PubMed Health⌕ Search

Biomedical subjects

B Epe

Publications and source records attributed to B Epe.

70 records · Page 4Linked to original sources
PubMed1988

DNA damage by oxygen radicals and excited state species: a comparative study using enzymatic probes in vitro.

Repair enzyme-containing extracts from a variety of cell types are used to analyse and compare DNA damage induced by oxygen radicals and excited molecules. The differing potentials of these extracts for recognising DNA damage leads to characteristic DNA damage profiles after treatment with superoxide (xanthine/xanthine oxidase), gamma-rays, chemically generated singlet oxygen, photosensitizers (rose bengal, methylene blue), UV254 and a 1,2-dioxetane. Three different types of damage profiles are distinguished and assigned to the predominant action of hydroxyl radicals, singlet oxygen or to the photoexcitation of thymine residues. The method applied in this study allows the analysis of DNA damage and the identification or exclusion of the participation of different ultimate reactive species without chemical identification of the lesions.

Bacteriophages↗
PubMed1987

Förster-type energy transfer. Simultaneous 'forward' and 'reverse' transfer between unlike fluorophores.

The general case of Förster-type energy transfer is that in which energy is exchanged in both directions between two unlike fluorophores. In such cases, energy is transferred from the conventionally defined donor to the conventionally defined acceptor (forward transfer) and at the same time from the acceptor to the donor (reverse transfer). Expressions are derived to describe the fluorescence intensities and lifetimes of fluorophores undergoing simultaneous forward and reverse transfer; these are compared with corresponding quantities for the case more usually considered, in which only forward transfer is significant. It is shown that the presence of reverse transfer removes the distinction between donor and acceptor, and allows such anomalous effects as 'acceptor quenching'. A confirmatory example is described. It is shown that the equations generally used in distance determination by steady-state fluorescence spectroscopy can also be applied in the presence of reverse transfer, if a correction term is included; however, for lifetime spectroscopy the correction is more complex.

Journal Article↗
PubMed1987

Induction of morphological transformation and micronuclei in Syrian hamster embryo fibroblasts by 1,2-dioxetanes. Correlation with single-strand breaks in HL-60 cells.

The photochemical genotoxic and cell-transforming potential of 4-hydroxymethyl-3,3,4-trimethyl-1,2-dioxetane (HTMD) and 3-(N-[4-pyridino]carbamoyl)methyl-3,4,4-trimethyl-1,2-dioxetane , (APD), in mammalian cell was studied. Both dioxetanes, which are efficient sources of triplet-excited ketones on thermal decomposition, induced morphological transformation in Syrian hamster embryo (SHE) fibroblasts. Unscheduled DNA synthesis in SHE and in HeLa cells could not be detected with these dioxetanes, but the number of micronuclei scored after the first mitosis was dose-dependently increased. Single-strand breaks but not Micrococcus luteus u.v.-endonuclease sensitive sites were observed by alkaline elution in HL-60 cells when treated with sub-lethal doses of HTMD and APD. A possible mechanism for the transformation mediated by DNA and chromosomal damage as well as the intermediacy of triplet carbonyls in these events are discussed.

Animals↗
PubMed1987

Site-specific covalent binding of stilbene-type and steroidal estrogens to tubulin following metabolic activation in vitro.

Both the steroidal estrogen, 2-hydroxy estradiol, and the stilbene-type estrogen, diethylstilbestrol, bind covalently and selectively to the C-terminal domain of beta-tubulin after peroxidative activation in vitro. The binding probably has to be attributed to quinonoid metabolites, as estrogens such as estradiol and hexestrol, which are unable to form quinones under these conditions, fail to bind. Albumin is not simultaneously modified, demonstrating the selectivity of the binding. The observed protein binding is discussed with respect to estrogen-induced aneuploidy and neoplastic cell transformation.

Binding Sites↗
PubMed1986

Synthesis and genotoxicity of acetoxyoxirane, the epoxide of vinyl acetate.

Acetoxyoxirane, the epoxide of vinyl acetate and a potential reactive intermediate, was synthesized and characterized by 13C-nuclear magnetic resonance (13C-NMR) and mass spectroscopy. The compound induced lesions (endonuclease-sensitive and alkali-labile sites) in supercoiled PM2 DNA in vitro and was directly mutagenic toward Salmonella typhimurium TA100. The mutagenicity of the epoxide in phosphate buffer (pH 7.4, 37 degrees C) decreased, with an initial half-life of 2.8 minutes, and mutagenicity was completely abolished by addition of S-9 mix. Acetoxyoxirane did not induce unscheduled DNA synthesis on incubation with Syrian hamster embryo fibroblasts (SHE cells). These findings may possibly be explained by an effective inactivation of acetoxyoxirane by esterases when these are present in the biological system. This view is consistent with the lack of acetoxyoxirane detected in rat liver microsomal incubations of vinyl acetate.

Animals↗
PubMed1986

Possible role of oxygen radicals in cell transformation by diethylstilbestrol and related compounds.

Diethylstilbestrol (DES) and four derivatives, viz. tetrafluoro-DES, 3'-hydroxy-DES, Z,Z-dienestrol and hexestrol, were examined for their abilities to form superoxide radicals and to induce DNA strand breaks in the presence of horseradish peroxidase/hydrogen peroxide metabolism in a cell-free system. Furthermore, the induction of strand breaks by these compounds was tested in Syrian hamster embryo (SHE) cells in vitro. Formation of superoxide radicals could be demonstrated by reduction of nitro blue tetrazolium for DES but not for its derivatives. With isolated superhelical DNA, induction of strand breaks in the presence of Fe3+ was observed for DES, tetrafluoro-DES and 3'-hydroxy-DES, while hexestrol and Z,Z-dienestrol were ineffective. In SHE cells, alkaline elution technique showed that DNA strand breaks were induced by DES and all derivatives tested, although only at cytotoxic concentrations. It is concluded that DES, under conditions of peroxidative metabolism, can give rise to superoxide generation and DNA strand breaks, and that these events may play a role in the process of DES-induced cell transformation.

Animals↗
PubMed1985

Nature of the macromolecular binding of diethylstilbestrol to DNA and protein following oxidation by peroxidase/hydrogen peroxide.

Incubation of [14C]diethylstilbestrol ([14C]DES) with horseradish peroxidase(HRP)/hydrogen peroxide in the presence of various polynucleotides and proteins led to macromolecular binding of radioactivity. Binding to DNA proved stable against ethanol precipitation, but was completely removed when the DNA was subjected to gel electrophoresis, caesium chloride density centrifugation, and mild hydrolysis. In contrast, binding to protein was stable in gel electrophoresis. The extent of binding did not differ significantly between proteins with and without thiol groups. These results imply that the products of peroxidase-mediated oxidation of DES bind to DNA in a strong but non-covalent manner, whereas binding to protein appears to be covalent and does not depend on the presence of thiol groups. The possible nature of the binding species is discussed.

Carbon Radioisotopes↗
PubMed1984

The binding of 6-demethylchlortetracycline to 70S, 50S and 30S ribosomal particles: a quantitative study by fluorescence anisotropy.

The binding of demeclocycline (6-demethylchlortetracycline) to ribosomes and ribosomal subunits from Escherichia coli was investigated by using the fluorescence anisotropy of the antibiotic to determine the extent of binding. Binding data obtained from 70S and 30S particles differed fundamentally from those obtained from 50S subunits: the first two showed a strong, specific interaction while the third did not. In addition, all three particles possessed weak, unspecific binding sites. Computer-aided least-squares analysis of the data yielded the following numbers of sites and equilibrium constants: for 30S, n1 = 1, K1 = 2.2 X 10(6) M-1, n2 K2 = 0.029 X 10(6) M-1; for 50S, n1 = 0, n2 K2 = 0.035 X 10(6) M-1; for 70S, n1 = 1, K1 = 3.2 X 10(6) M-1, n2 K2 = 0.082 X 10(6) M-1. These data resolve current disagreement in the literature and are a prerequisite for quantitative studies of the mechanism of inhibition by tetracycline of protein biosynthesis.

Computers↗
PubMed1984

Location of protein S4 on the small ribosomal subunit of E. coli and B. stearothermophilus with protein- and hapten-specific antibodies.

In spite of considerable effort there is still serious disagreement in the literature about the question of whether epitopes of ribosomal protein S4 are accessible for antibody binding on the intact small ribosomal subunit. We have attempted to resolve this issue using three independent approaches: (i) a re-investigation of the exposure and the location of epitopes of ribosomal protein S4 on the surface of the 30S subunit and 30S core particles of the E. coli ribosome, including rigorous controls of antibody specificity, (ii) a similar investigation of protein S4 from Bacillus stearothermophilus and (iii) the labelling of residue Cys-31 of E. coli S4 with a fluorescein derivative the accessibility of which towards a fluorescein-specific antibody was demonstrated directly by fluorimetry. In each of the three cases the antigen (E. coli S4, B. stearothermophilus S4 or fluorescein) was found to reside on the small lobe.

Antibodies, Bacterial↗
PubMed1984

Peroxidase-mediated binding of diethylstilbestrol analogs to DNA in vitro: a possible role for a phenoxy radical.

In order to investigate the role of peroxidase-mediated metabolic activation in the mechanism of carcinogenicity of diethylstilbestrol (DES), a series of 14C-labelled analogs of DES was synthesized and their binding to DNA upon oxidation by peroxidases from horseradish or mouse uterus was studied in vitro. The compounds chosen for this study were the erythro and threo form of hexestrol (HES), the E,E- and Z,Z-isomer of dienestrol (DIES) and the mono- and dimethyl ether of DES. Non-extractable binding to DNA was observed for all compounds with at least one free hydroxyl group independent of the stilbene structure. The extent of binding was highest for the HES isomers and for E,E-DIES, whereas Z,Z-DIES and the monomethyl ether were bound to about the extent of DES. These findings imply that the formation of a phenoxy free radical is sufficient for non-extractable DNA binding and the stilbene structure is not required for peroxidase-mediated activation of DES.

Animals↗
PubMed1983

Distance measurement by energy transfer. Ribosomal proteins L6, L10 and L11 of Escherichia coli.

Ribosomal proteins L6, L11 and the complex [(L12)4 X L10] were labelled specifically at their respective single thiol groups, either with the acetylaminoethyl-dansyl or with the acetamidofluorescein fluorophore. The labelled proteins were then reconstituted, singly or in pairs, into ribosomal 50S subunits; the presence of the label had no observable effect on the composition, shape or activity of the reconstituted subunits. The distances between the labelled thiol groups were measured by a fluorescence energy transfer method detailed elsewhere [Epe, B. et al. (1983) Proc. Natl Acad. Sci. USA, 80, 2579-2583] and were found to be: for L6-L10, 60 A (6.0 nm); for L6-L11, 46 A (4.6 nm); for L10-L11, 56 A (5.6 nm). Reversal of the direction of energy transfer by exchanging labels gave duplicate distances which differed, on average, by about 4%. The distance between the fluorescent labels on L10 and L11 in the [23 S-RNA X L10 X L11 X (L12)4] ribonucleoprotein complex was the same as in the 50S subunit, but all three distances were greater in 50S subunits which had been reconstituted without the final activation step (incubation at 50 degrees C). This suggests a tightening of the L6/L10/L11 domain of the 50S subunit during the activation step.

Bacterial Proteins↗
PubMed1983

Immunoelectron microscopy of ribosomes carrying a fluorescence label in a defined position. Location of proteins S17 and L6 in the ribosome of Escherichia coli.

By coupling fluorescein to a defined amino acid of a single ribosomal protein and incorporating this protein into the ribosome, we have obtained ribosomes labelled at a single, defined position. A fluorescein-specific antibody preparation was used to locate the fluorescein residues bound to the two cysteines at positions 58 and 63 of protein S17 and to the cysteine at position 86 of protein L6. This study demonstrates the advantages which accrue from the combination of electron microscopy and fluorimetry.

Antibodies↗
PubMed1983

Theory of measurement of Förster-type energy transfer in macromolecules.

We describe the theoretical basis of an unconventional method for the determination of the amount of energy transferred between two fluorophores by the Förster mechanism. The method involves an internal comparison made by separation of the fluorophores in situ (i.e., in the optical cell), for example, by means of enzymic digestion; it eliminates several important sources of error and it simplifies calculation while making maximal use of the information contained in the fluorescence spectra. The validity of the method is demonstrated by determination of the known distance between two modifiable sites on the transfer RNA molecule, and its usefulness is exemplified by its application to triangulation of the ribosome of Escherichia coli.

Journal Article↗
PubMed1982

Structure of ribosomal protein L6 from Escherichia coli. A fluorescence study.

Protein L6 from the 50-S ribosomal subunit has been investigated using fluorimetric techniques. The intrinsic fluorophore Trp-61 and fluorescent labels (acetylaminoethyl-dansyl and acetylaminofluorescein) attached to the residue Cys-124 were used. It proved possible to incorporate fluorescence-labelled L6 into the 50-S ribosome. Trp-61 is exposed to solvent, as shown by its emission wavelength and by quenching experiments; the latter also show that it lies in a pocket with a high positive charge due to the basic residues in the N-terminal fragment. Cys-124 lies in a less strongly positive region. Upon incorporation into the 50-S subunit, the label on Cys-124 becomes less accessible for quenching but its positive potential rises, showing the absence of direct contact with 23-S RNA. Analysis of anisotropy data indicates a considerable degree of asphericity of free L6. Energy transfer between Trp-61 and the dansyl label on Cys-124, measured by donor quenching and acceptor enhancement, reveals a separation of 3.5 +/- 0.4 nm (35 +/- 4 A) between fluorophores.

Bacterial Proteins↗
PubMed1982

Distance measurement by energy transfer: the 3' end of 16-S RNA and proteins S4 and S17 of the ribosome of Escherichia coli.

Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl-dansyl and/or bimane fluorophores. Each formed a complex with 16-S RNA and, when the other 30-S ribosomal proteins were added, a complete 30-S subunit with at least partial activity. If the 3' end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Förster-type energy transfer. The result for S4 was 6.0 nm (60 A) in the ribonucleoprotein complex and 5.6 nm (56 A) in the 30-S subunit, and for S17 6.3 nm (63 A) in the complex and 6.2 nm (62 A) in the subunit. There is no evidence for a major change in the relative disposition of the 3' and 5' ends of the 16-S RNA during formation of the 30-S subunit. Sources of error are discussed, including the question of multiple labelling. In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper.

Bacterial Proteins↗