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Biomedical subjects

M Brendel

Publications and source records attributed to M Brendel.

At least 91 records · Page 5Linked to original sources
PubMed1989

Formation and stability of interstrand cross-links induced by cis- and trans-diamminedichloroplatinum (II) in the DNA of Saccharomyces cerevisiae strains differing in repair capacity.

Four haploid yeast strains differing in proficiency for DNA repair were treated with cis- or transDDP. The wild type was least sensitive while the excision-deficient mutants rad1, rad2 and snm1 exhibited higher sensitivities to either platinum compound. In all four strains tested cisDDP showed a two- to five-fold higher cytotoxicity than equimolar concentrations of transDDP. DNA interstrand cross-linking was caused by both agents in all strains. However, transDDP introduced more DNA cross-links at exposure times up to 6 h while cisDDP was the more active cross-linking agent at longer times. There was no clear-cut correlation of the number of DNA interstrand cross-links with survival. Formaldehyde-treated cells showed DNA with lower buoyant density due to proteinase K sensitive DNA-protein cross-linking; this effect was not observed after treatment with either platinum compound. Post-treatment incubation of wild-type cells exposed to cisDDP led to degradation of DNA by single and double-strand breaks, parallel with further increase of DNA interstrand cross-linking. DNA from transDDP-treated cells did not show extensive degradation although interstrand cross-links were lost during liquid holding.

Centrifugation, Density Gradient↗
PubMed1989

Molecular characterization of the two genes SNQ and SFA that confer hyperresistance to 4-nitroquinoline-N-oxide and formaldehyde in Saccharomyces cerevisiae.

The genes SNQ and SFA confer hyperresistance to 4-NQO and FA when present on a multi-copy plasmid in yeast. Both are non-essential genes since transplacement of SNQ by a disrupted snq-0::LEU2 yielded stable and viable haploid integrants. Southern analysis revealed that SNQ and SFA are single-loci genes, and OFAGE analysis showed that they are located on chromosome XIII and IV, respectively. Northern blot analysis of SNQ and SFA revealed poly(A)+ RNA transcripts of 2 kb and 1.7 kb, respectively. Nuclease S1 mapping showed SNQ to have a coding region of 1.6 kb and SFA, one of 1.3 kb. The 5' coding regions were determined for both genes, while the 3' end could only be determined for gene SNQ. Both genes do not appear to contain introns. The SFA locus was also mapped by transposon mutagenesis. Tn10-LUK integrants disrupted the SFA gene function at sites that were determined by subcloning to lie within the SFA transcription unit.

4-Nitroquinoline-1-oxide↗
PubMed1989

Reassessing the genotoxic potential of 8-MOP + UVA-induced DNA damage in the yeast Saccharomyces cerevisiae.

Two different UVA irradiation systems were initially biologically calibrated with two haploid yeast strains proficient and deficient, respectively, in nucleotide excision repair. The number of DNA lesions introduced into the cell's genome by the photoactivated bifunctional furocoumarin 8-MOP was then calculated by means of the applied UVA exposure doses. At LD37 the repair-proficient wild type had about 14 ICL and 34 furan-side monoadducts in its DNA, while doubly blocked repair mutant rad3-12 pso1-1 had 2 ICL and 3 monoadducts. Locus-specific reversion of lys1-1 followed two-hit kinetics in the repair-proficient wild type and one-hit kinetics in an excision-deficient rad2-20 mutant, as would be expected if ICL was the main type of mutagenic lesion in the wild type and monoadducts the main mutagenic lesion type in the excision-deficient strain. Quantitative comparison of 8-MOP + UVA-induced ICL with those induced by bifunctional mustard revealed the former to have a much higher genotoxicity.

DNA Damage↗
PubMed1989

UVA-induced binding of 8-methoxypsoralen to cells of Saccharomyces cerevisiae: separation and characterization of DNA photoadducts.

We present methods for the determination of UVA-induced binding of 8-methoxypsoralen (8-MOP) to nucleic acids and protein and for a quantitative assay of radioactively labelled 8-MOP plus UVA induced DNA photoproducts in the yeast Saccharomyces cerevisiae. For the dose range up to 60 kJ m-2, with a wild-type survival of 1% or higher, binding to DNA is 100-fold and to RNA 10- to 20-fold more efficient than that to protein. Between 20% and 65% of the 8-MOP binds to macromolecules that are neither nucleic acids nor protein. The number of DNA-bound 8-MOP molecules for the haploid genome rises from 14 (unirradiated control) to 349 at the highest UVA exposure dose (60 kJ m-2). Gel chromatography reveals three types of DNA thymine photoproduct, the pyrone-side monoadducts, the furan-side monoadducts and the diadducts. Among these, pyrone-side monoadducts always constitute the smallest fraction, regardless of whether the treatment is with in vitro or in vivo 8-MOP plus UVA.

Chromatography, Liquid↗
PubMed1989

Molecular dosimetry of 8-MOP + UVA-induced DNA photoadducts in Saccharomyces cerevisiae: correlation of lesions number with genotoxic potential.

Acid hydrolysis of purified DNA extracted from cells of a haploid repair-proficient (RAD) yeast strain that had been treated with 8-MOP + UVA revealed the existence of two major and one minor thymine photoproduct. At survival levels of the RAD strain between 100% and 1% furanside monoadducts constituted the major DNA lesion, followed by diadducts that, at the lowest survival level, nearly reached 50% of the thymine photoproducts; pyrone-side monoadducts were only detectable at the highest UVA exposure dose applied and clearly constitute a minority photoproduct. The number of induced diadducts was verified by determination of interstrand cross-links via denaturation and renaturation of 8-MOP + UVA-treated DNA from RAD and rad2 yeast strain. 8-MOP + UVA was shown to induce two types of locus-specific mutations: reversion of the lys1-1 ochre allele was between 20- to 50-fold higher than that of the his4-38 frameshift allele. Mutant yield for the lys 1-1 reversion was the same in RAD and excision repair-deficient rad2-20 strains whereas frameshift mutagenesis was about eightfold higher in the rad2-20 background.

Cross-Linking Reagents↗
PubMed1989

Bovine islets of Langerhans. Potential source for transplantation?

Two methods are described for large-scale isolation of islets from the bovine pancreas. With the single-endpoint technique the average number of islets isolated from a 73.3-g pancreas sample was 150,560, representing an estimated total islet volume of 0.391 ml. Pancreases processed by the continuous digestion-filtration method yielded an average of 246,229 islets from a 93.9-g pancreas sample, representing an estimated total islet volume of 0.458 ml. Isolated bovine pancreatic islets demonstrated a well-preserved endocrine cell ultrastructure, responded to acute stimulation with propionic acid, and temporarily reversed experimental diabetes in mice.

Animals↗
PubMed1989

[Islands of Langerhans of cattle--potential donor tissue for clinical islet transplantation?].

A standard method for bovine islet isolation has been developed. The mean islet yield per gram pancreas obtained from the last twelve consecutive glands was 2,098 +/- 292 representing an insulin recovery of 36.1 +/- 6.5%. The static incubation of freshly isolated bovine islets in the presence of glucose resulted in a less distinct increase of insulin secretion than in the presence of free fatty acids. Nevertheless, xenotransplantation of bovine islets to immunosuppressed C57BL6J mice did reverse streptozotocin-induced diabetes for 2 to 6 days.

Animals↗
PubMed1988

Genetic characterization of hyperresistance to formaldehyde and 4-nitroquinoline-N-oxide in the yeast Saccharomyces cerevisiae.

The hyperresistance to 4-nitroquinoline-N-oxide (4-NQO) and formaldehyde (FA) of yeast strains transformed with the multi-copy plasmids pAR172 and pAR184, respectively, is due to the two genes, SNQ and SFA, which are present on these plasmids. Restriction analysis revealed the maximal size of SFA as 2.7 kb and of SNQ as 2.2 kb, including transcription control elements. The presence of the smallest 2.7 kb subclone carrying SFA increased hyperresistance to formaldehyde fivefold over that of the original pAR184 isolate. No such increase in hyperresistance to 4-NQO was seen with the smaller subclones of the pAR172 isolate. Disruption of the SFA gene led to a threefold increase in sensitivity to FA as compared with the wild type. Expression of gene SNQ introduced on a multi-copy vector into haploid yeast mutants rad2, rad3, and snm1 did not complement these mutations that block excision repair.

4-Nitroquinoline-1-oxide↗
PubMed1988

Effects of excess thymidylate on thymidylate low-requiring strains of Saccharomyces cerevisiae: high mutagenicity and absence of DNA strand breaks.

dTMP exposure concentrations of 0.1 mM or higher are genotoxic in exponentially growing cells of thymidylate low-requiring mutants of Saccharomyces cerevisiae. Mutagenicity of excess dTMP is highest in an exposure concentration 10-fold of that needed for external supplementation of endogenously blocked thymidylate synthesis. Still higher dTMP concentrations are primarily cytotoxic. The canavanine forward-mutation system shows excess dTMP to be as potent a mutagen as irradiation by ultraviolet light. Mutagenicity of excess dTMP, however, differs from that of direct DNA-attacking mutagens in that it is highest in the absence of significant toxicity. Alkaline sucrose gradient centrifugation shows that excess dTMP does not induce significant numbers of DNA single- or double-strand breaks, while conditions of thymidylate deprivation lead to DNA-strand breaks and thymineless death.

Canavanine↗
PubMed1986

Hyperresistance to DNA damaging agents in yeast.

In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13. Genetic variants hyperresistant to 4-nitroquinoline-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid. Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA. By transfer to E. coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.

4-Nitroquinoline-1-oxide↗
PubMed1985

Mutation induction by excess deoxyribonucleotides in Saccharomyces cerevisiae.

Excess dTMP is toxic and mutagenic with exponentially growing dTMP efficient uptaking yeast strains 831 rho+ and 833 rho. The respiratory deficient strain 833 exhibits a tenfold sensitivity to the genotoxicity of excess dTMP. Mutant yield in the forward mutation system CAN1----can1 after dTMP excess is comparable to that found after irradiation with UV254nm. Excess dTMP is a poor mutagen in stationary phase cells of both strains. Mutagenicity of excess dTMP is not found in an ochre mutant allele (ade2-1). Exposure of exponentially growing cells to other deoxyribonucleotides (dCMP, dAMP, and dGMP) reveal these nucleotides to have mutagenic potential as well.

Canavanine↗
PubMed1983

Analysis of mutagenic DNA repair in a thermoconditional repair mutant of Saccharomyces cerevisiae. I. Influence of cycloheximide on UV-irradiated stationary phase rev2ts cells.

Using the thermoconditional yeast mutant rev2ts that controls an apparently site-specific step of mutagenic DNA repair it was possible to measure the time course of REV2 dependent UV-induced reversion of the ochre allele his5-2 and recovery of survival for UV-treated stationary phase cells: due to the rev2ts coded protein being active at 23 degrees C, survival and mutation frequencies increased with duration of incubation under permissive conditions in growth medium before the temperature was shifted to 36 degrees C (restrictive temperature). This increase was abolished in the presence of the protein synthesis inhibitor, cycloheximide. Furthermore, the REV2 dependent recovery of survival could be blocked or nearly blocked by cycloheximide added at any time during repair. Therefore, REV2 dependent repair can be characterized as a process requiring concomitant protein synthesis. These findings give further support to the concept that in yeast, mutagenesis involves UV inducible components of DNA repair.

Cycloheximide↗
PubMed1983

Analysis of mutagenic DNA repair in a thermoconditional repair mutant of Saccharomyces cerevisiae. II. Influence of cycloheximide on UV-irradiated exponentially growing rev2ts cells.

The time course of REV2 dependent recovery from prelethal UV damage and UV-induced locus-specific reversion of the his5-2 allele was determined in temperature-shift experiments by use of a thermoconditional allele of the rev2 gene (rad5-8, rev2ts). In UV-irradiated, exponentially growing rev2ts cells the REV2 dependent repair activity persists for up to 8 h at permissive temperature (23 degrees C), while the REV2 dependent mutagenic process is mostly completed within 2 h. The REV2 dependent process in exponentially growing cells is highly impaired by inhibition of protein synthesis. However, a REV2 dependent repair activity independent of de novo synthesis is detectable, even in the presence of up to 200 micrograms/ml cycloheximide, a response not found in stationary phase cells. Thus, the REV2 dependent process seems to be partially constitutive in exponentially growing cells. Additionally, exponentially growing rev2ts cells were considerably more UV-sensitive at restrictive temperature (36 degrees C) than were stationary phase cells.

Cell Cycle↗
PubMed1983

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate. Quantitative comparison of the mutagenic potency in Neurospora crassa and Saccharomyces cerevisiae.

Extending previous work with E. coli and mammalian cells in culture, forward-mutation frequencies induced by ethyl methanesulfonate (EMS) were quantitatively compared in Neurospora crassa and Saccharomyces cerevisiae under standardized conditions. Concomitantly, the actual dose to DNA was measured by determining the amount of radioactivity bound to DNA after treatment with tritium-labeled EMS. After exposure to EMS (2.5-50 mM), alkylation levels in N. crassa and S. cerevisiae were similar to those previously determined in E. coli and cultured mammalian cells. Consistently, there was a slightly less than proportional increase of the DNA alkylation level with the exposure concentration of the mutagen. Forward mutagenesis induced in yeast and N. crassa showed exponential kinetics with exponents of 1.5 and 2.6, respectively. These results are similar to those previously reported with E. coli, which differed from the results with cultured mammalian cells, where a linear dose-effect relationship between exposure and genetic effect was observed. These differences may reflect differences in the fate of EMS-induced adducts by cellular DNA repair systems, but are not due to initial differences in DNA alkylation levels. The fate and persistence of specific DNA adducts potentially responsible for pre-mutagenic changes are under investigation.

Animals↗
PubMed1982

Toxicity, interstrand cross-links and DNA fragmentation induced by 'activated' cyclophosphamide in yeast: comparative studies on 4-hydroperoxy-cyclophosphamide, its monofunctional analogon, acrolein, phosphoramide mustard, and nor-nitrogen mustard.

Activated cyclophosphamide (CP) is known to achieve its cytotoxic and alkylating capacity upon spontaneous hydrolytic breakdown of the oxazaphosphorine ring structure. Treatment of yeast cells with the chemically activated form of CP (4-hydroperoxy-CP, 4-OOH-CP) and with several potentially toxic cleavage products revealed that cytotoxicity is closely linked to the formation of DNA interstrand cross-links and to DNA fragmentation. While this holds true for 4-OOH-CP and its bifunctional alkylating breakdown products, phosphoramide mustard (PM) and nor-nitrogen mustard (NNM), equimolar concentrations of acrolein and the monofunctional analogon of activated CP were inactive. NNM, the ultimate cleavage product within the successive degradation of the oxazaphosphorine structure was five times more toxic than 4-OOH-CP, whereas the cytotoxic action of PM was only slightly enhanced. The high cytotoxicity of NNM was matched by its ability to induce DNA interstrand cross-links: at concentrations and treatment times producing equal cell killing, 4-OOH-CP and NNM produced the same extent of cross-linking and DNA fragmentation. Biochemical potency of NNM is in contrast to data found with the NBP colorimetric assay which suggest that NNM loses its alkylating activity at neutral pH. 4-OOH-CP and PM are much more stable than predicted from half-life measurements performed via the NBP colorimetric assay: they retain a considerable fraction of their cytotoxic and cross-linking activity in spite of a 12-h preincubation at pH 7 and 36 degrees C.

Acrolein↗
PubMed1982

Nucleic acid metabolism in yeast, V. Excretion of thymidylate.

It is shown that highly efficient utilisers of exogenous dTMP of the yeast Saccharomyces cerevisiae are able to excrete the nucleotide with similar efficiency. Strains Pi-repressible in acid phosphatase/nucleotidase excrete dTMP at extracellular high Pi; strains constitutive for this enzymic activity excrete dThd. Excretion of thymidylate and dThd, unlike uptake of exogenous dTMP, seems to be unaffected by the extracellular pH, by the extracellular presence of dTMP, and to be rather independent of the extracellular presence of a metabolisable carbohydrate such as D(+)-glucose. A model of the yeast dTMP-incorporation principle (TIP) is presented suggesting that it is also responsible for export of endogenous thymidylate.

Cell Membrane↗