Biomedical subjects
B Singer
Publications and source records attributed to B Singer.
[Wallenberg's syndrome due to a dissecting aneurysm of the vertebral artery].
A 54 year old man without pathologic past history but mild hypertension, obesity and gastric ulcer, presented with a syndrome of Wallenberg. He had complained for five days of progressive and diffuse headache. The neurological condition improved initially, but the patient died suddenly two weeks later. Pathological examination showed no significant alteration except for left ventricular enlargement and mild arteriosclerosis. There was a hemodissection (dissecting aneurysm) of the left vertebral artery next to the inferior oliva. It induced a lateral infarct and a limited dorsal infarct at the middle third level of medulla oblongata. Although the location of the arterial changes is usual, their nature is exceptional. The cause of the arterial hemodissection could not be ascertained: fibrous arterial dysplasia, atherosclerosis or congenital abnormalities of internal elastic layer may be discussed. But no definite conclusion can be reached.
Correlations between sites of chemical modification of DNA, repair, and carcinogenesis.
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Effect of tautomeric shift on mutation: N4-methoxycytidine forms hydrogen bonds with adenosine in polymers.
N4-Methoxycytidine (mo4C), previously found to act only as uridine (U) in transcription [Singer, B., & Spengler, S. (1981) Biochemistry 20, 1127], was tested for its ability to base pair as U in copolymers of (U,mo4C) annealed with poly(A) or transcribed with ATP and DNA-dependent RNA polymerase. Mixing curves have now indicated that the derivative is retained in a poly(U,39% mo4C).poly(A) helix, unlike unmodified C in poly(U,35% C). The presence of 13-39% mo4C in U polymers lowered the melting temperature, Tm, observed in annealed complexes both with poly(A) and after transcription with ATP. However, complexes isolated after transcription had a large hyperchromicity and melted cooperatively, which indicated that they are hydrogen bonded. The decreased Tm for poly(U,mo4C).poly(A) compared to that for poly(U).poly(A) can be attributed to stacking changes and adjacent base-pair disruption by mo4C. The greater cooperative melting of transcribed poly(U,39% mo4C) as compared to the annealed complex may indicate that the methoxy substituent is normally a mixture of rotamers and that the syn rotamer is required for transcription. The interference of the methoxy substituent was also shown by the loss of helix formation by poly(C,mo4C) in acid solution. mo4C decreased the Tm much more than A, which stacks well in acid. U, which neither stacks nor participates in an acid structure, caused more distortion than either of the other bases. It is inferred that mo4C has the base-pairing ability of U but that the planarity of the substituent is lost.
Ambiguity and transcriptional errors as a result of modification of exocyclic amino groups of cytidine, guanosine, and adenosine.
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Transcriptional errors and ambiguity resulting from the presence of 1,N6-ethenoadenosine or 3,N4-ethenocytidine in polyribonucleotides.
1,N6-Ethenoadenosine (epsilon A) and 3,N4-Ethenocytidine (epsilon C) in copolymers with unmodified nucleosides were transcribed using DNA-dependent RNA polymerase in the presence of Mn2+. Nearest neighbor analysis of the products showed that epsilon A directed incorporation of A much greater than U greater than C while epsilon C directed the incorporation of U greater than or equal to A much greater than C Neither directed G into the complementary polymer. Such misincorporations resulting from epsilon A and epsilon C, compounds that are formed in vivo by the carcinogen vinyl chloride, may have a biological role as promutagens.
Human lymphoblasts contain DNA glycosylase activity excising N-3 and N-7 methyl and ethyl purines but not O6-alkylguanines or 1-alkyladenines.
Cultured human lymphoblasts (CCRF-CEM line) have DNA glycosylase activities, the specificities of which were investigated by using high-performance liquid chromatography. In addition to 3-methyladenine, 3-ethyladenine, 7-methylguanine, 7-ethylguanine, 3-methylguanine, and 3-ethylguanine also were excised from alkylated double-stranded DNA and deoxypolynucleotides, but 1-methyladenine, 1-ethyladenine, O6-methylguanine, and O6-ethylguanine were not. The glycosylase activity was generally greater for the methylated than for the corresponding ethylated purines and was also greater toward 3-alkyladenine than toward 3-alkyladenine than toward 3-alkylguanine. 7-Methylguanine and 7-ethylguanine were excised to similar but low extents. However, in molar terms, the release of 7-methylguanine was similar to that of 3-methyladenine.
Tissue-dependent enzyme-mediated repair or removal of O-ethyl pyrimidines and ethyl purines in carcinogen-treated rats.
Treatment of perinatal rats with N-ethyl-N-nitrosourea (EtNU) leads predominantly to brain tumors. The DNA in tissues of 10-day-old BD IX rats is alkylated by this ultimate carcinogen at the same sites as is DNA in mammalian cell cultures or DNA in solution. Similar proportions of the derivatives quantitated (O6-EtG, 7-EtG, 3-EtA, O2-EtT, O4EtT, O2EtC, and ethyl phosphotriesters) are found in each tissue examined 1 h after treatment with EtNU. Most of the ethylated bases are poorly removed (or, in the case of O4-EtT, not at all) from DNA in the brain, the target tissue of oncogenicity. A pool of five other tissues, excluding liver, exhibits a similar pattern of ethyl base persistence over a 75 h period. In contrast, liver apparently contains enzymes capable of removing all of the ethylated bases. In all tissues used, ethyl phosphotriesters are very stable. The observed kinetics imply that removal of ethylated bases would be complete within 10 days in liver, while over 50% of the chemically ethylated stable bases would persist in other tissues, including brain, for many weeks. We propose that any or all persistent promutagenic derivatives (O6-EtG, O2EtT, O4-EtT, O2-EtC) can be important in the initiation of carcinogenesis by somatic mutation, given that the damage DNA is expressed. The differing rates of removal of the ethyl purines and pyrimidines in brain, liver and pooled tissues imply that mammals possess multiple independent repair systems.
Infectivity and reconstitution of TMV RNA modified with N-acetoxy-2-acetylaminofluorene or benzol [a] pyrene 7,8-dihydrodiol 9,10 oxide.
TMV RNA was modified by two bulky carcinogens, N-acetoxy-2-acetylamino-fluorene (AAAF) and (+/-)-7beta, 8alpha- dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (BPDE), and the effects of such substituents on biological and physical properties was studied. For both types of modification, the loss of infectivity was directly proportional to the number of chemical modifications indicating that all modifications are lethal. Neither AAAF nor BPDE produced measurable mutations. Reconstitution of modified RNA with TMV protein was partially inhibited, but such inhibition occurred to similar extents with either carcinogen and a varying levels of modification. The data suggest that both types of substitution of TMV RNA generally permit the TMV coat protein to aggregate normally around the RNA, but that AAAF and BPDE may induce some conformational change in the initiation region that inhibits the initiation step.
Effect of introduction of small alkyl groups on mRNA function.
Treatment of RNA with dimethyl sulfate methylates only nitrogens, preferentially the 7 position of guanosine, whereas treatment with ethylnitrosourea ethylates mainly oxygens, preferentially the phosphodiester groups. Two plant viral mRNAs were modified with these two reagents at levels of 4-28 alkylations per molecule. The ability of alkylated RNAs to stimulate amino acid incorporation in the wheat germ system was somewhat diminished by both types of modification, but the predominant protein made, as ascertained by polyacrylamide gel electrophoresis, was the typical gene product of the respective mRNA. These data suggest that random alkylations, mostly of either the guanosine N-7s or the phosphates, do not interfere with peptide chain elongation, but that the ability to initiate translation properly is affected by these substitutions, presumably through their effect on the conformation of the RNAs.
[Clinical experiments with Dequonal, a new oral antiseptic].
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Ambiguity and transcriptional errors as a result of methylation of N-1 of purines and N-3 of pyrimidines.
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Influence of hydrogen bonding in DNA and polynucleotides on reaction of nitrogens and oxygens toward ethylnitrosourea.
The reactivity of ethylnitrosourea toward hydrogen-bonded sites in double-stranded DNA or oly(rA).poly(rU) was compared with those sites in single-stranded DNA, RNA, or poly(rA). Alkylation of the N-1 of A in poly(rA).poly(rU) was almost suppressed at 5 degrees C but could be markedly increased by raining the reaction temperature to 25 degrees C, well below the Tm of 56 degrees C. In contrast, the N-7 and N-6 of A, which are not hydrogen bonded, reacted to the same extent at temperatures ranging from 5 to 65 degrees C. The extent of reaction at the N-3 of A varied inversely with the reactivity of the N-1 of A, indicating that of these two nitrogens the N-1 of A is the most reactive. The proportion of reaction at the various nitrogens in poly(rA) was not affected by temperature. Hydrogen-bonded oxygens in double-stranded DNA are the O-6 of G, the O-4 of T, and the O2 of C. All are equally reactive at 5, 25, and 51 degrees C. It is concluded that the observed temperature independence is due to these oxygens having an electron pair not involved in hydrogen bonding and, thus, available for reaction. In contrast, the electron pair of the N-1 of A (or the N-3 of C) is involved in hydrogen bonding, and the extent of their reactivity is dependent on thermal fluctuation providing transiently open base pairs at temperatures far below the Tm.
Evidence for removal at different rates of O-ethyl pyrimidines and ethylphosphotriesters in two human fibroblast cell lines.
The potent carcinogen, ethylnitrosourea, has been shown to ethylate oxygens, in preference to nitrogens, in the DNA of cultured cells. We have now studied the removal of seven ethyl derivatives in replicating cells. The following findings are reported. 1) The absolute amounts of 02-EtT, 04-EtT and 02-EtC are decreased in cellular DNA after correction for cell growth. However the rate of decrease diminishes after approximately 20 hr and after more than two cell doublings 20--40% of each derivative persists. This decrease is presumed to be due to enzymes since these derivatives are stable in isolated DNA. 2) The amount of ethyl phosphotriesters remains almost unchanged during 72 hr of cell culture. 3) The unstable purine derivatives, 7-EtG and 3-EtA, are both removed from cellular DNA with a rate faster than can be accounted for by the lability of the glycosyl bond. 4) Both GM 637 fibroblasts and Xeroderma pigmentosum fibroblasts (12-RO) (XP-12) have similar ability to remove ethyl products, except for O6-ethyl G which persists to a greater extent in XP12 cells. 5) The implications of the in vivo persistence of ethylated bases is discussed in regard to recent demonstrations that O2-EtT, O4-ET, O2-EtC and O6-EtG are all mutagenic.
Influence of different levels of 2-thiocytidine on physical and template properties of cytidine--2-thiocytidine copolymers.
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Participation of modified nucleosides in translation and transcription.
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Synthesis and coding properties of dinucleoside diphosphates containing alky pyrimidines which are formed by the action of carcinogens on nucleic acids.
Dinucleoside diphosphates of the general type pGpN have been prepared enzymatically using ribonuclease N1. Alkylated uridines or cytidines, which are products of carcinogens acting on nucleic acids, were tested in dinucleoside diphosphates for their ability to stimulate the binding of Ala- or Val-tRNA to ribosomes. O2-Ethyl C and 3-methyl C functioned as U, but not as C. In contrast, 3-methyl U behaved as C, but not as U. Both O2 and O4-ethyl U could be recognized as C or U, although binding in both cases was weak. Thus, modifications of the hydrogen-bonding sites of U or C causes miscoding and could be considered to represent mutagenic reactions.
N-nitroso alkylating agents: formation and persistence of alkyl derivatives in mammalian nucleic acids as contributing factors in carcinogenesis.
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