PubMed Health⌕ Search

Biomedical subjects

G D Frenkel

Publications and source records attributed to G D Frenkel.

At least 37 records · Page 2Linked to original sources
PubMed1989

Involvement of cellular sulfhydryl compounds in the inhibition of RNA synthesis by selenite.

Selenite has been shown previously to inhibit cellular RNA synthesis. Based upon our previous observation that selenite inhibits purified RNA polymerase only in the presence of a sulfhydryl compound (Frenkel et al., Mol Pharmacol 31: 112-116, 1987), we hypothesized that the inhibition of cellular RNA synthesis by selenite involves endogenous sulfhydryl compounds. We found that depletion of cells of endogenous sulfhydryl compounds, by exposure to diethylmaleate (DEM), virtually eliminated the inhibitory effect of a 1-hr exposure of cells to selenite. This inhibition was restored to normal or higher levels when the selenite was reacted with glutathione or cysteamine prior to addition to the DEM-treated cells. RNA synthesis in DEM-treated cells was inhibited after a 4-hr exposure to higher concentrations of selenite. In contrast to the effect of DEM, specific depletion of the cells of glutathione, by exposure to buthionine sulfoximine, had no effect on the inhibition of RNA synthesis by selenite. These results demonstrate the involvement of endogenous cellular sulfhydryl compounds in the inhibition of RNA synthesis by selenite, but indicate that glutathione, in particular, is not involved in this inhibition.

HeLa Cells↗
PubMed1989

Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation.

We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds [Frenkel, Walcott, and Middleton, Molecular Pharmacology 31, 112 (1987)]. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.

Cysteamine↗
PubMed1988

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei.

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.

Amanitins↗
PubMed1988

Methyl mercury stimulates chain elongation by purified HeLa RNA polymerase II.

Methyl mercury (MeHg) inhibited the overall RNA synthetic reaction of HeLa RNA polymerase II. However, when RNA chain initiation was allowed to occur in its absence, MeHg stimulated the rate of the subsequent elongation stage of the reaction. Chain elongation with both double-stranded and single-stranded DNA templates was stimulated. This stimulatory effect was specific for MeHg; both p-hydroxymercuribenzoate and HgCl2 inhibited chain elongation (to about the same degree as they inhibited the overall reaction). The stimulatory effect was also specific for the HeLa polymerase; with Escherichia coli RNA polymerase, MeHg inhibited elongation (to the same degree as it inhibited the overall reaction).

Escherichia coli↗
PubMed1988

Evidence for the involvement of sulfhydryl compounds in the inhibition of cellular DNA synthesis by selenite.

Previous studies have demonstrated that selenite inhibits cellular DNA synthesis. We have now found that endogenous cellular sulfhydryl compounds are involved in this effect of selenite. Treatment of cells with diethylmaleate, which produces a nonspecific depletion of cellular sulfhydryl compounds, resulted in a significant decrease in the sensitivity of DNA synthesis to inhibition by selenite. This decrease was eliminated by exogenous cysteine, but not by cystine. Similarly, DNA synthesis in nuclei isolated from diethylmaleate-treated cells was much less sensitive to inhibition by selenite than was synthesis in nuclei isolated from control cells. In contrast, treatment with buthionine sulfoximine, which specifically depletes the cells of glutathione, had no effect on the inhibition of DNA synthesis by selenite, indicating that cellular glutathione is not involved in the inhibition. Nevertheless, glutathione is able to compensate to some extent for the decreased level of sulfhydryl compounds in nuclei isolated from DEM-treated cells (although not as well as cysteine). Thus, although glutathione is able to potentiate the inhibition of cellular DNA synthesis by selenite, it apparently does not function in this capacity in the cell.

Buthionine Sulfoximine↗
PubMed1987

Effects of lead acetate on DNA and RNA synthesis by intact HeLa cells, isolated nuclei and purified polymerases.

The effects of lead acetate on DNA and RNA synthesis have been investigated with intact HeLa cells, isolated nuclei, and purified DNA and RNA polymerases. No inhibition of DNA or RNA synthesis in intact cells was found even after exposure to 0.5 mM lead acetate for 18 hr. In contrast, both DNA and RNA synthesis in isolated nuclei were inhibited by lead (with 50% inhibition at approximately 150 and 80 microM respectively). Similarly, both HeLa DNA polymerase alpha and RNA polymerase II were inhibited, with 50% inhibition obtained at approximately 150 and 20 microM lead acetate respectively. The inhibition of nucleic acid synthesis in isolated nuclei can thus be accounted for by inhibition of the polymerases. The sensitivity of Escherichia coli DNA polymerase I to lead acetate was found to be significantly greater than the HeLa DNA polymerase alpha (50% inhibition at only 10 microM), but the sensitivity of the E. coli RNA polymerase was the same as that of the HeLa enzyme.

DNA Replication↗
PubMed1987

The enhanced rate of transcription of methyl mercury-exposed DNA by RNA polymerase is not sufficient to explain the stimulatory effect of methyl mercury on RNA synthesis in isolated nuclei.

Previous work demonstrated two stimulatory effects of methyl mercury on nucleic acid synthesis: (1) in isolated nuclei, methyl mercury stimulates RNA synthesis which is catalyzed by RNA polymerase II [Frenkel and Randles, J. Biol. Chem. 257, 6275-6279 (1982)]. (2) Brief exposure of purified DNA to methyl mercury increases the rate of its transcription by purified RNA polymerase II [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)]. The latter effect was considered as a possible mechanism of the former. Two lines of evidence are presented here which demonstrate that the latter effect is not a sufficient explanation for the former. (1) Mercuric perchlorate has been found to increase the rate of DNA transcription by purified polymerase and the template properties of the mercuric perchlorate-exposed DNA have been found to resemble those of methyl mercury-exposed DNA. Nevertheless, mercuric perchlorate has been shown not to stimulate RNA synthesis in isolated HeLa nuclei. (2) In isolated nuclei of the B50 rat neuroblastoma cell line, RNA synthesis has been found to be stimulated only minimally by methyl mercury. Nevertheless, RNA polymerase II purified from the B50 cells has been found to transcribe methyl mercury-exposed DNA at a higher rate than unexposed control DNA.

Cell Nucleus↗
PubMed1987

Inhibition of RNA and DNA polymerases by the product of the reaction of selenite with sulfhydryl compounds.

Sodium selenite has previously been shown to inhibit DNA and RNA synthesis in both intact cells and isolated nuclei. Nevertheless, DNA and RNA polymerases, the enzymes responsible for this synthesis, are insensitive to inhibition by selenite. Several DNA and RNA polymerases have now been shown to be inhibited by selenite in the presence of sulfhydryl compounds. This inhibition is due to the reaction of selenite with the sulfhydryl compounds to form selenotrisulfide derivatives which inhibit the enzymes. The selenotrisulfides decrease the Vmax of the polymerase reaction and increase the apparent Km for the triphosphates, but do not alter the apparent Km of the enzyme for the DNA template. There are differences in potency between selenotrisulfides formed from similar sulfhydryl compounds such as mercaptoethanol and mercaptoethylamine. There are also differences in the sensitivity of different polymerases to inhibition by the selenotrisulfides.

Chemical Phenomena↗
PubMed1986

Effects of exposure of DNA to methyl mercury on its activity as a template-primer for DNA polymerases.

A previous publication [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)] described the observation that double-stranded DNA which was briefly exposed to methyl mercury (MeHg) and purified to remove free methyl mercury was transcribed at a higher rate by RNA polymerase II from wheat germ. The specificity of this phenomenon has now been investigated by examining the activity of this MeHg-exposed DNA as a template-primer for DNA polymerases. DNA synthesis by the bacteriophage T4-induced DNA polymerase was higher with the MeHg-exposed DNA as a template-primer than with control DNA. In contrast, the rate of DNA synthesis by E. coli DNA polymerase I was lower with the MeHg-exposed DNA as template-primer. With both enzymes (as well as with RNA polymerase II), after denaturation of the MeHg-exposed and control DNAs the differences in template activity were either eliminated or markedly reduced. The enzymes are thus able to detect a MeHg-induced alteration in DNA. In contrast, circular dichroism, a physical method that is sensitive to conformational changes in DNA, did not detect any difference between the MeHg-exposed and control DNAs.

DNA↗
PubMed1985

Exposure of DNA to methyl mercury results in an increase in the rate of its transcription by RNA polymerase II.

Double-stranded DNA which was exposed to methyl mercury at concentrations of 1 mM and above, and purified by ethanol precipitation and dialysis, was transcribed at a higher rate by RNA polymerase II than was control DNA. The rate of transcription of single-stranded DNA was not affected by similar exposure to methyl mercury. The higher rate of transcription of methyl mercury-treated double-stranded DNA appears to result from a decreased Km of the enzyme for this DNA. This does not appear to result from extensive denaturation, nor from formation of a large number of single-stranded breaks in the DNA.

Bacteriophage lambda↗
PubMed1985

Effects of sodium selenite and selenate on DNA and RNA synthesis in vitro.

The effects of sodium selenite and sodium selenate on DNA and RNA synthesis have been examined using intact HeLa cells, isolated nuclei and extracted polymerases. Selenate had no effect on any of the systems examined. Selenite inhibited DNA synthesis in intact cells and in isolated nuclei, and to a limited extent also inhibited DNA polymerase alpha. Selenite also inhibited RNA synthesis in intact cells and alpha-amanitin resistant RNA synthesis in isolated nuclei (i.e., synthesis catalyzed by RNA polymerase I and III). It had no effect on alpha-amanitin sensitive synthesis (catalyzed by RNA polymerase II) at concentrations up to 500 microM. However, transcription of exogenous DNA by extracted RNA polymerase II (as well as by polymerase I and III) was inhibited by selenite.

Catalysis↗
PubMed1984

A comparative study of the effects of mercury compounds on cell viability and nucleic acid synthesis in HeLa cells.

The effects of various mercury compounds on HeLa cell viability and DNA and RNA syntheses in intact cells and in isolated nuclei have been studied. The compounds examined were: methylmercuric chloride, ethylmercuric chloride, dimethylmercury, phenylmercuric acetate, p-hydroxymercuribenzoate, p-hydroxymercuribenzenesulfonate, HgCl2, HgSO4 , Hg(ClO4)2 and Hg2(ClO4)2. All of the compounds except dimethylmercury inhibited colony formation as well as DNA synthesis in intact cells and in isolated nuclei. RNA synthesis in intact cells was inhibited by all the compounds except dimethylmercury, p-hydroxymercuribenzoate and Hg(ClO4)2. In isolated nuclei, alpha-amanitin-resistant RNA synthesis was inhibited by all the compounds except dimethylmercury, alpha-Amanitin-sensitive RNA synthesis was stimulated by some compounds, inhibited by some, and unaffected by others. The effects of two non-mercurial sulfhydryl reagents, N-ethylmaleimide and iodoacetic acid, were also examined. These compounds showed a pattern of effects on nucleic acid synthesis which differed considerably from that of the mercury compounds. Neither compound significantly inhibited alpha-amanitin-resistant RNA synthesis in isolated nuclei, although both inhibited RNA synthesis in intact cells. Iodoacetic acid had no inhibitory effect on DNA synthesis in isolated nuclei but strongly inhibited DNA synthesis in intact cells.

Cell Survival↗
PubMed1984

Application of an in vitro keratinization assay to extracts of soot from a fire in a polychlorinated biphenyl-containing transformer.

A fire in the State Office Building in Binghamton, New York, involving a polychlorinated biphenyl-containing electrical transformer, resulted in contamination of the structure with soot containing 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran. Benzene extracts of soot collected from various areas of the building were tested for in vitro keratinization-inducing activity by the method of J. C. Knutson and A. Poland (Cell 22, 27-36, 1980). The results, in terms of relative keratinization-inducing activity, are compared to a high-resolution gas chromatographic/mass spectrometric analysis for total polychlorinated dibenzofurans in the same samples. This comparison showed a good correlation and suggests that the in vitro keratinization model has potential for use as a semiquantitative assay for dioxinlike activity.

Air Pollutants↗
PubMed1983

Studies on the mechanism of the stimulation of polymerase II-catalyzed RNA synthesis by mercury compounds.

The specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated nuclei by methyl mercury (Frenkel, G. D., and Randles, K. (1982) J. Biol. Chem. 257, 6275-6279) has been further investigated. Using the method of alkaline hydrolysis/uridine analysis to determine the number of RNA chains growing in vitro, it was found that the stimulation could not be accounted for by an increase in the number of growing chains. The stimulatory effect of heparin (Coupar, B. E. H., and Chesterton, C. J. (1977) Eur. J. Biochem. 79, 525-533), was found to be additive with that of methyl mercury at saturating concentrations of the latter. Various detergents were found to affect RNA synthesis per se and to modify the stimulatory effect of methyl mercury, suggesting that the stimulation by methyl mercury requires a degree of structural integrity of some nuclear components. The ability of a number of other mercury compounds to stimulate RNA synthesis was investigated. None of the inorganic compounds examined, i.e. HgCl2, HgSO4, and Hg(ClO4)2, stimulated synthesis. Among the alkyl organic compounds tested in addition to methyl mercury, ethyl mercury also stimulated RNA synthesis, but dimethylmercury did not. Among the aryl compounds tested, phenylmercury did not stimulate synthesis whereas p-hydroxymercuribenzoate and p-hydroxymercuribenzenesulfonate did. N-Ethylmaleimide, a nonmercurous sulfhydryl reagent, was found to have only weak ability to stimulate RNA synthesis, compared to a comparable mercury-containing sulfhydryl reagent, p-hydroxymercuribenzoate. The stimulatory effect of the latter was, however, effectively competed out by the former, indicating that sulfhydryl binding is necessary for the stimulation but not sufficient. This conclusion was reinforced by experiments which utilized a model system to measure the ability of various mercury compounds to compete with N-ethylmaleimide in binding to cysteine. The results showed that even compounds such as phenylmercury and the inorganic mercurials, which are unable to stimulate RNA synthesis, are able to bind to a sulfhydryl group.

Amanitins↗
PubMed1983

DNase inhibition by the adenovirus DNA-binding protein exhibits specificity for the enzyme but not for the secondary structure of the DNA.

The adenovirus-specific DNA-binding protein (DBP) has been shown to inhibit the hydrolysis of single-stranded DNA by a DNase isolated from KB cells, (Nass, K., and Frenkel, G.D. (1980). J. Virol. 35, 314-319). The specificity of the inhibition has now been investigated. The DBP inhibits the hydrolysis of single-stranded DNA by several different DNases (DNase II, KB DNase, S1 nuclease) under a variety of reaction conditions, but it has no effect on DNase I-catalyzed hydrolysis of single-stranded DNA. The DBP also inhibits the rate of hydrolysis of double-stranded DNA by KB DNase and DNase II, but has no effect on DNase I-catalyzed hydrolysis of this substrate. The DBP also inhibits the dephosphorylation of 5'-phosphoryl-terminated DNA by bacterial alkaline phosphatase but stimulates the phosphorylation of 5'-hydroxyl-terminated DNA by polynucleotide kinase.

Adenoviridae↗
PubMed1982

Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury.

Methyl mercury has previously been shown to inhibit DNA and RNA synthesis in intact HeLa cells (Gruenwedel, D. W., and Cruikshank, M. K. (1979) Biochem. Pharmacol. 28, 651-655). Its effect on nucleic acid synthesis in vitro in isolated nuclei has now been investigated. DNA synthesis in nuclei was inhibited by methyl mercury; 50% inhibition was obtained with a concentration of 8 microM, compared to approximately 30 microM which is required in vivo. In contrast, RNA synthesis in HeLa nuclei was stimulated by methyl mercury. This stimulation is specific for alpha-amanitin-sensitive RNA synthesis (catalyzed by RNA polymerase II); in contrast, alpha-amanitin-resistant synthesis (catalyzed by polymerases I and III) was inhibited. Transcription of calf thymus DNA by solubilized RNA polymerases was inhibited by ethyl mercury. However, if the enzymes were preincubated with the DNA prior to addition of the methyl mercury, subsequent synthesis by RNA polymerase II was stimulated, whereas the inhibition of the other enzymes was unaffected. The most likely explanation for these results is that methyl mercury specifically stimulates chain elongation by RNA polymerase II whereas it inhibits elongation by polymerases I and III, and initiation by all three enzymes.

Amanitins↗
PubMed1981

Purification and properties of a new DNase activity from KB cells.

A deoxyribonuclease activity with specificity towards single-stranded DNA has been purified approximately four hundred-fold from KB cells, by chromatography on DEAE-cellulose, phosphocellulose and hydroxylapatite. The last step of the purification results in separation of the enzyme from a DNase activity which has been described previously (Wang, E.C., Furth, J.J. and Rose, J.A., (1978) Biochemistry 17: 544-549). The properties of the new DNase activity are significantly different from those of the enzymes which have previously been identified in these cells. The activity sediments at approximately 7.5S in a glycerol gradient. The DNase activity is optimal at pHs between 6.0 and 6.5. It cleaves DNA endonucleolytically and hydrolyzes single-stranded DNA at about 11 times the rate of double-stranded DNA and at twice the rate of Poly (dA). The activity is moderately sensitive to inhibition by N-ethylmaleimide and is inhibited 80% by 50 mM NaCl. It is stimulated twenty-fold by Mn++ at an optimal concentration of approximately 0.7 mM. It is stimulated by a lesser extent by Mg++, but not by Ca++.

Carcinoma↗