. The trends of using o-quinone methides in the synthesis of various heterocycles are analyzed and the heterocyclization reactions involving these compounds are classified. The bibliography includes 395 references.

  10. Structure Dependent Binding of Arylimidamides to the DNA Minor Groove

    PubMed Central

    Chai, Yun; Munde, Manoj; Kumar, Arvind; Mickelson, Leah; Lin, Sen; Campbell, Nancy H.; Banerjee, Moloy; Akay, Senol; Liu, Zongying; Farahat, Abdelbasset A.; Nhili, Raja; Depauw, Sabine; David-Cordonnier, Marie-Hélène; Neidle, Stephen

    2014-01-01

    Heterocyclic diamidines are strong DNA minor groove binders and have excellent antiparasitic activity. To extend the biological activity of these compounds, a series of arylimidamides (AIAs) analogs, which have better uptake properties in Leishmania and T. cruizi than diamidines, was prepared. The binding of the AIAs to DNA was investigated by Tm, fluorescence displacement titration, circular dichroism, DNase I footprinting, biosensor surface plasmon resonance, X-ray Crystallography and molecular modeling. These compounds form 1:1 complexes with AT sequences in the DNA minor groove and the binding strength varies with substituent size, charge and polarity. This substituent dependent structure and properties provide a SAR that can be used to estimate K values for binding to DNA in this series. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for AIAs. PMID:24323836

  11. Evaluation of purification procedures of DNA from maize-meal samples by exploiting different analytical techniques for the assessment of DNA quality.

    PubMed

    Vanni, Adriano; Anfossi, Laura; Giovannoli, Cristina; Oddenino, Leila; Giraudi, Gianfranco

    2004-04-01

    Two different approaches generally applied to achieve purification of DNA extracted from cells were compared: precipitation by organic solvents and enzymatic treatments. We investigated various experimental protocols reported in literature by evaluating DNA purity, integrity and yield. Reliability of analytical techniques normally employed to assess DNA purity and quantity was studied and comments and conclusions were suggested by comparing results obtained by different analytical techniques. Enzymatic treatments prove to be unable of increasing DNA purity while determining a significant degradation. In contrast, optimised conditions for solvent precipitation enabled a sharp increase of DNA purity to be obtained, associated with the maintenance of the initial DNA integrity. The application of the optimised protocol to maize-meal samples allowed us to achieve a good PCR amplification even with those samples which gave poor amplification by following the protocol recommended by the Italian legislation in force for GMO detection in food.

  12. TATA Binding Protein Discriminates between Different Lesions on DNA, Resulting in a Transcription Decrease

    PubMed Central

    Coin, Frédéric; Frit, Philippe; Viollet, Benoit; Salles, Bernard; Egly, Jean-Marc

    1998-01-01

    DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position −29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage. PMID:9632775

  13. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories.

    PubMed

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej; Ferlińska, Magdalena; Palus, Jadwiga; Teixeira, João Paulo; Costa, Solange; Jones, George D D; Higgins, Jennifer A; Kain, Johanna; Möller, Lennart; Forchhammer, Lykke; Loft, Steffen; Lorenzo, Yolanda; Collins, Andrew R; van Schooten, Frederik-Jan; Laffon, Blanca; Valdiglesias, Vanessa; Cooke, Marcus; Mistry, Vilas; Karbaschi, Mahsa; Phillips, David H; Sozeri, Osman; Routledge, Michael N; Nelson-Smith, Kirsty; Riso, Patrizia; Porrini, Marisa; López de Cerain, Adela; Azqueta, Amaya; Matullo, Giuseppe; Allione, Alessandra; Møller, Peter

    2014-07-01

    This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp). © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Applying the stochastic difference equation to DNA conformational transitions: a study of B-Z and B-A DNA transitions.

    PubMed

    Lim, Wilber; Feng, Yuan Ping

    2005-06-15

    Despite the existence of numerous models to account for the B-Z DNA transition, experimenters have not yet arrived at a conclusive answer to the structural and dynamical features of the B-Z transition. By applying the stochastic difference equation to simulate the B-Z DNA transition, we have shown that the stretched intermediate model of the B-Z transition is more probable than other B-Z transition models such as the Harvey model. This is accomplished by comparing potential energy profiles of various B-Z DNA transition models and calculating relative probabilities based on the stochastic difference equation with respect to length (SDEL) formalism. The results garnered in this article allow for new approaches in determining the structural transition of B-DNA to Z-DNA experimentally. We have also simulated the B-A DNA transition using the stochastic difference equation. Unlike the B-Z DNA transition, the mechanism for the B-A DNA transition is well established. The variation in the pseudorotation angle during the transition is in good agreement with experimental results. Qualitative features of the simulated B-A transition also agree well with experimental data. The SDEL approach is thus a suitable numerical technique to compute long-time molecular dynamics trajectory for DNA molecules.

  15. Analysis of Telomere-Homologous DNA with Different Conformations Using 2D Agarose Electrophoresis and In-Gel Hybridization.

    PubMed

    Zhang, Zepeng; Hu, Qian; Zhao, Yong

    2017-01-01

    In mammalian cells, in addition to double-stranded telomeric DNA at chromosome ends, extra telomere-homologous DNA is present that adopts different conformations, including single-stranded G- or C-rich DNA, extrachromosomal circular DNA (T-circle), and telomeric complex (T-complex) with an unidentified structure. The formation of such telomere-homologous DNA is closely related to telomeric DNA metabolism and chromosome end protection by telomeres. Conventional agarose gel electrophoresis is unable to separate DNA based on conformation. Here, we introduce the method of two-dimensional (2D) agarose electrophoresis in combination with in-gel native/denatured hybridization to determine different conformations formed by telomere-homologous DNA.

  16. Differences in sequence selectivity of DNA alkylation by isomeric intercalating aniline mustards.

    PubMed

    Prakash, A S; Denny, W A; Wakelin, L P

    1990-01-01

    Two DNA-targeted mustard derivatives, N,N-bis(2-chloroethyl)-4-(5-[9-acridinylamino]-pentamido)aniline and 4-(9-[acridinylamino]butyl 4-(N,N-bis[2-chloroethyl]-aminobenzamide, which are isomeric compounds where the mustard is linked to the DNA-binding 9-aminoacridine moiety by either a -CONH- or a -NHCO- group, show significant differences in the sequence selectivity of their alkylation of DNA. The CONH isomer is a more efficient alxylating agent than the NHCO compound by an order of magnitude, consistent with the larger electron release of the CONH group to the aniline ring. However, the pattern of alkylation by the two compounds is also very different, with the CONH isomer preferring alkylation of guanines adjacent to 3'- or 5'-adenines and cytosines (for example those in sequences 5'-CGC, 5'-AGC, 5'-CGG and 5'-AGA) while the isomeric NHCO compound shows preference for guanines in runs of Gs. In addition, both isomers alkylate 3'-adenines in runs of adenines. Both compounds also show completely different patterns of alkylation to their untargeted mustard counterparts, since 4-MeCONH-aniline mustard alkylates all guanines and adenines in runs of adenines, while 4-Me2NCO-aniline mustard fails to alkylate DNA at all. These differences in alkylation patterns between the CONH- and its isomeric NHCO- compounds and their relationships between the alkylation patterns of the isomers and their biological activities are discussed.

  17. General and efficient one-pot synthesis of novel sugar/heterocyclic(aryl) 1,2-diketones from sugar terminal alkynes by Sonogashira/tetra-n- butylammonium permanganate oxidation.

    PubMed

    Zhang, Fuyi; Wu, Xiaopei; Wang, Liming; Liu, Hong; Zhao, Yufen

    2015-11-19

    A new approach for one-pot synthesis of novel sugar/heterocyclic(aryl) 1,2-diketones has been achieved by the reaction of various sugar terminal alkynes with heterocyclic(aryl) iodides at room temperature. This one-pot protocol includes Sonogashira coupling and mild n-Bu4NMnO4 oxidation reaction. This method is mild, general and efficient. Fifty-six examples have been given and the sugar/heterocyclic(aryl) 1,2-diketones were obtained in 71-94% yields. The sugar terminal alkynes include 9 structurally different sugars in pyranose, furanose, and acyclic form which have various protecting groups, sensitive groups, and sterically bulky substituents. The heterocyclic(aryl) iodides include sterically bulky heterocyclic compounds and iodobenzenes with electron-donating, electron-neutral, and electron-withdrawing substituents.

  18. Prediction model of biocrude yield and nitrogen heterocyclic compounds analysis by hydrothermal liquefaction of microalgae with model compounds.

    PubMed

    Sheng, Lili; Wang, Xin; Yang, Xiaoyi

    2017-08-05

    The model of biocrude yield and the nitrogen heterocyclic compounds in biocrude of microalgae hydrothermal liquefaction are two of the most concerned issues in this field at present. This study explored a hydrothermal liquefaction biocrude yield model involved in the interaction among biochemical compounds in microalgae and analysed nitrogen heterocyclic compounds in biocrude. The model compound (castor oil, soya protein and glucose) and Nanochloropsis were liquefied at 280°C for 1h. The products were analyzed by GC-MS, element analysis and FTIR. The results suggested that interactions among different components in microalgae enhanced biocrude yield. The biocrude yield prediction model involved cross-interactions performed more accurate than previous models.When the ratio of protein and carbohydrate around 3, the cross-interaction and nitrogen heterocyclic compounds in biocrude would both reach the highest extent. Copyright © 2017. Published by Elsevier Ltd.

  19. Different effects of eubacterial and eukaryotic DNA topoisomerase II inhibitors on chloroplasts ofEuglena gracilis

    NASA Astrophysics Data System (ADS)

    Krajčovič, Juraj; Ebringer, Libor

    1990-03-01

    Inhibitors of eubacterial and eukaryotic DNA topoisomerases type II exhibited different effects on chloroplasts of the flagellateEuglena gracilis. Antibacterial agents (cinoxacin, nalidixic and oxolinic acids, ciprofloxacin, enoxacin, norfloxacin and ofloxacin) from the group of quinolones and coumarins (coumermycin A1, clorobiocin and novobiocin) — all inhibitors of prokaryotic DNA topoisomerase II — were very potent eliminators of chloroplasts fromE. gracilis. In contrast, antitumor drugs (adriamycin, etoposide, teniposide and mitoxantrone) — antagonists of the eukaryotic counterpart — did not affect these semiautonomous photosynthetic organelles. These findings point out again the close evolutionary relationships between eubacteria and chloroplasts and are in agreement with the hypothesis of an endosymbiotic origin of chloroplasts.

  20. Genetic diversity in different populations of sloths assessed by DNA fingerprinting.

    PubMed

    Moraes, N; Morgante, J S; Miyaki, C Y

    2002-08-01

    In this study we analyzed a population of Bradypus torquatus with individuals originally distributed in different localities of Bahia, and two populations of B. variegatus with individuals from Bahia and São Paulo States. Using the DNA fingerprinting method, we assessed the genetic variability within and between populations. Analysis of the DNA profiles revealed genetic similarity indices ranging from 0.34 +/- 0.07 to 0.87 +/- 0.04. Similar low levels of genetic variability were found only in isolated mammalian populations or among related individuals. This study presents the first analyses of genetic diversity in sloth populations.

  1. The Paradoxical Effects of Different Hepatitis C Viral Loads on Host DNA Damage and Repair Abilities

    PubMed Central

    Li, Chia-Yang; Chiang, Chi-Shiun; Yu, Guann-Yi; Sakamoto, Naoya; Tu, Wen-Yu; Hsieh, Meng-Hsuan; Huang, Jee-Fu; Chuang, Wan-Long; Dai, Chia-Yen

    2017-01-01

    Hepatitis C virus (HCV)-induced hepatic stress is associated with increased oxidative DNA damage and has been implicated in hepatic inflammation. However, HCV infection and replication are uneven and vary among individual hepatocytes. To investigate the effect of the viral load on host DNA damage, we used an Enhanced Yellow Fluorescent Protein gene (EYFP)-tagged HCV virus to distinguish between HCV intracellular high viral load (HVL) cells and low viral load (LVL) cells. The cell sorting efficiency was confirmed by the high expression of the HCV polyprotein. We found DNA damage γ-H2AX foci in the HVL population. Comet assays demonstrated that HVL was related to the extent of the DNA strand breaks. Surprisingly, the DNA qPCR arrays and western blotting showed that the damage-related genes GPX2, MRE11, phospho-ATM, and OGG1 were significantly up-regulated in LVL cells but inversely down-regulated or consistently expressed in HVL cells. The colony survival assay to examine the repair abilities of these cells in response to irradiation showed that the LVL cells were more resistant to irradiation and had an increased ability to repair radiation-induced damage. This study found that intracellular viral loads drove cellular DNA damage levels but suppressed damage-related gene expression. However, the increase in damage-related gene expression in the LVL cells may be affected by ROS from the HVL cells. These findings provide new insights into the distinct DNA damage and repair responses resulting from different viral loads in HCV-infected cells. PMID:28052067

  2. The Paradoxical Effects of Different Hepatitis C Viral Loads on Host DNA Damage and Repair Abilities.

    PubMed

    Wang, Shu-Chi; Lai, Kuan-Ru; Li, Chia-Yang; Chiang, Chi-Shiun; Yu, Guann-Yi; Sakamoto, Naoya; Tu, Wen-Yu; Hsieh, Meng-Hsuan; Huang, Jee-Fu; Chuang, Wan-Long; Dai, Chia-Yen; Yu, Ming-Lung

    2017-01-01

    Hepatitis C virus (HCV)-induced hepatic stress is associated with increased oxidative DNA damage and has been implicated in hepatic inflammation. However, HCV infection and replication are uneven and vary among individual hepatocytes. To investigate the effect of the viral load on host DNA damage, we used an Enhanced Yellow Fluorescent Protein gene (EYFP)-tagged HCV virus to distinguish between HCV intracellular high viral load (HVL) cells and low viral load (LVL) cells. The cell sorting efficiency was confirmed by the high expression of the HCV polyprotein. We found DNA damage γ-H2AX foci in the HVL population. Comet assays demonstrated that HVL was related to the extent of the DNA strand breaks. Surprisingly, the DNA qPCR arrays and western blotting showed that the damage-related genes GPX2, MRE11, phospho-ATM, and OGG1 were significantly up-regulated in LVL cells but inversely down-regulated or consistently expressed in HVL cells. The colony survival assay to examine the repair abilities of these cells in response to irradiation showed that the LVL cells were more resistant to irradiation and had an increased ability to repair radiation-induced damage. This study found that intracellular viral loads drove cellular DNA damage levels but suppressed damage-related gene expression. However, the increase in damage-related gene expression in the LVL cells may be affected by ROS from the HVL cells. These findings provide new insights into the distinct DNA damage and repair responses resulting from different viral loads in HCV-infected cells.

  3. Coaxial atomic force microscope probes for dielectrophoresis of DNA under different buffer conditions

    NASA Astrophysics Data System (ADS)

    Tao, Yinglei; Kumar Wickramasinghe, H.

    2017-02-01

    We demonstrate a coaxial AFM nanoprobe device for dielectrophoretic (DEP) trapping of DNA molecules in Tris-EDTA (TE) and phosphate-buffered saline (PBS) buffers. The DEP properties of 20 nm polystyrene beads were studied with coaxial probes in media with different conductivities. Due to the special geometry of our DEP probe device, sufficiently high electric fields were generated at the probe end to focus DNA molecules with positive DEP. DEP trapping for both polystyrene beads and DNA molecules was quantitatively analyzed over the frequency range from 100 kHz to 50 MHz and compared with the Clausius-Mossotti theory. Finally, we discussed the negative effect of medium salinity during DEP trapping.

  4. Electrophoresis of small DNA molecules in agaroses with different electroendosmotic properties.

    PubMed

    Pramatarova, A; Hamelin, C

    1993-01-01

    The migration rate of DNA standards in agaroses with different electroendosmotic (EEO) properties was compared in order to find an alternative to polyacrylamide slab gels for the separation of small restriction fragments by electrophoresis. Slower migration of DNA molecules only a few hundred of base pairs in length was observed after raising the concentration of unmodified low EEO agarose in gels up to 6%. Resolution of low-molecular-weight DNA fragments was best achieved, however, with hydroxyethylated agaroses showing high or low EEO properties. An immediate application of the above results was to confirm the presence of the human cytomegalovirus BamH I-P subgenomic fragment (7.2 kb) in a recombinant pAT153 plasmid by restriction endonuclease analysis.

  5. Transition from B to Z DNA: Contribution of Internal Fluctuations to the Configurational Entropy Difference

    NASA Astrophysics Data System (ADS)

    Irikura, K. K.; Tidor, B.; Brooks, B. R.; Karplus, M.

    1985-08-01

    The internal motions of the double-stranded DNA oligomer (dCdG)3 (dC, deoxycytidylate; dG, deoxyguanylate) in the B and Z forms have been calculated in the harmonic approximation. A complete vibrational analysis has been made, and the resulting normal mode frequencies have been used to evaluate the vibrational entropy of B and Z DNA. The greater flexibility of the B DNA hexamer leads to an entropic stabilization relative to the stiffer Z DNA hexamer of 22 calories per mole per kelvin at 300 K. The calculated value is of the same order as that (21 to 27 calories per mole per kelvin) obtained from nuclear magnetic resonance measurements on the methylated duplexes (m5dCdG)3 and (dCdGm5dCdGdCdG). This result demonstrates the importance of internal motions, which have been neglected in earlier studies of the transition from B to Z DNA, in the stability of different nucleic acid conformers.

  6. Oxaliplatin and Its Enantiomer Induce Different Condensation Dynamics of Single DNA Molecules

    PubMed Central

    Zhang, Hong-Yan; Liu, Yu-Ru; Ji, Chao; Li, Wei; Dou, Shuo-Xing; Xie, Ping; Wang, Wei-Chi; Zhang, Ling-Yun; Wang, Peng-Ye

    2013-01-01

    The interactions of DNA with oxaliplatin (Pt(R,R-DACH)) or its enantiomer (Pt(S,S-DACH)) were investigated using magnetic tweezers and atomic force microscope. In the process of DNA condensation induced by Pt-DACH, only diadducts and micro-loops are formed at low Pt-DACH concentrations, while at high Pt-DACH concentrations, besides the diadducts and micro-loops, long-range cross-links are also formed. The diadduct formation rate of Pt(R,R-DACH) is higher than that of Pt(S,S-DACH). However, the proportions of micro-loops and long-range cross-links for Pt(S,S-DACH) are higher than those for Pt(R,R-DACH). We propose a model to explain these differences between the effect of Pt(R,R-DACH) and that of Pt(S,S-DACH) on DNA condensation. The study has strong implications for the understanding of the effect of chirality on the interaction between Pt-DACH and DNA and the kinetics of DNA condensation induced by platinum complexes. PMID:23951187

  7. Assessment of DNA Damage in Leukoplakia Patients with Different Degrees of Dysplasia.

    PubMed

    Vellappally, Sajith; Binmgren, Mohammed A; Huraib, Sahar Bin; Hashem, Mohamed I; Patil, Sh'ankargouudda; Anil, Sukumaran

    2015-12-01

    Single cell gel electrophoresis (SCGE) assay also known as comet assay is a rapid and highly sensitive fluorescent molecular technique for detecting various forms of deoxyribonucleic acid (DNA) damage at individual cellular level. The present study was done to detect the extent of DNA damage in oral leukoplakia (OL) and compare with normal individuals. The sample population was obtained from an outpatient clinic of a tertiary teaching dental institute. A total of 36 consecutive patients with leukoplakia and 10 healthy normal volunteers were recruited for the study and assessed for the extent of DNA damage using SCGE following clinical diagnosis and histological grading. Peripheral blood was obtained by venipuncture and SCGE assay was performed. Mean comet tail length was recorded and analyzed statistically to compare the extent of damage in each group. The mean comet tail length seen in leukoplakia patients with moderate to severe dysplasia was 1.25 ± 0.14 mm while for the control subjects, it was 0.31 ± 0.10 mm. The difference was statistically significant (p = 0.000). On comparing within the grades of leukoplakia, a progressive trend of increasing tail length was observed with increasing grades of dysplasia. Deoxyribonucleic acid damage as measured by SCGE is seen in leukoplakia. A stepwise increase in DNA damage levels from healthy controls, through patients with non-dysplastic epithelium to varying grades of dysplasia has been observed indicating the extent of DNA damage in this high risk group.

  8. Humans and chimpanzees differ in their cellular response to DNA damage and non-coding sequence elements of DNA repair-associated genes.

    PubMed

    Weis, E; Galetzka, D; Herlyn, H; Schneider, E; Haaf, T

    2008-01-01

    Compared to humans, chimpanzees appear to be less susceptible to many types of cancer. Because DNA repair defects lead to accumulation of gene and chromosomal mutations, species differences in DNA repair are one plausible explanation. Here we analyzed the repair kinetics of human and chimpanzee cells after cisplatin treatment and irradiation. Dot blots for the quantification of single-stranded (ss) DNA repair intermediates revealed a biphasic response of human and chimpanzee lymphoblasts to cisplatin-induced damage. The early phase of DNA repair was identical in both species with a peak of ssDNA intermediates at 1 h after DNA damage induction. However, the late phase differed between species. Human cells showed a second peak of ssDNA intermediates at 6 h, chimpanzee cells at 5 h. One of four analyzed DNA repair-associated genes, UBE2A, was differentially expressed in human and chimpanzee cells at 5 h after cisplatin treatment. Immunofluorescent staining of gammaH2AX foci demonstrated equally high numbers of DNA strand breaks in human and chimpanzee cells at 30 min after irradiation and equally low numbers at 2 h. However, at 1 h chimpanzee cells had significantly less DNA breaks than human cells. Comparative sequence analyses of approximately 100 DNA repair-associated genes in human and chimpanzee revealed 13% and 32% genes, respectively, with evidence for an accelerated evolution in promoter regions and introns. This is strikingly contrasting to the 3% of DNA repair-associated genes with positive selection in the coding sequence. Compared to the rhesus macaque as an outgroup, chimpanzees have a higher accelerated evolution in non-coding sequences than humans. The TRF1-interacting, ankyrin-related ADP-ribose polymerase (TNKS) gene showed an accelerated intraspecific evolution among humans. Our results are consistent with the view that chimpanzee cells repair different types of DNA damage faster than human cells, whereas the overall repair capacity is similar in

  9. Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers.

    PubMed

    Scholz, Claudia; Wagner, Ernst

    2012-07-20

    Gene therapy offers great opportunities for the treatment of severe diseases including cancer. In recent years the design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of plasmid DNA (pDNA) have brought up a variety of gene delivery vehicles. The more recently emerged gene silencing strategy by the intracellular delivery of small interfering RNA (siRNA) takes benefit from existing expertise in pDNA transfer. Despite common properties however, delivery of siRNA also faces distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action. This review emphasizes the common aspects and main differences between pDNA and siRNA delivery, taking into consideration a wide spectrum of polymer-based, lipidic and peptide carriers. Challenges and opportunities which result from these differences as well as the recent progress made in the optimization of carrier design are presented.

  10. Nanodosimetric Simulation of Direct Ion-Induced DNA Damage Using Different Chromatin Geometry Models.

    PubMed

    Henthorn, N T; Warmenhoven, J W; Sotiropoulos, M; Mackay, R I; Kirkby, K J; Merchant, M J

    2017-08-09

    Monte Carlo based simulation has proven useful in investigating the effect of proton-induced DNA damage and the processes through which this damage occurs. Clustering of ionizations within a small volume can be related to DNA damage through the principles of nanodosimetry. For simulation, it is standard to construct a small volume of water and determine spatial clusters. More recently, realistic DNA geometries have been used, tracking energy depositions within DNA backbone volumes. Traditionally a chromatin fiber is built within the simulation and identically replicated throughout a cell nucleus, representing the cell in interphase. However, the in vivo geometry of the chromatin fiber is still unknown within the literature, with many proposed models. In this work, the Geant4-DNA toolkit was used to build three chromatin models: the solenoid, zig-zag and cross-linked geometries. All fibers were built to the same chromatin density of 4.2 nucleosomes/11 nm. The fibers were then LET proton irradiated (5-80 keV/μm) or LET alpha-particle irradiated (63-226 keV/μm). Nanodosimetric parameters were scored for each fiber after each LET and used as a comparator among the models. Statistically significant differences were observed in the double-strand break backbone size distributions among the models, although nonsignificant differences were noted among the nanodosimetric parameters. From the data presented in this article, we conclude that selection of the solenoid, zig-zag or cross-linked chromatin model does not significantly affect the calculated nanodosimetric parameters. This allows for a simulation-based cell model to make use of any of these chromatin models for the scoring of direct ion-induced DNA damage.

  11. Insights into the formation of inorganic heterocycles via cyclocondensation of primary amines with group 15 and 16 halides.

    PubMed

    Chivers, Tristram; Laitinen, Risto S

    2017-01-31

    Cyclocondensation is a major preparative route for the generation of inorganic heterocycles especially in the case of ring systems involving a Group 15 or 16 element linked to nitrogen. This Perspective will consider recent experimental and computational studies involving the reactions of primary amines (or their synthetic equivalents) with pnictogen and chalcogen halides. The major focus will be a discussion of the identity and role of acyclic intermediates in the reaction pathways to ring formation, as well as the nature of the heterocycles so formed. The similarities and differences between the chemistry of group 15 and 16 systems are emphasised with a view to providing signposts for further investigations.

  12. Metabolic polymorphisms and carcinogen-DNA adduct formation in human populations.

    PubMed

    Kaderlik, K R; Kadlubar, F F

    1995-01-01

    Metabolic polymorphisms have long been recognized as important determinants of carcinogen susceptibility and recent efforts have shown that interindividual differences in specific cytochromes P450, acetyltransferases, sulfotransferases and glutathione S-transferases are often disproportionately represented in epidemiological studies between cancer cases and controls. Concomitantly, biomonitoring of carcinogen-DNA adducts in human tissues using immunochemical, 32P-postlabelling, fluorescence, and mass spectrometric methods have recently provided direct evidence of human exposure to genotoxic aromatic and heterocyclic amines, polycyclic hydrocarbons, alkylating agents, and endogenous products. However, a combined approach is now needed in order to assess the relevance of these findings to cancer etiology, to identify high-risk individuals, and to provide better health monitoring, earlier diagnosis, and cancer prevention. Using this paradigm, results are presented that implicate specific aromatic amines, heterocyclic amines, and polycyclic aromatic hydrocarbons in the etiology of human urinary bladder, colon, and laryngeal cancers.

  13. The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.

    PubMed

    Strand, Janne M; Scheffler, Katja; Bjørås, Magnar; Eide, Lars

    2014-06-01

    The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. DNA entropy reveals a significant difference in complexity between housekeeping and tissue specific gene promoters.

    PubMed

    Thomas, David; Finan, Chris; Newport, Melanie J; Jones, Susan

    2015-10-01

    The complexity of DNA can be quantified using estimates of entropy. Variation in DNA complexity is expected between the promoters of genes with different transcriptional mechanisms; namely housekeeping (HK) and tissue specific (TS). The former are transcribed constitutively to maintain general cellular functions, and the latter are transcribed in restricted tissue and cells types for specific molecular events. It is known that promoter features in the human genome are related to tissue specificity, but this has been difficult to quantify on a genomic scale. If entropy effectively quantifies DNA complexity, calculating the entropies of HK and TS gene promoters as profiles may reveal significant differences. Entropy profiles were calculated for a total dataset of 12,003 human gene promoters and for 501 housekeeping (HK) and 587 tissue specific (TS) human gene promoters. The mean profiles show the TS promoters have a significantly lower entropy (p<2.2e-16) than HK gene promoters. The entropy distributions for the 3 datasets show that promoter entropies could be used to identify novel HK genes. Functional features comprise DNA sequence patterns that are non-random and hence they have lower entropies. The lower entropy of TS gene promoters can be explained by a higher density of positive and negative regulatory elements, required for genes with complex spatial and temporary expression. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. [Spectral manifestations of different types of acriflavine binding to DNA in ultraviolet and visible region].

    PubMed

    Zhadin, N N; Rapoport, V L

    1975-01-01

    Difference absorption spectra (complex-sum of the initial reagents) are obtained in the visible and longwave UV region for the system of actiflavine and DNA in a number of cases differing in initial and final degrees of DNA filling by the dye, in particular separately for two types of dye binding to DNA. For these binding types conventional absorption spectra are calculated. In the visible region for the first binding type ("strong" binding) red shift of the absorption band is observed; for the second type ("weak" binding) we observed splitting of the band, short wavelength component being highly prevailing, and hypochromism. In the UV region for both binding types the spectra changed in approximately similar way; a slight blue shift and a rather remarkable hypochromism are observed. It is shown that the dye brings the main contribution into the spectral changes in the UV region. If to take into account the spectral properties of molecular aggregates the data obtained are compatible with the intercalation model for "strong" binding and dye stacking on DNA for "weak" binding.

  16. Stereoselectively fluorinated N-heterocycles: a brief survey.

    PubMed

    Hu, Xiang-Guo; Hunter, Luke

    2013-11-29

    The stereoselective incorporation of fluorine atoms into N-heterocycles can lead to dramatic changes in the molecules' physical and chemical properties. These changes can be rationally exploited for the benefit of diverse fields such as medicinal chemistry and organocatalysis. This brief review will examine some of the effects that fluorine substitution can have in N-heterocycles, including changes to the molecules' stability, their conformational behaviour, their hydrogen bonding ability, and their basicity. Finally, some methods for the synthesis of stereoselectively fluorinated N-heterocycles will also be reviewed.

  17. Biosynthesis of oxygen and nitrogen-containing heterocycles in polyketides.

    PubMed

    Hemmerling, Franziska; Hahn, Frank

    2016-01-01

    This review highlights the biosynthesis of heterocycles in polyketide natural products with a focus on oxygen and nitrogen-containing heterocycles with ring sizes between 3 and 6 atoms. Heterocycles are abundant structural elements of natural products from all classes and they often contribute significantly to their biological activity. Progress in recent years has led to a much better understanding of their biosynthesis. In this context, plenty of novel enzymology has been discovered, suggesting that these pathways are an attractive target for future studies.

  18. Biosynthesis of oxygen and nitrogen-containing heterocycles in polyketides

    PubMed Central

    Hemmerling, Franziska

    2016-01-01

    Summary This review highlights the biosynthesis of heterocycles in polyketide natural products with a focus on oxygen and nitrogen-containing heterocycles with ring sizes between 3 and 6 atoms. Heterocycles are abundant structural elements of natural products from all classes and they often contribute significantly to their biological activity. Progress in recent years has led to a much better understanding of their biosynthesis. In this context, plenty of novel enzymology has been discovered, suggesting that these pathways are an attractive target for future studies. PMID:27559404

  19. Biocatalysts for the formation of three- to six-membered carbo- and heterocycles.

    PubMed

    Lechner, Horst; Pressnitz, Desiree; Kroutil, Wolfgang

    2015-01-01

    During the last decade, the number of different types of enzymes applicable for organic synthesis as biocatalysts has significantly increased. Consequently, the spectrum of reactions has significantly expanded also for cyclisations. This review highlights heterologously expressable biocatalysts transforming non-natural substrates for the formation of three- to six-membered carbo- and heterocycles, excluding terpene cyclases as well as SAM-dependent enzymes. The review focuses on the non-natural substrate scope and the mechanism of the selected enzymes.

  20. Detection of Histomonas meleagridis DNA in different organs after natural and experimental infections of meat turkeys.

    PubMed

    Hauck, R; Lüschow, D; Hafez, H M

    2006-03-01

    Histomonas meleagridis infection of turkeys is usually accompanied by a severe disease with unspecific clinical symptoms but with distinct pathological lesions in the ceca and liver. In the literature some macro- and microscopic evidence of the spread of histomonads to the other organs has been provided. The aim of the present investigations was to use real-time polymerase chain reaction (PCR) to demonstrate the dissemination of H. meleagridis DNA to different organs after natural and experimental infection of meat turkeys. Samples from several organs were collected from a meat-turkey flock, which proved to be naturally infected with histomoniasis, and examined for histomonad DNA by real-time PCR. Histomonad DNA was detected in all investigated ceca, livers, spleens, kidneys, and pooled brain swabs. Additionally it was found in 75% of investigated samples from bursae of Fabricius, in 50% of investigated duodenums, and in 40% of investigated jejunum samples. After experimental intracloacal infection of 3-wk-old turkey poults with 147,500 histomonads, similar samples were collected from all turkeys that died. After a 3-wk observation period the surviving birds, as well as the noninfected control group, were euthanatized and samples were taken. During the entire experimental period, 10 birds out the 20 infected birds died. Histomonad DNA was detected in all investigated ceca, livers, lungs, and hearts (100%) and almost all kidneys (90%) and bursae of Fabricius (80%). On the other hand, only 30% of examined spleens and 10% of brain samples revealed positive results. Surviving infected birds were euthanatized and necropsied; histomonad DNA was found in one out of 10 livers but not in any ceca. Also, histomonad DNA could not be detected in examined cecal and lung samples from the noninfected control group.

  1. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.

    PubMed

    Pavšič, Jernej; Devonshire, Alison; Blejec, Andrej; Foy, Carole A; Van Heuverswyn, Fran; Jones, Gerwyn M; Schimmel, Heinz; Žel, Jana; Huggett, Jim F; Redshaw, Nicholas; Karczmarczyk, Maria; Mozioğlu, Erkan; Akyürek, Sema; Akgöz, Müslüm; Milavec, Mojca

    2017-04-01

    Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

  2. Characteristic differences in the X-ray photoelectron spectrum between B-DNA and M-DNA monolayers on gold.

    PubMed

    Dinsmore, Michael J; Lee, Jeremy S

    2008-08-01

    Duplex DNA monolayers were self-assembled on gold through a disulfide linkage and both B- and M-DNA conformations were studied using X-ray photoelectron spectroscopy (XPS). The film thickness, density, elemental composition and ratios for samples were analyzed and compared. The DNA surface coverage, calculated from both XPS and electrochemical measurements, was approximately 1.2 x 10(13)molecules/cm(2) for B-DNA. All samples showed distinct peaks for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA. On addition of Zn(2+) to form M-DNA the C 1s, P 2p and S 2p showed only small changes while both the N 1s and O 1s spectra changed considerably. This result is consistent with Zn(2+) interacting with oxygen on the phosphate backbone as well as replacing the imino protons of thymine (T) and guanine (G) in M-DNA. Analysis of the Zn 2p spectra also demonstrated that the concentration of Zn(2+) present under M-DNA conditions is consistent with Zn(2+) binding to both the phosphate backbone as well as replacing the imino protons of T or G in each base pair. After the M-DNA monolayer is washed with a buffer containing only Na(+) the Zn(2+) bound to the phosphate backbone is removed while the Zn(2+) bound internally still remains.

  3. Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform.

    PubMed

    Ward, Matthew S; Silva, Isba; Martinez, Walfre; Jefferson, Jameka; Rahman, Shakila; Garcia, Jeanie M; Kanichar, Divya; Roppiyakuda, Lance; Kosmowska, Ewa; Faust, Michelle A; Tran, Kim P; Chow, Felicia; Buglo, Elena; Zhou, Feimeng; Groziak, Michael P; Xu, H Howard

    2016-08-01

    One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collection of cell clones in an Escherichia coli mutant (AS19) host with increased outer membrane permeability. Five known antibiotics/inhibitors and 28 boron heterocycles were tested by TIPA II assay, in conjunction with the original assay TIPA. The TIPA II was more sensitive than TIPA because eight boron heterocycles previously found to be inactive to AG1 cells in TIPA assays exhibited activity to AS19 cells. For 15 boron heterocycles, resistant colonies were observed within the zones of inhibition only on the inducing plates in TIPA II assays. DNA sequencing confirmed that resistant clones harbor plasmids with fabI gene as insert, indicating that these boron heterocycles all target enoyl ACP reductase. Additionally, cell-based assays and dose response curved obtained indicated that for two boron heterocycle inhibitors, the fabI cell clone in AG1 (wild-type) host cells exhibited at least 11 fold more resistance under induced conditions than under non-induced conditions. Moreover, TIPA II also identified cellular targets of known antibacterial inhibitors triclosan, phosphomycin, trimethoprim, diazaborine and thiolactomycin, further validating the utility of the new system.

  4. 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)

  5. 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)

  6. DNA analysis of molluscs from a museum wet collection: a comparison of different extraction methods.

    PubMed

    Jaksch, Katharina; Eschner, Anita; Rintelen, Thomas V; Haring, Elisabeth

    2016-07-18

    DNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S). Overall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did. Museum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The

  7. Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

    PubMed

    Speit, Günter; Schütz, Petra; Bausinger, Julia

    2016-06-01

    The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.

  8. Lability to acid hydrolysis in some different DNA-protein complexes of spermatozoa.

    PubMed

    Silva, M J; Mello, M L

    1986-01-01

    The Feulgen hydrolysis kinetics was investigated in spermatozoa with different composition in DNA-protein complexes. The species used were: Bos taurus (arginine rich nuclear protein also containing cystine residues), Pichroplus bergi, Triatoma infestans (arginine-rich nuclear protein), Lytechinus variegatus and Apis mellifera (lysine-rich nuclear protein). The spermatozoa were subjected to Feulgen's reaction, after varying the fixative type and the hydrolysis times. Feulgen-DNA values were obtained with an automatic scanning cytophotometric procedure. Differences were demonstrated in the hydrolysis kinetics as a function of differences in composition of the DNA-protein complexes being present in the spermatozoon chromatin. Differences in the profiles of Feulgen hydrolysis curves, having for basis the fixation, were rather clear for bull, grasshopper, and blood-sucking insect spermatozoa than for the sea-urchin and bee spermatozoa. The different hydrolysis kinetics of chromatin of blood-sucking insect spermatozoa compared to that of grasshopper, sea-urchin, and bee sperm cells suggests that, although the first 2 materials contain an arginine-rich "germinative" protein and the latter 2 ones contain a lysine-rich protein, these differ to each other. The DNA depurination was obtained more quickly for T. infestans (20 min) and P. bergi (10 min) spermatozoa when they were fixed in the ethanol-acetic acid (EA) mixture. Morphologically anomalous bull spermatozoa fixed in the EA mixture presented a quicker depurination (30 min) as compared to the normal cells (1 h). The fast lability to acid hydrolysis in the abnormal cells is certainly due to anomalies in their basic nuclear "germinative" protein. In the formalin fixed materials the maximal depurination was obtained earlier in bull spermatozoa (30 min) followed by sperm cells of P. bergi, T. infestans, L. variegatus (all of them one-hour hydrolysis) and finally Apis mellifera (2 h hydrolysis). The presence of secondary

  9. 1,3-Dien-5-ynes: Versatile Building Blocks for the Synthesis of Carbo- and Heterocycles.

    PubMed

    Aguilar, Enrique; Sanz, Roberto; Fernández-Rodríguez, Manuel A; García-García, Patricia

    2016-07-27

    1,3-Dien-5-ynes have been extensively used as starting materials for the synthesis of a wide number of different carbo- and heterocycles. The aim of this review is to give an overview of their utility in organic synthesis, highlighting the variety of compounds that can be directly accessed from single reactions over these systems. Thus, cycloaromatization processes are initially commented, followed by reactions directed toward the syntheses of five-membered rings, other carbocycles and, finally, heterocycles. The diverse methodologies that have been developed for the synthesis of each of these types of compounds from 1,3-dien-5-ynes are presented, emphasizing the influence of the reaction conditions and the use of additional reagents in the outcome of the transformations.

  10. Fluorous Mixture Synthesis of Two Libraries with Novel Hydantoin- and Benzodiazepinedione-Fused Heterocyclic Scaffolds

    PubMed Central

    Zhang, Wei; Lu, Yimin; Chen, Christine Hiu-Tung; Zeng, Lu; Kassel, Daniel B.

    2007-01-01

    Diversity-oriented synthesis (DOS) and fluorous mixture synthesis (FMS) are two aspects of combinatorial chemistry. DOS generates library scaffolds with skeletal, substitution, and stereochemistry variations, whereas FMS is a highly efficient tool for library production. The combination of these two aspects in solution-phase synthesis of two novel heterocyclic compound libraries is presented in this paper. Mixtures of different fluorous amino acids undergo [3+2] cycloadditions followed by post-condensation reactions. The mixtures are then demixed by fluorous HPLC. Fluorous tags are removed by cyclization to afford hydantoin- and benzodiazepinedione-fused heterocyclic compounds as individual, pure and structurally defined molecules. The application of MS-directed HPLC and parallel four-channel LC/MS analysis further increases the efficiency of FMS. PMID:16961407

  11. Physicochemical and Nonlinear Optical Properties of Novel Environmentally Benign Heterocyclic Azomethine Dyes: Experimental and Theoretical Studies

    PubMed Central

    Afzal, S. M.; Razvi, M. A. N.; Khan, Salman A.; Osman, Osman I.; Bakry, Ahmed H.; Asiri, Abdullah M.

    2016-01-01

    Novel heterocyclic azomethine dyes were prepared by the reaction of anthracene-9-carbaldehyde with different heterocyclic amines under microwave irradiation. Structures of the azomethine dyes were confirmed by the elemental analysis, mass spectrometry and several spectroscopic techniques. We studied absorbance and fluorescence spectra of the azomethine dyes in various solvents. They are found to be good absorbers and emitters. We also report photophysical properties like, extinction coefficient, oscillator strength, stokes shift and transition dipole moment. This reflects physicochemical behaviors of synthesized dyes. In addition, their intramolecular charge transfer and nonlinear optical properties, supported by natural bond orbital technique, were also studied computationally by density functional theory. The negative nonlinear refractive index and nonlinear absorption coefficient were measured for these dyes using the closed and open aperture Z-scan technique with a continuous wave helium-neon laser. These are found to vary linearly with solution concentration. PMID:27631371

  12. [Synthesis and biological evaluation of novel pleuromutilin derivatives with nitrogen-containing heterocycles as antibacterial agents].

    PubMed

    Wang, Xin-yang; Chen, Min; Wang, Duo; Chen, Xiang-dong; Ling, Yong; Wang, Xiao-li; Wang, Hui

    2015-10-01

    A series of new pleuromutilins derivatives were designed and synthesized through coupling 2-aminothiazole ring of WL001 with different nitrogen-containing substituted heterocycles in the side chain. Their biological activities were evaluated against both Gram-positive and Gram-negative clinical bacteria in vitro Most new compounds displayed specificity to certain strain of bacteria. Particularly, compounds with saturated nitrogen-containing heterocycles exhibited significant antibacterial activities (0.062 5-8 µg · mL(-1)) superior or similar to those of amoxicillin, tiamulin and levofloxcin. Furthermore, treatment with 15a and 15b having piperidine or morpholine residues also could effectively inhibit Gram-negative bacteria. Therefore, our novel findings may provide a new insight into the design of novel pleuromutilin derivatives and lay the basis for further studies on the treatment of drug-resistance of pathogenic bacteria.

  13. Physicochemical and Nonlinear Optical Properties of Novel Environmentally Benign Heterocyclic Azomethine Dyes: Experimental and Theoretical Studies.

    PubMed

    Afzal, S M; Razvi, M A N; Khan, Salman A; Osman, Osman I; Bakry, Ahmed H; Asiri, Abdullah M

    2016-01-01

    Novel heterocyclic azomethine dyes were prepared by the reaction of anthracene-9-carbaldehyde with different heterocyclic amines under microwave irradiation. Structures of the azomethine dyes were confirmed by the elemental analysis, mass spectrometry and several spectroscopic techniques. We studied absorbance and fluorescence spectra of the azomethine dyes in various solvents. They are found to be good absorbers and emitters. We also report photophysical properties like, extinction coefficient, oscillator strength, stokes shift and transition dipole moment. This reflects physicochemical behaviors of synthesized dyes. In addition, their intramolecular charge transfer and nonlinear optical properties, supported by natural bond orbital technique, were also studied computationally by density functional theory. The negative nonlinear refractive index and nonlinear absorption coefficient were measured for these dyes using the closed and open aperture Z-scan technique with a continuous wave helium-neon laser. These are found to vary linearly with solution concentration.

  14. Sex and strain differences in the hepatocyte primary culture/DNA repair test

    SciTech Connect

    McQueen, C.A.; Way, B.M. )

    1991-01-01

    The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B{sub 1} (AFB{sub 1}). No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

  15. Extreme difference in rate of mitochondrial and nuclear DNA evolution in a large ectotherm, Galápagos tortoises.

    PubMed

    Caccone, Adalgisa; Gentile, Gabriele; Burns, Catherine E; Sezzi, Erminia; Bergman, Windsong; Ruelle, Morgan; Saltonstall, Kristin; Powell, Jeffrey R

    2004-05-01

    We sequenced approximately 4.5 kb of mtDNA from 161 individuals representing 11 named taxa of giant Galápagos tortoises (Geochelone nigra) and about 4 kb of non-coding nuclear DNA from fewer individuals of these same 11 taxa. In comparing mtDNA and nucDNA divergences, only silent substitutions (introns, ITS, mtDNA control region, and synonymous substitutions in coding sequences) were considered. mtDNA divergence was about 30 times greater than that for nucDNA. This rate discrepancy for mtDNA and nucDNA is the greatest yet documented and is particularly surprising for large ectothermic animals that are thought to have relatively low rates of mtDNA evolution. This observation may be due to the somewhat unusual reproductive biology and biogeographic history of these organisms. The implication is that the ratio of effective population size of nucDNA/mtDNA is much greater than the usually assumed four. The nearly neutral theory of molecular evolution predicts this would lead to a greater difference between rates of evolution.

  16. Intragenic DNA methylation status down-regulates bovine IGF2 gene expression in different developmental stages.

    PubMed

    Huang, Yong-Zhen; Zhan, Zhao-Yang; Sun, Yu-Jia; Cao, Xiu-Kai; Li, Ming-Xun; Wang, Jing; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Chen, Hong

    2014-01-25

    DNA methylation is a key epigenetic modification in mammals and has an essential and important role in muscle development. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to evaluate the expression of IGF2 and the methylation pattern on the differentially methylated region (DMR) of the last exon of IGF2 in six tissues with two different developmental stages. The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The quantitative real-time PCR (qPCR) analysis indicated that IGF2 has a broad tissue distribution and the adult bovine group showed significant lower mRNA expression levels than that in the fetal bovine group (P<0.05 or P<0.01). Moreover, the DNA methylation level analysis showed that the adult bovine group exhibited a significantly higher DNA methylation levels than that in the fetal bovine group (P<0.05 or P<0.01). These results indicate that IGF2 expression levels were negatively associated with the methylation status of the IGF2 DMR during the two developmental stages. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a marker-assisted selection for muscle developmental in beef cattle breeding program and as a model for studies in other species.

  17. Impact of different running distances on muscle and lymphocyte DNA damage in amateur marathon runners.

    PubMed

    Ryu, Jae Hoon; Paik, Il Young; Woo, Jin Hee; Shin, Ki Ok; Cho, Su Youn; Roh, Hee Tae

    2016-01-01

    [Purpose] The aim of this study was to investigate the impact of different marathon running distances (10 km, 21 km, and 42.195 km) on muscle and lymphocyte DNA damage in amateur marathon runners. [Subjects and Methods] Thirty male amateur runners were randomly assigned to 10 km, 21 km, and 42 km groups, with 10 subjects in each group. Blood samples were collected before and after the races and on the 3rd day of recovery to examine levels of muscle damage (creatine kinase and lactate dehydrogenase) and lymphocyte DNA damage (DNA in the tail, tail length, and tail moment). [Results] Serum creatine kinase, serum lactate dehydrogenase, and tail moment were significantly higher after the races compared with before the races in all groups. In addition, the 42 km group showed significantly higher levels of creatine kinase, lactate dehydrogenase, and tail moment than the 10 km and 21 km groups after the races. [Conclusion] Strenuous endurance exercise can cause muscle and lymphocyte DNA damage, and the extent of such damage can increase as running distance increases.

  18. Theory of phase segregation in DNA assemblies containing two different base-pair sequence types

    NASA Astrophysics Data System (ADS)

    (O’ Lee, Dominic J.; Wynveen, Aaron; Kornyshev, Alexei A.

    2017-01-01

    Spontaneous pairing of homologous DNA sequences—a challenging subject in molecular biophysics, often referred to as ‘homology recognition’—has been observed in vitro for several DNA systems. One of these experiments involved liquid crystalline quasi-columnar phases formed by a mixture of two kinds of double stranded DNA oligomer. Both oligomer types were of the same length and identical stoichiometric base-pair composition, but the base-pairs followed a different order. Phase segregation of the two DNA types was observed in the experiments, with the formation of boundaries between domains rich in molecules of one type (order) of base pair sequence. We formulate here a modified ‘X–Y model’ for phase segregation in such assemblies, obtain approximate solutions of the model, compare analytical results to Monte Carlo simulations, and rationalise past experimental observations. This study, furthermore, reveals the factors that affect the degree of segregation. Such information could be used in planning new versions of similar segregation experiments, needed for deepening our understanding of forces that might be involved, e.g., in gene–gene recognition.

  19. Neuronal DNA content variation (DCV) with regional and individual differences in the human brain

    PubMed Central

    Westra, Jurjen W.; Rivera, Richard R.; Bushman, Diane M.; Yung, Yun C.; Peterson, Suzanne E.; Barral, Serena; Chun, Jerold

    2010-01-01

    It is widely assumed that the human brain contains genetically identical cells through which post-genomic mechanisms contribute to its enormous diversity and complexity. The relatively recent identification of neural cells throughout the neuraxis showing somatically generated mosaic aneuploidy indicates that the vertebrate brain can be genomically heterogeneous (Rehen et al., 2001; Rehen et al., 2005; Westra et al., 2008; Yurov et al., 2007). The extent of human neural aneuploidy is currently unknown because of technically limited sample sizes, but is reported to be small (Iourov et al., 2006). During efforts to interrogate larger cell populations using DNA content analyses, a surprising result was obtained: human frontal cortex brain cells were found to display “DNA content variation (DCV)” characterized by an increased range of DNA content both in cell populations and within single cells. On average, DNA content increased by ~250 megabases often representing a substantial fraction of cells within a given sample. DCV within individual human brains showed regional variation, with increased prevalence in the frontal cortex and less variation in the cerebellum. Further, DCV varied between individual brains. These results identify DCV as a new feature of the human brain, encompassing and further extending genomic alterations produced by aneuploidy, which may contribute to neural diversity in normal and pathophysiological states, altered functions of normal and disease-linked genes, and differences amongst individuals. PMID:20737596

  20. Cocaine induces DNA damage in distinct brain areas of female rats under different hormonal conditions.

    PubMed

    de Souza, Marilise F; Gonçales, Tierre A; Steinmetz, Aline; Moura, Dinara J; Saffi, Jenifer; Gomez, Rosane; Barros, Helena M T

    2014-04-01

    We evaluated levels of neuronal DNA damage after acute or repeated cocaine treatment in different brain areas of female rats after ovariectomy or sham surgery. Rats in the control and acute groups were given saline i.p., whereas in the repeated group were given 15 mg/kg, i.p., cocaine for 8 days. After a 10 day washout period, the control group was given saline i.p., whereas rats in the acute and repeated groups were given a challenge dose of 15 mg/kg, i.p., cocaine. After behavioural assessment, rats were killed and the cerebellum, hippocampus, hypothalamus, prefrontal cortex and striatum were dissected for the Comet assay. Acute cocaine exposure induced DNA damage in all brain areas. This effect persisted after repeated administration, except in the hypothalamus, where repeated treatment did not cause increased DNA damage. Sexual hormones exhibited a neuroprotective effect, decreasing cocaine-induced DNA damage in cycling rats in all brain areas. © 2014 Wiley Publishing Asia Pty Ltd.

  1. DNA Repair and Cytokines: TGF-β, IL-6, and Thrombopoietin as Different Biomarkers of Radioresistance

    PubMed Central

    Centurione, Lucia; Aiello, Francesca B.

    2016-01-01

    Double strand breaks (DSBs) induced by radiotherapy are highly cytotoxic lesions, leading to chromosomal aberrations and cell death. Ataxia-telangiectasia-mutated (ATM)-dependent DNA-damage response, non-homologous end joining, and homologous recombination pathways coordinately contribute to repairing DSBs in higher eukaryotes. It is known that the expression of DSB repair genes is increased in tumors, which is one of the main reasons for radioresistance. The inhibition of DSB repair pathways may be useful to increase tumor cell radiosensitivity and may target stem cell-like cancer cells, known to be the most radioresistant tumor components. Commonly overexpressed in neoplastic cells, cytokines confer radioresistance by promoting proliferation, survival, invasion, and angiogenesis. Unfortunately, tumor irradiation increases the expression of various cytokines displaying these effects, including transforming growth factor-beta and interleukin-6. Recently, the capabilities of these cytokines to support DNA repair pathways and the ATM-dependent DNA response have been demonstrated. Thrombopoietin, essential for megakaryopoiesis and very important for hematopoietic stem cell (HSC) homeostasis, has also been found to promote DNA repair in a highly selective manner. These findings reveal a novel mechanism underlying cytokine-related radioresistance, which may be clinically relevant. Therapies targeting specific cytokines may be used to improve radiosensitivity. Specific inhibitors may be chosen in consideration of different tumor microenvironments. Thrombopoietin may be useful in fending off irradiation-induced loss of HSCs. PMID:27500125

  2. Comparison of different methods for the recovery of DNA from spores of mycotoxin-producing moulds in spiked food samples.

    PubMed

    Grube, S; Schönling, J; Prange, A

    2015-06-01

    Several food samples were spiked with fungal conidia to test the efficiency of different cell disruption methods and DNA extraction kits for subsequent molecular detection. For disrupting the firm cell walls of the spores, two different pretreatment methods, namely sonication and bead beating, were tested against no pretreatment. The subsequent DNA extraction and purification was performed using three different DNA extraction methods, which are based on a diverse combination of extraction principles, such as precipitation, thermic-enzymatic lysis, pH-enhancement and bonding with a silica membrane. The aim of the study was to find out the suitable pretreatment and DNA extraction method for the recovery of detectable amounts of fungal DNA from different food matrices. Significance and impact of the study: The choice of 'ready-to-use' commercial kits and methods has been of great importance regarding the recovery of extracted DNA. However, these commercially available kits are neither effective nor time-efficient when extracting DNA from fungal spores embedded in complex food matrices. Different extraction principles were compared and their effectiveness tested using real-time PCR. The combination of different principles for the extraction and purification of DNA was found as the most efficient method (quantity and purity) to obtain DNA from moulds and their spores from food samples. © 2015 The Society for Applied Microbiology.

  3. Intraspecific DNA Content Variability in Festuca pallens on Different Geographical Scales and Ploidy Levels

    PubMed Central

    ŠMARDA, PETR; BUREŠ, PETR

    2006-01-01

    • Background and Aims Intraspecific genome size variability of Festuca pallens occurring on relict rocky steppes in Central Europe was studied on two ploidy levels and three geographical scales: (1) local scale of 24 populations, (2) landscape scale of three transects in river canyons or hill systems, and (3) global scale of 160 samples covering the whole distribution area. • Methods DAPI flow cytometry of homogeneously cultivated samples (≥1 year), measured randomly with two internal standards, Lycopersicon esculentum and Pisum sativum. Differences in DNA content were confirmed (1) by the double peaks of simultaneously measured samples, (2) based on measurements carried out in different seasons, and (3) by additional measurements with propidium iodide. • Key Results On a global scale, the relative DNA content ranged between 1·170-fold in diploids and 1·164-fold in tetraploids. A maximum difference of 1·088-fold between the mean relative DNA content of nearby populations was found. In 16 of 24 populations significant variability was shown (P < 0·001, 1·121-fold as maximum). For both ploidy levels, the relative genome size had the same range and geographical pattern, correlated with geographical coordinates (P < 0·01). Diploids with larger genomes occur on relict habitats (P < 0·01), and in areas of periglacial steppes (20 000 years ago; P < 0·02). In tetraploids, the relative DNA content differs among the three previously recognized geographical types (Alpine, Pannonian and Scabrifolia, P < 0·001). Tetraploids have a relative DNA content smaller than twice that of the diploids (P < 0·001). An influence of microhabitat on DNA content variation was not confirmed. • Conclusions Genome size variability occurs over all spatial scales: intrapopulation, landscape and global. Correlation between geographical coordinates and palaeovegetation type, concomitant with diploids and tetraploids, and no influence of microhabitat were found. Genome size

  4. Calculation of direct effects of 60Co gamma rays on the different DNA structural levels: A simulation study using the Geant4-DNA toolkit

    NASA Astrophysics Data System (ADS)

    Tajik, Marjan; Rozatian, Amir S. H.; Semsarha, Farid

    2015-03-01

    In this study, simple single strand breaks (SSB) and double strand breaks (DSB) due to direct effects of the secondary electron spectrum of 60Co gamma rays on different organizational levels of a volume model of the B-DNA conformation have been calculated using the Geant4-DNA toolkit. Result of this study for the direct DSB yield shows a good agreement with other theoretical and experimental results obtained by both photons and their secondary electrons; however, in the case of SSB a noticeable difference can be observed. Moreover, regarding the almost constant yields of the direct strand breaks in the different structural levels of the DNA, calculated in this work, and compared with some theoretical studies, it can be deduced that the direct strand breaks yields depend mainly on the primary double helix structure of the DNA and the higher-order structures cannot have a noticeable effect on the direct DNA damage inductions by 60Co gamma rays. In contrast, a direct dependency between the direct SSB and DSB yields and the volume of the DNA structure has been found. Also, a further study on the histone proteins showed that they can play an important role in the trapping of low energy electrons without any significant effect on the direct DNA strand breaks inductions, at least in the range of energies used in the current study.

  5. Serine-based gemini surfactants with different spacer linkages: from self-assembly to DNA compaction.

    PubMed

    Silva, Sandra G; Oliveira, Isabel S; do Vale, M Luísa C; Marques, Eduardo F

    2014-12-14

    Cationic gemini surfactants have strong potential as compaction agents of nucleic acids for efficient non-viral gene delivery. In this work, we present the aggregation behavior of three novel cationic serine-based gemini surfactants as well as their ability to compact DNA per se and mixed with a helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). All the surfactants have a 12-12-12 configuration, i.e. two main 12-carbon alkyl chains linked to the nitrogen atom of the amino acid residue and a 12 methylene spacer, but they differ in the nature of the spacer linkage: for (12Ser)2N12, an amine bond; for (12Ser)2CON12, an amide bond; and for (12Ser)2COO12, an ester bond. Interestingly, while the amine-based gemini aggregates into micelles, the amide and ester ones spontaneously form vesicles, which denotes a strong influence of the type of linkage on the surfactant packing parameter. The size, ζ-potential and stability of the vesicles have been characterized by light microscopy, cryogenic scanning electron microscopy (cryo-SEM) and dynamic light scattering (DLS). The interaction of the gemini aggregates with DNA at different charge ratios and in the absence and presence of DOPE has been studied by DLS, fluorescence spectroscopy and cryo-SEM. All the compounds are found to efficiently compact DNA (complexation > 90%), but relevant differences are obtained in terms of the size, ζ-potential and stability of the lipoplexes formed. Results are rationalized in terms of headgroup differences and the type of aggregates present prior to DNA condensation.

  6. Heterocycles in Peptidomimetics and Pseudopeptides: Design and Synthesis †

    PubMed Central

    Cerminara, Iole; Chiummiento, Lucia; Funicello, Maria; Guarnaccio, Ambra; Lupattelli, Paolo

    2012-01-01

    This minireview provides a brief outline of the peculiar aspects of the preparation of peptidomimetic and pseudopeptidic structures containing heterocycles. In particular novel tricyclic structures are investigated as potential drugs. PMID:24281380

  7. SPECTROSCOPIC STUDY OF SORPTION OF NITROGEN HETEROCYCLIC COMPOUNDS ON PHYLLOSILICATES

    EPA Science Inventory

    The present study focused on understanding the sorption characteristics of acridine (AcN)and acridine-9-carboxylic acid (AcNCOOH), two typical nitrogen heterocyclic compounds (NHCs), on well-characterized phyllosilicates (hectorite, saponite, and muscovite). Results presented in...

  8. Evaluation of Different DNA Vaccines against Porcine Reproductive and Respiratory Syndrome (PRRS) in Pigs

    PubMed Central

    Petrini, Stefano; Ramadori, Giorgio; Villa, Riccardo; Borghetti, Paolo; de Angelis, Elena; Cantoni, Anna Maria; Corradi, Attilio; Amici, Augusto; Ferrari, Maura

    2013-01-01

    In veterinary medicine, there have been different experiences with the plasmid DNA vaccination. In this area and with the hypothesis to demonstrate the effectiveness of different plasmids encoding porcine respiratory and reproductive syndrome (PRRS), five DNA vaccines against PRRS were evaluated for their innocuity and efficacy in pigs. Eighteen animals were divided into five groups which were injected with five (A, B, C, D, E) different DNA vaccines. Albeit, none of the proposed vaccines were able to protect the animals against PRRS virus. Only vaccines A and B were able to reduce the clinical signs of the infection. ELISA IgM were detected 30 days after the first vaccination in the pigs injected by Vaccine A or B. ELISA IgG were detected 90 days after the first vaccination in the pigs injected by Vaccine B or C. Neutralizing antibody were detected Post Challenge Days 61 (PCD) in all groups. In the pigs inoculated with Vaccine C, IFN-γ were detected 90 days after first vaccination, and after challenge exposure they increased. In the other groups, the IFN-γ were detected after challenge infection. Pigs injected with each of the vaccines A, B, C, D and E showed a significantly higher level of CD4−CD8+ lymphocytes (p < 0.001) after infection in comparison with their controls. PMID:26344342

  9. Toward Multiple Conductance Pathways with Heterocycle-Based Oligo(phenyleneethynylene) Derivatives.

    PubMed

    Miguel, Delia; Álvarez de Cienfuegos, Luis; Martín-Lasanta, Ana; Morcillo, Sara P; Zotti, Linda A; Leary, Edmund; Bürkle, Marius; Asai, Yoshihiro; Jurado, Rocío; Cárdenas, Diego J; Rubio-Bollinger, Gabino; Agraït, Nicolás; Cuerva, Juan M; González, M Teresa

    2015-11-04

    In this paper, we have systematically studied how the replacement of a benzene ring by a heterocyclic compound in oligo(phenyleneethynylene) (OPE) derivatives affects the conductance of a molecular wire using the scanning tunneling microscope-based break junction technique. We describe for the first time how OPE derivatives with a central pyrimidine ring can efficiently link to the gold electrode by two pathways presenting two different conductance G values. We have demonstrated that this effect is associated with the presence of two efficient conductive pathways of different length: the conventional end-to-end configuration, and another with one of the electrodes linked directly to the central ring. This represents one of the few examples in which two defined conductive states can be set up in a single molecule without the aid of an external stimulus. Moreover, we have observed that the conductance through the full length of the heterocycle-based OPEs is basically unaffected by the presence of the heterocycle. All these results and the simplicity of the proposed molecules push forward the development of compounds with multiple conductance pathways, which would be a breakthrough in the field of molecular electronics.

  10. Relaxase DNA Binding and Cleavage Are Two Distinguishable Steps in Conjugative DNA Processing That Involve Different Sequence Elements of the nic Site*

    PubMed Central

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-01-01

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR2). Mutation analysis in the nic region indicated that recognition of the IR2 proximal arm and the nucleotides located between IR2 and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR2 cruciform and recognition of the distal IR2 arm and loop were not necessary for these reactions to take place. On the other hand, IR2 was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR2-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place. PMID:20061574

  11. Relaxase DNA binding and cleavage are two distinguishable steps in conjugative DNA processing that involve different sequence elements of the nic site.

    PubMed

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-03-19

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR(2)). Mutation analysis in the nic region indicated that recognition of the IR(2) proximal arm and the nucleotides located between IR(2) and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR(2) cruciform and recognition of the distal IR(2) arm and loop were not necessary for these reactions to take place. On the other hand, IR(2) was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR(2)-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

  12. Investigation on interaction of DNA and several cationic surfactants with different head groups by spectroscopy, gel electrophoresis and viscosity technologies.

    PubMed

    Guo, Qing; Zhang, Zhaohong; Song, Youtao; Liu, Shuo; Gao, Wei; Qiao, Heng; Guo, Lili; Wang, Jun

    2017-02-01

    In this study, the interaction between DNA and several cationic surfactants with different head groups such as ethyl hexadecyl dimethyl ammonium bromide (EHDAB), hexadecyl dimethyl benzyl ammonium chloride (HDBAC), and cetyl pyridinium bromide (CPB) were investigated by UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopy, gel electrophoresis, and viscosity technologies. The results show that these cationic surfactants can interact with DNA and major binding modes are electrostatic and hydrophobic. Also, CPB and HDBAC molecules interact with DNA by partial intercalation, and CPB has slightly stronger intercalation than HDBAC, while EHDAB interacts with DNA by non-intercalation. The different head groups of the surfactant molecules can influence the interaction strength. CPB has the stronger interaction with DNA than the others. Moreover, surfactant concentration, the ratio of DNA and fluorescence probe, ionic strength can influence the interaction. The surfactants may interact with DNA by the competition reactions with BR for DNA-BR. The increase of ionic strength may favor the surface binding between DNA and surfactants to some extent. This work provides deep mechanistic insight on the toxicity of cationic surfactants with different head groups to DNA molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Assessment of the quality and quantity of genomic DNA recovered from canine blood samples by three different extraction methods.

    PubMed

    Clements, Dylan N; Wood, Shona; Carter, Stuart D; Ollier, William E R

    2008-08-01

    The ideal method for genomic DNA (gDNA) extraction should recover high quantities of pure, integral gDNA from the original sample source with minimal co-extraction of inhibitors of downstream processes. Canine ethylenediamine tetra-acetic acid (EDTA) treated and clotted blood samples were extracted by three different methods (a silica column method, a phenol-chloroform method and a modified salt precipitation method). Phenol-chloroform and modified salt precipitation based extractions demonstrated similar relative recovery of gDNA with EDTA preserved blood, but were less efficient at recovering gDNA from clotted blood. Spectrophotometer measurement of phenol-chloroform based extractions tended to overestimate the quantity of gDNA recovered from extractions, and was associated with the greater co-extraction of PCR inhibitors. The silica column method recovered gDNA with equal efficiency, purity and integrity irrespective of the sample type or method of quantification.

  14. A novel method for heterocyclic amide–thioamide transformations

    PubMed Central

    Ali, Ibrahim A I; Pazdera, Pavel

    2017-01-01

    In this paper, we introduce a novel and convenient method for the transformation of heterocyclic amides into heteocyclic thioamides. A two-step approach was applied for this transformation: Firstly, we applied a chlorination of the heterocyclic amides to afford the corresponding chloroheterocycles. Secondly, the chloroherocycles and N-cyclohexyl dithiocarbamate cyclohexylammonium salt were heated in chloroform for 12 h at 61 °C to afford heteocyclic thioamides in excellent yields. PMID:28228858

  15. Multicomponent reactions: A simple and efficient route to heterocyclic phosphonates

    PubMed Central

    2016-01-01

    Summary Multicomponent reactions (MCRs) are one of the most important processes for the preparation of highly functionalized organic compounds in modern synthetic chemistry. As shown in this review, they play an important role in organophosphorus chemistry where phosphorus reagents are used as substrates for the synthesis of a wide range of phosphorylated heterocycles. In this article, an overview about multicomponent reactions used for the synthesis of heterocyclic compounds bearing a phosphonate group on the ring is given. PMID:27559377

  16. A novel method for heterocyclic amide-thioamide transformations.

    PubMed

    Fathalla, Walid; Ali, Ibrahim A I; Pazdera, Pavel

    2017-01-01

    In this paper, we introduce a novel and convenient method for the transformation of heterocyclic amides into heteocyclic thioamides. A two-step approach was applied for this transformation: Firstly, we applied a chlorination of the heterocyclic amides to afford the corresponding chloroheterocycles. Secondly, the chloroherocycles and N-cyclohexyl dithiocarbamate cyclohexylammonium salt were heated in chloroform for 12 h at 61 °C to afford heteocyclic thioamides in excellent yields.

  17. N-heterocyclic carbene-catalyzed rearrangements of vinyl sulfones.

    PubMed

    Atienza, Roxanne L; Roth, Howard S; Scheidt, Karl A

    2011-01-01

    N-heterocyclic carbenes catalyze the rearrangement of 1,1-bis(arylsulfonyl)ethylene to the corresponding trans-1,2-bis(phenylsulfonyl) under mild conditions. Tandem rearrangement/cycloadditions have been developed to capitalize on this new process and generate highly substituted isoxazolines and additional heterocyclic compounds. Preliminary mechanistic studies support a new conjugate addition/Umpolung process involving the ejection and subsequent unusual re-addition of a sulfinate ion.

  18. Genetic differences at four DNA typing loci in Finnish, Italian, and mixed Caucasian populations.

    PubMed Central

    Krane, D E; Allen, R W; Sawyer, S A; Petrov, D A; Hartl, D L

    1992-01-01

    Highly polymorphic segments of the human genome containing variable numbers of tandem repeats (VNTRs) have been widely used to establish DNA profiles of individuals for use in forensics. Methods of estimating the probability of occurrence of matching DNA profiles between two randomly selected individuals have been subject to extensive debate regarding the possibility of significant substructure occurring within the major races. We have sampled two Caucasian subpopulations, Finns and Italians, at four commonly used VNTR loci to determine the extent to which the subgroups differ from each other and from a mixed Caucasian database. The data were also analyzed for the occurrence of linkage disequilibrium among the loci. The allele frequency distributions of some loci were found to differ significantly among the subpopulations in a manner consistent with population substructure. Major differences were also found in the probability of occurrence of matching DNA profiles between two individuals chosen at random from the same subpopulation. With respect to the Finnish and Italian subpopulations, the conventional product rule for estimating the probability of a multilocus VNTR match using a mixed Caucasian database consistently yields estimates that are artificially small. Systematic errors of this type were not found using the interim ceiling principle recently advocated in the National Research Council's report [National Research Council (1992) DNA Technology in Forensic Science (Natl. Acad. Sci., Washington)]. The interim ceiling principle is based on currently available racial or ethnic databases and sets an arbitrary lower limit on each VNTR allele frequency. In the future the ceiling frequencies are expected to be established from more adequate data acquired for relevant VNTR loci from multiple subpopulations. Images PMID:1438254

  19. Differences in DNA methylation by extent of breast cancer family history in unaffected women.

    PubMed

    Delgado-Cruzata, Lissette; Wu, Hui-Chen; Liao, Yuyan; Santella, Regina M; Terry, Mary Beth

    2014-02-01

    Breast cancer clusters within families but genetic factors identified to date explain only a portion of this clustering. Lower global DNA methylation in white blood cells (WBC) has been associated with increased breast cancer risk. We examined whether WBC DNA methylation varies by extent of breast cancer family history in unaffected women from high-risk breast cancer families. We evaluated DNA methylation levels in LINE-1, Alu and Sat2 in 333 cancer-free female family members of the New York site of the Breast Cancer Family Registry, the minority of which were known BRCA1 or BRCA2 mutation carriers. We used generalized estimated equation models to test for differences in DNA methylation levels by extent of their breast cancer family history after adjusting for age. All unaffected women had at least one sister affected with breast cancer. LINE-1 and Sat2 DNA methylation levels were lower in individuals with 3 or more (3+) first-degree relatives with breast cancer relative to women with only one first-degree relative. For LINE-1, Alu, and Sat2, having 3+ affected first-degree relatives was associated with a decrease of 23.4% (95%CI = -46.8%, 0.1%), 17.9% (95%CI = -39.5%, 3.7%) and 11.4% (95% CI = -20.3%, -2.5%), respectively, relative to individuals with only one affected first-degree relative, but the results were only statistically significant for Sat2. Individuals having an affected mother had 17.9% lower LINE-1 DNA methylation levels (95% CI = -28.8%, -7.1%) when compared with those not having an affected mother. No associations were observed for Alu or Sat2 by maternal breast cancer status. If replicated, these results indicate that lower global WBC DNA methylation levels in families with extensive cancer histories may be one explanation for the clustering of cancers in these families. Family clustering of disease may reflect epigenetic as well as genetic and shared environmental factors.

  20. Cidofovir selectivity is based on the different response of normal and cancer cells to DNA damage

    PubMed Central

    2013-01-01

    Background Cidofovir (CDV) proved efficacious in treatment of human papillomaviruses (HPVs) hyperplasias. Antiproliferative effects of CDV have been associated with apoptosis induction, S-phase accumulation, and increased levels of tumor suppressor proteins. However, the molecular mechanisms for the selectivity and antitumor activity of CDV against HPV-transformed cells remain unexplained. Methods We evaluated CDV drug metabolism and incorporation into cellular DNA, in addition to whole genome gene expression profiling by means of microarrays in two HPV+ cervical carcinoma cells, HPV- immortalized keratinocytes, and normal keratinocytes. Results Determination of the metabolism and drug incorporation of CDV into genomic DNA demonstrated a higher rate of drug incorporation in HPV+ tumor cells and immortalized keratinocytes compared to normal keratinocytes. Gene expression profiling clearly showed distinct and specific drug effects in the cell types investigated. Although an effect on inflammatory response was seen in all cell types, different pathways were identified in normal keratinocytes compared to immortalized keratinocytes and HPV+ tumor cells. Notably, Rho GTPase pathways, LXR/RXR pathways, and acute phase response signaling were exclusively activated in immortalized cells. CDV exposed normal keratinocytes displayed activated cell cycle regulation upon DNA damage signaling to allow DNA repair via homologous recombination, resulting in genomic stability and survival. Although CDV induced cell cycle arrest in HPV- immortalized cells, DNA repair was not activated in these cells. In contrast, HPV+ cells lacked cell cycle regulation, leading to genomic instability and eventually apoptosis. Conclusions Taken together, our data provide novel insights into the mechanism of action of CDV and its selectivity for HPV-transformed cells. The proposed mechanism suggests that this selectivity is based on the inability of HPV+ cells to respond to DNA damage, rather than on a

  1. Differences in DNA methylation by extent of breast cancer family history in unaffected women

    PubMed Central

    Delgado-Cruzata, Lissette; Wu, Hui-Chen; Liao, Yuyan; Santella, Regina M; Terry, Mary Beth

    2014-01-01

    Breast cancer clusters within families but genetic factors identified to date explain only a portion of this clustering. Lower global DNA methylation in white blood cells (WBC) has been associated with increased breast cancer risk. We examined whether WBC DNA methylation varies by extent of breast cancer family history in unaffected women from high-risk breast cancer families. We evaluated DNA methylation levels in LINE-1, Alu and Sat2 in 333 cancer-free female family members of the New York site of the Breast Cancer Family Registry, the minority of which were known BRCA1 or BRCA2 mutation carriers. We used generalized estimated equation models to test for differences in DNA methylation levels by extent of their breast cancer family history after adjusting for age. All unaffected women had at least one sister affected with breast cancer. LINE-1 and Sat2 DNA methylation levels were lower in individuals with 3 or more (3+) first-degree relatives with breast cancer relative to women with only one first-degree relative. For LINE-1, Alu, and Sat2, having 3+ affected first-degree relatives was associated with a decrease of 23.4% (95%CI = −46.8%, 0.1%), 17.9% (95%CI = −39.5%, 3.7%) and 11.4% (95% CI = −20.3%, −2.5%), respectively, relative to individuals with only one affected first-degree relative, but the results were only statistically significant for Sat2. Individuals having an affected mother had 17.9% lower LINE-1 DNA methylation levels (95% CI = −28.8%, −7.1%) when compared with those not having an affected mother. No associations were observed for Alu or Sat2 by maternal breast cancer status. If replicated, these results indicate that lower global WBC DNA methylation levels in families with extensive cancer histories may be one explanation for the clustering of cancers in these families. Family clustering of disease may reflect epigenetic as well as genetic and shared environmental factors. PMID:24172832

  2. Two Drosophila retrotransposon gypsy subfamilies differ in ability to produce new DNA copies via reverse transcription in Drosophila cultured cells.

    PubMed Central

    Lyubomirskaya, N V; Avedisov, S N; Surkov, S A; Ilyin, Y V

    1993-01-01

    Plasmid DNA constructs containing 5' end truncated retrotransposon gypsy were introduced into Drosophila cultured cells. Appearance of new complete DNA copies with reconstructed via reverse transcription 5'LTR were detected by PCR after transient expression and by Southern blot analysis of genome DNA of stably transformed cells. Two gypsy subfamilies supposed to be different in transpositional activity were analyzed in terms of their ability to produce new DNA copies via reverse transcription in D. hydei cultured cells. It was demonstrated that both gypsy variants undergo retrotransposition but with different efficiency. Images PMID:7688116

  3. Metabolism and Biomarkers of Heterocyclic Aromatic Amines in Molecular Epidemiology Studies: Lessons Learned from Aromatic Amines

    PubMed Central

    2011-01-01

    Aromatic amines and heterocyclic aromatic amines (HAAs) are structurally related classes of carcinogens that are formed during the combustion of tobacco or during the high-temperature cooking of meats. Both classes of procarcinogens undergo metabolic activation by N-hydroxylation of the exocyclic amine group, to produce a common proposed intermediate, the arylnitrenium ion, which is the critical metabolite implicated in toxicity and DNA damage. However, the biochemistry and chemical properties of these compounds are distinct and different biomarkers of aromatic amines and HAAs have been developed for human biomonitoring studies. Hemoglobin adducts have been extensively used as biomarkers to monitor occupational and environmental exposures to a number of aromatic amines; however, HAAs do not form hemoglobin adducts at appreciable levels and other biomarkers have been sought. A number of epidemiologic studies that have investigated dietary consumption of well-done meat in relation to various tumor sites reported a positive association between cancer risk and well-done meat consumption, although some studies have shown no associations between well-done meat and cancer risk. A major limiting factor in most epidemiological studies is the uncertainty in quantitative estimates of chronic exposure to HAAs and, thus, the association of HAAs formed in cooked meat and cancer risk has been difficult to establish. There is a critical need to establish long-term biomarkers of HAAs that can be implemented in molecular epidemioIogy studies. In this review article, we highlight and contrast the biochemistry of several prototypical carcinogenic aromatic amines and HAAs to which humans are chronically exposed. The biochemical properties and the impact of polymorphisms of the major xenobiotic-metabolizing enzymes on the biological effects of these chemicals are examined. Lastly, the analytical approaches that have been successfully employed to biomonitor aromatic amines and HAAs, and

  4. The effects of mitochondrial DNA deletion and copy number variations on different exercise intensities in highly trained swimmers.

    PubMed

    Baykara, O; Sahin, S K; Akbas, F; Guven, M; Onaran, I

    2016-10-31

    It has been suggested that heavy exercise might increase oxidative stress, causing mitochondrial DNA (mtDNA) mutations as well as DNA mutations and changes in the mtDNA copy number in cells. mtDNA4977 deletion is one of the most common deletions seen on mitochondria. We hypothesize association between exercise induced oxidative stress and mtDNA damage in peripheral blood lymphocytes (PBLs) of highly trained swimmers. Therefore we studied the mtDNA4977 deletion level, mtDNA copy number and their relationship with cellular ATP and oxidative stress status in PBLs of swimmers. 8 highly trained and 8 normal trained swimmers and 8 non-athlete subjects were included in the study. The mtDNA4977 deletion and amount of mtDNA were measured using RT-PCR method whereas dichlorohydrofluoroscein (DCF) assay method was used to assess cellular oxidative stress and ATP levels were measured using bioluminescence method. Even though an increase in mtDNA4977 deletion was found in all study groups, the difference was not statistically significant (p=0.98). The mtDNA copy numbers were found to be surprisingly high in highly trained swimmers compared to normal trained swimmers and non-athlete subjects by 4.03 fold (p= 0.0002) and 5.58 fold (p=0.0003), respectively. No significant differences were found between groups by means of intracellular ATP levels (p=0.406) and oxidative stress (p=0.430).  No correlation was found between mtDNA copy number and intracellular ATP content of the PBLs (p=0.703). Our results suggest that heavy training does not have a specific effect on mtDNA4977 deletion but it may be affecting mitochondrial copy numbers which may act as a compensatory mechanism related to ATP levels in blood.

  5. In situ detection of neuronal DNA strand breaks using the Klenow fragment of DNA polymerase I reveals different mechanisms of neuron death after global cerebral ischemia.

    PubMed

    Jin, K; Chen, J; Nagayama, T; Chen, M; Sinclair, J; Graham, S H; Simon, R P

    1999-03-01

    Ischemic cell injury in the brain may involve a cascade of programmed cell death. DNA damage may be either a catalyst or a consequence of this cascade. Therefore, the induction of DNA strand breaks in the rat brain following transient global ischemia was examined using (a) the Klenow labeling assay, identifying DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) with protruding 5' termini, and (b) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), detecting DNA DSBs with protruding 3' termini or blunt ends. Klenow-positive staining occurred within 2 h of reperfusion and increased with increasing durations of reperfusion. DNA damage detected with the Klenow labeling assay preceded that of TUNEL expression in the caudate putamen, reticular thalamus, thalamus, and cortex. However, in CA1, DNA SSBs were not detected until 72 h of reperfusion and occurred simultaneously with DSBs. Thus, the time course and fragmentation characteristics of DNA damage differ between the hippocampal CA1 and other selectively vulnerable brain regions. This distinct pattern suggests that the delayed neuronal death in CA1 following transient global ischemia may occur via an apoptotic mechanism different from that of other brain regions.

  6. Yields of clustered DNA damage induced by charged-particle radiations of similar kinetic energy per nucleon: LET dependence in different DNA microenvironments

    SciTech Connect

    Keszenman, D.J.; Sutherland, B. M.

    2010-08-01

    To determine the linear energy transfer (LET) dependence of the biological effects of densely ionizing radiation in relation to changes in the ionization density along the track, we measured the yields and spectrum of clustered DNA damages induced by charged particles of different atomic number but similar kinetic energy per nucleon in different DNA microenvironments. Yeast DNA embedded in agarose in solutions of different free radical scavenging capacity was irradiated with 1 GeV protons, 1 GeV/nucleon oxygen ions, 980 MeV/nucleon titanium ions or 968 MeV/nucleon iron ions. The frequencies of double-strand breaks (DSBs), abasic sites and oxypurine clusters were quantified. The total DNA damage yields per absorbed dose induced in non-radioquenching solution decreased with LET, with minor variations in radioquenching conditions being detected. However, the total damage yields per particle fluence increased with LET in both conditions, indicating a higher efficiency per particle to induce clustered DNA damages. The yields of DSBs and non-DSB clusters as well as the damage spectra varied with LET and DNA milieu, suggesting the involvement of more than one mechanism in the formation of the different types of clustered damages.

  7. The tumorigenic diversity of the three PLAG family members is associated with different DNA binding capacities.

    PubMed

    Hensen, Karen; Van Valckenborgh, Isabelle C C; Kas, Koen; Van de Ven, Wim J M; Voz, Marianne L

    2002-03-01

    Pleomorphic adenoma gene (PLAG) 1, the main translocation target in pleomorphic adenomas of the salivary glands, is a member of a new subfamily of zinc finger proteins comprising the tumor suppressor candidate PLAG-like1 (also called ZAC1 or lost on transformation 1) and PLAGL2. In this report, we show that NIH3T3 cells overexpressing PLAG1 or PLAGL2 display the typical markers of neoplastic transformation: (a) the cells lose cell-cell contact inhibition; (b) show anchorage-independent growth; and (c) are able to induce tumors in nude mice. In contrast, PLAGL1 has been shown to prevent the proliferation of tumor cells by inducing cell cycle arrest and apoptosis. This difference in function is also reflected in their DNA binding, as we show here that the three PLAG proteins, although highly homologous in their DNA-binding domain, bind different DNA sequences in a distinct fashion. Interestingly, the PLAG1- and PLAGL2-induced transformation is accompanied by a drastic up-regulation of insulin-like growth factor-II, which we prove is a target of PLAG1 and PLAGL2. This strongly suggests that the oncogenic capacity of PLAG1 and PLAGL2 is mediated at least partly by activating the insulin-like growth factor-II mitogenic pathway.

  8. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells

    SciTech Connect

    Ahamed, Maqusood; Karns, Michael; Goodson, Michael; Rowe, John; Hussain, Saber M.; Schlager, John J.

    2008-12-15

    Silver nanoparticles (Ag NPs) have recently received much attention for their possible applications in biotechnology and life sciences. Ag NPs are of interest to defense and engineering programs for new material applications as well as for commercial purposes as an antimicrobial. However, little is known about the genotoxicity of Ag NPs following exposure to mammalian cells. This study was undertaken to examine the DNA damage response to polysaccharide surface functionalized (coated) and non-functionalized (uncoated) Ag NPs in two types of mammalian cells; mouse embryonic stem (mES) cells and mouse embryonic fibroblasts (MEF). Both types of Ag NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and phosphorylated-H2AX expression. Furthermore both of them induced cell death as measured by the annexin V protein expression and MTT assay. Our observations also suggested that the different surface chemistry of Ag NPs induce different DNA damage response: coated Ag NPs exhibited more severe damage than uncoated Ag NPs. The results suggest that polysaccharide coated particles are more individually distributed while agglomeration of the uncoated particles limits the surface area availability and access to membrane bound organelles.

  9. On-chip DNA preconcentration in different media conductivities by electrodeless dielectrophoresis.

    PubMed

    Li, Shunbo; Ye, Ziran; Hui, Yu Sanna; Gao, Yibo; Jiang, Yusheng; Wen, Weijia

    2015-09-01

    Electrodeless dielectrophoresis is the best choice to achieve preconcentration of nanoparticles and biomolecules due to its simple, robust, and easy implementation. We designed a simple chip with microchannels and nano-slits in between and then studied the trapping of DNA in high conductive medium and low conductive medium, corresponding to positive and negative dielectrophoresis (DEP), respectively. It is very important to investigate the trapping in media with different conductivities since one always has to deal with the sample solutions with different conductivities. The trapping process was analyzed by the fluorescent intensity changes. The results showed that DNA could be trapped at the nano-slit in both high and low conductive media in a lower electric field strength (10 V/cm) compared to the existing methods. This is a significant improvement to suppress the Joule heating effect in DEP related experiments. Our work may give insight to researchers for DNA trapping by a simple and low cost device in the Lab-on-a-Chip system.

  10. Assessment of ultraviolet-radiation-induced DNA damage within melanocytes in skin of different constitutive pigmentation.

    PubMed

    Del Bino, S; Sok, J; Bernerd, F

    2013-05-01

    Melanoma incidence and pigmentary disorders are known to be related to the degree of skin pigmentation, but few data exist on the specific impact of ultraviolet radiation (UVR) on melanocytes in skin of different constitutive pigmentation. To analyse UVR-induced DNA damage within melanocytes in different skin-colour types. Skin samples were objectively classified into light, intermediate, tan, brown and dark skin according to their individual typology angle (°ITA), based on colorimetric parameters. Samples were exposed to increasing doses of solar simulated radiation. Detection of DNA damage specifically in melanocytes was achieved by cyclobutane thymine dimer (CPD)-tyrosinase-related protein 1 double staining. For light, intermediate and tan skin, accumulation of CPDs in melanocytes was detected at the lowest dose, with a steep increase with dose. At estimated erythemally equivalent doses, around 80-100% of melanocytes were positive for CPDs in tan, intermediate and light skin types. In contrast, in dark and brown skin types, CPDs were found in only approximately 15% of melanocytes at the highest dose. This work demonstrates that melanocytes from constitutively highly pigmented skin types are less impacted in terms of UVR-induced DNA damage than those from lighter skin types, even those that are moderately pigmented. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

  11. Detection of human papillomavirus DNA in pterygia from different geographical regions

    PubMed Central

    Piras, F; Moore, P S; Ugalde, J; Perra, M T; Scarpa, A; Sirigu, P

    2003-01-01

    Background/aims: The aetiology and pathogenesis of pterygia remain unclear and the involvement of human papillomavirus (HPV) is controversial. 41 pterygia from two geographic locations were evaluated for the presence of HPV DNA. Methods: 41 pterygium biopsies (17 from Italy and 24 from Ecuador) were analysed using the L1C1 and PU-1ML primer sets by polymerase chain reaction (PCR) and DNA sequence analysis. Results: 22 of the 41 pterygia (54%) were positive for HPV, including all 17 Italian cases and 5/24 (21%) Ecuadorean cases. DNA sequencing of the 22 positive cases showed that 11 were HPV type 52, four were type 54, five were candHPV90, and two of unknown genotype. Conclusions: The major differences in the frequency of HPV in geographically distant populations might suggest a possible explanation for the vast differences in the reported detection rates. Three subtypes of HPV were found in this sample of pterygia. None the less, these results suggest that HPV may have a pathogenic role in pterygium. PMID:12812887

  12. Quantification of Cell-Free DNA in Red Blood Cell Units in Different Whole Blood Processing Methods.

    PubMed

    Shih, Andrew W; Bhagirath, Vinai C; Heddle, Nancy M; Acker, Jason P; Liu, Yang; Eikelboom, John W; Liaw, Patricia C

    2016-01-01

    Background. Whole blood donations in Canada are processed by either the red cell filtration (RCF) or whole blood filtration (WBF) methods, where leukoreduction is potentially delayed in WBF. Fresh WBF red blood cells (RBCs) have been associated with increased in-hospital mortality after transfusion. Cell-free DNA (cfDNA) is released by neutrophils prior to leukoreduction, degraded during RBC storage, and is associated with adverse patient outcomes. We explored cfDNA levels in RBCs prepared by RCF and WBF and different storage durations. Methods. Equal numbers of fresh (stored ≤14 days) and older RBCs were sampled. cfDNA was quantified by spectrophotometry and PicoGreen. Separate regression models determined the association with processing method and storage duration and their interaction on cfDNA. Results. cfDNA in 120 RBC units (73 RCF, 47 WBF) were measured. Using PicoGreen, WBF units overall had higher cfDNA than RCF units (p = 0.0010); fresh WBF units had higher cfDNA than fresh RCF units (p = 0.0093). Using spectrophotometry, fresh RBC units overall had higher cfDNA than older units (p = 0.0031); fresh WBF RBCs had higher cfDNA than older RCF RBCs (p = 0.024). Conclusion. Higher cfDNA in fresh WBF was observed compared to older RCF blood. Further study is required for association with patient outcomes.

  13. Quantification of Cell-Free DNA in Red Blood Cell Units in Different Whole Blood Processing Methods

    PubMed Central

    Bhagirath, Vinai C.; Heddle, Nancy M.; Liu, Yang; Eikelboom, John W.; Liaw, Patricia C.

    2016-01-01

    Background. Whole blood donations in Canada are processed by either the red cell filtration (RCF) or whole blood filtration (WBF) methods, where leukoreduction is potentially delayed in WBF. Fresh WBF red blood cells (RBCs) have been associated with increased in-hospital mortality after transfusion. Cell-free DNA (cfDNA) is released by neutrophils prior to leukoreduction, degraded during RBC storage, and is associated with adverse patient outcomes. We explored cfDNA levels in RBCs prepared by RCF and WBF and different storage durations. Methods. Equal numbers of fresh (stored ≤14 days) and older RBCs were sampled. cfDNA was quantified by spectrophotometry and PicoGreen. Separate regression models determined the association with processing method and storage duration and their interaction on cfDNA. Results. cfDNA in 120 RBC units (73 RCF, 47 WBF) were measured. Using PicoGreen, WBF units overall had higher cfDNA than RCF units (p = 0.0010); fresh WBF units had higher cfDNA than fresh RCF units (p = 0.0093). Using spectrophotometry, fresh RBC units overall had higher cfDNA than older units (p = 0.0031); fresh WBF RBCs had higher cfDNA than older RCF RBCs (p = 0.024). Conclusion. Higher cfDNA in fresh WBF was observed compared to older RCF blood. Further study is required for association with patient outcomes. PMID:27774338

  14. Iron chelators hydroxyurea and bathophenanthroline disulfonate inhibit DNA synthesis by different pathways.

    PubMed

    Alcaín, F J; Löw, H; Crane, F L; Navas, P

    1994-09-01

    We previously showed that thrombin-stimulated DNA synthesis in CCL 39 cells was inhibited by hydroxyurea (HU) and bathophenanthroline disulfonate (BPS) (Proc. Natl. Acad. Sci. USA, in press). A clear difference exists between these two inhibitors. Inhibition mediated by HU was immediate and must be present in the culture medium. BPS was equally effective when it was present in the medium or after preincubation, but it required at least 12 h to achieve maximal effect. The permeable form 1,10 phenanthroline had the same inhibitory effect in short-term incubations that BPS. Moreover, 1,10 phenanthroline was cytotoxic in long-term incubations indicating that the site of BPS inhibition was outside the cell. Further, long-term incubations with HU did not affect the ability of the cell to reinitiate DNA synthesis after removal of the chelator.

  15. Distinct differences in metal ion specificity of RNA and DNA G-quadruplexes.

    PubMed

    Guiset Miserachs, Helena; Donghi, Daniela; Börner, Richard; Johannsen, Silke; Sigel, Roland K O

    2016-12-01

    RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li(+), Na(+), K(+), Rb(+), Cs(+), NH4(+), Mg(2+), Ca(2+), Sr(2+), and Ba(2+). Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na(+), K(+), Rb(+), NH4(+), as well as Sr(2+) and Ba(2+) in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K(+) and Sr(2+) lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na(+) ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.

  16. A comprehensive evaluation of the toxicology of cigarette ingredients: heterocyclic nitrogen compounds.

    PubMed

    Coggins, Christopher R E; Merski, Jerome A; Oldham, Michael J

    2011-06-01

    Three heterocyclic nitrogen compounds, 2,3-diethylpyrazine (DEP), 2,3,5,6-tetramethylpyrazine (TMP), and 2-acetyl pyridine (AP), are naturally present in tobacco and are also added to tobacco as flavor ingredients. A battery of tests was used to compare the toxicity of mainstream smoke from experimental cigarettes containing the three heterocyclic nitrogen compounds added individually at three different levels. The lowest target inclusion level of the ingredient was 10 ppm, and the highest was 10,000 ppm. Smoke from experimental and control cigarettes was evaluated in analytical smoke chemistry, in vitro cytotoxicity, and mutagenicity assays. The cigarettes with added DEP produced some minor (approximately 10%) changes in smoke chemistry when compared with the cigarettes containing no DEP. Smoke chemistry was effectively unchanged by the addition of either AP or TMP. Cytotoxicity, assessed by the neutral red uptake assay using both gas-vapor and particulate phases of smoke, was unaffected by the addition of any of the test ingredients. Mutagenicity, assessed in five strains of Salmonella treated with mainstream cigarette smoke condensate, also was unaffected by any of the test ingredients. Despite the exaggerated ingredient levels relative to commercial-use levels, there was a lack of a toxicological response for the three heterocyclic nitrogen compounds in the test systems used.

  17. Theoretical study of ionization and one-electron oxidation potentials of N-heterocyclic compounds.

    PubMed

    Sviatenko, Liudmyla K; Gorb, Leonid; Hill, Frances C; Leszczynski, Jerzy

    2013-05-15

    A number of density functionals was utilized to predict gas-phase adiabatic ionization potentials (IPs) for nitrogen-rich heterocyclic compounds. Various solvation models were applied to the calculation of difference in free energies of solvation of oxidized and reduced forms of heterocyclic compounds in acetonitrile (AN) for correct reproduction of their standard oxidation potentials. We developed generally applicable protocols that could successfully predict the gas-phase adiabatic ionization potentials of nitrogen-rich heterocyclic compounds and their standard oxidation potentials in AN. This approach is supported by a MPW1K/6-31+G(d) level of theory which uses SMD(UA0) approximation for estimation of solvation energy of neutral molecules and PCM(UA0) model for ionized ones. The mean absolute derivation (MAD) and root mean square error (RMSE) of the current theoretical models for IP are equal to 0.22 V and 0.26, respectively, and for oxidation potentials MAD = 0.13 V and RMSE = 0.17.

  18. Detection theory in identification of RNA-DNA sequence differences using RNA-sequencing.

    PubMed

    Toung, Jonathan M; Lahens, Nicholas; Hogenesch, John B; Grant, Gregory

    2014-01-01

    Advances in sequencing technology have allowed for detailed analyses of the transcriptome at single-nucleotide resolution, facilitating the study of RNA editing or sequence differences between RNA and DNA genome-wide. In humans, two types of post-transcriptional RNA editing processes are known to occur: A-to-I deamination by ADAR and C-to-U deamination by APOBEC1. In addition to these sequence differences, researchers have reported the existence of all 12 types of RNA-DNA sequence differences (RDDs); however, the validity of these claims is debated, as many studies claim that technical artifacts account for the majority of these non-canonical sequence differences. In this study, we used a detection theory approach to evaluate the performance of RNA-Sequencing (RNA-Seq) and associated aligners in accurately identifying RNA-DNA sequence differences. By generating simulated RNA-Seq datasets containing RDDs, we assessed the effect of alignment artifacts and sequencing error on the sensitivity and false discovery rate of RDD detection. Overall, we found that even in the presence of sequencing errors, false negative and false discovery rates of RDD detection can be contained below 10% with relatively lenient thresholds. We also assessed the ability of various filters to target false positive RDDs and found them to be effective in discriminating between true and false positives. Lastly, we used the optimal thresholds we identified from our simulated analyses to identify RDDs in a human lymphoblastoid cell line. We found approximately 6,000 RDDs, the majority of which are A-to-G edits and likely to be mediated by ADAR. Moreover, we found the majority of non A-to-G RDDs to be associated with poorer alignments and conclude from these results that the evidence for widespread non-canonical RDDs in humans is weak. Overall, we found RNA-Seq to be a powerful technique for surveying RDDs genome-wide when coupled with the appropriate thresholds and filters.

  19. Changes in the Infrared Microspectroscopic Characteristics of DNA Caused by Cationic Elements, Different Base Richness and Single-Stranded Form

    PubMed Central

    Mello, Maria Luiza S.; Vidal, B. C.

    2012-01-01

    Background The infrared (IR) analysis of dried samples of DNA and DNA-polypeptide complexes is still scarce. Here we have studied the FT-IR profiles of these components to further the understanding of the FT-IR signatures of chromatin and cell nuclei. Methodology/Principal Findings Calf thymus and salmon testis DNA, and complexes of histone H1, protamine, poly-L-lysine and poly-L-arginine (histone-mimic macromolecules) with DNA were analyzed in an IR microspectroscope equipped with an attenuated total reflection diamond objective and Grams software. Conditions including polypeptides bound to the DNA, DNA base composition, and single-stranded form were found to differently affect the vibrational characteristics of the chemical groups (especially, PO2−) in the nucleic acid. The antisymmetric stretching (νas) of the DNA PO2− was greater than the symmetric stretching (νs) of these groups and increased in the polypeptide-DNA complexes. A shift of the νas of the DNA PO2− to a lower frequency and an increased intensity of this vibration were induced especially by lysine-rich histones. Lysine richness additionally contributed to an increase in the vibrational stretching of the amide I group. Even in simple molecules such as inorganic phosphates, the vibrational characteristics of the phosphate anions were differently affected by different cations. As a result of the optimization of the DNA conformation by binding to arginine-rich polypeptides, enhancements of the vibrational characteristics in the FT-IR fingerprint could be detected. Although different profiles were obtained for the DNA with different base compositions, this situation was no longer verified in the polypeptide-DNA complexes and most likely in isolated chromatin or cell nuclei. However, the νas PO2−/νs PO2− ratio could discriminate DNA with different base compositions and DNA in a single-stranded form. Conclusions/Significance FT-IR spectral profiles are a valuable tool for establishing the

  20. GC-Rich DNA Fragments and Oxidized Cell-Free DNA Have Different Effects on NF-kB and NRF2 Signaling in MSC.

    PubMed

    Sergeeva, Vasilina A; Kostyuk, Svetlana V; Ershova, Elizaveta S; Malinovskaya, Elena M; Smirnova, Tatiana D; Kameneva, Larisa V; Veiko, Natalia N

    2016-01-01

    It has been established that cell-free DNA circulating in the bloodstream affects cells. The characteristics of cfDNA depend on the physiological state of the organism. As we showed previously, diseases can cause either GC-enrichment of the cell-free DNA pool or its oxidation. Thus, in cases of cerebral atherosclerosis, heart attack and rheumatic arthritis the cell-free DNA pool is GC-enriched and, in the case of cancer, both GC-enriched and oxidized. Herein we investigated the time-dependent effect of oxidized and GC-rich cell-free DNA on NF-kB and NRF2 signaling pathways in human mesenchymal stem cells and showed that they affect cells in different ways. Oxidized DNA drastically increases expression of NRF2 in a short period of time, but the effect does not last long. GC-rich DNA causes a prolonged increase in mRNA levels of NF-kB and NRF2 which lasts 48 and 24 h, respectively.

  1. [Analysis of the level and pattern of genomic DNA methylation in different ploidy watermelons by MSAP (Citrullus lanatus)].

    PubMed

    Wang, Chun Guo; Gu, Yu; Chen, Cheng Bin; Jiao, Ding Liang; Xue, Zhen Yi; Song, Wen Qin

    2009-04-01

    DNA methylation is one of the major epigenetic modifications. It is very important to the regulation of gene expression. In present study, an autoploidy series (2x, 3x and 4x) in watermelon (Citrullus lanatus) was constructed and MSAP (Methylation-Sensitive Amplification Polymorphism) analysis was conducted to elucidate the level and pattern of DNA methylation at CCGG sites in different ploidy watermelons. Totally, 1883 genetic loci were produced by 23 pairs of selective primers, of which 647, 655 and 581 sites were detected in diploid, autotriploid and autotetraploid, respectively. The methylation sites were 181, 150 and 159, and the corresponding total methylation ratios were 28.0%, 22.9% and 27.4% in 2x, 3x and 4x, respectively, of which the fully methylation sites were 121, 80 and 82, and the corresponding fully methylation ratios were 18.7%, 12.2% and 14.1%. Further analysis of the pattern of DNA methylation suggested that compared 4x with 2x, about half of detected sites (54.4%) shown changes of DNA methylation patterns. Similarly, compared 4x with 3x, 45.4% sites also shown changes of DNA methylation patterns. Moreover, the trend of DNA methylation adjustment mainly involved increase of DNA methylation levels in 4x. However, compared 3x with 2x or 4x, although the changes of DNA methylation pattern also widely occurred, which involved 41.6% (compared 3x with 2x) and 45.4% (compared 3x with 4x) sites, respectively, the trend of DNA methylation adjustment mainly involved decrease of DNA methylation levels in 3x. All these results indicated that DNA methylation events were widely existed in different ploidy watermelons. However, not only based on the total DNA methylation ratio or fully DNA methylation ratio, the results both implied that the DNA methylation levels were not closely associated with the autopolyploidy level in watermelon. Autotriploid watermelon shows obvious low level of DNA methylation. Analysis of DNA methylation patterns also suggested that

  2. Intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial DNA.

    PubMed Central

    Horai, S; Hayasaka, K

    1990-01-01

    Nucleotide sequences of the major noncoding region of human mitochondrial DNA (mtDNA) from 95 human placentas have been determined. These sequences include at least a 482-bp-long region encompassing most of the D-loop-forming region. Comparisons of these sequences with those previously determined have revealed remarkable features of nucleotide substitutions and insertion/deletion events. The nucleotide diversity among the sequences is estimated as 1.45%, which is three- to fourfold higher than the corresponding value estimated from restriction-enzyme analysis of whole mtDNA genome. A hypervariable region has also been defined. In this 14-bp region, 17 different sequences were detected. More than 97% of the base changes are transitions. A significantly nonrandom distribution of nucleotide substitutions and sequence length variations were also noted. The phylogenetic analysis indicates that diversity among the negroids is much larger than that among the caucasoids or the mongoloids. In fact, part of the negroids first diverged from other humans in the phylogenetic tree. A striking finding in the phylogenetic analysis is that the mongoloids can be separated into two distinct groups. Divergence of part of the mongoloids follows the earliest divergence of part of the negroids. The remainder of the mongoloids subsequently diverged together with the caucasoids. This observation confirmed our earlier study, which clearly demonstrated, by the restriction-enzyme analysis, existence of two distinct groups in the Japanese. Images Figure 3 PMID:2316527

  3. Directly labeled DNA probes using fluorescent nucleotides with different length linkers.

    PubMed Central

    Zhu, Z; Chao, J; Yu, H; Waggoner, A S

    1994-01-01

    Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR. Images PMID:8078779

  4. Aneuploidogenic effects and DNA oxidation induced in vitro by differently sized gold nanoparticles

    PubMed Central

    Di Bucchianico, Sebastiano; Fabbrizi, Maria Rita; Cirillo, Silvia; Uboldi, Chiara; Gilliland, Douglas; Valsami-Jones, Eugenia; Migliore, Lucia

    2014-01-01

    Gold nanoparticles (Au NPs) are used in many fields, including biomedical applications; however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms is available. For this reason, experiments in human primary lymphocytes and murine macrophages (Raw264.7) were performed exposing cells to spherical citrate-capped Au NPs with two different nominal diameters (5 nm and 15 nm). The proliferative activity, mitotic, apoptotic, and necrotic markers, as well as chromosomal damage were assessed by the cytokinesis-block micronucleus cytome assay. Fluorescence in situ hybridization with human and murine pancentromeric probes was applied to distinguish between clastogenic and aneuploidogenic effects. Our results indicate that 5 nm and 15 nm Au NPs are able to inhibit cell proliferation by apoptosis and to induce chromosomal damage, in particular chromosome mis-segregation. DNA strand breaks were detected by comet assay, and the modified protocol using endonuclease-III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that pyrimidines and purines were oxidatively damaged by Au NPs. Moreover, we show a size-independent correlation between the cytotoxicity of Au NPs and their tested mass concentration or absolute number, and genotoxic effects which were more severe for Au NP 15 nm compared to Au NP 5 nm. Results indicate that apoptosis, aneuploidy, and DNA oxidation play a pivotal role in the cytotoxicity and genotoxicity exerted by Au NPs in our cell models. PMID:24855356

  5. Arsenic and the epigenome: interindividual differences in arsenic metabolism related to distinct patterns of DNA methylation.

    PubMed

    Bailey, Kathryn A; Wu, Michael C; Ward, William O; Smeester, Lisa; Rager, Julia E; García-Vargas, Gonzalo; Del Razo, Luz-Maria; Drobná, Zuzana; Stýblo, Miroslav; Fry, Rebecca C

    2013-02-01

    Biotransformation of inorganic arsenic (iAs) is one of the factors that determines the character and magnitude of the diverse detrimental health effects associated with chronic iAs exposure, but it is unknown how iAs biotransformation may impact the epigenome. Here, we integrated analyses of genome-wide, gene-specific promoter DNA methylation levels of peripheral blood leukocytes with urinary arsenical concentrations of subjects from a region of Mexico with high levels of iAs in drinking water. These analyses revealed dramatic differences in DNA methylation profiles associated with concentrations of specific urinary metabolites of arsenic (As). The majority of individuals in this study had positive indicators of As-related disease, namely pre-diabetes mellitus or diabetes mellitus (DM). Methylation patterns of genes with known associations with DM were associated with urinary concentrations of specific iAs metabolites. Future studies will determine whether these DNA methylation profiles provide mechanistic insight into the development of iAs-associated disease, predict disease risk, and/or serve as biomarkers of iAs exposure in humans. © 2013 Wiley Periodicals, Inc.

  6. Interspecies comparative genome hybridization and interspecies representational difference analysis reveal gross DNA differences between humans and great apes.

    PubMed

    Toder, R; Xia, Y; Bausch, E

    1998-09-01

    Comparative chromosome G-/R-banding, comparative gene mapping and chromosome painting techniques have demonstrated that only few chromosomal rearrangements occurred during great ape and human evolution. Interspecies comparative genome hybridization (CGH), used here in this study, between human, gorilla and pygmy chimpanzee revealed species-specific regions in all three species. In contrast to the human, a far more complex distribution of species-specific blocks was detected with CGH in gorilla and pygmy chimpanzee. Most of these blocks coincide with already described heterochromatic regions on gorilla and chimpanzee chromosomes. Representational difference analysis (RDA) was used to subtract the complex genome of gorilla against human in order to enrich gorilla-specific DNA sequences. Gorilla-specific clones isolated with this technique revealed a 32-bp repeat unit. These clones were mapped by fluorescence in situ hybridization (FISH) to the telomeric regions of gorilla chromosomes that had been shown by interspecies CGH to contain species-specific sequences.

  7. Double-stranded DNA viruses: 20 families and only five different architectural principles for virion assembly.

    PubMed

    Krupovic, Mart; Bamford, Dennis H

    2011-08-01

    The number of viral particles in the biosphere is enormous. Virus classification helps to comprehend the virosphere and to understand the relationship between different virus groups. However, the evolutionary reach of the currently employed sequence-based approaches in virus taxonomy is rather limited, producing a fragmented view of the virosphere. As a result, viruses are currently classified into 87 different families. However, studies on virion architectures have unexpectedly revealed that their structural diversity is far more limited. Here we describe structures of the major capsid proteins of double-stranded DNA viruses infecting hosts residing in different domains of life. We note that viruses belonging to 20 different families fall into only five distinct structural groups, suggesting that optimal virus classification approach should equally rely on both sequence and structural information.

  8. The DNA methylome and transcriptome of different brain regions in schizophrenia and bipolar disorder.

    PubMed

    Xiao, Yun; Camarillo, Cynthia; Ping, Yanyan; Arana, Tania Bedard; Zhao, Hongying; Thompson, Peter M; Xu, Chaohan; Su, Bin Brenda; Fan, Huihui; Ordonez, Javier; Wang, Li; Mao, Chunxiang; Zhang, Yunpeng; Cruz, Dianne; Escamilla, Michael A; Li, Xia; Xu, Chun

    2014-01-01

    Extensive changes in DNA methylation have been observed in schizophrenia (SC) and bipolar disorder (BP), and may contribute to the pathogenesis of these disorders. Here, we performed genome-scale DNA methylation profiling using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) on two brain regions (including frontal cortex and anterior cingulate) in 5 SC, 7 BP and 6 normal subjects. Comparing with normal controls, we identified substantial differentially methylated regions (DMRs) in these two brain regions of SC and BP. To our surprise, different brain regions show completely distinct distributions of DMRs across the genomes. In frontal cortex of both SC and BP subjects, we observed widespread hypomethylation as compared to normal controls, preferentially targeting the terminal ends of the chromosomes. In contrast, in anterior cingulate, both SC and BP subjects displayed extensive gain of methylation. Notably, in these two brain regions of SC and BP, only a few DMRs overlapped with promoters, whereas a greater proportion occurs in introns and intergenic regions. Functional enrichment analysis indicated that important psychiatric disorder-related biological processes such as neuron development, differentiation and projection may be altered by epigenetic changes located in the intronic regions. Transcriptome analysis revealed consistent dysfunctional processes with those determined by DMRs. Furthermore, DMRs in the same brain regions from SC and BP could successfully distinguish BP and/or SC from normal controls while differentially expressed genes could not. Overall, our results support a major role for brain-region-dependent aberrant DNA methylation in the pathogenesis of these two disorders.

  9. The DNA Methylome and Transcriptome of Different Brain Regions in Schizophrenia and Bipolar Disorder

    PubMed Central

    Xiao, Yun; Camarillo, Cynthia; Ping, Yanyan; Arana, Tania Bedard; Zhao, Hongying; Thompson, Peter M.; Xu, Chaohan; Su, Bin Brenda; Fan, Huihui; Ordonez, Javier; Wang, Li; Mao, Chunxiang; Zhang, Yunpeng; Cruz, Dianne; Escamilla, Michael A.; Li, Xia; Xu, Chun

    2014-01-01

    Extensive changes in DNA methylation have been observed in schizophrenia (SC) and bipolar disorder (BP), and may contribute to the pathogenesis of these disorders. Here, we performed genome-scale DNA methylation profiling using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) on two brain regions (including frontal cortex and anterior cingulate) in 5 SC, 7 BP and 6 normal subjects. Comparing with normal controls, we identified substantial differentially methylated regions (DMRs) in these two brain regions of SC and BP. To our surprise, different brain regions show completely distinct distributions of DMRs across the genomes. In frontal cortex of both SC and BP subjects, we observed widespread hypomethylation as compared to normal controls, preferentially targeting the terminal ends of the chromosomes. In contrast, in anterior cingulate, both SC and BP subjects displayed extensive gain of methylation. Notably, in these two brain regions of SC and BP, only a few DMRs overlapped with promoters, whereas a greater proportion occurs in introns and intergenic regions. Functional enrichment analysis indicated that important psychiatric disorder-related biological processes such as neuron development, differentiation and projection may be altered by epigenetic changes located in the intronic regions. Transcriptome analysis revealed consistent dysfunctional processes with those determined by DMRs. Furthermore, DMRs in the same brain regions from SC and BP could successfully distinguish BP and/or SC from normal controls while differentially expressed genes could not. Overall, our results support a major role for brain-region-dependent aberrant DNA methylation in the pathogenesis of these two disorders. PMID:24776767

  10. Comet assay: a reliable tool for the assessment of DNA damage in different models.

    PubMed

    Dhawan, Alok; Bajpayee, Mahima; Parmar, Devendra

    2009-02-01

    New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans.

  11. Methamidophos alters sperm function and DNA at different stages of spermatogenesis in mice

    SciTech Connect

    Urióstegui-Acosta, Mayrut; Hernández-Ochoa, Isabel; Sánchez-Gutiérrez, Manuel; Piña-Guzmán, Belem; Rafael-Vázquez, Leticia; Solís-Heredia, M.J.; Martínez-Aguilar, Gerardo; Quintanilla-Vega, Betzabet

    2014-09-15

    Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5 mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis–vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43–57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5 mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5 mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5 mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation. - Highlights: • Methamidophos alters sperm cell function at different stages of spermatogenesis. • Testicular stages of spermatogenesis are more sensitive to

  12. Polycationic ligands of different chemical classes stimulate DNA strand displacement between short oligonucleotides in a protein-free system.

    PubMed

    Volodin, Alexander A; Bocharova, Tatiana N; Smirnova, Elena A

    2016-09-01

    The ability of polycationic ligands to stimulate DNA strand displacement between short oligonucleotides in a protein-free system is demonstrated. We show that two ligands, tetracationic aliphatic amine (spermine) and a dicationic intercalating drug (chloroquine), promote strand displacement in a concentration-dependent manner. At low concentrations both ligands decelerate spontaneous strand displacement because of their impact on the stability of the DNA duplex. At elevated concentrations they accelerate strand displacement via formation of intermediate structures containing three DNA strands. The rate of the last process does not correlate with the thermal dissociation rate of the entire DNA duplex. It indicates that, possibly, the action of these agents cannot be explained by their influence on the stability of the DNA duplex. In general, our results suggest that the ability to stimulate DNA strand displacement appears to be a common feature of polycations of different chemical and structural classes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 633-641, 2016.

  13. Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans.

    PubMed

    Ruíz-Estévez, Mercedes; López-León, M Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2013-09-01

    B chromosomes are considered to be genetically inert elements. However, some of them are able to show nucleolus organizer region (NOR) activity, as detected by both cytological and molecular means. The grasshopper Eyprepocnemis plorans shows a B chromosome polymorphism characterized by the existence of many B variants. One of them, B24, shows NOR activity in about half of B-carrying males in the Torrox population. Molecular data have suggested the recent origin for B chromosomes in this species, and on this basis it would be expected that NOR activity was widespread among the different B variants. Here we test this hypothesis in four different B chromosome variants (B1, B2, B5, and B24) from 11 natural populations of the grasshopper E. plorans covering the south and east of the Iberian Peninsula plus the Balearic Islands. We used two different approaches: (1) the cytological observation of nucleoli attached to the distal region of the B chromosome (where the rDNA is located), and (2) the molecular detection of the rDNA transcripts carrying an adenine insertion characteristic of B chromosome ITS2 sequences. The results showed NOR expression not only for B24 but also for the B1 and B2 variants. However, the level of B-NOR expression in these latter variants, measured by the proportion of cells showing nucleoli attached to the B chromosomes, was much lower than that previously reported for B24. This suggests the possibility that structural or genetic background conditions are enhancing the expressivity of the rDNA in the B24 variant.

  14. Gene Expression Differences in Infected and Noninfected Middle Ear Complementary DNA Libraries

    PubMed Central

    Kerschner, Joseph E.; Horsey, Edward; Ahmed, Azad; Erbe, Christy; Khampang, Pawjai; Cioffi, Joseph; Hu, Fen Ze; Post, James Christopher; Ehrlich, Garth D.

    2010-01-01

    Objectives To investigate genetic differences in middle ear mucosa (MEM) with nontypeable Haemophilus influenzae (NTHi) infection. Genetic upregulation and downregulation occurs in MEM during otitis media (OM) pathogenesis. A comprehensive assessment of these genetic differences using the techniques of complementary DNA (cDNA) library creation has not been performed. Design The cDNA libraries were constructed from NTHi-infected and noninfected chinchilla MEM. Random clones were picked, sequenced bidirectionally, and submitted to the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags database, where they were assigned accession numbers. These numbers were used with the basic local alignment search tool (BLAST) to align clones against the nonredundant nucleotide database at NCBI. Results Analysis with the Web-based statistical program FatiGO identified several biological processes with significant differences in numbers of represented genes. Processes involved in immune, stress, and wound responses were more prevalent in the NTHi-infected library. S100 calcium-binding protein A9 (S100A9); secretory leukoprotease inhibitor (SLPI); β2-microglobulin (B2M); ferritin, heavy-chain polypeptide 1 (FTH1); and S100 calcium-binding protein A8 (S100A8) were expressed at significantly higher levels in the NTHi-infected library. Calcium-binding proteins S100A9 and S100A8 serve as markers for inflammation and have antibacterial effects. Secretory leukoprotease inhibitor is an antibacterial protein that inhibits stimuli-induced MUC1, MUC2, and MUC5AC production. Conclusions A number of genes demonstrate changes during the pathogenesis of OM, including SLPI, which has an impact on mucin gene expression; this expression is known to be an important regulator in OM. The techniques described herein provide a framework for future investigations to more thoroughly understand molecular changes in the middle ear, which will likely be important in developing new

  15. The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

    SciTech Connect

    Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.; DeBeer, Madeleine A.P.; Lopper, Matthew E.

    2010-05-25

    Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 {angstrom} resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.

  16. SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content.

    PubMed

    Schoft, Vera K; Chumak, Nina; Bindics, János; Slusarz, Lucyna; Twell, David; Köhler, Claudia; Tamaru, Hisashi

    2015-03-01

    Key message: Purification of pollen nuclei. Germ cell epigenetics is a critical topic in plants and animals. The male gametophyte (pollen) of flowering plants is an attractive model to study genetic and epigenetic reprogramming during sexual reproduction, being composed of only two sperm cells contained within, its companion, vegetative cell. Here, we describe a simple and efficient method to purify SYBR Green-stained sperm and vegetative cell nuclei of Arabidopsis thaliana pollen using fluorescence-activated cell sorting to analyze chromatin and RNA profiles. The method obviates generating transgenic lines expressing cell-type-specific fluorescence reporters and facilitates functional genomic analysis of various mutant lines and accessions. We evaluate the purity and quality of the sorted pollen nuclei and analyze the technique's molecular basis. Our results show that both DNA and RNA contents contribute to SYBR Green-activated nucleus sorting and RNA content differences impact on the separation of sperm and vegetative cell nuclei. We demonstrate the power of the approach by sorting wild-type and polyploid mutant sperm and vegetative cell nuclei from mitotic and meiotic mutants, which is not feasible using cell-type-specific transgenic reporters. Our approach should be applicable to pollen nuclei of crop plants and possibly to cell/nucleus types and cell cycle phases of different species containing substantially different amounts of DNA and/or RNA.

  17. MICROBIAL DEGRADATION OF NITROGEN, OXYGEN AND SULFUR HETEROCYCLIC COMPOUNDS UNDER ANAEROBIC CONDITIONS: STUDIES WITH AQUIFER SAMPLES

    EPA Science Inventory

    The potential for anaerobic biodegradation of 12 heterocyclic model compounds was studied. Nine of the model compounds were biotransformed in aquifer slurries under sulfate-reducing or methanogenic conditions. The nitrogen and oxygen heterocyclic compounds were more susceptible t...

  18. MICROBIAL DEGRADATION OF NITROGEN, OXYGEN AND SULFUR HETEROCYCLIC COMPOUNDS UNDER ANAEROBIC CONDITIONS: STUDIES WITH AQUIFER SAMPLES

    EPA Science Inventory

    The potential for anaerobic biodegradation of 12 heterocyclic model compounds was studied. Nine of the model compounds were biotransformed in aquifer slurries under sulfate-reducing or methanogenic conditions. The nitrogen and oxygen heterocyclic compounds were more susceptible t...

  19. Ruthenium olefin metathesis catalysts featuring unsymmetrical N-heterocyclic carbenes.

    PubMed

    Paradiso, Veronica; Bertolasi, Valerio; Costabile, Chiara; Grisi, Fabia

    2016-01-14

    New ruthenium Grubbs' and Hoveyda-Grubbs' second generation catalysts bearing N-alkyl/N-isopropylphenyl N-heterocyclic carbene (NHC) ligands with syn or anti backbone configuration were obtained and compared in model olefin metathesis reactions. Different catalytic efficiencies were observed depending on the size of the N-alkyl group (methyl or cyclohexyl) and on the backbone configuration. The presence of an N-cyclohexyl substituent determined the most significant reactivity differences between catalysts with syn or anti phenyl groups on the backbone. In particular, anti catalysts proved highly efficient, especially in the ring-closing metathesis (RCM) of encumbered diolefins, while syn catalysts showed low efficiency in the RCM of less hindered diolefins. This peculiar behavior, rationalized through DFT studies, was found to be related to the high propensity of these catalysts to give nonproductive metathesis events. Enantiopure anti catalysts were also tested in asymmetric metathesis reactions, where moderate enantioselectivities were observed. The steric and electronic properties of unsymmetrical NHCs with the N-cyclohexyl group were then evaluated using the corresponding rhodium complexes. While steric factors proved unimportant for both syn and anti NHCs, a major electron-donating character was found for the unsymmetrical NHC with anti phenyl substituents on the backbone.

  20. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    NASA Astrophysics Data System (ADS)

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-01

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  1. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions.

    PubMed

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-11

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  2. Widespread differences in cortex DNA methylation of the "language gene" CNTNAP2 between humans and chimpanzees.

    PubMed

    Schneider, Eberhard; El Hajj, Nady; Richter, Steven; Roche-Santiago, Justin; Nanda, Indrajit; Schempp, Werner; Riederer, Peter; Navarro, Bianca; Bontrop, Ronald E; Kondova, Ivanela; Scholz, Claus Jürgen; Haaf, Thomas

    2014-04-01

    CNTNAP2, one of the largest genes in the human genome, has been linked to human-specific language abilities and neurodevelopmental disorders. Our hypothesis is that epigenetic rather than genetic changes have accelerated the evolution of the human brain. To compare the cortex DNA methylation patterns of human and chimpanzee CNTNAP2 at ultra-high resolution, we combined methylated DNA immunoprecipitation (MeDIP) with NimbleGen tiling arrays for the orthologous gene and flanking sequences. Approximately 1.59 Mb of the 2.51 Mb target region could be aligned and analyzed with a customized algorithm in both species. More than one fifth (0.34 Mb) of the analyzed sequence throughout the entire gene displayed significant methylation differences between six human and five chimpanzee cortices. One of the most striking interspecies differences with 28% methylation in human and 59% in chimpanzee cortex (by bisulfite pyrosequencing) lies in a region 300 bp upstream of human SNP rs7794745 which has been associated with autism and parent-of-origin effects. Quantitative real-time RT PCR revealed that the protein-coding splice variant CNTNAP2-201 is 1.6-fold upregulated in human cortex, compared with the chimpanzee. Transcripts CNTNAP2-001, -002, and -003 did not show skewed allelic expression, which argues against CNTNAP2 imprinting, at least in adult human brain. Collectively, our results suggest widespread cortex DNA methylation changes in CNTNAP2 since the human-chimpanzee split, supporting a role for CNTNAP2 fine-regulation in human-specific language and communication traits.

  3. Temozolomide chemoresistance heterogeneity in melanoma with different treatment regimens: DNA damage accumulation contribution.

    PubMed

    Boeckmann, Lars; Nickel, Ann-Christin; Kuschal, Christiane; Schaefer, Annika; Thoms, Kai-Martin; Schön, Michael P; Thomale, Jürgen; Emmert, Steffen

    2011-06-01

    The efficacy of temozolomide in melanoma treatment is low (response rate <20%) and may depend on the activity of O-methylguanine DNA methyltransferase (MGMT) and mismatch repair. We identified melanoma cell lines with different sensitivities to single versus prolonged clinical dosing regimens of temozolomide treatment and assessed a variety of potential resistance mechanisms using this model. We measured mRNA expression and promoter methylation of MGMT and essential mismatch repair genes (MLH1, MSH2). Cell cycle distribution, apoptosis/necrosis induction, O-methylguanine-adduct formation, and ABCB1 gene expression were assessed. We found that three cell lines, MelA, MelB, and MelC, were more sensitive to a single dose regimen than to a prolonged regimen, which would be expected to exhibit higher cytotoxicity. KAII and LIBR cell sensitivity was higher with regard to the prolonged treatment regimen, as expected. Only MelC expressed MGMT. Gene expression correlated well with promoter methylation. Temozolomide exposure did not alter mRNA expression. Different sensitivities to temozolomide were caused neither by delayed apoptosis induction due to early cell cycle arrest nor by O-methylguanine-adduct formation or efflux transporter expression. MelC was the most resistant cell line with rapid elimination of O-methylguanine adducts. This was in good agreement with its MGMT expression. The sensitive cell lines KAII and LIBR accumulated O-methylguanine adducts after a second treatment cycle with temozolomide in contrast with the other three cell lines. We conclude that MGMT expression and DNA adduct accumulation are relevant factors in temozolomide chemosensitivity. Considering individualized temozolomide treatment regimens either by quantification of DNA adducts or by chemosensitivity testing seems worthwhile clinically.

  4. DNA-directed aniline mustards with high selectivity for adenine or guanine bases: mutagenesis in a variety of Salmonella typhimurium strains differing in DNA-repair capability.

    PubMed

    Ferguson, L R; Denny, W A; Boritzki, T J

    1994-04-01

    Two closely-related aniline monomustards (1 and 2), linked to a DNA-targeting acridine chromophore by a linker chain of different length, show high selectivity for alkylation of polymer DNA. The shorter-chain derivative (2) alkylates mainly at guanine N7 sites, while the longer-chain analogue (1) reacts almost exclusively at adenine N1. The biological effects of these compounds have been studied in standard Ames Salmonella typhimurium strains in order to determine the mutagenic consequences of such well-defined DNA lesions, and the effect of DNA-repair systems on them. Both compounds caused detectable mutations in strains TA1537, TA98 or TA100 and some related strains. Mutation rates were greatly enhanced in strains carrying either a uvrB deletion or the plasmid pKM101. Frameshift mutagenesis by both compounds was completely eliminated by recA deletion, in both the presence or absence of the plasmid. The adenine-selective compound (1) appeared more sensitive to the DNA-repair defects than the guanine-selective derivative (2). Additionally, only the adenine-selective compound (1) caused statistically significant levels of detectable mutation in the repair-proficient strains TA102, TA4001 or TA4006. The bacterial mutagenesis evidence suggests that a bulky, major groove-residing adenine lesion may be more readily recognised by DNA-repair systems, and more likely to lead to a wider range of mutagenic events, than a similar guanine lesion.

  5. Prediction of retention characteristics of heterocyclic compounds.

    PubMed

    Nesměrák, Karel; Toropov, Andrey A; Toropova, Alla P; Yildiz, Ilkay; Yalcin, Ismail; Brozikova, Marketa; Klimešová, Vera; Waisser, Karel

    2015-12-01

    The CORAL software ( http://www.insilico.eu/coral ) was used to build up quantitative structure-property relationships (QSPRs) for the retention characteristics of 93 derivatives of three groups of heterocyclic compounds: 2-phenyl-1,3-benzoxazoles, 4-benzylsulfanylpyridines, and benzoxazines. The QSPRs are one-variable models based on the optimal descriptors calculated from the molecular structure represented by simplified molecular input-line entry systems (SMILES). Each symbol (or two undivided symbols) of SMILES is characterized by correlation weight. The optimal descriptor is the sum of the correlation weights. The numerical data on the correlation weights were calculated with the Monte Carlo method by the manner which provides best correlation between endpoint and optimal descriptor for the calibration set. The predictive ability of the model is checked with the validation set (compounds invisible during building up of the model). The approach has been checked with three random splits into the training, calibration, and validation sets: all models have apparent predictive potential. The mechanistic interpretation of the molecular features extracted from SMILES as the promoters of increase or decrease of examined endpoints is suggested.

  6. Computational design of fused heterocyclic energetic materials

    NASA Astrophysics Data System (ADS)

    Tsyshevskiy, Roman; Pagoria, Philip; Batyrev, Iskander; Kuklja, Maija

    A continuous traditional search for effective energetic materials is often based on a trial and error approach. Understanding of fundamental correlations between the structure and sensitivity of the materials remains the main challenge for design of novel energetics due to the complexity of the behavior of energetic materials. State of the art methods of computational chemistry and solid state physics open new compelling opportunities in simulating and predicting a response of the energetic material to various external stimuli. Hence, theoretical and computational studies can be effectively used not only for an interpretation of sensitivity mechanisms of widely used explosives, but also for identifying criteria for material design prior to its synthesis and experimental characterization. We report here, how knowledge on thermal stability of recently synthesized materials of LLM series is used for design of novel fused heterocyclic energetic materials, including DNBTT (2,7-dinitro-4H,9H-bis([1, 2, 4"]triazolo)[1,5-b:1',5'-e][1, 2, 4, 5]tetrazine), compound with high thermal stability, which is on par or better than that of TATB. This research is supported by ONR (Grant N00014-12-1-0529), NSF XSEDE resources (Grant DMR-130077) and DOE NERSC resources (Contract DE-AC02-05CH11231).

  7. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    PubMed Central

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  8. Ancient DNA reveals differences in behaviour and sociality between brown bears and extinct cave bears.

    PubMed

    Fortes, Gloria G; Grandal-d'Anglade, Aurora; Kolbe, Ben; Fernandes, Daniel; Meleg, Ioana N; García-Vázquez, Ana; Pinto-Llona, Ana C; Constantin, Silviu; de Torres, Trino J; Ortiz, Jose E; Frischauf, Christine; Rabeder, Gernot; Hofreiter, Michael; Barlow, Axel

    2016-10-01

    Ancient DNA studies have revolutionized the study of extinct species and populations, providing insights on phylogeny, phylogeography, admixture and demographic history. However, inferences on behaviour and sociality have been far less frequent. Here, we investigate the complete mitochondrial genomes of extinct Late Pleistocene cave bears and middle Holocene brown bears that each inhabited multiple geographically proximate caves in northern Spain. In cave bears, we find that, although most caves were occupied simultaneously, each cave almost exclusively contains a unique lineage of closely related haplotypes. This remarkable pattern suggests extreme fidelity to their birth site in cave bears, best described as homing behaviour, and that cave bears formed stable maternal social groups at least for hibernation. In contrast, brown bears do not show any strong association of mitochondrial lineage and cave, suggesting that these two closely related species differed in aspects of their behaviour and sociality. This difference is likely to have contributed to cave bear extinction, which occurred at a time in which competition for caves between bears and humans was likely intense and the ability to rapidly colonize new hibernation sites would have been crucial for the survival of a species so dependent on caves for hibernation as cave bears. Our study demonstrates the potential of ancient DNA to uncover patterns of behaviour and sociality in ancient species and populations, even those that went extinct many tens of thousands of years ago. © 2016 John Wiley & Sons Ltd.

  9. Mismatched DNTP Incorporation By DNA Polymerase Beta Does Not Proceed Via Globally Different Conformational Pathways

    SciTech Connect

    Tang, K.-H.; Niebuhr, M.; Tung, C.-S.; Chan, H.-c.; Chou, C.-C.; Tsai, M.-D.

    2009-05-26

    Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase {beta} (Pol {beta}) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln{sup 260}, Tyr{sup 296}, Glu{sup 295} and Arg{sup 258}, freeing up Asp{sup 192} to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.

  10. Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells

    PubMed Central

    Kong, Xiangduo; Mohanty, Samarendra K.; Stephens, Jared; Heale, Jason T.; Gomez-Godinez, Veronica; Shi, Linda Z.; Kim, Jong-Soo; Yokomori, Kyoko; Berns, Michael W.

    2009-01-01

    Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed. PMID:19357094

  11. DNA stretching on the wall surfaces in curved microchannels with different radii

    NASA Astrophysics Data System (ADS)

    Hsieh, Shou-Shing; Wu, Fong-He; Tsai, Ming-Ju

    2014-08-01

    DNA molecule conformation dynamics and stretching were made on semi-circular surfaces with different radii (500 to 5,000 μm) in microchannels measuring 200 μm × 200 μm in cross section. Five different buffer solutions - 1× Tris-acetate-EDTA (TAE), 1× Tris-borate-EDTA (TBE), 1× Tris-EDTA (TE), 1× Tris-phosphate-EDTA (TPE), and 1× Tris-buffered saline (TBS) solutions - were used with a variety of viscosity such as 40, 60, and 80 cP, with resultant 10-4 ≤ Re ≤ 10-3 and the corresponding 5 ≤ Wi ≤ 12. The test fluids were seeded with JOJO-1 tracer particles for flow visualization and driven through the test channels via a piezoelectric (PZT) micropump. Micro particle image velocimetry (μPIV) measuring technique was applied for the centered-plane velocity distribution measurements. It is found that the radius effect on the stretch ratio of DNA dependence is significant. The stretch ratio becomes larger as the radius becomes small due to the larger centrifugal force. Consequently, the maximum stretch was found at the center of the channel with a radius of 500 μm.

  12. Practical and innate carbon-hydrogen functionalization of heterocycles.

    PubMed

    Fujiwara, Yuta; Dixon, Janice A; O'Hara, Fionn; Funder, Erik Daa; Dixon, Darryl D; Rodriguez, Rodrigo A; Baxter, Ryan D; Herlé, Bart; Sach, Neal; Collins, Michael R; Ishihara, Yoshihiro; Baran, Phil S

    2012-12-06

    Nitrogen-rich heterocyclic compounds have had a profound effect on human health because these chemical motifs are found in a large number of drugs used to combat a broad range of diseases and pathophysiological conditions. Advances in transition-metal-mediated cross-coupling have simplified the synthesis of such molecules; however, C-H functionalization of medicinally important heterocycles that does not rely on pre-functionalized starting materials is an underdeveloped area. Unfortunately, the innate properties of heterocycles that make them so desirable for biological applications--such as aqueous solubility and their ability to act as ligands--render them challenging substrates for direct chemical functionalization. Here we report that zinc sulphinate salts can be used to transfer alkyl radicals to heterocycles, allowing for the mild (moderate temperature, 50 °C or less), direct and operationally simple formation of medicinally relevant C-C bonds while reacting in a complementary fashion to other innate C-H functionalization methods (Minisci, borono-Minisci, electrophilic aromatic substitution, transition-metal-mediated C-H insertion and C-H deprotonation). We prepared a toolkit of these reagents and studied their reactivity across a wide range of heterocycles (natural products, drugs and building blocks) without recourse to protecting-group chemistry. The reagents can even be used in tandem fashion in a single pot in the presence of water and air.

  13. Synthesis of medicinally privileged heterocycles through dielectric heating.

    PubMed

    Bandyopadhyay, Debasish; Banik, Bimal K

    2017-02-23

    This review summarizes the potential application of microwave irradiation (dielectric heating) to synthesize biologically important heterocyclic small molecules in the recent past. A huge number of heterocyclic compounds are present in various natural sources like plant, marine microbe or other organisms and many of them possess unique biological activity. In addition to nature-derived heterocyclic compounds, a large number of synthetic heterocycles are being used as medicines. This review describes the relevant recent examples of microwave irradiation to accomplish various chemical transformations accelerated by a variety of catalysts which include, but not limited to, Lewis acids, other metal containing catalysts, organocatalysts, heterogeneous catalysts, phase-transfer catalysts, solid-supported catalysts, inorganic catalysts (bases, acids and salts) and so on. Although there are an increasing number of reports on application of dielectric heating in various other fields, this review is focused on a large number of new and novel strategies related to synthetic organic chemistry. The discussion is mostly organized by the disease type although some reactions/molecules can certainly be placed in multiple sections. Since green chemistry is an extremely emerging and comparatively new field of research, attempts to stimulate more activities on green medicinal chemistry are provided. Discussion related to the concurrent effect of microwaves, catalysts and/or solvents, supports to constitute expeditious and general route for the syntheses of medicinally important heterocyclic compounds and pharmacophores has also been included. While every effort has been made to include all pertinent reports in this field, any omission is unintentional.

  14. Recent advances in the electrochemical construction of heterocycles.

    PubMed

    Francke, Robert

    2014-01-01

    Due to the fact that the major portion of pharmaceuticals and agrochemicals contains heterocyclic units and since the overall number of commercially used heterocyclic compounds is steadily growing, heterocyclic chemistry remains in the focus of the synthetic community. Enormous efforts have been made in the last decades in order to render the production of such compounds more selective and efficient. However, most of the conventional methods for the construction of heterocyclic cores still involve the use of strong acids or bases, the operation at elevated temperatures and/or the use of expensive catalysts and reagents. In this regard, electrosynthesis can provide a milder and more environmentally benign alternative. In fact, numerous examples for the electrochemical construction of heterocycles have been reported in recent years. These cases demonstrate that ring formation can be achieved efficiently under ambient conditions without the use of additional reagents. In order to account for the recent developments in this field, a selection of representative reactions is presented and discussed in this review.

  15. Recent advances in the electrochemical construction of heterocycles

    PubMed Central

    2014-01-01

    Summary Due to the fact that the major portion of pharmaceuticals and agrochemicals contains heterocyclic units and since the overall number of commercially used heterocyclic compounds is steadily growing, heterocyclic chemistry remains in the focus of the synthetic community. Enormous efforts have been made in the last decades in order to render the production of such compounds more selective and efficient. However, most of the conventional methods for the construction of heterocyclic cores still involve the use of strong acids or bases, the operation at elevated temperatures and/or the use of expensive catalysts and reagents. In this regard, electrosynthesis can provide a milder and more environmentally benign alternative. In fact, numerous examples for the electrochemical construction of heterocycles have been reported in recent years. These cases demonstrate that ring formation can be achieved efficiently under ambient conditions without the use of additional reagents. In order to account for the recent developments in this field, a selection of representative reactions is presented and discussed in this review. PMID:25550752

  16. Apples and oranges: avoiding different priors in Bayesian DNA sequence analysis

    PubMed Central

    2010-01-01

    Background One of the challenges of bioinformatics remains the recognition of short signal sequences in genomic DNA such as donor or acceptor splice sites, splicing enhancers or silencers, translation initiation sites, transcription start sites, transcription factor binding sites, nucleosome binding sites, miRNA binding sites, or insulator binding sites. During the last decade, a wealth of algorithms for the recognition of such DNA sequences has been developed and compared with the goal of improving their performance and to deepen our understanding of the underlying cellular processes. Most of these algorithms are based on statistical models belonging to the family of Markov random fields such as position weight matrix models, weight array matrix models, Markov models of higher order, or moral Bayesian networks. While in many comparative studies different learning principles or different statistical models have been compared, the influence of choosing different prior distributions for the model parameters when using different learning principles has been overlooked, and possibly lead to questionable conclusions. Results With the goal of allowing direct comparisons of different learning principles for models from the family of Markov random fields based on the same a-priori information, we derive a generalization of the commonly-used product-Dirichlet prior. We find that the derived prior behaves like a Gaussian prior close to the maximum and like a Laplace prior in the far tails. In two case studies, we illustrate the utility of the derived prior for a direct comparison of different learning principles with different models for the recognition of binding sites of the transcription factor Sp1 and human donor splice sites. Conclusions We find that comparisons of different learning principles using the same a-priori information can lead to conclusions different from those of previous studies in which the effect resulting from different priors has been neglected. We

  17. Apples and oranges: avoiding different priors in Bayesian DNA sequence analysis.

    PubMed

    Keilwagen, Jens; Grau, Jan; Posch, Stefan; Grosse, Ivo

    2010-03-22

    One of the challenges of bioinformatics remains the recognition of short signal sequences in genomic DNA such as donor or acceptor splice sites, splicing enhancers or silencers, translation initiation sites, transcription start sites, transcription factor binding sites, nucleosome binding sites, miRNA binding sites, or insulator binding sites. During the last decade, a wealth of algorithms for the recognition of such DNA sequences has been developed and compared with the goal of improving their performance and to deepen our understanding of the underlying cellular processes. Most of these algorithms are based on statistical models belonging to the family of Markov random fields such as position weight matrix models, weight array matrix models, Markov models of higher order, or moral Bayesian networks. While in many comparative studies different learning principles or different statistical models have been compared, the influence of choosing different prior distributions for the model parameters when using different learning principles has been overlooked, and possibly lead to questionable conclusions. With the goal of allowing direct comparisons of different learning principles for models from the family of Markov random fields based on the same a-priori information, we derive a generalization of the commonly-used product-Dirichlet prior. We find that the derived prior behaves like a Gaussian prior close to the maximum and like a Laplace prior in the far tails. In two case studies, we illustrate the utility of the derived prior for a direct comparison of different learning principles with different models for the recognition of binding sites of the transcription factor Sp1 and human donor splice sites. We find that comparisons of different learning principles using the same a-priori information can lead to conclusions different from those of previous studies in which the effect resulting from different priors has been neglected. We implement the derived prior is

  18. Specific Inhibition of the Eubacterial DNA Ligase by Arylamino Compounds

    PubMed Central

    Ciarrocchi, Giovanni; MacPhee, Donald G.; Deady, Les W.; Tilley, Leann

    1999-01-01

    All known DNA ligases catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA via very similar mechanisms. The ligase family can, however, be divided into two classes: eubacterial ligases, which require NAD+ as a cofactor, and other ligases, from viruses, archaea, and eukaryotes, which use ATP. Drugs that discriminate between DNA ligases from different sources may have antieubacterial activity. We now report that a group of arylamino compounds, including some commonly used antimalarial and anti-inflammatory drugs and a novel series of bisquinoline compounds, are specific inhibitors of eubacterial DNA ligases. Members of this group of inhibitors have different heterocyclic ring systems with a common amino side chain in which the two nitrogens are separated by four carbon atoms. The potency, but not the specificity of action, is influenced by the DNA-binding characteristics of the inhibitor, and the inhibition is noncompetitive with respect to NAD+. The arylamino compounds appear to target eubacterial DNA ligase in vivo, since a Salmonella Lig− strain that has been rescued with the ATP-dependent T4 DNA ligase is less sensitive than the parental Salmonella strain. PMID:10543760

  19. Detection Theory in Identification of RNA-DNA Sequence Differences Using RNA-Sequencing

    PubMed Central

    Toung, Jonathan M.; Lahens, Nicholas; Hogenesch, John B.; Grant, Gregory

    2014-01-01

    Advances in sequencing technology have allowed for detailed analyses of the transcriptome at single-nucleotide resolution, facilitating the study of RNA editing or sequence differences between RNA and DNA genome-wide. In humans, two types of post-transcriptional RNA editing processes are known to occur: A-to-I deamination by ADAR and C-to-U deamination by APOBEC1. In addition to these sequence differences, researchers have reported the existence of all 12 types of RNA-DNA sequence differences (RDDs); however, the validity of these claims is debated, as many studies claim that technical artifacts account for the majority of these non-canonical sequence differences. In this study, we used a detection theory approach to evaluate the performance of RNA-Sequencing (RNA-Seq) and associated aligners in accurately identifying RNA-DNA sequence differences. By generating simulated RNA-Seq datasets containing RDDs, we assessed the effect of alignment artifacts and sequencing error on the sensitivity and false discovery rate of RDD detection. Overall, we found that even in the presence of sequencing errors, false negative and false discovery rates of RDD detection can be contained below 10% with relatively lenient thresholds. We also assessed the ability of various filters to target false positive RDDs and found them to be effective in discriminating between true and false positives. Lastly, we used the optimal thresholds we identified from our simulated analyses to identify RDDs in a human lymphoblastoid cell line. We found approximately 6,000 RDDs, the majority of which are A-to-G edits and likely to be mediated by ADAR. Moreover, we found the majority of non A-to-G RDDs to be associated with poorer alignments and conclude from these results that the evidence for widespread non-canonical RDDs in humans is weak. Overall, we found RNA-Seq to be a powerful technique for surveying RDDs genome-wide when coupled with the appropriate thresholds and filters. PMID:25396741

  20. EVALUATION OF DIFFERENT METHODS FOR THE EXTRACTION OF DNA FROM FUNGAL CONIDIA BY QUANTITATIVE COMPETITIVE PCR ANALYSIS

    EPA Science Inventory

    Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartaru...

  1. EVALUATION OF DIFFERENT METHODS FOR THE EXTRACTION OF DNA FROM FUNGAL CONIDIA BY QUANTITATIVE COMPETITIVE PCR ANALYSIS

    EPA Science Inventory

    Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartaru...

  2. Construction of Nine-Membered Heterocycles through Palladium-Catalyzed Formal [5+4] Cycloaddition.

    PubMed

    Yang, Li-Cheng; Rong, Zi-Qiang; Wang, Ya-Nong; Tan, Zher Yin; Wang, Min; Zhao, Yu

    2017-03-06

    The first catalytic formal [5+4] cycloaddition to prepare nine-membered heterocycles is presented. Under palladium catalysis, the reaction of N-tosyl azadienes and substituted vinylethylene carbonates (VECs) proceeds smoothly to produce benzofuran-fused heterocycles in uniformly high efficiency. Highly diastereoselective functionalization of the nine-membered heterocycles through peripheral attack is also demonstrated.

  3. Iron-Catalyzed Arylation of Heterocycles via Directed C–H Bond Activation

    PubMed Central

    2015-01-01

    The iron-catalyzed arylation of aromatic heterocycles, such as pyridines, thiophenes, and furans, has been achieved. The use of an imine directing group allowed for the ortho functionalization of these heterocycles with complete conversion in 15 min at 0 °C. Yields up to 88% were observed in the synthesis of 15 heterocyclic biaryls. PMID:24450989

  4. Genome-wide gene expression and DNA methylation differences in abnormally cloned and normally natural mating piglets.

    PubMed

    Zou, C; Fu, Y; Li, C; Liu, H; Li, G; Li, J; Zhang, H; Wu, Y; Li, C

    2016-08-01

    Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways (e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer.

  5. Difference between ²JC2H3 and ²JC3H2 spin-spin couplings in heterocyclic five- and six-membered rings as a probe for studying σ-ring currents: a quantum chemical analysis.

    PubMed

    Contreras, Rubén H; dos Santos, Francisco P; Ducati, Lucas C; Tormena, Cláudio F

    2010-12-01

    Adequate analyses of canonical molecular orbitals (CMOs) can provide rather detailed information on the importance of different σ-Fermi contact (FC) coupling pathways (FC term transmitted through the σ-skeleton). Knowledge of the spatial distribution of CMOs is obtained by expanding them in terms of natural bond orbitals (NBOs). Their relative importance for transmitting the σ-FC contribution to a given spin-spin coupling constants (SSCCs) is estimated by resorting to the expression of the FC term given by the polarisation propagator formalism. In this way, it is possible to classify the effects affecting such couplings in two different ways: delocalisation interactions taking place in the neighbourhood of the coupling nuclei and 'round the ring' effects. The latter, associated with σ-ring currents, are observed to yield significant differences between the FC terms of (2)J(C2H3) and (2)J(C3H2) SSCCs which, consequently, are taken as probes to gauge the differences in σ-ring currents for the five-membered rings (furan, thiophene, selenophene and pyrrol) and also for the six-membered rings (benzene, pyridine, protonated pyridine and N-oxide pyridine) used in the present study. Copyright © 2010 John Wiley & Sons, Ltd.

  6. Mitochondrial DNA levels in blood and tissue samples from breast cancer patients of different stages.

    PubMed

    Xia, Peng; Wang, Hui-Juan; Geng, Ting-Ting; Xun, Xiao-Jie; Zhou, Wen-Jing; Jin, Tian-Bo; Chen, Chao

    2014-01-01

    Alterations in mitochondrial DNA (mtDNA) have been implicated in carcinogenesis and tumor progression. We here evaluated the diagnostic and prognostic potential of mtDNA as a biomarker for breast cancer. Using multiplex real-time polymerase chain reaction, nuclear DNA (nDNA) and mtDNA levels in serum, buffy coat, tumor, and tumor-adjacent tissue samples from 50 breast cancer patients were determined and assessed for associations with clinicopathological features. To evaluate mtDNA as a biomarker for distinguishing between the four sample types, we created receiver operating characteristic (ROC) curves. The mtDNA levels in buffy coat were significantly lower than in other sample types. Relative to tumor-adjacent tissue, reduced levels of mtDNA were identified in buffy coat and tumor tissue but not in serum. According to ROC curve analysis, mtDNA levels could be used to distinguish between buffy coat and tumor-adjacent tissue samples with good sensitivity (77%) and specificity (83%). Moreover, mtDNA levels in serum and tumor tissue were positively associated with cancer TMN stage. The mtDNA levels in blood samples may represent a promising, non-invasive biomarker in breast cancer patients. Additional, large-scale validation studies are required to establish the potential use of mtDNA levels in the early diagnosis and monitoring of breast cancer.

  7. Activation of different split functionalities on re-association of RNA-DNA hybrids

    NASA Astrophysics Data System (ADS)

    Afonin, Kirill A.; Viard, Mathias; Martins, Angelica N.; Lockett, Stephen J.; Maciag, Anna E.; Freed, Eric O.; Heldman, Eliahu; Jaeger, Luc; Blumenthal, Robert; Shapiro, Bruce A.

    2013-04-01

    Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of `smart' nucleic acid-based nanoparticles and switches for various biomedical applications.

  8. DNA-mediated adjuvant immunotherapy extends survival in two different mouse models of myeloid malignancies

    PubMed Central

    Le Pogam, Carole; Patel, Satyananda; Gorombei, Petra; Guerenne, Laura; Krief, Patricia; Omidvar, Nader; Tekin, Nilgun; Bernasconi, Elena; Sicre, Flore; Schlageter, Marie-Helene; Chopin, Martine; Noguera, Maria-Elena; West, Robert; Abu, Ansu; Mathews, Vikram; Pla, Marika; Fenaux, Pierre; Chomienne, Christine; Padua, Rose Ann

    2015-01-01

    We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers. PMID:26378812

  9. DNA-mediated adjuvant immunotherapy extends survival in two different mouse models of myeloid malignancies.

    PubMed

    Le Pogam, Carole; Patel, Satyananda; Gorombei, Petra; Guerenne, Laura; Krief, Patricia; Omidvar, Nader; Tekin, Nilgun; Bernasconi, Elena; Sicre, Flore; Schlageter, Marie-Helene; Chopin, Martine; Noguera, Maria-Elena; West, Robert; Abu, Ansu; Mathews, Vikram; Pla, Marika; Fenaux, Pierre; Chomienne, Christine; Padua, Rose Ann

    2015-10-20

    We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers.

  10. Chalcogen containing heterocyclic scaffolds: New hybrids with antitumoral activity.

    PubMed

    Alcolea, Verónica; Plano, Daniel; Encío, Ignacio; Palop, Juan Antonio; Sharma, Arun K; Sanmartín, Carmen

    2016-11-10

    In this work, 27 novel hybrid derivatives containing diverse substituents with chalcogen atoms (selenium or sulfur) and several active heterocyclic scaffolds have been synthesized. Compounds were tested against two human cancer cells lines (MCF7 and PC-3) and a normal human mammary epithelial cell line (184B5) in order to determine their activity and selectivity against malignant cells. Ten compounds showed GI50 values below 10 μM in at least one of the cancer cell lines and six of them exhibited a selectivity index higher than 9. In general, selenium-containing compounds were more active than their corresponding sulfur analogs but we found some thiocyanate derivatives with comparable or higher activity and selectivity. Among the different substituents, the seleno- and thio-cyanate groups showed the most promising results. On the basis of their potent activity and high selectivity index, compounds 7e and 8f (containing a thiocyanate and a selenocyanate group, respectively) were selected for further biological evaluation. Both the compounds induced caspase-dependent cell death and cell cycle arrest in G2/M phase. In addition, these compounds do not violate any of the Lipinski's Rule of Five and thus possess good potential to become drugs, compound 7e being particularly promising. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Unconventional interactions between water and heterocyclic nitrogens in protein structures.

    PubMed

    Stollar, Elliott J; Gelpí, Jose Luis; Velankar, Sameer; Golovin, Adel; Orozco, Modesto; Luisi, Ben F

    2004-10-01

    We report an unusual interaction in which a water molecule approaches the heterocyclic nitrogen of tryptophan and histidine along an axis that is roughly perpendicular to the aromatic plane of the side chain. The interaction is distinct from the well-known conventional aromatic hydrogen-bond, and it occurs at roughly the same frequency in protein structures. Calculations indicate that the water-indole interaction is favorable energetically, and we find several cases in which such contacts are conserved among structural orthologs. The indole-water interaction links side chains and peptide backbone in turn regions, connects the side chains in beta-sheets, and bridges secondary elements from different domains. We suggest that the water-indole interaction can be indirectly responsible for the quenching of tryptophan fluorescence that is observed in the folding of homeodomains and, possibly, many other proteins. We also observe a similar interaction between water and the imidazole nitrogens of the histidine side chain. Taken together, these observations suggest that the unconventional water-indole and water-imidazole interactions provide a small but favorable contribution to protein structures.

  12. Effect of Different Human Papillomavirus Serological and DNA Criteria on Vaccine Efficacy Estimates

    PubMed Central

    Lang Kuhs, Krystle A.; Porras, Carolina; Schiller, John T.; Rodriguez, Ana Cecilia; Schiffman, Mark; Gonzalez, Paula; Wacholder, Sholom; Ghosh, Arpita; Li, Yan; Lowy, Douglas R.; Kreimer, Aimée R.; Poncelet, Sylviane; Schussler, John; Quint, Wim; van Doorn, Leen-Jan; Sherman, Mark E.; Sidawy, Mary; Herrero, Rolando; Hildesheim, Allan; Safaeian, Mahboobeh; Lang Kuhs, Krystle A.; Schiller, John T.; Schiffman, Mark; Wacholder, Sholom; Lowy, Douglas R.; Kreimer, Aimée R.; Sherman, Mark E.; Hildesheim, Allan; Safaeian, Mahboobeh; Porras, Carolina; Rodriguez, Ana Cecilia; Gonzalez, Paula; Herrero, Rolando; Gonzalez, Paula; Herrero, Rolando; Ghosh, Arpita; Li, Yan; Poncelet, Sylviane; Schussler, John; Quint, Wim; van Doorn, Leen-Jan; Sidawy, Mary; Self, Steve; Benavides, Adriana; Calzada, Luis Diego; Karron, Ruth; Nayar, Ritu; Roach, Nancy; Cain, Joanna; Davey, Diane; DeMets, David; Fuster, Francisco; Gershon, Ann; Holly, Elizabeth; Raventós, Henriette; Rida, Wasima; Rosero-Bixby, Luis; Suthers, Kristen; Lara, Silvia; Thomas, Sarah; Alfaro, Mario; Barrantes, Manuel; Concepción Bratti, M.; Cárdenas, Fernando; Cortés, Bernal; Espinoza, Albert; Estrada, Yenory; González, Paula; Guillén, Diego; Herrero, Roland; Jiménez, Silvia E.; Morales, Jorge; Villegas, Luis; Morera, Lidia Ana; Pérez, Elmer; Porras, Carolina; Rodríguez, Ana Cecilia; Rivas, Libia; Freer, Enrique; Bonilla, José; García-Piñeres, Alfanso; Silva, Sandra; Atmella, Ivannia; Ramírez, Margarita; Hildesheim, Allan; Kreimer, Aimée R.; Lowy, Douglas R.; Macklin, Nora; Schiffman, Mark; Schiller, John T.; Sherman, Mark; Solomon, Diane; Wacholder, Sholom; Pinto, Ligia; Kemp, Troy; Eklund, Claire; Hutchinson, Martha; Sidawy, Mary; Quint, Wim; van Doorn, Leen-Jan

    2014-01-01

    Two trials of clinically approved human papillomavirus (HPV) vaccines, Females United to Unilaterally Reduce Endo/Ectocervical Disease (FUTURE I/II) and the Papilloma Trial Against Cancer in Young Adults (PATRICIA), reported a 22% difference in vaccine efficacy (VE) against cervical intraepithelial neoplasia grade 2 or worse in HPV-naïve subcohorts; however, serological testing methods and the HPV DNA criteria used to define HPV-unexposed women differed between the studies. We applied previously described methods to simulate these HPV-naïve subcohorts within the Costa Rica HPV16/18 Vaccine Trial and assessed how these criteria affect the estimation of VE. We applied 2 enzyme-linked immunosorbent assay (ELISA) thresholds for HPV16 and HPV18 seropositivity (8 and 7 ELISA units/mL, respectively, for PATRICIA; 54 and 65 ELISA units/mL, respectively, for FUTURE I/II (to approximate the competitive Luminex immunoassay)) and 2 criteria for HPV DNA positivity (12 oncogenic HPV types, plus HPV66 and 68/73 for PATRICIA; or plus HPV6 and 11 for FUTURE I/II). VE was computed in the 2 naïve subcohorts. Using the FUTURE I/II and PATRICIA criteria, VE estimates against cervical intraepithelial neoplasia grade 2 or worse, regardless of HPV type, were 69.0% (95% confidence interval: 40.3%, 84.9%) and 80.8% (95% confidence interval: 52.6%, 93.5%), respectively (P = 0.1). Although the application of FUTURE I/II criteria to our cohort resulted in the inclusion of more sexually experienced women, methodological differences did not fully explain the VE differences. PMID:25139208

  13. Effect of different human papillomavirus serological and DNA criteria on vaccine efficacy estimates.

    PubMed

    Lang Kuhs, Krystle A; Porras, Carolina; Schiller, John T; Rodriguez, Ana Cecilia; Schiffman, Mark; Gonzalez, Paula; Wacholder, Sholom; Ghosh, Arpita; Li, Yan; Lowy, Douglas R; Kreimer, Aimée R; Poncelet, Sylviane; Schussler, John; Quint, Wim; van Doorn, Leen-Jan; Sherman, Mark E; Sidawy, Mary; Herrero, Rolando; Hildesheim, Allan; Safaeian, Mahboobeh

    2014-09-15

    Two trials of clinically approved human papillomavirus (HPV) vaccines, Females United to Unilaterally Reduce Endo/Ectocervical Disease (FUTURE I/II) and the Papilloma Trial Against Cancer in Young Adults (PATRICIA), reported a 22% difference in vaccine efficacy (VE) against cervical intraepithelial neoplasia grade 2 or worse in HPV-naïve subcohorts; however, serological testing methods and the HPV DNA criteria used to define HPV-unexposed women differed between the studies. We applied previously described methods to simulate these HPV-naïve subcohorts within the Costa Rica HPV16/18 Vaccine Trial and assessed how these criteria affect the estimation of VE. We applied 2 enzyme-linked immunosorbent assay (ELISA) thresholds for HPV16 and HPV18 seropositivity (8 and 7 ELISA units/mL, respectively, for PATRICIA; 54 and 65 ELISA units/mL, respectively, for FUTURE I/II (to approximate the competitive Luminex immunoassay)) and 2 criteria for HPV DNA positivity (12 oncogenic HPV types, plus HPV66 and 68/73 for PATRICIA; or plus HPV6 and 11 for FUTURE I/II). VE was computed in the 2 naïve subcohorts. Using the FUTURE I/II and PATRICIA criteria, VE estimates against cervical intraepithelial neoplasia grade 2 or worse, regardless of HPV type, were 69.0% (95% confidence interval: 40.3%, 84.9%) and 80.8% (95% confidence interval: 52.6%, 93.5%), respectively (P = 0.1). Although the application of FUTURE I/II criteria to our cohort resulted in the inclusion of more sexually experienced women, methodological differences did not fully explain the VE differences. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  14. Boron-Heteroatom Addition Reactions via Borylative Heterocyclization: Oxyboration, Aminoboration, and Thioboration.

    PubMed

    Issaian, Adena; Tu, Kim N; Blum, Suzanne A

    2017-09-21

    oxyboration to form borylated lactones from o-alkynyl esters is then described. This class of reactions takes advantage of bifunctional ClBcat as a carbophilic carbon-carbon π-bond activator and eventual dealkylating agent. We describe our motivation in developing this new class of catalyst-free borylation reactions and subsequently applying the formal borylation strategy to the thioboration of o-alkynylthioanisole substrates to form borylated benzothiophenes. We then proceed to describe our investigations into the details of the mechanism of the formal thioboration reaction. These collaborative mechanistic studies included experimental and computational findings that elucidated the rate-determining step and intermediates of the reaction. These studies further compared different boron sources as electrophiles, including those used in other known reactions, providing fundamental knowledge about the capabilities of commercially available boron reagents toward borylative heterocyclization. Our findings provide guiding principles for reaction design and information leading toward the design of a diverse set of boron-heteroatom addition reactions and their formal equivalents that proceed through borylative heterocyclization.

  15. Development and validation of method for heterocyclic compounds in wine: optimization of HS-SPME conditions applying a response surface methodology.

    PubMed

    Burin, Vívian Maria; Marchand, Stéphanie; de Revel, Gilles; Bordignon-Luiz, Marilde T

    2013-12-15

    Considering the importance of the heterocyclic compounds in terms of wine flavor, this study aims to propose a new rapid and solvent free method to quantify different classes of heterocyclic compounds, such as furans, thiophenes, thiazols and pyrazines, which are products of the Maillard reaction, in wines. The use of a central composite design and the response surface methodology to determine the best conditions allows the optimum combination of analytical variables (pH, NaCl and extraction time) to be identified. The validation was carried out using several types of wine as matrices. The method shows satisfactory repeatability (2.7%different heterocyclic compounds were determined, mainly for red wines. © 2013 Elsevier B.V. All rights reserved.

  16. Syntheses and Reactions of Chalcogen-containing Heterocycles.

    PubMed

    Sashida, Haruki

    2016-01-01

    The advances in my laboratory for the past 20-25 years concerning the chemistry of chalcogen-containing heterocycles are reviewed. The intramolecular cyclization of the chalcogenols (-TeH, -SeH, -SH) into a triple bond or appropriate leaving group produced various chalcogen-containing heterocycles. The reactions of the obtained products were examined: the reactions of 1-benzo- and 2-benzopyrylium salts containing a tellurium or selenium element with several nucleophiles, including alkoxides, amines, the cyanide ion, an active methyl compound (acetone), Grignard reagents, copper reagents, and tin reagents, along with hydrogenation and hydrolysis reactions, provided corresponding chromes or isochromes having various functional groups at the 2- or 1-C position. Isothiocyanate and isoselenocyanate were used as chalcogen sources for the preparation of five- or six-membered heterocycles. In addition, double intramolecular cyclization, ring-expansion reactions, electrophilic cyclization and iodocyclization were also carried out.

  17. Heterocyclic Curcumin Derivatives of Pharmacological Interest: Recent Progress.

    PubMed

    Martinez-Cifuentes, Maximiliano; Weiss-Lopez, Boris; Santos, Leonardo S; Araya-Maturana, Ramiro

    2015-01-01

    Curcumin, a natural yellow polyphenol, is isolated from the herb Curcuma longa L. (turmeric), a member of the ginger family. It has been extensively studied due to their multiple pharmacological properties. Nevertheless, curcumin has disadvantages such as poor water solubility, poor bioavailability and rapid metabolism, which has prompted the search for analogues that overcome these shortcomings while maintaining or improving their good pharmacological properties. Among the main curcumin analogues that have been developed, the heterocyclic curcuminoids show a high interest. In this review, we describe recent progress in the synthesis and pharmacological properties of new heterocyclic curcumin derivatives. The most recent developments in anti-cancer, anti-Alzheimer, anti-bacterial and anti-oxidants heterocyclic curcumin derivatives are covered.

  18. Selective DNA methylation of BDNF promoter in bipolar disorder: differences among patients with BDI and BDII.

    PubMed

    D'Addario, Claudio; Dell'Osso, Bernardo; Palazzo, Maria Carlotta; Benatti, Beatrice; Lietti, Licia; Cattaneo, Elisabetta; Galimberti, Daniela; Fenoglio, Chiara; Cortini, Francesca; Scarpini, Elio; Arosio, Beatrice; Di Francesco, Andrea; Di Benedetto, Manuela; Romualdi, Patrizia; Candeletti, Sanzio; Mari, Daniela; Bergamaschini, Luigi; Bresolin, Nereo; Maccarrone, Mauro; Altamura, A Carlo

    2012-06-01

    The etiology of bipolar disorder (BD) is still poorly understood, involving genetic and epigenetic mechanisms as well as environmental contributions. This study aimed to investigate the degree of DNA methylation at the promoter region of the brain-derived neurotrophic factor (BDNF) gene, as one of the candidate genes associated with major psychoses, in peripheral blood mononuclear cells isolated from 94 patients with BD (BD I=49, BD II=45) and 52 healthy controls. A significant BDNF gene expression downregulation was observed in BD II 0.53±0.11%; P<0.05), but not in BD I (1.13±0.19%) patients compared with controls (CONT: 1±0.2%). Consistently, an hypermethylation of the BDNF promoter region was specifically found in BD II patients (CONT: 24.0±2.1%; BDI: 20.4±1.7%; BDII: 33.3±3.5%, P<0.05). Of note, higher levels of DNA methylation were observed in BD subjects on pharmacological treatment with mood stabilizers plus antidepressants (34.6±4.2%, predominantly BD II) compared with those exclusively on mood-stabilizing agents (21.7±1.8%; P<0.01, predominantly BD I). Moreover, among the different pharmacological therapies, lithium (20.1±3.8%, P<0.05) and valproate (23.6±2.9%, P<0.05) were associated with a significant reduction of DNA methylation compared with other drugs (35.6±4.6%). Present findings suggest selective changes in DNA methylation of BDNF promoter in subjects with BD type II and highlight the importance of epigenetic factors in mediating the onset and/or susceptibility to BD, providing new insight into the mechanisms of gene expression. Moreover, they shed light on possible mechanisms of action of mood-stabilizing compounds vs antidepressants in the treatment of BD, pointing out that BDNF regulation might be a key target for their effects.

  19. Epigenomic profiling of men exposed to early-life stress reveals DNA methylation differences in association with current mental state.

    PubMed

    Khulan, B; Manning, J R; Dunbar, D R; Seckl, J R; Raikkonen, K; Eriksson, J G; Drake, A J

    2014-09-23

    Early-life stress (ELS) is known to be associated with an increased risk of neuropsychiatric and cardiometabolic disease in later life. One of the potential mechanisms underpinning this is through effects on the epigenome, particularly changes in DNA methylation. Using a well-phenotyped cohort of 83 men from the Helsinki Birth Cohort Study, who experienced ELS in the form of separation from their parents during childhood, and a group of 83 matched controls, we performed a genome-wide analysis of DNA methylation in peripheral blood. We found no differences in DNA methylation between men who were separated from their families and non-separated men; however, we did identify differences in DNA methylation in association with the development of at least mild depressive symptoms over the subsequent 5-10 years. Notably, hypomethylation was identified at a number of genes with roles in brain development and/or function in association with depressive symptoms. Pathway analysis revealed an enrichment of DNA methylation changes in pathways associated with development and morphogenesis, DNA and transcription factor binding and programmed cell death. Our results support the concept that DNA methylation differences may be important in the pathogenesis of psychiatric disease.

  20. Epigenomic profiling of men exposed to early-life stress reveals DNA methylation differences in association with current mental state

    PubMed Central

    Khulan, B; Manning, J R; Dunbar, D R; Seckl, J R; Raikkonen, K; Eriksson, J G; Drake, A J

    2014-01-01

    Early-life stress (ELS) is known to be associated with an increased risk of neuropsychiatric and cardiometabolic disease in later life. One of the potential mechanisms underpinning this is through effects on the epigenome, particularly changes in DNA methylation. Using a well-phenotyped cohort of 83 men from the Helsinki Birth Cohort Study, who experienced ELS in the form of separation from their parents during childhood, and a group of 83 matched controls, we performed a genome-wide analysis of DNA methylation in peripheral blood. We found no differences in DNA methylation between men who were separated from their families and non-separated men; however, we did identify differences in DNA methylation in association with the development of at least mild depressive symptoms over the subsequent 5–10 years. Notably, hypomethylation was identified at a number of genes with roles in brain development and/or function in association with depressive symptoms. Pathway analysis revealed an enrichment of DNA methylation changes in pathways associated with development and morphogenesis, DNA and transcription factor binding and programmed cell death. Our results support the concept that DNA methylation differences may be important in the pathogenesis of psychiatric disease. PMID:25247593

  1. Tissue-specific differences of C-banded heterochromatin in human embryos: The possible role of DNA methylation

    SciTech Connect

    Nazarenko, S.A.; Karageorgii, N.M.

    1995-11-01

    Sizes of C-band heterochromatin regions were estimated in embryonic and extraembryonic lineages. These regions in chromosomes 1 and 16, containing mainly satellite 2 DNA, were significantly longer in extraembryonic tissues than in embryonic tissues. The differences in length between chromosome 9 C-band heterochromatin, which contained satellite 3 DNA, and Y chromosome C-band heterochromatin, which contained all four classical satellite types, were not significant between the two tissues. Our data, together with other findings on the correlation between chromosome compactization and DNA methylation, indicated that the observed variations in C-band length reflected a tissue-specific pattern of heterochromatin methylation. Satellite 2 DNA probably was the most sensitive target for the decondensation effect of DNA methylation compared with other satellite types. 24 refs., 1 tab.

  2. Mitochondrial DNA in the sea urchin Arbacia lixula: nucleotide sequence differences between two polymorphic molecules indicate asymmetry of mutations.

    PubMed

    De Giorgi, C; De Luca, F; Saccone, C

    1991-07-22

    Two polymorphic forms of mitochondrial DNA (mtDNA) extracted from Arbacia lixula eggs were cloned and the nucleotide sequences of specific regions determined. A comparison of the sequences of the sense strand of the two molecules demonstrates that all the differences are transitions and only of the A----G type. A change such as G----A (or A----G) on the sense mtDNA strand results from either a direct G----A (or A----G) mutation on that strand or a C----T (or T----C) on the complementary strand. None of the C----T (or T----C) changes were detected on the sense strand, which implies that the A----G mutation bias on the sense strand is not reversed for the other strand. Our observation indicates the existence of mechanisms acting asymmetrically on the two mtDNA strands, possibly during mtDNA replication.

  3. Do the Four Clades of the mtDNA Haplogroup L2 Evolve at Different Rates?

    PubMed Central

    Torroni, Antonio; Rengo, Chiara; Guida, Valentina; Cruciani, Fulvio; Sellitto, Daniele; Coppa, Alfredo; Calderon, Fernando Luna; Simionati, Barbara; Valle, Giorgio; Richards, Martin; Macaulay, Vincent; Scozzari, Rosaria

    2001-01-01

    Forty-seven mtDNAs collected in the Dominican Republic and belonging to the African-specific haplogroup L2 were studied by high-resolution RFLP and control-region sequence analyses. Four sets of diagnostic markers that subdivide L2 into four clades (L2a–L2d) were identified, and a survey of published African data sets appears to indicate that these clades encompass all L2 mtDNAs and harbor very different geographic/ethnic distributions. One mtDNA from each of the four clades was completely sequenced by means of a new sequencing protocol that minimizes time and expense. The phylogeny of the L2 complete sequences showed that the two mtDNAs from L2b and L2d seem disproportionately derived, compared with those from L2a and L2c. This result is not consistent with a simple model of neutral evolution with a uniform molecular clock. The pattern of nonsynonymous versus synonymous substitutions hints at a role for selection in the evolution of human mtDNA. Regardless of whether selection is shaping the evolution of modern human mtDNAs, the population screening of L2 mtDNAs for the mutations identified by our complete sequence study should allow the identification of marker motifs of younger age with more restricted geographic distributions, thus providing new clues about African prehistory and the origin and relationships of African ethnic groups. PMID:11595973

  4. Different methylation of oestrogen receptor DNA in human breast carcinomas with and without oestrogen receptor.

    PubMed Central

    Piva, R.; Rimondi, A. P.; Hanau, S.; Maestri, I.; Alvisi, A.; Kumar, V. L.; del Senno, L.

    1990-01-01

    The methylation of the human oestrogen receptor (ER) gene was analysed by restriction enzymes in normal and neoplastic human breast tissues and cell lines. CCGG sequences in regions inside the gene, which are methylated both in normal breast and in tissues that are not the target of the oestrogen, are hypomethylated in 30% of tumours, both ER+ and ER- carcinomas. Moreover, 5' sequences of the gene, which are hypomethylated in normal breast and not in tissues not the target of oestrogen, are methylated to a lower degree in ER+ carcinomas, whereas they are methylated to a greater degree in ER- carcinomas. However, the same region is equally hypomethylated in both ER+ and ER- cancer cell lines. Our results indicate that in breast carcinomas ER DNA methylation is deranged, and in cancer cell lines is different from that observed in primary tumours. Furthermore, the abnormal methylation in the 5' end seems to be related to abnormal expression, namely diffuse hypomethylation in carcinomas with high ER content and hypermethylation in carcinomas without ER. These findings support our previous hypothesis that DNA methylation could be involved in the control of ER gene expression and demonstrate that abnormal ER gene methylation is a typical feature of breast cancers. Images Figure 1 Figure 2 Figure 3 PMID:2155643

  5. Protein and DNA oxidation in different anatomic regions of rat brain in a mimetic ageing model.

    PubMed

    Yanar, Karolin; Aydın, Seval; Cakatay, Ufuk; Mengi, Murat; Buyukpınarbaşılı, Nur; Atukeren, Pınar; Sitar, Mustafa E; Sönmez, Aslı; Uslu, Ezel

    2011-12-01

    It has been reported that d-galactose administration causes an increase in oxidative and osmotic stresses in several tissues of rodents. In this study, we established a brain ageing model by using d-galactose and investigated the concentrations of oxidative stress markers on the hippocampus, parietal and frontal lobes of male Sprague-Dawley rats. A mimetic ageing model was established by injecting d-galactose (60 mg/kg/day/i.p.) in the experimental group for 42 days. At the end of this period, we tested spatial memory using the Morris water maze test. To investigate the magnitude of oxidative damage in proteins, lipids and DNA, we studied the concentrations of various oxidative stress parameters in the hippocampus, parietal and frontal lobes of the brain. Glial and neuronal cell oxidative damage was observed in each of the three anatomic regions. It was found that protein carbonyl groups and advanced oxidation product concentrations in the d-galactose applied group were significantly high in each of the three brain lobes compared with the control group. Thiol concentration was found to be decreased in the parietal lobe. A concurrent increase in lipid hydroperoxides was also observed in this lobe. On the other hand, 8-hydroxy-2'-deoxyguanosine concentration was significantly increased in the hippocampal lobe of rats in the experimental group when compared with the controls. The results obtained from the mimetic ageing model rats showed that various anatomical regions of brain have different susceptibility to oxidative damage of proteins, lipids and DNA.

  6. ELK1 uses different DNA binding modes to regulate functionally distinct classes of target genes.

    PubMed

    Odrowaz, Zaneta; Sharrocks, Andrew D

    2012-01-01

    Eukaryotic transcription factors are grouped into families and, due to their similar DNA binding domains, often have the potential to bind to the same genomic regions. This can lead to redundancy at the level of DNA binding, and mechanisms are required to generate specific functional outcomes that enable distinct gene expression programmes to be controlled by a particular transcription factor. Here we used ChIP-seq to uncover two distinct binding modes for the ETS transcription factor ELK1. In one mode, other ETS transcription factors can bind regulatory regions in a redundant fashion; in the second, ELK1 binds in a unique fashion to another set of genomic targets. Each binding mode is associated with different binding site features and also distinct regulatory outcomes. Furthermore, the type of binding mode also determines the control of functionally distinct subclasses of genes and hence the phenotypic response elicited. This is demonstrated for the unique binding mode where a novel role for ELK1 in controlling cell migration is revealed. We have therefore uncovered an unexpected link between the type of binding mode employed by a transcription factor, the subsequent gene regulatory mechanisms used, and the functional categories of target genes controlled.

  7. Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

    NASA Astrophysics Data System (ADS)

    Brandt, Petra; Gerdts, Gunnar; Boersma, Maarten; Wiltshire, Karen H.; Wichels, Antje

    2010-12-01

    Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp . and Temora sp . from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.

  8. Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum▿

    PubMed Central

    Muniz, Mauro de Medeiros; Morais e Silva Tavares, Patrícia; Meyer, Wieland; Nosanchuk, Joshua Daniel; Zancope-Oliveira, Rosely Maria

    2010-01-01

    Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited. PMID:20453140

  9. Mutagenic activity and heterocyclic amine content of the human diet

    SciTech Connect

    Knize, M.G.; Dolbeare, F.A.; Cunningham, P.L.; Felton, J.S.

    1993-01-15

    The mutagenic activity and the mass amount of heterocyclic amines responsible for the mutagenic activity have been measured in some cooked foods. Cooked meats are the predominant source of mutagenic activity in the diet with values ranging from 0 to 10,000 revertants per gram reported in the Ames/Salmonelia test with strain TA98. Several heterocyclic amines are present and have been quantified using solid-phase extraction followed by HPLC. Frying at higher temperatures and for longer times produces the greatest mutagenic response, and concomitantly, the largest amounts of heterocyclic amines. Most of the mutagenic activity in fried meat samples can be accounted for by MelQx, DiMelQx and IQ, although other heterocylic amines are present and PHIP mutagenic activity becomes significant at higher temperatures. Non-meat products such as baked breads can also form significant mutagenic activity, particularly when overcooked. Commercially prepared hamburgers made from meat substitutes such as tofu, wheat gluten or tempeh and fried at 210{degrees}C have up to 10% of the mutagenic activity of a fried beef patty cooked under the same conditions. When detected, amounts of heterocyclic amines in fried beef patties range from a total of 0.35 ng/g for commercial beef hamburgers to 142 ng/g for a beef patty cooked over a barbecue. Dietary intake is expected to have a large range, from less than one microgram per day to over 50 micrograms per day based on current knowledge of known heterocyclic amine chemicals and heterocyclic amine-containing foods.

  10. Substantial DNA methylation differences between two major neuronal subtypes in human brain

    PubMed Central

    Kozlenkov, Alexey; Wang, Minghui; Roussos, Panos; Rudchenko, Sergei; Barbu, Mihaela; Bibikova, Marina; Klotzle, Brandy; Dwork, Andrew J.; Zhang, Bin; Hurd, Yasmin L.; Koonin, Eugene V.; Wegner, Michael; Dracheva, Stella

    2016-01-01

    The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons—GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification. PMID:26612861

  11. No effects of chlorophyllin on IQ (2-amino-3-methylimidazo[4,5-f]-quinoline)-genotoxicity and -DNA adduct formation in Drosophila.

    PubMed

    Negishi, Tomoe; Shinoda, Aki; Ishizaki, Nao; Hayatsu, Hikoya; Sugiyama, Chitose

    2004-02-01

    Previously we demonstrated that chlorophyllin suppressed the genotoxicities of many carcinogens. However, the genotoxicity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline), a carcinogenic heterocyclic amine, was not suppressed in Drosophila. On the contrary, it has been reported that chrolophyllin suppressed the genotoxicity of IQ in rodents, rainbow trout and Salmonella. We demonstrated that the chlorophyllin-induced suppression of MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline)-genotoxicity was associated with a decrease in MeIQx-DNA adduct formation in Drosophila larval DNA. MeIQx represents another type of heterocyclic amine and is similar to IQ in structure. In this study we utilized (32)P-postlabeling to examine whether chlorophyllin reduced IQ-DNA adduct formation in Drosophila DNA in the same way as MeIQx. The results revealed that the formation of IQ-DNA adducts was unaffected by treatment with chlorophyllin. This was consistent with the absence of any inhibitory effect on genotoxicity as observed in the Drosophila repair test. These results suggest that IQ-behavior in Drosophila is not affected by chlorophyllin, indicating that the process of IQ-DNA adduct formation followed by expression of genotoxicity in Drosophila may be different from that in other organisms.

  12. Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils.

    PubMed

    Töwe, Stefanie; Wallisch, Stefanie; Bannert, Andrea; Fischer, Doreen; Hai, Brigitte; Haesler, Felix; Kleineidam, Kristina; Schloter, Michael

    2011-03-01

    We developed an improved protocol, allowing the simultaneous extraction of DNA and RNA from soil using phenol-chloroform with subsequent column-based separation of DNA and RNA (PCS). We compared this new approach with the well established protocol published by Griffiths et al. (2000), where DNA and RNA are separated by selective enzymatic digestions and two commercial kits used for DNA or RNA extraction, respectively, using four different agricultural soils. We compared yield and purity of the nucleic acids as well as abundance and diversity profiles of the soil bacterial communities targeting the nosZ gene via quantitative real-time PCR and terminal restriction fragment length polymorphism on DNA and RNA level. The newly developed protocol provided purer nucleic acid extracts compared to the used kit-based protocols. All protocols were suitable for DNA- and RNA-based gene quantification, however high variations between replicates were obtained for RNA samples using the original Griffiths protocol. Diversity patterns of nosZ were highly influenced by the extraction protocol used both on the DNA and RNA level. Finally, our data showed that the new protocol allows a simultaneous and reproducible extraction and separation of DNA and RNA, which were suitable for reliable analyses of gene and transcript copy numbers and diversity pattern. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Immunoaffinity purification of dietary heterocyclic amine carcinogens

    SciTech Connect

    Vanderlaan, M.; Hwang, M.; Djanegara, T. )

    1993-03-01

    Cooking meats produces a family of heterocyclic aromatic amines that are carcinogens in rodents and genotoxic in many short-term assays. Concern that these compounds may be human carcinogens has prompted us to develop immunochemical methods for quantifying these compounds in the human diet and for identifying the parent compounds and metabolites in urine and feces. Previously reported monoclonal antibodies to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 6-phenyl-2-amino-1-methylimidazo[4,5-f]pyridine (PhIP) were used to purify by immunoaffinity these known mutagens and cross-reacting structural analogs from well-done cooked beef and urine samples. Materials recovered from the immunoaffinity columns were subsequently separated by HPLC to purify the known mutagens from cross-reacting chemicals that co-purify by immunoaffinity. Immunoaffinity chromatography was found to be a rapid means of quantifying individual known mutagens, with a minimum of precolumn sample clean-up required. In addition, this procedure has yielded several new mutagens present in cooked meats that are apparently structural analogs of PhIP. Immunoaffinity techniques were also used to purify metabolites from the urine of rats and humans exposed to MeIQx or PhIP. For MeIQx-exposed rats, the combination antibodies immunoconcentrated 75% of the total urinary radioactivity. Analysis of PhIP metabolites recovered from antibody columns is facilitated by the intrinsic fluorescence of PhIP and its metabolites, providing sufficient sensitivity to monitor individuals for the levels of PhIP excreted following consumption of typical western diets. 6 refs., 3 figs.

  14. DNA methylation profiling reveals differences in the 3 human monocyte subsets and identifies uremia to induce DNA methylation changes during differentiation

    PubMed Central

    Zawada, Adam M.; Schneider, Jenny S.; Michel, Anne I.; Rogacev, Kyrill S.; Hummel, Björn; Krezdorn, Nicolas; Müller, Soeren; Rotter, Björn; Winter, Peter; Obeid, Rima; Geisel, Jürgen; Fliser, Danilo; Heine, Gunnar H.

    2016-01-01

    ABSTRACT Human monocytes are a heterogeneous cell population consisting of 3 subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. Via poorly characterized mechanisms, intermediate monocyte counts rise in chronic inflammatory diseases, among which chronic kidney disease is of particular epidemiologic importance. DNA methylation is a central epigenetic feature that controls hematopoiesis. By applying next-generation Methyl-Sequencing we now tested how far the 3 monocyte subsets differ in their DNA methylome and whether uremia induces DNA methylation changes in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. In vitro, uremia induced dysregulation of DNA methylation in differentiating monocytes, which affected several transcription regulators important for monocyte differentiation (e.g., FLT3, HDAC1, MNT) and led to enhanced generation of intermediate monocytes. As potential mediator, the uremic toxin and methylation inhibitor S-adenosylhomocysteine induced shifts in monocyte subsets in vitro, and associated with monocyte subset counts in vivo. Our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets that occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by accumulation of uremic toxins that mediate epigenetic dysregulation. PMID:27018948

  15. DNA methylation profiling reveals differences in the 3 human monocyte subsets and identifies uremia to induce DNA methylation changes during differentiation.

    PubMed

    Zawada, Adam M; Schneider, Jenny S; Michel, Anne I; Rogacev, Kyrill S; Hummel, Björn; Krezdorn, Nicolas; Müller, Soeren; Rotter, Björn; Winter, Peter; Obeid, Rima; Geisel, Jürgen; Fliser, Danilo; Heine, Gunnar H

    2016-04-02

    Human monocytes are a heterogeneous cell population consisting of 3 subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. Via poorly characterized mechanisms, intermediate monocyte counts rise in chronic inflammatory diseases, among which chronic kidney disease is of particular epidemiologic importance. DNA methylation is a central epigenetic feature that controls hematopoiesis. By applying next-generation Methyl-Sequencing we now tested how far the 3 monocyte subsets differ in their DNA methylome and whether uremia induces DNA methylation changes in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. In vitro, uremia induced dysregulation of DNA methylation in differentiating monocytes, which affected several transcription regulators important for monocyte differentiation (e.g., FLT3, HDAC1, MNT) and led to enhanced generation of intermediate monocytes. As potential mediator, the uremic toxin and methylation inhibitor S-adenosylhomocysteine induced shifts in monocyte subsets in vitro, and associated with monocyte subset counts in vivo. Our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets that occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by accumulation of uremic toxins that mediate epigenetic dysregulation.

  16. DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs.

    PubMed

    Niewolik, Doris; Pannicke, Ulrich; Lu, Haihui; Ma, Yunmei; Wang, Ling-Chi Vicky; Kulesza, Peter; Zandi, Ebrahim; Lieber, Michael R; Schwarz, Klaus

    2006-11-10

    During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5' and 3' overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5' and 3' overhang substrates for the endonucleolytic function of Artemis.

  17. The Bag320 satellite DNA family in Bacillus stick insects (Phasmatodea): different rates of molecular evolution of highly repetitive DNA in bisexual and parthenogenic taxa.

    PubMed

    Mantovani, B; Tinti, F; Bachmann, L; Scali, V

    1997-12-01

    The Bag320 satellite DNA (satDNA) family was studied in seven populations of the stick insects Bacillus atticus (parthenogenetic, unisexual) and Bacillus grandii (bisexual). It was characterized as widespread in all zymoraces of B. atticus and in all subspecies of B. grandii. The copy number of this satellite is higher in the bisexual B. grandii (15%-20% of the genome) than in the parthenogenetic B. atticus (2%-5% of the genome). The nucleotide sequences of 12 Bag320 clones from B. atticus and 17 from B. grandii differed at 13 characteristic positions by fixed nucleotide substitutions. Thus, nucleotide sequences from both species cluster conspecifically in phylogenetic dendrograms. The nucleotide sequences derived from B. grandii grandii could be clearly discriminated from those of B. grandii benazzii and B. grandii maretimi on the basis of 25 variable sites, although all taxa come from Sicily. In contrast, the Bag320 sequences from B. atticus could not be discriminated accordingly, although they derive from geographically quite distant populations of its three zymoraces (the Italian and Greek B. atticus atticus, the Greek and Turkish B. atticus carius, and the Cyprian B. atticus cyprius). The different rate of evolutionary turnover of the Bag320 satDNA in both species can be related to their different modes of reproduction. This indicates that meiosis and chromosome segregation affect processes in satDNA diversification.

  18. Presence and potential of cell free DNA in different types of forensic samples.

    PubMed

    Vandewoestyne, Mado; Van Hoofstat, David; Franssen, Aimée; Van Nieuwerburgh, Filip; Deforce, Dieter

    2013-02-01

    Extracellular or cell free DNA has been found to exist in many biological media such as blood and saliva. To check whether cell free DNA is present in the supernatant which is normally discarded during several DNA extraction processes, such as Chelex(®) extraction, DNA profiles of cell pellet and concentrated supernatant from 30 artificial case like samples and from 100 real forensic samples were compared. Presence of cell free DNA was shown in all investigated sample types. Moreover, in some samples additional alleles, not detected during analysis of the cell pellet, were detected, offering valuable information which would normally have been discarded together with the supernatant. The results presented here indicate that cell free DNA deserves further consideration since it has the potential to increase the DNA yield in forensic casework samples in general and in contact traces in particular.

  19. Transition metal-free one-pot synthesis of nitrogen-containing heterocycles.

    PubMed

    Kumari, Simpal; Kishore, Dharma; Paliwal, Sarvesh; Chauhan, Rajani; Dwivedi, Jaya; Mishra, Aakanksha

    2016-02-01

    One-pot heterocyclic synthesis is an exciting research area as it can open routes for the development of otherwise complex transformations in organic synthesis. Heterocyclic compounds show wide spectrum of applications in medicinal chemistry, chemical biology, and materials science. These heterocycles can be generated very efficiently through highly economical and viable routes using one-pot synthesis. In particular, the metal-free one-pot synthetic protocols are highly fascinating due to several advantages for the industrial production of heterocyclic frameworks. This comprehensive review is devoted to the transition metal-free one-pot synthesis of nitrogen-containing heterocycles from the period 2010-2013.

  20. Sequence variability of the MspI satellite DNA family of the pinewood nematode Bursaphelenchus xylophilus at different geographic scales.

    PubMed

    Vieira, Paulo; Castagnone, Chantal; Mallez, Sophie; Espada, Margarida; Navas, Alfonso; Mota, Manuel; Castagnone-Sereno, Philippe

    2014-01-01

    Tandemly repeated sequences known as satellite DNA (satDNA) generally exhibit complex evolutionary patterns of concerted evolution in which mutations are homogenized and fixed in a stochastic process of molecular drive. Here, the nucleotidic variability of the MspI satDNA family of the pinewood nematode Bursaphelenchus xylophilus is analyzed in order to understand the evolutionary dynamics of satDNA at the intraspecific level. A total of 425 MspI monomer units, either PCR-amplified from isolates of local (Peninsula of Setúbal, Portugal) or worldwide origin, or retrieved from the B. xylophilus genome sequence, were characterized and compared. Whatever their origin, sliding window analysis of sequence variability patterns among monomers revealed low, moderate and highly variant domains, indicating that variable levels of evolutionary constraint may act upon the entire monomers. The phylogenetic inference based on the different sets of MspI satDNA family for this species shows a broad polymorphism of the individual monomers, which were distributed into four main clusters. However, such clustering appeared independent from the geographic origin of the nematodes, and could not discriminate isolates or groups of geographically close isolates. Rather, the formation of different phylogenetic groups within this satDNA family suggests an a priori embodying of a set of diverging repeats from a common ancestor satDNA library, which have been differently amplified along the evolutionary pathway of this species. The present work improves knowledge on the evolutionary dynamics of satDNA at the intraspecific level, and provides new information on satDNA sequence variability among natural populations sampled at a local geographic scale.

  1. Exceptional motifs in different Markov chain models for a statistical analysis of DNA sequences.

    PubMed

    Schbath, S; Prum, B; de Turckheim, E

    1995-01-01

    Identifying exceptional motifs is often used for extracting information from long DNA sequences. The two difficulties of the method are the choice of the model that defines the expected frequencies of words and the approximation of the variance of the difference T(W) between the number of occurrences of a word W and its estimation. We consider here different Markov chain models, either with stationary or periodic transition probabilities. We estimate the variance of the difference T(W) by the conditional variance of the number of occurrences of W given the oligonucleotides counts that define the model. Two applications show how to use asymptotically standard normal statistics associated with the counts to describe a given sequence in terms of its outlying words. Sequences of Escherichia coli and of Bacillus subtilis are compared with respect to their exceptional tri- and tetranucleotides. For both bacteria, exceptional 3-words are mainly found in the coding frame. E. coli palindrome counts are analyzed in different models, showing that many overabundant words are one-letter mutations of avoided palindromes.

  2. Reverse Transcription Errors and RNA–DNA Differences at Short Tandem Repeats

    PubMed Central

    Fungtammasan, Arkarachai; Tomaszkiewicz, Marta; Campos-Sánchez, Rebeca; Eckert, Kristin A.; DeGiorgio, Michael; Makova, Kateryna D.

    2016-01-01

    Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to nongenetically encoded transcript variants, or RNA–DNA differences (RDDs). Such variation has been understudied, in part because its detection is obscured by reverse transcription (RT) and sequencing errors. It has only been evaluated for intertranscript base substitution differences. Here, we investigated transcript sequence variation for short tandem repeats (STRs). We developed the first maximum-likelihood estimator (MLE) to infer RT error and RDD rates, taking next generation sequencing error rates into account. Using the MLE, we empirically evaluated RT error and RDD rates for STRs in a large-scale DNA and RNA replicated sequencing experiment conducted in a primate species. The RT error rates increased exponentially with STR length and were biased toward expansions. The RDD rates were approximately 1 order of magnitude lower than the RT error rates. The RT error rates estimated with the MLE from a primate data set were concordant with those estimated with an independent method, barcoded RNA sequencing, from a Caenorhabditis elegans data set. Our results have important implications for medical genomics, as STR allelic variation is associated with >40 diseases. STR nonallelic transcript variation can also contribute to disease phenotype. The MLE and empirical rates presented here can be used to evaluate the probability of disease-associated transcripts arising due to RDD. PMID:27413049

  3. Reverse Transcription Errors and RNA-DNA Differences at Short Tandem Repeats.

    PubMed

    Fungtammasan, Arkarachai; Tomaszkiewicz, Marta; Campos-Sánchez, Rebeca; Eckert, Kristin A; DeGiorgio, Michael; Makova, Kateryna D

    2016-10-01

    Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to nongenetically encoded transcript variants, or RNA-DNA differences (RDDs). Such variation has been understudied, in part because its detection is obscured by reverse transcription (RT) and sequencing errors. It has only been evaluated for intertranscript base substitution differences. Here, we investigated transcript sequence variation for short tandem repeats (STRs). We developed the first maximum-likelihood estimator (MLE) to infer RT error and RDD rates, taking next generation sequencing error rates into account. Using the MLE, we empirically evaluated RT error and RDD rates for STRs in a large-scale DNA and RNA replicated sequencing experiment conducted in a primate species. The RT error rates increased exponentially with STR length and were biased toward expansions. The RDD rates were approximately 1 order of magnitude lower than the RT error rates. The RT error rates estimated with the MLE from a primate data set were concordant with those estimated with an independent method, barcoded RNA sequencing, from a Caenorhabditis elegans data set. Our results have important implications for medical genomics, as STR allelic variation is associated with >40 diseases. STR nonallelic transcript variation can also contribute to disease phenotype. The MLE and empirical rates presented here can be used to evaluate the probability of disease-associated transcripts arising due to RDD. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Phylogenetic Analysis of Different Ploidy Saccharum spontaneum Based on rDNA-ITS Sequences.

    PubMed

    Liu, Xinlong; Li, Xujuan; Liu, Hongbo; Xu, Chaohua; Lin, Xiuqin; Li, Chunjia; Deng, Zuhu

    2016-01-01

    Saccharum spontaneum L. is a crucial wild parent of modern sugarcane cultivars whose ploidy clones have been utilized successfully in improving the stress resistance and yield related traits of sugarcane cultivars. To establish knowledge regarding the genetic variances and evolutional relationships of ploidy clones of Saccharum spontaneum collected in China, the rDNA-ITS sequences of 62 ploidy clones including octaploid clones (2n = 64), nonaploid clones (2n = 72), decaploid clones (2n = 80), and dodecaploid clones (2n = 96), were obtained and analyzed. The rDNA-ITS sequences of four species from Saccharum and Sorghum bicolor selected as controls. The results showed that decaploid clones (2n = 80) possess the most abundant variances with 58 variable sites and 20 parsim-informative sites in ITS sequences, which were then followed by octaploid clones with 43 variable sites and 17 parsim-informative sites. In haplotype diversity, all four population exhibited high diversity, especially nonaploid and decaploid populations. By comparing the genetic distances among four ploidy populations, the dodecaploid population exhibited the closest relationship with the nonaploid population, and then the relationship strength decreased successively for the decaploid population and then for the octaploid population. Population differentiation analysis showed that the phenomena of population differentiation were not found among different ploidy populations, and low coefficient of gene differentiation(Gst) and high gene flow(Nm) occur among these populations possessing close genetic relationship. These results mentioned above will contribute to the understanding of the evolution of different ploidy populations of Saccharum spontaneum and provide vital knowledge for their utilization in sugarcane breeding and innovation.

  5. Impact of different ChIP-Seq protocols on DNA integrity and quality of bioinformatics analysis results.

    PubMed

    Felsani, Armando; Gudmundsson, Bjarki; Nanni, Simona; Brini, Elena; Moles, Anna; Thormar, Hans Guttormur; Estibeiro, Peter; Gaetano, Carlo; Capogrossi, Maurizio; Farsetti, Antonella; Jonsson, Jon Johannes; Guffanti, Alessandro

    2015-03-01

    Different ChIP-Seq protocols may have a significant impact on the final outcome in terms of quality, number and distribution of called peaks. Sample DNA undergoes a long procedure before the final sequencing step, and damaged DNA can result in excessive mismatches in the alignment with reference genome. In this letter, we present the effect of well-defined modifications (timing of formaldehyde crosslink reversal, brand of the sonicator) of standard ChIP-Seq protocol on parallel samples derived from the same cell line correlating the initial DNA quality control metrics to the final bioinformatics analysis results.

  6. Comparison of four different methods for extraction of Stachybotrys chartarum spore DNA and verification by real-time PCR.

    PubMed

    Black, J A; Foarde, K K

    2007-07-01

    A comparison of four different methods for the extraction of spore DNA from Stachybotrys chartarum was conducted. Spore DNA was extracted and purified using either one of three different commercial kits or water. All preparations utilized bead milling. Genomic DNA extracted from 10(1) to 10(7) spores was assessed by both agarose gel electrophoresis and real-time quantitative polymerase chain reaction (qPCR) performed against multi-copy (rRNA) and single-(tubulin) gene targets. The spore isolation technique we employed was verified to be pure by light microscopy. Although all preparatory methods led to successful detection by qPCR, S. chartarum spore DNA prepared using the Qiagen Plant kit was notably better over the extraction range.

  7. Alteration of the Total Nuclear DNA Ploidy in Different Histopathological Liver Tissues Negative and Positive for HCV RNA.

    PubMed

    Tabll, Ashraf A; Kishta, Sara S; Farrag, Abdel Razik H; El Din, Noha G Bader; Mohamed, Mervat S; El Abd, Yasmine S; El Esawy, Basem H; Kishta, Sobhy A; Dawood, Reham M; Ismail, Allaa; El Awady, Mostafa K

    2015-01-01

    Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma (HCC). The molecular mechanisms of HCV-associated carcinogenesis are unknown. We aim to investigate the alteration of the total nuclear DNA content (ploidy) in different histopathological liver tissues infected with HCV and their relation to the seropositivity of HCV RNA. Blood and liver tissues were collected from 26 patients. Diagnosis was carried out according to clinical and pathological examinations by specialized physicians. HCV RNA was detected in patients' sera and tissue samples by RT-PCR. To examine nuclear DNA ploidy, liver tissues were stained with blue Fulgen using the image analysis techniques. Finally, the patients' DNA content was examined by histochemical analysis depending on the optical density of DNA from liver biopsies using the grey image menu in each specimen. The HCV RT-PCR results demonstrated that 13/26 (50%) patients had detectable HCV RNA in their sera samples while 18/26 (69%) had detectable HCV RNA in liver tissues. The DNA content from those patients measured by image cytometry showed a high level of alteration of nuclear DNA ploidy and proliferation in liver tissues with HCC, less alteration of nuclear DNA ploidy in cirrhotic patients, and least proliferation nearly normal in liver fibrosis patients. Moreover, the results of histochemical analysis confirmed the DNA image cytometry results and showed that positive HCV RNA liver tissues had more DNA ploidy than negative HCV RNA liver tissues with statistical significance (p-value < 0.05). HCV positive liver tissue had alterations in DNA content (ploidy) which may lead to liver disease progression, malignant transformation of the liver cells and development of hepatocellular carcinoma.

  8. Differences in nuclear DNA organization between lymphocytes, Hodgkin and Reed-Sternberg cells revealed by structured illumination microscopy.

    PubMed

    Righolt, Christiaan H; Guffei, Amanda; Knecht, Hans; Young, Ian T; Stallinga, Sjoerd; van Vliet, Lucas J; Mai, Sabine

    2014-08-01

    Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first time, the organization of nuclear DNA and that of DNA-free space in control lymphocytes, Hodgkin cells and Reed-Sternberg cells using 3D structured illumination microscopy (SIM). We have observed detail in these SIM images that was not observed in conventional widefield images. We have measured the size distribution of the DNA structure using granulometry and noted a significant, progressive increase in the amount of sub-micron structures from control lymphocytes to Hodgkin cells to Reed-Sternberg cells. The DNA-free space changes as well; "holes" in the DNA distribution start to appear in the malignant cells. We have studied whether these "holes" are nucleoli by staining for upstream binding factor (UBF), a protein associated with the nucleolus. We have found that the relative UBF content progressively and significantly decreases-or is absent-in the DNA-free space when measured as either the Pearson correlation coefficient with the DNA-free space or as the number of "holes" that contain UBF. Similar differences exist within the population of Reed-Sternberg cells between binucleated and multinucleated cells with four or more subnuclei. To our knowledge, this is the first study that investigates the changes of the nuclear DNA structure in any disease with superresolution light microscopy.

  9. Different mechanisms between copper and iron in catecholamines-mediated oxidative DNA damage and disruption of gene expression in vitro.

    PubMed

    Nishino, Yoshihiko; Ando, Motozumi; Makino, Rena; Ueda, Koji; Okamoto, Yoshinori; Kojima, Nakao

    2011-07-01

    Catechols produce reactive oxygen species (ROS) and induce oxidative DNA damage through reduction-oxidation reactions with metals such as copper. Here, we examined oxidative DNA damage by neurotransmitter catecholamines in the presence of copper or iron and evaluated the effects of this damage on gene expression in vitro. Dopamine induced strand breaks and base oxidation in calf thymus DNA in the presence of Cu(II) or Fe(III)-NTA (nitrilotriacetic acid). The extent of this damage was greater for Cu(II) than for Fe(III)-NTA. For the DNA damage induced by dopamine, the responsible reactive species were hydrogen peroxide and Cu(I) for Cu(II) and hydroxyl radicals and Fe(II) for Fe(III)-NTA. Cu(II) induced DNA conformational changes, but Fe(III)-NTA did not in the presence of dopamine. These differences indicate different modes of action between Cu and Fe-NTA with regard to the induction of DNA damage. Expression of the lacZ gene coded on plasmid DNA was inhibited depending on the extent of the oxidative damage and strand breaks. Endogenous catecholamines (dopamine, adrenaline, and noradrenaline) were more potent than catechols (no aminoalkyl side chains) or 3,4-dihydroxybenzylamine (aminomethyl side chain). These results suggest that the metal-mediated DNA damage induced by dopamine disrupts gene expression, and leukoaminochromes (further oxidation products of O-quinones having aminoethyl side chain) are involved in the DNA damage. These findings indicate a possibility that metal (especially iron and copper)-mediated oxidation of catecholamines plays an important role in the pathogenesis of neurodegenerative disorders including Parkinson's disease.

  10. Controlling self-assembly of diphenylalanine peptides at high pH using heterocyclic capping groups

    PubMed Central

    Martin, Adam D.; Wojciechowski, Jonathan P.; Robinson, Andrew B.; Heu, Celine; Garvey, Christopher J.; Ratcliffe, Julian; Waddington, Lynne J.; Gardiner, James; Thordarson, Pall

    2017-01-01

    Using small angle neutron scattering (SANS), it is shown that the existence of pre-assembled structures at high pH for a capped diphenylalanine hydrogel is controlled by the selection of N-terminal heterocyclic capping group, namely indole or carbazole. At high pH, changing from a somewhat hydrophilic indole capping group to a more hydrophobic carbazole capping group results in a shift from a high proportion of monomers to self-assembled fibers or wormlike micelles. The presence of these different self-assembled structures at high pH is confirmed through NMR and circular dichroism spectroscopy, scanning probe microscopy and cryogenic transmission electron microscopy. PMID:28272523

  11. Controlling self-assembly of diphenylalanine peptides at high pH using heterocyclic capping groups

    NASA Astrophysics Data System (ADS)

    Martin, Adam D.; Wojciechowski, Jonathan P.; Robinson, Andrew B.; Heu, Celine; Garvey, Christopher J.; Ratcliffe, Julian; Waddington, Lynne J.; Gardiner, James; Thordarson, Pall

    2017-03-01

    Using small angle neutron scattering (SANS), it is shown that the existence of pre-assembled structures at high pH for a capped diphenylalanine hydrogel is controlled by the selection of N-terminal heterocyclic capping group, namely indole or carbazole. At high pH, changing from a somewhat hydrophilic indole capping group to a more hydrophobic carbazole capping group results in a shift from a high proportion of monomers to self-assembled fibers or wormlike micelles. The presence of these different self-assembled structures at high pH is confirmed through NMR and circular dichroism spectroscopy, scanning probe microscopy and cryogenic transmission electron microscopy.

  12. Multicenter Comparison of Three Different Analytical Systems for Evaluation of DNA Banding Patterns from Cryptococcus neoformans

    PubMed Central

    Cardinali, Gianluigi; Martini, Alessandro; Preziosi, Roberta; Bistoni, Francesco; Baldelli, Franco

    2002-01-01

    The enormous improvement of molecular typing techniques for epidemiological and clinical studies has not always been matched by an equivalent effort in applying optimal criteria for the analysis of both phenotypic and molecular data. In spite of the availability of a large collection of statistical and phylogenetic methods, the vast majority of commercial packages are limited by using only the unweighted pair group method with arithmetic mean algorithm to construct trees and by considering electrophoretic pattern only as migration distances. The latter method has serious drawbacks when different runs (separate gels) of the same molecular analysis are to be compared. This work presents a multicenter comparison of three different systems of banding pattern analysis on random amplified polymorphic DNA, (GACA)4, and contour-clamped homogeneous electric field patterns from strains of Cryptococcus neoformans var. neoformans isolated in different clinical and geographical situations and a standard Saccharomyces cerevisiae strain employed as an outgroup. The systems considered were evaluated for their actual ability to(i) recognize identities, (ii) define complete differences (i.e., the ability to place S. cerevisiae out of the C. neoformans cluster), and (iii) estimate the extent of similarity among different strains. The ability to cluster strains according to the patient from which they were isolated was also evaluated. The results indicate that different algorithms do indeed produce divergent trees, both in overall topology and in clustering of individual strains, thus suggesting that care must be taken by individual investigators to use the most appropriate procedure and by the scientific community in defining a consensus system. PMID:12037071

  13. The influence of the structure of some aromatic heterocyclic derivatives on their retention in reversed-phase high-performance liquid chromatography

    NASA Astrophysics Data System (ADS)

    Kurbatova, S. V.; Saifutdinov, B. R.; Larionov, O. G.; Meshkovaya, V. V.

    2009-03-01

    The chromatographic behavior of aromatic heterocyclic derivatives in reversed-phase high-performance liquid chromatography was investigated. The retention characteristics of the substances under the conditions of chromatography with water-acetonitrile mobile phases (retention factors, relative retentions, distribution coefficients, Henry adsorption constants, differences in the differential molar energy of sorption, and Gibbs energies of sorption) were determined. It was shown that the chromatographic retention of the sorbates depended on their molecular structure. The influence of the nature of heteroatoms and their number on the sorption of heterocyclic compounds was discussed.

  14. Association of DNA Methylation Differences With Schizophrenia in an Epigenome-Wide Association Study.

    PubMed

    Montano, Carolina; Taub, Margaret A; Jaffe, Andrew; Briem, Eirikur; Feinberg, Jason I; Trygvadottir, Rakel; Idrizi, Adrian; Runarsson, Arni; Berndsen, Birna; Gur, Ruben C; Moore, Tyler M; Perry, Rodney T; Fugman, Doug; Sabunciyan, Sarven; Yolken, Robert H; Hyde, Thomas M; Kleinman, Joel E; Sobell, Janet L; Pato, Carlos N; Pato, Michele T; Go, Rodney C; Nimgaonkar, Vishwajit; Weinberger, Daniel R; Braff, David; Gur, Raquel E; Fallin, Margaret Daniele; Feinberg, Andrew P

    2016-05-01

    DNA methylation may play an important role in schizophrenia (SZ), either directly as a mechanism of pathogenesis or as a biomarker of risk. To scan genome-wide DNA methylation data to identify differentially methylated CpGs between SZ cases and controls. Epigenome-wide association study begun in 2008 using DNA methylation levels of 456 513 CpG loci measured on the Infinium HumanMethylation450 array (Illumina) in a consortium of case-control studies for initial discovery and in an independent replication set. Primary analyses used general linear regression, adjusting for age, sex, race/ethnicity, smoking, batch, and cell type heterogeneity. The discovery set contained 689 SZ cases and 645 controls (n = 1334), from 3 multisite consortia: the Consortium on the Genetics of Endophenotypes in Schizophrenia, the Project among African-Americans To Explore Risks for Schizophrenia, and the Multiplex Multigenerational Family Study of Schizophrenia. The replication set contained 247 SZ cases and 250 controls (n = 497) from the Genomic Psychiatry Cohort. Identification of differentially methylated positions across the genome in SZ cases compared with controls. Of the 689 case participants in the discovery set, 477 (69%) were men and 258 (37%) were non-African American; of the 645 controls, 273 (42%) were men and 419 (65%) were non-African American. In our replication set, cases/controls were 76% male and 100% non-African American. We identified SZ-associated methylation differences at 923 CpGs in the discovery set (false discovery rate, <0.2). Of these, 625 showed changes in the same direction including 172 with P < .05 in the replication set. Some replicated differentially methylated positions are located in a top-ranked SZ region from genome-wide association study analyses. This analysis identified 172 replicated new associations with SZ after careful correction for cell type heterogeneity and other potential confounders. The overlap with previous genome

  15. Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus.

    PubMed

    Wiley, Kenneth L; Treadwell, Edward; Manigaba, Kayihura; Word, Beverly; Lyn-Cook, Beverly D

    2013-02-01

    Systemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy. Real-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlash(TM) methylated quantification colorimetric assay. Significant increase in DNMT3A (p < 0.001) was shown in lupus patients when compared to age-matched healthy controls. This increase was associated with a higher SLEDI index. More striking was that expression levels for African American (AA) women were higher than European American women in the lupus populations. A subset of AA women on DHEA therapy showed a significant decrease (p < 0.05) in DNMT3A expression in comparison to lupus patients not on the therapy. DHEA is an androgenic steroid found in low levels in the serum of lupus patients. Supplementation of this hormone has been shown to be beneficial to some lupus patients. DHEA was not shown to effect DNMT1 or DNMT3B expression. Increased expression was also noted in DNMT3B (p < 0.05) in lupus patients compared to age-matched healthy controls. However, no significant difference was noted in DNMT1 (p = 0.2148) expression between lupus patients and healthy controls. Although increases were detected in de novo methyltransferases, a global decrease (p < 0.001) in 5-methycytosine was observed in

  16. Molecular and Bioenergetic Differences between Cells with African versus European Inherited Mitochondrial DNA Haplogroups: Implications for Population Susceptibility to Diseases

    PubMed Central

    Kenney, M. Cristina; Chwa, Marilyn; Atilano, Shari R.; Falatoonzadeh, Payam; Ramirez, Claudio; Malik, Deepika; Tarek, Mohamed; Cáceres del Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Vawter, Marquis P.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin

    2015-01-01

    The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases. PMID:24200652

  17. Insights into the O-Acetylation Reaction of Hydroxylated Heterocyclic Amines by Human Arylamine N-Acetyltransferases: A Computational Study

    SciTech Connect

    Lau, E Y; Felton, J S; Lightstone, F C

    2006-06-06

    A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclic amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.

  18. Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status.

    PubMed

    Lyckesvärd, Madeleine Nordén; Delle, Ulla; Kahu, Helena; Lindegren, Sture; Jensen, Holger; Bäck, Tom; Swanpalmer, John; Elmroth, Kecke

    2014-07-01

    Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ((211)At), concentrated in the thyroid by the same mechanism as (131)I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ((60)Co) and alpha particles from (211)At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to (211)At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1Gy (211)At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative effectiveness of alpha particles. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51-DNA filaments.

    PubMed

    Sanchez, Humberto; Kertokalio, Aryandi; van Rossum-Fikkert, Sari; Kanaar, Roland; Wyman, Claire

    2013-07-09

    Essential genome transactions, such as homologous recombination, are achieved by concerted and dynamic interactions of multiple protein components with DNA. Which proteins do what and how, will be reflected in their relative arrangements. However, obtaining high-resolution structural information on the variable arrangements of these complex assemblies is a challenge. Here we demonstrate the versatility of a combined total internal reflection fluorescence and scanning force microscope (TIRF-SFM) to pinpoint fluorescently labeled human homologous recombination protein RAD54 interacting with presynaptic (ssDNA) and postsynaptic (dsDNA) human recombinase RAD51 nucleoprotein filaments. Labeled proteins were localized by superresolution imaging on complex structures in the SFM image with high spatial accuracy. We observed some RAD54 at RAD51 filament ends, as expected. More commonly, RAD54 interspersed along RAD51-DNA filaments. RAD54 promotes RAD51-mediated DNA strand exchange and has been described to both stabilize and destabilize RAD51-DNA filaments. The different architectural arrangements we observe for RAD54 with RAD51-DNA filaments may reflect the diverse roles of this protein in homologous recombination.

  20. PCR-fingerprint profiles of mitochondrial and genomic DNA extracted from Fetus cervi using different extraction methods.

    PubMed

    Ai, Jinxia; Wang, Xuesong; Gao, Lijun; Xia, Wei; Li, Mingcheng; Yuan, Guangxin; Niu, Jiamu; Zhang, Lihua

    2016-06-01

    The use of Fetus cervi, which is derived from the embryo and placenta of Cervus Nippon Temminck or Cervs elaphus Linnaeus, has been documented for a long time in China. There are abundant species of deer worldwide. Those recorded by China Pharmacopeia (2010 edition) from all the species were either authentic or adulterants/counterfeits. Identification of their origins or authenticity became a key in the preparation of the authentic products. The traditional SDS alkaline lysis and salt-outing methods were modified to extract mt DNA and genomic DNA from fresh and dry Fetus cervi in addition to Fetus from false animals, respectively. A set of primers were designed by bioinformatics to target the intra-and inter-variation. The mt DNA and genomic DNA extracted from Fetus cervi using the two methods meet the requirement for authenticity. Extraction of mt DNA by SDS alkaline lysis is more practical and accurate than extraction of genomic DNA by salt-outing method. There were differences in length and number of segments amplified by PCR between mt DNA from authentic Fetus cervi and false animals Fetus. The distinctive PCR-fingerprint patterns can distinguish the Fetus cervi from adulterants and counterfeit animal Fetus.

  1. Umpolung of Michael acceptors catalyzed by N-heterocyclic carbenes.

    PubMed

    Fischer, Christian; Smith, Sean W; Powell, David A; Fu, Gregory C

    2006-02-08

    N-Heterocyclic carbenes can catalyze beta-alkylations of a range of alpha,beta-unsaturated esters, amides, and nitriles that bear pendant leaving groups to form a variety of ring sizes. In this process, the nucleophilic catalyst transiently transforms the normally electrophilic beta carbon into a nucleophilic site through an unanticipated addition-tautomerization sequence.

  2. Straightforward synthesis of iron cyclopentadienone N-heterocyclic carbene complexes.

    PubMed

    Cingolani, Andrea; Cesari, Cristiana; Zacchini, Stefano; Zanotti, Valerio; Cassani, Maria Cristina; Mazzoni, Rita

    2015-11-28

    Novel iron complexes bearing both cyclopentadienone and N-heterocyclic carbene ancillary ligands were obtained by a straightforward synthesis from Fe2(CO)9. The preparation represents a rare example of silver transmetallation involving iron. The reaction is general and occurs in the presence of variously functionalized NHC and cyclopentadienones.

  3. A Comprehensive Review of N-Heterocycles as Cytotoxic Agents.

    PubMed

    Kumar, Dinesh; Jain, Subheet Kumar

    2016-01-01

    Scientific community is striving to understand the role of heterocycles and fused heterocycles in drug discovery programme due to its impact on multi-drug resistance (MDR) of anticancer drugs. Architecting of various scaffolds for cancer treatment has become gradually increased in many years. Till now there is no treatment which is so proficient that it can cure the cancer from the roots. Hence, it is very necessary to design novel anticancer agents with minimum side effects. Synthesis of hybrids from natural leads is one of the rationale approaches in medicinal chemistry. It remains a big challenge to invent new efficient drugs to beat cancer. The design and synthesis of fused molecules as anticancer agents is one of the great innovations of modern era. In drug discovery archetype, a variety of heterocycles have been considered for the development of novel lead compounds. This article presents some recent advancements in the field of anticancer heterocyclic agents all around the world and also attracted the structure activity relationship along with the structure of the most promising molecules along with IC50 values against various human cancer cell lines.

  4. Synthesis and properties of heterocyclic type I photoinitiators

    NASA Astrophysics Data System (ADS)

    Liska, R.; Knaus, S.; Wendrinsky, J.

    1999-05-01

    The synthesis and properties of a series of new heterocyclic hydroxyalkylphenone-analogous photoinitiators (PIs) is described. The PIs are obtained by reaction of aromatic organolithium compounds with nitriles or by Friedel-Craft's-acylation. Preliminary photocalorimetric tests and UV absorption data are included.

  5. N-heterocyclic carbene catalyzed direct carbonylation of dimethylamine.

    PubMed

    Li, Xiaonian; Liu, Kun; Xu, Xiaoliang; Ma, Lei; Wang, Hong; Jiang, Dahao; Zhang, Qunfeng; Lu, Chunshan

    2011-07-21

    N-Heterocyclic carbene (NHC) catalyzed direct carbonylation of dimethylamine leading to the formation of DMF was successfully accomplished under metal-free conditions. The catalytic efficiency was investigated and the turnover numbers can reach as high as >300. The possible mechanism was also proposed.

  6. Greener Alternatives to Expedient Synthesis of Heterocycles and Nanomaterial

    EPA Science Inventory

    A brief account of reactions involving microwave (MW) exposure of neat reactants or catalyzed by mineral support surfaces, such as alumina, silica, clay, or their ‘doped’ versions, for the rapid one-pot assembly of heterocyclic compounds [1] from in situ generated reactive interm...

  7. Greener Alternatives to Expedient Synthesis of Heterocycles and Nanomaterial

    EPA Science Inventory

    A brief account of reactions involving microwave (MW) exposure of neat reactants or catalyzed by mineral support surfaces, such as alumina, silica, clay, or their ‘doped’ versions, for the rapid one-pot assembly of heterocyclic compounds [1] from in situ generated reactive interm...

  8. Acyl anion free N-heterocyclic carbene organocatalysis.

    PubMed

    Ryan, Sarah J; Candish, Lisa; Lupton, David W

    2013-06-21

    Reaction discovery using N-heterocyclic carbene organocatalysis has been dominated by the chemistry of acyl anion equivalents. Recent studies demonstrate that NHCs are far more diverse catalysts, with a variety of reactions discovered that proceed without acyl anion equivalent formation. In this tutorial review selected examples of acyl anion free NHC catalysis using carbonyl compounds are presented.

  9. Greener and Expeditious Synthesis of Bioactive Heterocycles using Microwave Irradiation

    EPA Science Inventory

    The utilization of green chemistry techniques is dramatically reducing chemical waste and reaction times as has recently been proven in several organic syntheses and chemical transformations. To illustrate these advantages in the synthesis of bio-active heterocycles, we have stud...

  10. GREENER SYNTHESIS OF HETEROCYCLIC COMPOUNDS USING MICROWAVE IRRADIATION

    EPA Science Inventory

    An introduction of our interest in the microwave-assisted greener synthesis of a variety of heterocyclic compounds will be presented. It involves microwave (MW) exposure of neat reactants (undiluted) catalyzed by the surfaces of recyclable mineral supports, such as alumina, sili...

  11. Greener Synthetic Alternatives to Heterocycles, Nanomaterials and Nanocomposites

    EPA Science Inventory

    Microwave (MW) expedited reaction of neat reactants or catalyzed by mineral support surfaces, such as alumina, silica, clay, or their ‘doped’ versions, for the rapid one-pot assembly of heterocyclic compounds from in situ generated reactive intermediates via enamines or using hyp...

  12. Greener Synthetic Alternatives to Heterocycles, Nanomaterials and Nanocomposites

    EPA Science Inventory

    Microwave (MW) expedited reaction of neat reactants or catalyzed by mineral support surfaces, such as alumina, silica, clay, or their ‘doped’ versions, for the rapid one-pot assembly of heterocyclic compounds from in situ generated reactive intermediates via enamines or using hyp...

  13. GREENER SYNTHESIS OF HETEROCYCLIC COMPOUNDS USING MICROWAVE IRRADIATION

    EPA Science Inventory

    An introduction of our interest in the microwave-assisted greener synthesis of a variety of heterocyclic compounds will be presented. It involves microwave (MW) exposure of neat reactants (undiluted) catalyzed by the surfaces of recyclable mineral supports, such as alumina, sili...

  14. Greener and Expeditious Synthesis of Bioactive Heterocycles using Microwave Irradiation

    EPA Science Inventory

    The utilization of green chemistry techniques is dramatically reducing chemical waste and reaction times as has recently been proven in several organic syntheses and chemical transformations. To illustrate these advantages in the synthesis of bio-active heterocycles, we have stud...

  15. Solvent-Free Synthesis of Heterocyclic Compounds Using Microwave Technology

    NASA Astrophysics Data System (ADS)

    Ahmad, Natiq Ghanim

    The synthesis of heterocyclic compounds containing pyrimidine, pyrazoline, isoxazoline and cyclohexanone ring from chalcone derivatives containing 3,4-dimethoxyphenyl group and furfuryl ring group under dry condition using microwaves. The structures of these compounds were confirmed by IR, H1 NMR and physical constants.

  16. Enantioselective N-heterocyclic carbene-catalyzed synthesis of trifluoromethyldihydropyridinones.

    PubMed

    Wang, Dong-Ling; Liang, Zhi-Qin; Chen, Kun-Quan; Sun, De-Qun; Ye, Song

    2015-06-05

    The enantioselective N-heterocyclic carbene-catalyzed [4 + 2] cyclocondensation of α-chloroaldehydes and trifluoromethyl N-Boc azadienes was developed, giving the corresponding 3,4-disubstituted-6-trifluoromethyldihydropyridin-2(1H)-ones in good yields with exclusive cis-selectivities and excellent enantioselectivities.

  17. Toxicity of six heterocyclic nitrogen compounds to Daphnia pulex

    USGS Publications Warehouse

    Perry, Cynthia M.; Smith, Stephen B.

    1988-01-01

    We determined the relative toxicities to the aquatic crustacean Daphniz pulex of six heterocyclic nitrogen compunds. These compounds were selected because they were detected in lake trout or walleyes and were commercially available. Stress to the daphnid populations may affect forage fish populations that depend either directly or indirectly on zooplankton as a food source in the Great Lakes.

  18. Synthesis and antitubercular activity of heterocycle substituted diphenyl ether derivatives

    USDA-ARS?s Scientific Manuscript database

    Despite being an ancient disease, tuberculosis (TB) remains the leading single-agent infectious disease killer in the world. The emerging serious problem due to TB control and clinical management prompted us to synthesize novel series of heterocyclic substituted diphenyl ether derivatives and determ...

  19. Comparison of three different DNA extraction methods from a highly degraded biological material.

    PubMed

    Kuś, M; Ossowski, A; Zielińska, G

    2016-05-01

    The identification of unknown victims is one of the most challenging tasks faced by forensic medicine. This is due to the rapid decomposition of tissues, beginning at the moment of death and caused by released enzymes and microbial activity. Decay is directly associated with the decomposition of soft tissues and also the degradation of genetic material inside cells. Decomposition rates vary depending on a number of environmental factors, including temperature, humidity, season, and soil properties. Decomposition also differs between bodies left in the open air or buried. To date, forensic medicine has identified mainly people who were the victims of various types of criminal offences. However, with advances in identification methods, increasingly frequent attempts are made to identify the victims of armed conflicts, crimes of totalitarian regimes, or genocide. The aim of the study was to compare three different methods for the extraction of nuclear DNA from material considered in forensic medicine as difficult to handle, i.e. fragments of bones and teeth, and to determine the performance of these methods and their suitability for identification procedures. Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  20. Different modes of interaction by TIAR and HuR with target RNA and DNA

    PubMed Central

    Kim, Henry S.; Wilce, Matthew C. J.; Yoga, Yano M. K.; Pendini, Nicole R.; Gunzburg, Menachem J.; Cowieson, Nathan P.; Wilson, Gerald M.; Williams, Bryan R. G.; Gorospe, Myriam; Wilce, Jacqueline A.

    2011-01-01

    TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2′-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways. PMID:21233170

  1. Different modes of interaction by TIAR and HuR with target RNA and DNA.

    PubMed

    Kim, Henry S; Wilce, Matthew C J; Yoga, Yano M K; Pendini, Nicole R; Gunzburg, Menachem J; Cowieson, Nathan P; Wilson, Gerald M; Williams, Bryan R G; Gorospe, Myriam; Wilce, Jacqueline A

    2011-02-01

    TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.

  2. Genome-Wide Differences in DNA Methylation Changes in Two Contrasting Rice Genotypes in Response to Drought Conditions

    PubMed Central

    Wang, Wensheng; Qin, Qiao; Sun, Fan; Wang, Yinxiao; Xu, Dandan; Li, Zhikang; Fu, Binying

    2016-01-01

    Differences in drought stress tolerance within diverse rice genotypes have been attributed to genetic diversity and epigenetic alterations. DNA methylation is an important epigenetic modification that influences diverse biological processes, but its effects on rice drought stress tolerance are poorly understood. In this study, methylated DNA immunoprecipitation sequencing and an Affymetrix GeneChip rice genome array were used to profile the DNA methylation patterns and transcriptomes of the drought-tolerant introgression line DK151 and its drought-sensitive recurrent parent IR64 under drought and control conditions. The introgression of donor genomic DNA induced genome-wide DNA methylation changes in DK151 plants. A total of 1190 differentially methylated regions (DMRs) were detected between the two genotypes under normal growth conditions, and the DMR-associated genes in DK151 plants were mainly related to stress response, programmed cell death, and nutrient reservoir activity, which are implicated to constitutive drought stress tolerance. A comparison of the DNA methylation changes in the two genotypes under drought conditions indicated that DK151 plants have a more stable methylome, with only 92 drought-induced DMRs, than IR64 plants with 506 DMRs. Gene ontology analyses of the DMR-associated genes in drought-stressed plants revealed that changes to the DNA methylation status of genotype-specific genes are associated with the epigenetic regulation of drought stress responses. Transcriptome analysis further helped to identify a set of 12 and 23 DMR-associated genes that were differentially expressed in DK151 and IR64, respectively, under drought stress compared with respective controls. Correlation analysis indicated that DNA methylation has various effects on gene expression, implying that it affects gene expression directly or indirectly through diverse regulatory pathways. Our results indicate that drought-induced alterations to DNA methylation may influence

  3. Presence of Extracellular DNA during Biofilm Formation by Xanthomonas citri subsp. citri Strains with Different Host Range

    PubMed Central

    Sena-Vélez, Marta; Redondo, Cristina; Graham, James H.; Cubero, Jaime

    2016-01-01

    Xanthomonas citri subsp. citri (Xcc) A strain causes citrus bacterial canker, a serious leaf, fruit and stem spotting disease of several Citrus species. X. alfalfae subsp. citrumelonis (Xac) is the cause of citrus bacterial spot, a minor disease of citrus nursery plants and X. campestris pv. campestris (Xc) is a systemic pathogen that causes black rot of cabbage. Xanthomonas spp. form biofilms in planta that facilitate the host infection process. Herein, the role of extracellular DNA (eDNA) was evaluated in the formation and stabilization of the biofilm matrix at different stages of biofilm development. Fluorescence and light microscopy, as well as DNAse treatments, were used to determine the presence of eDNA in biofilms and bacterial cultures. DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development, as well as disrupted preformed biofilm. By comparison, no significant effect of the DNAse was detected for biofilm formation by Xc. DNAse effects on biofilm formation or disruption varied among Xcc strains and Xanthomonas species which suggest different roles for eDNA. Variation in the structure of fibers containing eDNA in biofilms, bacterial cultures, and in twitching motility was also visualized by microscopy. The proposed roles for eDNA are as an adhesin in the early stages of biofilm formation, as an structural component of mature bacterial aggregates, and twitching motility structures. PMID:27248687

  4. A 28,000 years old Cro-Magnon mtDNA sequence differs from all potentially contaminating modern sequences.

    PubMed

    Caramelli, David; Milani, Lucio; Vai, Stefania; Modi, Alessandra; Pecchioli, Elena; Girardi, Matteo; Pilli, Elena; Lari, Martina; Lippi, Barbara; Ronchitelli, Annamaria; Mallegni, Francesco; Casoli, Antonella; Bertorelle, Giorgio; Barbujani, Guido

    2008-07-16

    DNA sequences from ancient specimens may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal) and early modern (Cro-Magnoid) Europeans. We typed the mitochondrial DNA (mtDNA) hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23) and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences. The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans.

  5. DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions.

    PubMed

    Chen, Xiaojun; Hu, Jihong; Zhang, Hongyuan; Ding, Yi

    2014-03-01

    Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility-fertility transition of PA64S under two different environmental conditions.

  6. Treatment of long-term stored DNA--comparison between different methods to obtain high-quality material.

    PubMed

    de Almeida, Máira Pedroso; do Nascimento, Carlos Souza; Périssé, Iuri Viotti; de Souza Duarte, Marcio; Veroneze, Renata; Facioni Guimarães, Simone E

    2013-11-01

    Long-term stored DNA can be sometimes the only source of genetic material of an organism that does not exist anymore, but a research interest still persists. However, there is a lack of information about useful methods to improve quality from such type of material. In this study, we compared four different protocols using DNA samples collected in 1998. Fresh DNA was also tested aiming to check the differences between these two material types. Sixteen samples of each DNA type treated with phenol-chloroform with PEG 5.0%, silica-gel membrane spin column, PEG 7.5%, and glass-fiber matrix spin column were submitted to spectrophotometer measurements, electrophoresis, PCR, and RFLP-PCR to assess the best method concerning yield, quality, and purity. Based on the results, purification with PEG 7.5% was considered the best method to treat aged DNA samples. In addition to the efficiency, this protocol has low cost. Analyzing the data, we also conclude that long-term stored DNA may be considered a reliable and potential resource for future molecular studies.

  7. Presence of Extracellular DNA during Biofilm Formation by Xanthomonas citri subsp. citri Strains with Different Host Range.

    PubMed

    Sena-Vélez, Marta; Redondo, Cristina; Graham, James H; Cubero, Jaime

    2016-01-01

    Xanthomonas citri subsp. citri (Xcc) A strain causes citrus bacterial canker, a serious leaf, fruit and stem spotting disease of several Citrus species. X. alfalfae subsp. citrumelonis (Xac) is the cause of citrus bacterial spot, a minor disease of citrus nursery plants and X. campestris pv. campestris (Xc) is a systemic pathogen that causes black rot of cabbage. Xanthomonas spp. form biofilms in planta that facilitate the host infection process. Herein, the role of extracellular DNA (eDNA) was evaluated in the formation and stabilization of the biofilm matrix at different stages of biofilm development. Fluorescence and light microscopy, as well as DNAse treatments, were used to determine the presence of eDNA in biofilms and bacterial cultures. DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development, as well as disrupted preformed biofilm. By comparison, no significant effect of the DNAse was detected for biofilm formation by Xc. DNAse effects on biofilm formation or disruption varied among Xcc strains and Xanthomonas species which suggest different roles for eDNA. Variation in the structure of fibers containing eDNA in biofilms, bacterial cultures, and in twitching motility was also visualized by microscopy. The proposed roles for eDNA are as an adhesin in the early stages of biofilm formation, as an structural component of mature bacterial aggregates, and twitching motility structures.

  8. Base flip in DNA studied by molecular dynamics simulationsof differently-oxidized forms of methyl-Cytosine.

    PubMed

    Helabad, Mahdi Bagherpoor; Kanaan, Natalia; Imhof, Petra

    2014-07-03

    Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme's active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.

  9. Genetic differences between Chibcha and Non-Chibcha speaking tribes based on mitochondrial DNA (mtDNA) haplogroups from 21 Amerindian tribes from Colombia

    PubMed Central

    Usme-Romero, Solangy; Alonso, Milena; Hernandez-Cuervo, Helena; Yunis, Emilio J.; Yunis, Juan J.

    2013-01-01

    We analyzed the frequency of four mitochondrial DNA haplogroups in 424 individuals from 21 Colombian Amerindian tribes. Our results showed a high degree of mtDNA diversity and genetic heterogeneity. Frequencies of mtDNA haplogroups A and C were high in the majority of populations studied. The distribution of these four mtDNA haplogroups from Amerindian populations was different in the northern region of the country compared to those in the south. Haplogroup A was more frequently found among Amerindian tribes in northern Colombia, while haplogroup D was more frequent among tribes in the south. Haplogroups A, C and D have clinal tendencies in Colombia and South America in general. Populations belonging to the Chibcha linguistic family of Colombia and other countries nearby showed a strong genetic differentiation from the other populations tested, thus corroborating previous findings. Genetically, the Ingano, Paez and Guambiano populations are more closely related to other groups of south eastern Colombia, as also inferred from other genetic markers and from archeological data. Strong evidence for a correspondence between geographical and linguistic classification was found, and this is consistent with evidence that gene flow and the exchange of customs and knowledge and language elements between groups is facilitated by close proximity. PMID:23885195

  10. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues

    USDA-ARS?s Scientific Manuscript database

    Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...

  11. A platinum complex that binds non-covalently to DNA and induces cell death via a different mechanism than cisplatin.

    PubMed

    Suntharalingam, Kogularamanan; Mendoza, Oscar; Duarte, Alexandra A; Mann, David J; Vilar, Ramon

    2013-05-01

    Cisplatin and some of its derivatives have been shown to be very successful anticancer agents. Their main mode of action has been proposed to be via covalent binding to DNA. However, one of the limitations of these drugs is their poor activity against some tumours due to intrinsic or acquired resistance. Therefore, there is interest in developing complexes with different binding modes and mode of action. Herein we present a novel platinum(ii)-terpyridine complex (1) which interacts non-covalently with DNA and induces cell death via a different mechanism than cisplatin. The interaction of this complex with DNA was studied by UV/Vis spectroscopic titrations, fluorescent indicator displacement (FID) assays and circular dichroism (CD) titrations. In addition, computational docking studies were carried out with the aim of establishing the complex's binding mode. These experimental and computational studies showed the complex to have an affinity constant for DNA of ∼10(4) M(-1), a theoretical free energy of binding of -10.83 kcal mol(-1) and selectivity for the minor groove of DNA. Long-term studies indicated that 1 did not covalently bind (or nick) DNA. The cancer cell antiproliferative properties of this platinum(ii) complex were probed in vitro against human and murine cell lines. Encouragingly the platinum(ii) complex displayed selective toxicity for the cancerous (U2OS and SH-SY5Y) and proliferating NIH 3T3 cell lines. Further cell based studies were carried out to establish the mode of action. Cellular uptake studies demonstrated that the complex is able to penetrate the cell membrane and localize to the nucleus, implying that genomic DNA could be a cellular target. Detailed immunoblotting studies in combination with DNA-flow cytometry showed that the platinum(ii) complex induced cell death in a manner consistent with necrosis.

  12. Endothelial Cell Bioenergetics and Mitochondrial DNA Damage Differ in Humans Having African or West Eurasian Maternal Ancestry

    PubMed Central

    Krzywanski, David M.; Moellering, Douglas R.; Westbrook, David G.; Dunham-Snary, Kimberly J.; Brown, Jamelle; Bray, Alexander W.; Feeley, Kyle P.; Sammy, Melissa J.; Smith, Matthew R.; Schurr, Theodore G.; Vita, Joseph A.; Ambalavanan, Namasivayam; Calhoun, David; Dell’Italia, Louis; Ballinger, Scott W.

    2016-01-01

    Background We hypothesized that endothelial cells having distinct mitochondrial genetic backgrounds would show variation in mitochondrial function and oxidative stress markers concordant with known differential cardiovascular disease susceptibilities. To test this hypothesis, mitochondrial bioenergetics were determined in endothelial cells from healthy individuals with African versus European maternal ancestries. Methods and Results Bioenergetics and mitochondrial DNA (mtDNA) damage were assessed in single donor human umbilical vein endothelial cells (HUVECs) belonging to mtDNA haplogroups H and L, representing West Eurasian and African maternal ancestry, respectively. HUVECs from haplogroup L utilized less oxygen for ATP production and had increased levels of mtDNA damage compared to those in haplogroup H. Differences in bioenergetic capacity were also observed in that HUVECs belonging to haplogroup L had decreased maximal bioenergetic capacities compared to haplogroup H. Analysis of peripheral blood mononuclear cells from age-matched healthy controls with West Eurasian or African maternal ancestries showed that haplogroups sharing an A to G mtDNA mutation at nucleotide pair (np) 10,398 had increased mtDNA damage compared to those lacking this mutation. Further study of angiographically proven coronary artery disease patients and age-matched healthy controls revealed that mtDNA damage was associated with vascular function and remodeling, and that age of disease onset was later in individuals from haplogroups lacking the A to G mutation at np 10,398. Conclusions Differences in mitochondrial bioenergetics and mtDNA damage associated with maternal ancestry may contribute to endothelial dysfunction and vascular disease. PMID:26787433

  13. Endothelial Cell Bioenergetics and Mitochondrial DNA Damage Differ in Humans Having African or West Eurasian Maternal Ancestry.

    PubMed

    Krzywanski, David M; Moellering, Douglas R; Westbrook, David G; Dunham-Snary, Kimberly J; Brown, Jamelle; Bray, Alexander W; Feeley, Kyle P; Sammy, Melissa J; Smith, Matthew R; Schurr, Theodore G; Vita, Joseph A; Ambalavanan, Namasivayam; Calhoun, David; Dell'Italia, Louis; Ballinger, Scott W

    2016-02-01

    We hypothesized that endothelial cells having distinct mitochondrial genetic backgrounds would show variation in mitochondrial function and oxidative stress markers concordant with known differential cardiovascular disease susceptibilities. To test this hypothesis, mitochondrial bioenergetics were determined in endothelial cells from healthy individuals with African versus European maternal ancestries. Bioenergetics and mitochondrial DNA (mtDNA) damage were assessed in single-donor human umbilical vein endothelial cells belonging to mtDNA haplogroups H and L, representing West Eurasian and African maternal ancestries, respectively. Human umbilical vein endothelial cells from haplogroup L used less oxygen for ATP production and had increased levels of mtDNA damage compared with those in haplogroup H. Differences in bioenergetic capacity were also observed in that human umbilical vein endothelial cells belonging to haplogroup L had decreased maximal bioenergetic capacities compared with haplogroup H. Analysis of peripheral blood mononuclear cells from age-matched healthy controls with West Eurasian or African maternal ancestries showed that haplogroups sharing an A to G mtDNA mutation at nucleotide pair 10398 had increased mtDNA damage compared with those lacking this mutation. Further study of angiographically proven patients with coronary artery disease and age-matched healthy controls revealed that mtDNA damage was associated with vascular function and remodeling and that age of disease onset was later in individuals from haplogroups lacking the A to G mutation at nucleotide pair 10398. Differences in mitochondrial bioenergetics and mtDNA damage associated with maternal ancestry may contribute to endothelial dysfunction and vascular disease. © 2016 American Heart Association, Inc.

  14. Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos.

    PubMed Central

    Dessain, S; Gross, C T; Kuziora, M A; McGinnis, W

    1992-01-01

    Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities. Images PMID:1347746

  15. DNA status in brain and heart as prominent co-determinant for life span? Assessing the different degrees of DNA damage, damage susceptibility, and repair capability in different organs of young and old mice.

    PubMed

    Zahn, R K; Jaud, S; Schröder, H C; Zahn-Daimler, G

    1996-08-15

    Alkaline filter elution has been modified by a freeze-grinding step that allows the evaluation of the DNA status of whole tissue, including mouse tail cross-sections, with only small additional artefacts. Four to seven different organs from individually coordinated female NMRI mice, rated as young when 2-3 months, and as old when 24-27 months, of age, have been used. Tissues of individual mice differ significantly in their DNA status. Alkali-labile sites are relatively rare and differ in amount in the different organs in the young. They show significant increases in the old, reaching the highest values in the brain and the heart. Proteinase K dependent DNA-protein cross-links are not prominent nor are they increased with age in some organs, except in the brain and the heart. DNA damage susceptibility was measured after application of 3.5 microM nitoquinolin-N-oxide to 15 mg fresh. tissue pieces for 90 min. The susceptibility is large and varying in wide ranges in the different organs. Upon 3 h post-exposure incubation in full medium all samples showed DNA repair, the young reach nearly complete repair in the lung, while repair, generally, in the old is significantly decreased. In old brain and heart it is even near zero. This together with high values in alkali-labile sites and protein-DNA cross-linking suggests that these two organs may act as pacemakers and play a role as prominent co-determinants for the life span of the species.

  16. Comparison of different methods for extraction and purification of human Papillomavirus (HPV) DNA from serum samples

    NASA Astrophysics Data System (ADS)

    Azizah, N.; Hashim, U.; Nadzirah, Sh.; Arshad, M. K. Md; Ruslinda, A. R.; Gopinath, Subash C. B.

    2017-03-01

    The affectability and unwavering quality of PCR for indicative and research purposes require effective fair systems of extraction and sanitization of nucleic acids. One of the real impediments of PCR-based tests is the hindrance of the enhancement procedure by substances exhibit in clinical examples. This examination considers distinctive techniques for extraction and cleaning of viral DNA from serum tests in view of recuperation productivity as far as yield of DNA and rate recouped immaculateness of removed DNA, and rate of restraint. The best extraction strategies were the phenol/chloroform strategy and the silica gel extraction methodology for serum tests, individually. Considering DNA immaculateness, extraction technique by utilizing the phenol/chloroform strategy delivered the most tasteful results in serum tests contrasted with the silica gel, separately. The nearness of inhibitors was overcome by all DNA extraction strategies in serum tests, as confirm by semiquantitative PCR enhancement.

  17. Different fates of oocytes with DNA double-strand breaks in vitro and in vivo.

    PubMed

    Lin, Fei; Ma, Xue-Shan; Wang, Zhen-Bo; Wang, Zhong-Wei; Luo, Yi-Bo; Huang, Lin; Jiang, Zong-Zhe; Hu, Meng-Wen; Schatten, Heide; Sun, Qing-Yuan

    2014-01-01

    In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

  18. [Characteristics of DNA adsorption on different sizes red soil colloidal particles].

    PubMed

    Liao, Min; Xie, Xiao-Mei; Fang, Shu; Qiu, Xiao-Bai; Chen, Na; Xu, Ya-Qian; Jiang, Chun-Yan; Chen, Xue-fang

    2013-03-01

    By using balance reaction method, this paper studied the adsorption characteristics and thermodynamic properties of DNA on four kinds of red soil colloids (organic matter-contained coarse clay, organic matter-removed coarse clay, organic matter-contained fine clay, and organic matter-removed fine clay). The DNA adsorption on the four red soil colloids was a process of fast reaction, and the adsorption isotherms were conformed to the Langmuir equation, with the corresponding correlation coefficient (r2) being 0.974, 0. 991, 0. 958, and 0. 975, respectively. The maximum adsorption amount of DNA on the colloidal particles followed the order of organic matter-contained fine clay > organic matter-removed fine clay > organic matter-contained coarse clay > organic matter-removed coarse clay, implying that the size and organic matter content of colloidal particles played an important role in DNA adsorption. Electrolyte concentration and type and adsorption system pH were the main factors affecting the DNA adsorption on the four soil colloids. Within a definite electrolyte concentration range (NaCl < 60 mmol . L-1 and CaCl2 <10 mmol L-1) , the adsorption amount of DNA on the red soil colloids increased significantly with the increase of electrolyte concentration. As compared with sodium ion, calcium ion had a greater promotion effect on the DNA adsorption, but the effect decreased significantly with the increase of adsorption system pH. The DNA adsorption on the organic matter-contained red soil colloids was an endothermic reaction, while the DNA adsorption on the organic matter-removed red soil colloids was an exothermic reaction. The DNA adsorption on the red soil colloids was a process of entropy increase.

  19. Comparison of different protocols for DNA preparation and PCR for the detection of fungal pathogens in vitro.

    PubMed

    Lugert, R; Schettler, C; Gross, U

    2006-07-01

    Although a large number of different PCR protocols for the detection of fungal DNA from clinical samples have been described, a generally recognised standardisation has not yet been developed. In a first step, we compared six different methods to isolate DNA under in vitro conditions from Aspergillus fumigatus, Candida albicans and Saccharomyces cerevisiae with respect to efficiency and expenditure of time. To this end, methods were tested that are based on both mechanical and enzymatic/thermic lysis. Thereby, enzymatic/thermic lysis were shown to be superior to mechanical lysis, although these methods of DNA isolation were more time consuming. The subsequent comparison of three different PCR protocols showed real-time PCR to be the most sensitive method.

  20. Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates.

    PubMed

    Shyma, K P; Gupta, S K; Gupta, J P; Singh, Ajit; Chaudhari, S S; Singh, Veer

    2016-09-01

    The differences or similarities among different isolates of Trypanosoma evansi through endonuclease profile was identified in the present study. The repetitive nuclear DNA of T. evansi isolated from infected cattle, buffalo and equine blood was initially amplified by PCR using specific primers. A panel of restriction enzymes, EcoRI, Eco91l, HindIII and PstI were for complete digestion of PCR products. Agarose gel electrophoresis of digested product did not show cleavage fragments and only single DNA band of the original size was visible in the ethidium bromide stained agarose gel. This indicated that the 227 bp PCR product from repetitive sequence had no site-specific cleavage sites for the REs used in this study. No heterogeneity in the repetitive nuclear DNA restriction endonuclease profile among the different isolates was recorded.

  1. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2.

    PubMed

    Jha, Jyoti K; Li, Mi; Ghirlando, Rodolfo; Miller Jenkins, Lisa M; Wlodawer, Alexander; Chattoraj, Dhruba

    2017-04-18

    Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid-the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication of Vibrio cholerae Chr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing

  2. Seasonal variations of atmospheric heterocyclic aromatic amines in Beijing, China

    NASA Astrophysics Data System (ADS)

    Dong, Xueling; Liu, Dameng; Gao, Shaopeng

    2013-02-01

    Heterocyclic aromatic amines (HAAs) belong to a group of substances associated with a high mutagenic and carcinogenic potential. This study reports that carcinogenic HAAs may be present in airborne particles. Airborne particles (PM10) were sampled from March 2005 to January 2006 at four urban sites in Beijing. Collected particulate matter was analyzed for six HAAs using high-performance liquid chromatography (HPLC) with fluorescence and UV detection. Clear seasonal variations of HAAs were observed with seasonal mass concentrations ranging from 0.66 ± 0.20 ng m- 3 (summer) to 19.76 ± 14.38 ng m- 3 (autumn). The carcinogenic amino-imidazo-azaarenes, including 2-Amino-3-methyl-3H-imidazo[4,5-f] quinoline (IQ), 2-Amino-3,4-dimethyl-3H-imidazo[4,5-f] quinoline (MeIQ), and 2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), were the major components with 75.2-87.0% of the total HAAs during the whole year except for summer. 3-Amino-1-methyl-5H-pyrido[3,4-b] indole (Trp-P-2), 2-Amino-9H-pyrido[2,3-b] indole (AαC), and 2-Amino-3-methyl-9H-pyrido[2,3-b] indole (MeAαC), with similar structures, were found to have similar seasonal patterns and strong correlations (r = 0.63-0.90) throughout the observation, which indicates that they most likely come from similar emission sources. Positive correlations between site-specific HAA concentrations and the relative humidity were observed. Of the different sites studied, the total HAA concentrations were most abundant at commercial sites and the smallest at residential sites. The combustion aerosols emitted from cooking, coal, and petroleum may be the sources of these carcinogens in the atmosphere, and cooking emissions may probably play an important role in Beijing's HAAs pollution.

  3. Different Evolutionary History for Basque Diaspora Populations in USA and Argentina Unveiled by Mitochondrial DNA Analysis.

    PubMed

    Baeta, Miriam; Núñez, Carolina; Cardoso, Sergio; Palencia-Madrid, Leire; Piñeiro-Hermida, Sergio; Arriba-Barredo, Miren; Villanueva-Millán, María Jesús; M de Pancorbo, Marian

    2015-01-01

    The Basque Diaspora in Western USA and Argentina represents two populations which have maintained strong Basque cultural and social roots in a completely different geographic context. Hence, they provide an exceptional opportunity to study the maternal genetic legacy from the ancestral Basque population and assess the degree of genetic introgression from the host populations in two of the largest Basque communities outside the Basque Country. For this purpose, we analyzed the complete mitochondrial DNA control region of Basque descendants living in Western USA (n = 175) and in Argentina (n = 194). The Diaspora populations studied here displayed a genetic diversity in their European maternal input which was similar to that of the Basque source populations, indicating that not important founder effects would have occurred. Actually, the genetic legacy of the Basque population still prevailed in their present-day maternal pools, by means of a haplogroup distribution similar to the source population characterized by the presence of autochthonous Basque lineages, such as U5b1f1a and J1c5c1. However, introgression of non-Basque lineages, mostly Native American, has been observed in the Diaspora populations, particularly in Argentina, where the quick assimilation of the newcomers would have favored a wider admixture with host populations. In contrast, a longer isolation of the Diaspora groups in USA, because of language and cultural differences, would have limited the introgression of local lineages. This study reveals important differences in the maternal evolutionary histories of these Basque Diaspora populations, which have to be taken into consideration in forensic and medical genetic studies.

  4. Different Evolutionary History for Basque Diaspora Populations in USA and Argentina Unveiled by Mitochondrial DNA Analysis

    PubMed Central

    Cardoso, Sergio; Palencia-Madrid, Leire; Piñeiro-Hermida, Sergio; Arriba-Barredo, Miren; Villanueva-Millán, María Jesús; M. de Pancorbo, Marian

    2015-01-01

    The Basque Diaspora in Western USA and Argentina represents two populations which have maintained strong Basque cultural and social roots in a completely different geographic context. Hence, they provide an exceptional opportunity to study the maternal genetic legacy from the ancestral Basque population and assess the degree of genetic introgression from the host populations in two of the largest Basque communities outside the Basque Country. For this purpose, we analyzed the complete mitochondrial DNA control region of Basque descendants living in Western USA (n = 175) and in Argentina (n = 194). The Diaspora populations studied here displayed a genetic diversity in their European maternal input which was similar to that of the Basque source populations, indicating that not important founder effects would have occurred. Actually, the genetic legacy of the Basque population still prevailed in their present-day maternal pools, by means of a haplogroup distribution similar to the source population characterized by the presence of autochthonous Basque lineages, such as U5b1f1a and J1c5c1. However, introgression of non-Basque lineages, mostly Native American, has been observed in the Diaspora populations, particularly in Argentina, where the quick assimilation of the newcomers would have favored a wider admixture with host populations. In contrast, a longer isolation of the Diaspora groups in USA, because of language and cultural differences, would have limited the introgression of local lineages. This study reveals important differences in the maternal evolutionary histories of these Basque Diaspora populations, which have to be taken into consideration in forensic and medical genetic studies. PMID:26659590

  5. No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA

    PubMed Central

    Mongkolchaipak, Suchada; Vutyavanich, Teraporn

    2013-01-01

    In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria, and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method (control), high magnification at ×6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6% vs. 1.7% P=0.032), with no significant difference in aneuploidy rate (0.8% vs 0.7% P=0.583), than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7% aneuploidy and 26.8% DNA fragmentation rates, respectively). In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference (0.9%) might not be clinically meaningful. Both methods were better than the conventional method of sperm selection. PMID:23435468

  6. No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA.

    PubMed

    Mongkolchaipak, Suchada; Vutyavanich, Teraporn

    2013-05-01

    In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria, and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method (control), high magnification at ×6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6% vs. 1.7%; P=0.032), with no significant difference in aneuploidy rate (0.8% vs 0.7%; P=0.583), than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7% aneuploidy and 26.8% DNA fragmentation rates, respectively). In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference (0.9%) might not be clinically meaningful. Both methods were better than the conventional method of sperm selection.

  7. Immobilization of denatured DNA to macroporous supports: I. Efficiency of different coupling procedures.

    PubMed

    Bünemann, H; Westhoff, P; Herrmann, R G

    1982-11-25

    Methods commonly used for covalent immobilization of single stranded DNA have been applied to several solid supports (Sephadex G-25 and Cellex 410) as well as to a number of macroporous materials (Sepharose C1-6B, C1-2B; Sephacryl S-500 and S-1000). Coupling efficiencies and stability of covalently bound DNA are compared for both classes of materials. The yields of the immobilization reaction for sonicated DNA are only 10-40% for G-25 and Cellex 410 in contrast to 60-80% for C1-6B and S-500. Under optimal conditions, up to 0.5 mg of DNA can be coupled initially per g of wet macroporous material. The immobilized DNAs are lost from the supports in a biphasic manner, with about 10-20% loss per day during the first 2-3 days at 45 degrees C, followed by only about 1% loss per day at the same temperature thereafter. The influence of the coupling procedure on the generation of mismatch effects has been studied in 2.4 M tetraethylammonium chloride solution for the hybrid formation between immobilized and mobile DNA. The degree of mismatch ranged from 0-3% and depended on the method of immobilization. The unspecific absorption of DNA on macroporous materials is sufficiently low to allow efficient hybrid selection. No size limitations have been observed when plastid mRNAs are selected by cloned fragments of plastid DNA immobilized to macroporous Sephacryl S-500.

  8. The glial transcription factor Sox10 binds to DNA both as monomer and dimer with different functional consequences.

    PubMed

    Peirano, R I; Wegner, M

    2000-08-15

    Sox10 is an important transcriptional regulator in the neural crest and various neural-crest derived lineages, such as the Schwann cells of the peripheral nervous system. Recently, we identified the gene for myelin Protein zero (P(0)) as a transcriptional target of Sox10 in Schwann cells, allowing for the first time a detailed analysis of Sox10 responsive elements and their functional interaction with Sox10. Here we show that Sox10 functions through two different types of DNA response elements, one that allows binding of monomers, and a second that favors cooperative binding of two molecules. This dimeric binding required the presence of two heptameric Sox binding sites in a specific orientation and spacing, and was mediated by an N-terminal region of Sox10 with high conservation in the related Sox9, which also exhibited dimeric binding. This argues that the conserved region has the capacity to function as a DNA-dependent dimerization domain. The interaction between Sox10 dimers and DNA differed dramatically from that of Sox10 monomers, as it drastically reduced the protein's off-rate and increased the protein-induced angle of DNA bending. These results indicate that functionally relevant interactions between Sox10 and DNA occur through completely different modes of binding.

  9. DNA Superresolution Structure of Reed-Sternberg Cells Differs Between Long-Lasting Remission Versus Relapsing Hodgkin's Lymphoma Patients.

    PubMed

    Righolt, Christiaan H; Knecht, Hans; Mai, Sabine

    2016-07-01

    Recent developments in microscopy have led to superresolution microscopy images of cells. Structured illumination microscopy was used before to reveal new details in the DNA structure and the structure of the DNA-free space in the DAPI-stained cell nuclei of the Hodgkin's lymphoma HDLM-2 cell line. This study extends this technology to primary pre-treatment classical Hodgkin's lymphoma samples of ten patients. Significant differences in both the DNA structure and the structure of the DNA-free space were detected between lymphocytes and malignant cells. Both types of structures were similar for lymphocytes of different patients. When the patients were un-blinded and grouped based on their clinical outcome, either non-relapsed or relapsed, a significant difference in the DNA structure of their Reed-Sternberg (RS) cells was found. Since, RS cells develop from mono-nucleated Hodgkin (H) cells, these data suggest distinct architectural restructuring of nuclei during RS cell formation in patients going to long-lasting remission versus relapse. J. Cell. Biochem. 117: 1633-1637, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  10. Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades.

    PubMed

    Stangherlin, Lucas Matheus; Castro, Fabiane Lucy Ferreira; Medeiros, Raphael Salles Scortegagna; Guerra, Juliana Mariotti; Kimura, Lidia Midori; Shirata, Neuza Kazumi; Nonogaki, Suely; Dos Santos, Claudia Januário; Carlan Silva, Maria Cristina

    2016-01-01

    Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties.

  11. Environmental DNA metabarcoding reveals primary chemical contaminants in freshwater sediments from different land-use types.

    PubMed

    Xie, Yuwei; Wang, Jizhong; Yang, Jianghua; Giesy, John P; Yu, Hongxia; Zhang, Xiaowei

    2017-04-01

    Land-use intensification threatens freshwater biodiversity. Freshwater eukaryotic communities are affected by multiple chemical contaminants with a land-use specific manner. However, biodiversities of eukaryotes and their associations with multiple chemical contaminants are largely unknown. This study characterized in situ eukaryotic communities in sediments exposed to mixtures of chemical contaminants and assessed relationships between various environmental variables and eukaryotic communities in sediments from the Nanfei River. Eukaryotic communities in the sediment samples were dominated by Annelida, Arthropoda, Rotifera, Ochrophyta, Chlorophyta and Ciliophora. Alpha-diversities (Shannon entropy) and structures of eukaryotic communities were significantly different between land-use types. According to the results of multiple statistical tests (PCoA, distLM, Mantel and network analysis), dissimilarity of eukaryotic community structures revealed the key effects of pyrethroid insecticides, manganese, zinc, lead, chromium and polycyclic aromatic hydrocarbons (PAHs) on eukaryotic communities in the sediment samples from the Nanfei River. Furthermore, taxa associated with land-use types were identified and several sensitive eukaryotic taxa to some of the primary contaminants were identified as potential indicators to monitor effects of the primary chemical contaminants. Overall, environmental DNA metabarcoding on in situ eukaryotic communities provided a powerful tool for biomonitoring and identifying primary contaminants and their complex effects on benthic eukaryotic communities in freshwater sediments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Direct estimation of biofilm density on different pipe material coupons using a specific DNA-probe.

    PubMed

    Chang, Young C; Le Puil, Michael; Biggerstaff, John; Randall, Andrew A; Schulte, Alfons; Taylor, James S

    2003-10-01

    A variety of approaches to quantify biomass in biofilms without disruption due to detachment have been developed over the years. One basic approach is the combination of advanced microscopy with molecular staining. However, many stains (e.g. 4',6-diamino-2-phenylindole, acridine orange or live-dead stains) can be non-specific when corrosion products, precipitates, and pipe material are present. In addition, some pipe materials cause high background when using epifluorescent microscopy. The new refinement discussed in this presentation used fluorescence spectroscopy to obtain the spectra from four common distribution system pipe materials: PVC, 'concrete' lined cast iron, cast iron, and galvanized steel. The emission maximum for all four materials was between 500 and 550 nm, but emissions radically decreased around 575-600 nm. A molecular probe, BO-PRO-3 (Molecular Probes, Inc., Eugene, OR, USA) was identified which has an emission intensity maximum at 599 nm (red), with emission intensity 200 times greater when it is bound to DNA. The BO-PRO-3 has greatly reduced non-specific staining and background problems. In the preliminary experiment, using diluted waste water, a significant exponential relationship was found between stained surface area/total area ratio and fixed biofilm inventory measurements from scraping heterotrophic plate counts (SHPC) on R2A medium. In addition, the biofilm inventory on different pipe material coupons from pilot distribution systems was also correlated to the stained surface area fraction and SHPC.

  13. DNA methylation profiling identifies two splenic marginal zone lymphoma subgroups with different clinical and genetic features.

    PubMed

    Arribas, Alberto J; Rinaldi, Andrea; Mensah, Afua A; Kwee, Ivo; Cascione, Luciano; Robles, Eloy F; Martinez-Climent, Jose A; Oscier, David; Arcaini, Luca; Baldini, Luca; Marasca, Roberto; Thieblemont, Catherine; Briere, Josette; Forconi, Francesco; Zamò, Alberto; Bonifacio, Massimiliano; Mollejo, Manuela; Facchetti, Fabio; Dirnhofer, Stephan; Ponzoni, Maurilio; Bhagat, Govind; Piris, Miguel A; Gaidano, Gianluca; Zucca, Emanuele; Rossi, Davide; Bertoni, Francesco

    2015-03-19

    Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

  14. Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades

    PubMed Central

    Medeiros, Raphael Salles Scortegagna; Guerra, Juliana Mariotti; Kimura, Lidia Midori; Shirata, Neuza Kazumi; Nonogaki, Suely; dos Santos, Claudia Januário; Carlan Silva, Maria Cristina

    2016-01-01

    Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties. PMID:27458810

  15. Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport bacterial DNA into the plant cell nucleus.

    PubMed

    Ream, Walt

    2009-07-01

    Agrobacterium tumefaciens and A. rhizogenes transport single-stranded DNA (ssDNA; T-strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour-inducing plasmid, attaches to the 5' end, and pilots T-strands into plant cells. Agrobacterium tumefaciens translocates ssDNA-binding protein VirE2 into plant cells where it targets T-strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T-strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full-length GALLS (GALLS-FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T-strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS-FL contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T-strands into the nucleus using the force generated by cooperative DNA binding. GALLS-FL accumulates inside the nucleus where its predicted ATP-dependent strand transferase may pull T-strands into the nucleus. These different mechanisms for nuclear import of T-strands may affect the efficiency and quality of transgenic events in plant biotechnology applications.

  16. Impedimetric genosensing of DNA polymorphism correlated to cystic fibrosis: a comparison among different protocols and electrode surfaces.

    PubMed

    Bonanni, Alessandra; Esplandiu, Maria José; del Valle, Manel

    2010-12-15

    In this work, a genosensor for the impedimetric detection of the triple base deletion in a cystic fibrosis-related DNA synthetic sequence is presented. Screen-Printed Carbon Electrodes containing Carboxyl functionalised multi-walled carbon nanotubes were used for the immobilization of an amino-modified oligonucleotide probe, complementary to the Cystic Fibrosis (CF) mutant gene. The complementary target (the mutant sequence) was then added and its hybridization allowed. The change of interfacial charge transfer resistance (R(ct)) between the solution and the electrode surface, experimented by the redox marker ferrocyanide/ferricyanide, confirmed the hybrid formation. A non-complementary DNA sequence and a three-mismatch sequence corresponding to the wild DNA gene (present in healthy people) were used as negative controls. A further step employing a signalling biotinylated probe was performed for signal amplification using streptavidin-modified gold nanoparticles (strept-AuNPs). In order to observe by SEM the presence and distribution of strept-AuNPs, a silver enhancement treatment was applied to electrodes already modified with DNA-nanoparticles conjugate. The developed protocol allowed the very sensitive detection of the triple base deletion in a label-free CF-related DNA sequence, achieving a LOD around 100 pM. Results were finally compared with those obtained using different protocols for immobilization of DNA capture probe. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Cyclization of carbonyl groups onto alkynes upon reaction with IPy2BF4 and their trapping with nucleophiles: a versatile trigger for assembling oxygen heterocycles.

    PubMed

    Barluenga, José; Vázquez-Villa, Henar; Ballesteros, Alfredo; González, José M

    2003-07-30

    Iodonium ions liberated from bis(pyridine)iodonium(I) tetrafluoroborate react with ortho-alkynyl-substituted carbonyl compounds and different nucleophiles to give valuable iodinated heterocycles at room temperature, through a new and metal-free reaction sequence. Interestingly, the nature of the nucleophile can be widely modified, and not only alcohols but also several carbon-based nucleophiles can be nicely used.

  18. On the uniqueness of quantitative DNA difference descriptors in 2D graphical representation models

    NASA Astrophysics Data System (ADS)

    Nandy, A.; Nandy, P.

    2003-01-01

    The rapid growth in additions to databases of DNA primary sequence data have led to searches for methods to numerically characterize these data and help in fast identification and retrieval of relevant sequences. The DNA descriptors derived from the 2D graphical representation technique have already been proposed to index chemical toxicity and single nucleotide polymorphic (SNP) genes but the inherent degeneracies in this representation have given rise to doubts about their suitability. We prove in this paper that such degeneracies will exist only in very restricted cases and that the method can be relied upon to provide unique descriptors for, in particular, the SNP genes and several other classes of DNA sequences.

  19. Direct, metal-free amination of heterocyclic amides/ureas with NH-heterocycles and N-substituted anilines in POCl3.

    PubMed

    Deng, Xiaohu; Roessler, Armin; Brdar, Ivana; Faessler, Roger; Wu, Jiejun; Sales, Zachary S; Mani, Neelakandha S

    2011-10-21

    A POCl(3)-mediated, direct amination reaction of heterocyclic amides/ureas with NH-heterocycles or N-substituted anilines is described. Compared to the existing methods, this operationally simple protocol provides unique reactivity and functional group compatibility because of the metal-free, acidic reaction conditions. The yields are generally excellent.

  20. A pyrimidopyrimidine Janus-AT nucleoside with improved base-pairing properties to both A and T within a DNA duplex: the stabilizing effect of a second endocyclic ring nitrogen.

    PubMed

    Largy, Eric; Liu, Wenbo; Hasan, Abid; Perrin, David M

    2014-02-03

    Janus bases are heterocyclic nucleic acid base analogs that present two different faces able to simultaneously hydrogen bond to nucleosides that form Watson-Crick base pairs. The synthesis of a Janus-AT nucleotide analogue, (N)JAT , that has an additional endocyclic ring nitrogen and is thus more capable of efficiently discriminating T/A over G/C bases when base-pairing in a standard duplex-DNA context is described. Conversion to a phosphoramidite ultimately afforded incorporation into an oligonucleotide. In contrast to the first generation of carbocyclic Janus heterocycles, it remains in its unprotonated state at physiological pH and, therefore, forms very stable Watson-Crick base pairs with either A or T bases. Biophysical and computational methods indicate that (N)JAT is an improved candidate for sequence-specific genome targeting.

  1. Novel inorganic heterocycles from dimetalated carboranylamidinates.

    PubMed

    Harmgarth, Nicole; Gräsing, Daniel; Dröse, Peter; Hrib, Cristian G; Jones, Peter G; Lorenz, Volker; Hilfert, Liane; Busse, Sabine; Edelmann, Frank T

    2014-04-07

    Mono- and dianionic carboranylamidinates are readily available in one-pot reactions directly from o-carborane (1). In situ-monolithiation of 1 followed by treatment with N,N'-diisopropylcarbodiimide, (i)PrN=C=N(i)Pr, or N,N'-dicyclohexylcarbodiimide, CyN=C=NCy, provided the lithium carboranylamidinates (o-C2B10H10C(NH(i)Pr)(=N(i)Pr)-κ(2)C,N)Li(DME) (2a) and (o-C2B10H10C(NH(i)Cy)(=N(i)Cy)-κ(2)C,N)Li(THF)2 (2b). Controlled hydrolysis of 2a,b afforded the free carboranylamidines o-C2B10H11C(NH(i)R)(=N(i)R) (3a: R = (i)Pr, 3b: R = Cy). The first dimetalated carboranylamidinates, o-C2B10H10C(N(i)Pr)(=N(i)Pr)Li2(DME)2 (4a) (DME = 1,2-dimethoxyethane) and o-C2B10H10C(N(i)Pr)(=N(i)Pr)Li2(THF)4 (4b), were prepared in high yield (83% yield) directly from 1 using a simple one-pot synthetic protocol. Treatment of 4b with 2 equiv. of Me3SiCl afforded the disilylated derivative o-C2B10H10-κ(2)C,N-[C(N(i)PrSiMe3)(=N(i)Pr)]SiMe3 (5). Dianionic 4b also served as an excellent precursor for novel inorganic heterocycles incorporating the closo-1,2-C2B10H10 cage, including the unsymmetrical distannene [o-C2B10H10C(N(i)Pr)(=N(i)Pr)-κ(2)C,N]Sn=Sn[((i)PrN)2C(n)Bu]2 (6) and the azaphosphole derivative [o-C2B10H10C(N(i)Pr)(=N(i)Pr)-κ(2)C,N]PPh (7). Surprisingly, it was found that the synthesis of new inorganic ring systems from dianionic carboranylamidinates can also be achieved by employing only 1 equiv. of n-butyllithium in the generation of the anionic carboranylamidinate intermediates. Using this straightforward one-pot synthetic protocol, the Group 14 metallacycles [o-C2B10H10C(NCy)(=NCy)-κ(2)C,N]SiR2 (R = Cl (8), Me (9), Ph (10)) and [o-C2B10H10C(NCy)([=NCy)-κ(2)C,N]GeCl2 (11) have become accessible. The same synthetic strategy could be successfully adapted to prepare the corresponding Group 4 metallocene derivatives Cp2Ti[o-C2B10H10C(NCy)(=NCy)-κ(2)C,N] (12) and Cp2Zr[o-C2B10H10C(NCy)(=NCy)-κ(2)C,N] (13). The molecular structures of 2b, 3b, 4b, 5, 6, 7, 10, 12, and 13 were

  2. Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA).

    PubMed

    Everts, R E; Versteeg, S A; Renier, C; Vignaux, F; Groot, P C; Rothuizen, J; van Oost, B A

    2000-09-01

    Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 differen