The binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated rat liver nuclei: effects of glutathione and methionine

The covalent binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated, intact rat liver nuclei was studied. The chemically synthesized 2-acetylaminofluorene-N-sulfate became covalently bound to DNA and protein to form adducts, 50% to 60% of which retained the N-acetyl group. Glutathione decreased the covalent binding of acetylated adducts to DNA by 18% and to protein by 50%. Methionine was more effective; it decreased DNA binding by 52% and protein binding by 79%. N-Hydroxy-2-acetylaminofluorene was deacetylated by the nuclear preparation. Almost exclusively, deacetylated 2-aminofluorene adducts to DNA and protein were formed. Glutathione decreased the covalent binding of deacetylated adducts to DNA by only 14%. Protein binding, however, was decreased by 57%. Methionine had no effect on the formation of these adducts to DNA and protein. Formation of 2-aminofluorene-glutathione conjugates was reduced by ascorbic acid by 65%. Covalent binding of deacetylated adducts to DNA and protein, however, was not decreased by ascorbic acid. These data suggest that "harder" nucleophiles like methionine can be used to protect macromolecules in vivo from damage by "hard" electrophiles such as those generated from the reactive 2-acetylaminofluorene-N-sulfate. However, such nucleophiles seem not to be effective with N-hydroxylamines, such as N-hydroxy-2-aminofluorene, formed by deacetylation of N-hydroxy-2-acetylaminofluorene.     

Linkage analysis of families with hereditary retinoblastoma: nonpenetrance of mutation, revealed by combined use of markers within and flanking the RB1 gene.

Nonpenetrance of the inherited mutation responsible for retinoblastoma has been reported. By DNA analysis in families with hereditary retinoblastoma, it is possible to identify healthy individuals in whom the mutation is nonpenetrant. This requires the use of DNA markers both within and flanking the retinoblastoma gene. We have analyzed the segregation of several markers in 19 families (69 meioses) with hereditary retinoblastoma. In two families a carrier was identified who showed nonpenetrance of the mutation predisposing to retinoblastoma. The intragenic markers were informative in 15 pedigrees. The use of flanking markers from the same chromosomal region caused an increase of the number of informative families to 18. No crossing-over within the gene was observed. In one family an inherited deletion involving one of the RB1 alleles was detected. Our findings emphasize the use of a combination of both intragenic and flanking markers to obtain both the highest reliability of carrier detection in families with hereditary retinoblastoma and an accurate estimate of the frequency of nonpenetrance.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

Metabolic cost of changes in diet and neutralization of allelochemics

Larvae ofPieris brassicae andMamestra brassicae use food on which they have been grown more efficiently than a novel food. This physiological adaptation to a certain food may be regarded as a functional reason underlying changes in food selection behaviour due to previous experiences (“preference induction”). Larvae ofManduca sexta show a reduced efficiency of digestion when the diet contains nicotine or atropine, alkaloids occurring in acceptable food plants. It is concluded that insects may spend a considerable amount of energy detoxifying host-plant specific allelochemics.

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Le Prix Métabolique du Changement de Diète et de la Neutralisaton de Produits Allélochimiques

Résumé Les chenilles dePieris brassicae et deMamestra brassicae digèrent la nourriture où elles ont poussé d'une manière plus efficace qu'un nouveau genre de nourriture. Cette adaption physiologique à la nourriture est probablement la raison fontionnelle qui est à la base de changements dans le comportement du choix alimentaire qui sont en rapport avec des expériences antérieures (“induction d'une préférence”). L'efficacité de la digestion dans les chenilles deManduca sexta subit une diminution, quand on ajoute à leur aliment artificiel de la nicotine ou de l'atropine, des alcalo?des qu'on trouve dans des plantes nutritives acceptables. Voilà pourquoi nous en venons à la conclusion que les insectes consacrent parfois une quantité considérable d'énergie à la détoxication de matières allélochimiques qui se trouvent dans leur plante-h?te.

     

The Influence of Antimicrobial Peptides and Mucolytics on the Integrity of Biofilms Consisting of Bacteria and Yeasts as Affecting Voice Prosthetic Air Flow Resistances

The integrity of biofilms on voice prostheses used to rehabilitate speech in laryngectomized patients causes unwanted increases in airflow resistance, impeding speech. Biofilm integrity is ensured by extracellular polymeric substances (EPS). This study aimed to determine whether synthetic salivary peptides or mucolytics, including N-acetylcysteine and ascorbic acid, influence the integrity of voice prosthetic biofilms. Biofilms were grown on voice prostheses in an artificial throat model and exposed to synthetic salivary peptides, mucolytics and two different antiseptics (chlorhexidine and Triclosan). Synthetic salivary peptides did not reduce the air flow resistance of voice prostheses after biofilm formation. Although both chlorhexidine and Triclosan reduced microbial numbers on the prostheses, only the Triclosan-containing positive control reduced the air flow resistance. Unlike ascorbic acid, the mucolytic N-acetylcysteine removed most EPS from the biofilms and induced a decrease in air flow resistance.     

Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage

We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%).The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.