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Proceedings of the National Academy of... Mar 1986In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7,...
In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells.
Topics: Acetoxyacetylaminofluorene; Apurinic Acid; Base Sequence; Benzopyrenes; Carcinogens; Chemical Phenomena; Chemistry; Gene Expression Regulation; Genes; Humans; Mutation; Nucleic Acid Hybridization; Oligonucleotides; Polynucleotides; Proto-Oncogene Proteins
PubMed: 3513171
DOI: 10.1073/pnas.83.5.1222 -
Future Oncology (London, England) Jan 2010Experiments on estrogen metabolism, formation of DNA adducts, mutagenicity, cell transformation and carcinogenicity have led to and supported the hypothesis that the... (Review)
Review
Experiments on estrogen metabolism, formation of DNA adducts, mutagenicity, cell transformation and carcinogenicity have led to and supported the hypothesis that the reaction of specific estrogen metabolites, mostly the electrophilic catechol estrogen-3,4-quinones, with DNA can generate the critical mutations to initiate breast and other human cancers. Analysis of depurinating estrogen-DNA adducts in urine demonstrates that women at high risk of, or with breast cancer, have high levels of the adducts, indicating a critical role for adduct formation in breast cancer initiation. Men with prostate cancer or non-Hodgkin lymphoma also have high levels of estrogen-DNA adducts. This knowledge of the first step in cancer initiation suggests the use of specific antioxidants that can block formation of the adducts by chemical and biochemical mechanisms. Two antioxidants, N-acetylcysteine and resveratrol, are prime candidates to prevent breast and other human cancers because in various M in vitro and in vivo experiments, they reduce the formation of estrogen-DNA adducts.
Topics: Acetylcysteine; Animals; Antineoplastic Agents; Antioxidants; Breast Neoplasms; DNA Adducts; Estrogens; Female; Humans; Male; Neoplasms; Resveratrol; Stilbenes
PubMed: 20021210
DOI: 10.2217/fon.09.137 -
Biochimica Et Biophysica Acta Jul 1982The influence of the spermidine, spermine and putrescine on the DNA depurination rate was studied. These polyamines protect DNA against depurination. The rate of Col EI...
The influence of the spermidine, spermine and putrescine on the DNA depurination rate was studied. These polyamines protect DNA against depurination. The rate of Col EI DNA depurination at pH 4.3 was decreased over 10-fold by addition of 10 mM polyamines.
Topics: DNA, Bacterial; Escherichia coli; Kinetics; Plasmids; Putrescine; Spermidine; Spermine
PubMed: 7052129
DOI: 10.1016/0167-4781(82)90190-7 -
Current Protocols in Nucleic Acid... Jun 2017Oligonucleotides carrying a variety of chemical modifications including conjugates are finding increasing applications in therapeutics, diagnostics, functional genomics,...
Oligonucleotides carrying a variety of chemical modifications including conjugates are finding increasing applications in therapeutics, diagnostics, functional genomics, proteomics, and as research tools in chemical and molecular biology. The successful synthesis of oligonucleotides primarily depends on the use of appropriately protected nucleoside building blocks including the exocyclic amino groups of the nucleobases, the hydroxyl groups at the 2'-, 3'-, and 5'-positions of the sugar moieties, and the internucleotide phospho-linkage. This unit is a thoroughly revised update of the previously published version and describes the recent development of various protecting groups that facilitate reliable oligonucleotide synthesis. In addition, various protecting groups for the imide/lactam function of thymine/uracil and guanine, respectively, are described to prevent irreversible nucleobase modifications that may occur in the presence of reagents used in oligonucleotide synthesis. © 2017 by John Wiley & Sons, Inc.
Topics: Acetylation; Deoxyribonucleosides; Photochemistry; Ribonucleosides
PubMed: 28628209
DOI: 10.1002/cpnc.32 -
Indian Journal of Experimental Biology Oct 2002An antiviral protein (25 kD) isolated from leaves of Celosia cristata (CCP 25) was tested for depurination study on ribosomal RNA from yeast. Ribosomal RNA yielded 360...
An antiviral protein (25 kD) isolated from leaves of Celosia cristata (CCP 25) was tested for depurination study on ribosomal RNA from yeast. Ribosomal RNA yielded 360 nucleotide base fragment after treatment with CCP 25 indicating that CCP 25 was a ribosome inactivating protein. CCP 25 also inhibited translation of brome mosaic virus (BMV) and pokeweed mosaic virus (PMV) RNAs in rabbit reticulocyte translation system. The radioactive assay showed that incorporation of [35S]-methionine was less in translation proteins of BMV nucleic acid when CCP 25 was added to translation system. This indicated that antiviral protein from Celosia cristata not only depurinated ribosomal RNA but also inhibited translation of viral RNA in vitro.
Topics: Antiviral Agents; Celosia; Mosaic Viruses; Protein Biosynthesis; Purines; RNA, Ribosomal; RNA, Viral
PubMed: 12693705
DOI: No ID Found -
Molecular Microbiology Dec 2008Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the...
Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the alpha-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the alpha-sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DeltaP1 and DeltaP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.
Topics: Gene Deletion; Phosphoproteins; Purines; RNA, Ribosomal; Ribosomal Proteins; Ribosomes; Ricin; Saccharomyces cerevisiae; Surface Plasmon Resonance
PubMed: 19019145
DOI: 10.1111/j.1365-2958.2008.06492.x -
ACS Applied Materials & Interfaces Jan 2016Quick and accurate on-site detection of active ricin has very important realistic significance in view of national security and defense. In this paper, optimized...
Quick and accurate on-site detection of active ricin has very important realistic significance in view of national security and defense. In this paper, optimized single-stranded oligodeoxynucleotides named poly(21dA), which function as a depurination substrate of active ricin, were screened and chemically attached on gold nanoparticles (AuNPs, ∼100 nm) via the Au-S bond [poly(21dA)-AuNPs]. Subsequently, poly(21dA)-AuNPs were assembled on a dihydrogen lipoic-acid-modified Si wafer (SH-Si), thus forming the specific surface-enhanced Raman spectroscopy (SERS) chip [poly(21dA)-AuNPs@SH-Si] for depurination of active ricin. Under optimized conditions, active ricin could specifically hydrolyze multiple adenines from poly(21dA) on the chip. This depurination-induced composition change could be conveniently monitored by measuring the distinct attenuation of the SERS signature corresponding to adenine. To improve sensitivity of this method, a silver nanoshell was deposited on post-reacted poly(21dA)-AuNPs, which lowered the limit of detection to 8.9 ng mL(-1). The utility of this well-controlled SERS chip was successfully demonstrated in food and biological matrices spiked with different concentrations of active ricin, thus showing to be very promising assay for reliable and rapid on-site detection of active ricin.
Topics: Gold; Nanoparticles; Oligodeoxyribonucleotides; Purines; Ricin; Spectrophotometry, Ultraviolet; Spectrum Analysis, Raman
PubMed: 26719952
DOI: 10.1021/acsami.5b12860 -
Annals of the New York Academy of... Dec 2004The first step in cancer initiation is the reaction of chemical carcinogens with DNA to form stable adducts, which remain in DNA unless removed by repair, and... (Review)
Review
The first step in cancer initiation is the reaction of chemical carcinogens with DNA to form stable adducts, which remain in DNA unless removed by repair, and depurinating adducts, which detach from DNA following destabilization of the glycosyl bond. Depurinating DNA adducts of polycyclic aromatic hydrocarbons play a major role in the initiation of cancer, as shown by the correlation between depurinating adducts and oncogenic mutations of the H-ras oncogene in mouse skin. Following these results, experiments on the metabolism of estrogens, formation of depurinating DNA adducts, carcinogenicity, mutagenicity, and cellular transformation have led us to the hypothesis that certain metabolites of endogenous estrogens--in particular, estradiol(estrone)-3,4-quinones--can react with DNA to form depurinating adducts at the N-3 of Ade and the N-7 of Gua. Depurination of these adducts can generate critical mutations by error-prone repair to initiate breast, prostate, and other cancers. The oxidation mechanism of catechols to quinone compounds includes not only natural estrogens, but also synthetic estrogens, such as hexestrol and the human carcinogen diethylstilbestrol, which react with DNA to form N3Ade and N7Gua adducts, presumably to initiate cancer. Furthermore, the initiating mechanism of the leukemogen benzene involves oxidation of catechol to catechol quinone, which reacts with DNA to form N3Ade and N7Gua adducts. The quinone of the neurotransmitter dopamine can also react with DNA to form N3Ade and N7Gua adducts. The subsequent mutations could be at the origin of Parkinson's and other neurodegenerative diseases. In summary, the apurinic sites produced in DNA from the loss of these depurinating adducts can be converted into mutations by error-prone repair, which may initiate cancer and other diseases.
Topics: Animals; Benzene; Breast Neoplasms; Carcinogens; Catechols; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; DNA; DNA Adducts; Dopamine; Estrogens; Humans; Hydrocarbons; Models, Chemical; Mutagenesis; Mutation; Neoplasms; Oxygen; Quinones
PubMed: 15650250
DOI: 10.1196/annals.1322.029 -
Nucleic Acids Research 2004Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved alpha-sarcin/ricin loop in the large rRNA. Here, using...
Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved alpha-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5' and the 3' ends of the PAP cDNA, we identified the amino acids important for ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although ribosomes were depurinated. These mutants depurinated ribosomes only during their translation and could not depurinate ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.
Topics: Adenine; Mutagenesis, Site-Directed; N-Glycosyl Hydrolases; Plant Proteins; Protein Biosynthesis; Protein Structure, Tertiary; RNA, Ribosomal; Ribosome Inactivating Proteins, Type 1; Ribosomes; Saccharomyces cerevisiae; Sequence Deletion; Transformation, Genetic
PubMed: 15304562
DOI: 10.1093/nar/gkh757 -
Molecules (Basel, Switzerland) Jan 2017The enzymes called ribosome-inactivating proteins (RIPs) that are able to depurinate ... (Review)
Review
The enzymes called ribosome-inactivating proteins (RIPs) that are able to depurinate nucleic acids and arrest vital cellular functions, including protein synthesis, are still a frontline research field, mostly because of their promising medical applications. The contributions of Stirpe to the development of these studies has been one of the most relevant. After a short biographical introduction, an overview is offered of the main results obtained by his investigations during last 55 years on his main research lines: hyperuricaemia, xanthine oxidoreductase and RIPs.
Topics: Animals; Famous Persons; Fructose; History, 20th Century; Humans; Hyperuricemia; Italy; Plant Proteins; Research; Ribosome Inactivating Proteins; Ricin; Translational Research, Biomedical; Xanthine Dehydrogenase
PubMed: 28134797
DOI: 10.3390/molecules22020206