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Biomedical subjects

D Grunberger

Publications and source records attributed to D Grunberger.

At least 91 records · Page 5Linked to original sources
PubMed1980

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.

2-Acetylaminofluorene↗
PubMed1980

Deficiency of nuclease activity in ribosomes of three tumor types.

Polysomes isolated from 3 types of neoplasms, mouse mammary adenocarcinoma designated DBAH, morris rat hepatoma 7777, and mouse chloroleukemia, do not undergo fast degradation upon incubation at 37 degrees C, unlike polysomes isolated from corresponding isogenic, normal tissues. Further,. 1M KCl extract of ribosomes from normal cells, in contrast to their neoplastic counterparts, stimulate degradation of poly(U). The nuclease activity in ribosomal preparations from neoplastic cells is not increased in the presence of p-chloromercuribenzoate, a blocking agent of RNAase inhibitor. This suggests that ribosomal preparations from tumor cells are free of nucleases rather than just being masked by an inhibitor. Ribosomes from neoplastic cells stimulate protein synthesis in vitro in the presence of endogenous or exogenous mRNA to a much higher degree than similar preparations from normal tissues. This event is in agreement with the deficiency of nuclease in polysomes from neoplastic cells.

Animals↗
PubMed1980

The effect of exogenous nutrients on the biosynthesis of Y base in tRNAPHe from Ehrlich ascites carcinoma.

In contrast to normal eukaryotic tissue, which contains a single isoaccepting species of tRNAPhe, the Ehrlich ascites carcinoma contains a spectrum of tRNAPhe species varying in their relative degree of posttranscriptional modification. The occurrence of multiple species involves an incomplete biosynthesis of the Y base, a complex hypermodified nucleoside located adjacent to the 3' end of the anticodon. Following intraperitoneal innoculation of a mixture of essential amino acids and vitamins into these mice, the majority of the tumor tRNAPhe behaved like the single liver tRNAPhe species. This suggests that under these conditions the modification of Y base was completed.

Amino Acids, Essential↗
PubMed1980

Circular dichroism and proton magnetic resonance studies of dApdG modified with 2-aminofluorene and 2-acetylaminofluorene.

The conformational properties of 2'-deoxyadenylyl-(3'-5')-2'-deoxyguanosine (dApdG) modified by the covalent binding of the carcinogens 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) have been investigated, utilizing circular dichroism and proton magnetic resonance spectroscopy. The attachment of AF residues to the C-8 position of guanosine introduced smaller changes in the circular dichroism spectra of dApdG than the binding of AAF residues. Similarly, binding of AF residues caused lower up-field shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with the neighboring A base in dApdG than AAF. Thus, AF residues bound on the C-8 position of guanine might induce less distortion in conformation of the modified regions than AAF residues.

2-Acetylaminofluorene↗
PubMed1980

Quantitation of benzo(a)pyrene-deoxyguanosine adducts by radioimmunoassay.

Calf thymus DNA was modified with the benzo(a)pyrene (BP) derivative, (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-) BPDE I], under conditions which yielded greater than 99% of the binding product in the form of trans-(7R)-N2-(10[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl) deoxyguanosine. Rabbits were immunized with modified DNA coupled to methylated bovine serum albumin, and the resulting antiserum was utilized in a competition radioimmunoassay for the quantitation of products of BP covalently bound to DNA. The antiserum was specific for both native and denatured immunogen DNA's as well as for the major isolated BP binding product, but it did not recognize BP, the tetrol of (+/-)BPDE I, or unmodified deoxyguanosine. The modified DNA was assayed in quantities as low as 2 pmol of adduct, a sensitivity sufficient to quantitate the extent of modification of cellular DNA when epidermal cell cultures were exposed either to BP or to (+/-)BPDE I. High-pressure liquid chromatographic analysis of DNA hydrolsates, obtained from epidermal cells exposed to BP or to (+/-)BPDE I, indicated that the major adduct was the same as than on the immunogen DNA. This approach should prove valuable for further studies on the mechanism of carcinogenesis and for monitoring human exposure to this ubiquitous carcinogen.

Animals↗
PubMed1979

Modification of deoxyribonucleic acid by a diol epoxide of benzo[a]pyrene. Relation to deoxyribonucleic acid structure and conformation and effects on transfectional activity.

The effects of secondary structure on DNA modification by (+/-)-7 beta, 9 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+/-)BPDE I] were investigated. No differences in the total extent of (+/-) BPDE I binding to double- and single-stranded calf thymus DNA were found. High-performance liquid chromatography (LC) of the nucleoside adducts obtained from hydrolysates of native and denatured calf thymus, as well as from superhelical and linear plasmid DNA, indicated that in all cases the major adduct (60--80% of total adducts) was formed by reaction of the (+) enantiomer of BPDE I with the N-2 position of dG residues in the DNA. A minor adduct formed from the reaction of the (-) enantiomer with dG residues was also detected and was present in greater amounts in denautred DNA than in native DNA. Small amounts of BPDE I--dA and BPDE I--dC adducts were also detected in both the single- and double-stranded DNAs. Restriction enzyme analysis of BPDE I modified SV40 and phage lambda DNA provided evidence that the modification of DNA by this carcinogen is fairly random with respect to nucleotide sequence. Partial hydrolysis of modified plasmid DNA by the single-strand-specific S1 nuclease and LC analysis of the nucleoside adducts in the digested and undigested fractions of the DNA revealed no preferential excision by the S1 nuclease of the different BPDE I--deoxynucleoside adducts. Functional changes in BPDE I modified DNA were demonstrated. With increasing extents of modification, there was a decrease in the ability of plasmid DNA to transfect a receptive Escherichia coli strain to antibiotic resistance.

Animals↗
PubMed1979

DNA--benzo[a]pyrene adducts formed in a Salmonella typhimurium mutagenesis assay system.

The DNA adducts formed in Salmonella typhimurium when bacteria are incubated with radioactive benzo[a]pyrene and liver microsomal enzymes from several sources has been investigated. When enzyme preparations from Aroclor I254 or 3-methylcholanthrene induced C57BL/6N (B6) mice were used to mediate activation, the predominant product was an adduct between the 10 position of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the N-2 position of deoxyguanosine. Similar results were obtained with human liver and with Aroclor-induced rat-liver enzyme preparations. This adduct is also the major DNA product previously found when human tissues or certain rodent cells were incubated with benzo[a]pyrene. On the other hand, when activation of benzo[a]pyrene was mediated by a phenobarbital-induced B6 mouse-liver enzyme preparation, the extent of binding was quite low and the profile of DNA adducts in S. typhimurium DNA was quite different. Thus, under appropriate conditions, the activation and DNA binding of benzo[a]pyrene inthe microsome mediated S. typhimurium mutagenesis assay generally resembles that seen in intact mammalian cells. Caution must be exercised, however, in the choice of microsome-activation systems.

Animals↗
PubMed1979

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.

Codon↗
PubMed1979

Incorporation of lysine into Y base of phenylalanine tRNA in Vero cells.

Vero cells, a line derived from African green monkey kidney, contains a hypermodified base, called Y, adjacent to the 3' end of the anticodon of tRNAPhe. Two types of evidence are presented suggesting that lysine is involved in biosynthesis of Y base in these cells. First, when Vero cells are starved for lysine, a new, early-eluting species of tRNAPhe which lacks the fully modified Y base can be detected by reversed phase chromatography (RPC-5). After addition of lysine to the medium, this new species disappears. Second, when these cells are grown in low-lysine medium and then exposed to [3H]lysine, radioactivity from the lysine comigrates with tRNAPhe. The Y base can be selectively excised from tRNAPhe by incubation at pH 2.9, and extracted into ethyl acetate. Thin-layer chromatography of acid-excised material from these cells reveals that lysine-derived radioactivity comigrates with genuine Y base from calf liver tRNAPhe and the acid-excised tRNA no longer contains radioactivity. These results are consistent with the model that lysine is a structural precursor of Y base in tRNAPhe of Vero cells.

Animals↗
PubMed1978

Formation of phenylalanine transfer RNA lacking the wye base in Vero cells during methionine starvation.

Vero, a cell line derived from African green monkey kidney, normally contains a single species of tRNAPhe (tRNA2Phe), containing a hypermodified base, wye (originally called Y), next to the 3' end of the anticodon. When methionine is removed from the growth medium, there appears a new tRNAPhe species (tRNA1Phe) lacking the wye base and eluting early from reversed phase chromatography columns. Its appearance is not due to the cessation of cell growth. Addition of methionine to cells containing both species of tRNAPhe leads to the disappearance of tRNA1Phe. When [methyl-3H5methionine is added in the presence of actinomycin D, which blocks new RNA synthesis, label appears in the wye base of tRNA2Phe. These results are consistent with the model that tRNA1Phe is an undermodifed precursor of tRNA2Phe and that methionine is required for modification to the mature form.

Cell Line↗
PubMed1978

Conformation of dinucleoside monophosphates modified with benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide as measured by circular dichroism.

The conformational properties of GpU modified with the reactive derivative of benzo[a]pyrene, (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, has been investigated utilizing circular dichroism spectroscopy. Binding of this carcinogen to the N2 of G residues in GpU resulted in the formation of four compounds (I to IV) representing two pairs of diastereoisomers. The molar ellipticity values of the modified dimers were approximately twofold higher than those of the modified guanosine monomers. These values were decreased appreciably when the spectra of the dimers were obtained at 80 degrees C or in methanol rather than at 25 degrees C in water, suggesting that under the latter conditions there is a stacking interaction between the carcinogen and the neighboring uridine residue. Based on these results, a conformation is proposed for modified GpU. It includes insertion of the benzo[a]pyrene moiety, by rotation of the modified guanine residue about its glycoside bond, coplanar to the neighboring uridine and perpendicular to the phosphodiester backbone.

Benzopyrenes↗
PubMed1977

Conformation of DNA modified with a dihydrodiol epoxide derivative of benzo[a]pyrene.

The conformation of calf thymus DNA modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, which binds covalently mainly to the 2-amino group of guanosine residues, was studied. With samples in which 1.5 or 2.2% of the bases were modified, there was a slight decrease in Tm during heat denaturation and a slight increase in susceptibility to the single strand specific nuclease S1. In a DNA sample in which 4.5% of the bases were modified, there was an appreciable decrease in Tm and a marked increase in susceptibility to S1 nuclease. The kinetics of the reaction of the modified DNAs with formaldehyde provided evidence for locally destabilized regions ranging from 1 to 7 base plates, depending on the extent of modification. Alkaline and neutral sucrose gradient analyses revealed no evidence for strand breakage in the 1.5 and 2.2% modified samples, although single-strand breaks were found in the 4.5% modified samples. Taken together, these results suggest that DNA molecules containing a covalently bound benzo[a]pyrene derivative have an altered conformation characterized by small localized regions which are destabilized and easily denatured. The conformational changes associated with the covalent binding of the benzo[a]pyrene derivative to native DNA appear to be different from, and less marked, than those associated with the covalent binding of N-2-acetylaminofluorene to native DNA.

Animals↗
PubMed1977

Template activity of calf thymus DNA modified by a dihydrodiol epoxide derivative of benzo[a]pyrene.

The purpose of the present study was to determine the effects of covalent binding to DNA of a reactive derivative of benzo[a]pyrene on template activity during in vitro transcription with RNA polymerase. Calf thymus deoxyribonucleic acid, modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, was transcribed with Escherichia coli DNA-dependent RNA polymerase. With increasing levels of modification, there was a progressive inhibition of transcription. The inhibition was much greater under conditions where continuous reinitiation of transcription occurred than under conditions where only one RNA chain was synthesized per initiation site. This suggested that the modified sites block the movement of polymerase along the template and prevent recycling of the enzyme. Consistent with this interpretation were analyses of RNA transcripts on sucrose density gradients which showed a progressive decrease in average RNA chain length as the extent of template modification increased. In contrast to the inhibitory effect on chain elongation, evidence was obtained that the modified DNA had an increase in the number of initiation sites for transcription. These results are consistent with separate physical studies indicating that modification of DNA by this benzo[a]pyrene derivative can induce small localized regions of denaturation.

Animals↗