Use of 8-methoxypsoralen and light (lambda--365 nm) for studying repair in yeasts]

The lethal effect of 8-metoxypsoralen (8/MOP) plus light (lambda = 365 nm) on the haploid radioresistant and UV-sensitive strains of Saccharomyces cerevisiae was studied. The mutation uvs1 increased the sensitivity to the lethal effect of 8-MOP more than 2.8 times as compared to radioresistant strain. The method of repeated irradiation allowed to study kinetics of excision of monoadducts induced by 8-MOP. The mutant uvs1 was characterized by the absence of excision of monoadducts. The radioresistant strain removed monoadducts very efficiently (80%) after the incubation in complete liquid medium for 2.5 hours at 28 degrees before repeated irradiation. After the incubation of this strain in buffer (pH 7.0) monoadducts were removed considerably less efficiently (30%).     

Dose-rate effects of 8-methoxypsoralen plus 365-NM irradiation on cell killing in Saccharomyces cerevisiae.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.     

Inactivation of transforming DNA by ultraviolet light. 3. Further observations on the effects of 365 nm radiation

In the doubly auxotrophic strain ad 3A 38701 inos 37401, nitrosoethylurethane (NEU) produces a storage effect for adenine reversions but not for inositol reversions. Shaking treated spores in water for several hours destroys their response to storage. Short heat-treatment during or before plating increases the frequency of adenine reversions but not that of inositol reversions. Storage and heat-treatment decrease the inositol-specificity of NEU and may reverse it into adenine-specificity. Comparison with similar results obtained for diepoxybutane (DEB) shows that the cellular effects of NEU are more complex than those of DEB.     

Lethal effects of pyrimidine dimers induced at 365 nm in strains of E. coli differing in repair capability

Photoreactivation (PR) after 365-nm inactivation was measured in four strains of Escherichia coli differing in repair capability. Photoreactivation was observed in the recA strains K12 and AB2480 and K12 AB2463 indicating a significant role of pyrimide dimers in the lethal action of 365-nm radiation in these strains. Significant PR was not observed in the uvrA strain, K12 AB1886, or in the repair proficient strain, K12 AB1157, after 265-nm inactivation. Biological evidence indicated that stationary phase cells had not lost the capacity for photo-enzymatic repair after fluences of 365-nm radiation of 2 × 10⁶ J/m⁻² or less. It is proposed that pyrimidine dimers, although induced, are not significant 365-nm lethal lesions in uvrA and wild-type strains because of their efficient dark repair.     

Reparation after the action of 8-methoxypsoralen and light (lambda=365 nm) on radiosensitive mutants of Saccharomyces cerevisiae yeasts]

The method of repeated irradiation allowed to study kinetics of excision of mono-adducts induced by 8-methoxypsoralen (8-MOP) plus light (lambda=365 nm) in DNA of UV-sensitive mutants rad4 and rad15 and X-ray sensitive mutants rad54, xrs2, xrs4. The survival of the mutant rad4 was not practically increased after incubation in complete liquid medium for 3 hours at 28 degrees C before the repeated irradiation. These data suggest that the mutant rad4 is characterized by nearly complete absence of the mono-adduct excision. The survival of mutants rad15 and rad54 in the same environment was increased less effectively than the survival of the control radioresistant strain, but the mutants xrs2 and xrs4 did not differ from the control strain. Possible causes of differences in survival between radiosensitive strains are discussed. The increased sensitivity of the excision defective strain (rad4) and of the postreplicative recombination defective strains (xrs2, xrs4, rad54) to the lethal effect of 8-MOP plus light (lambda=365 nm) suggests that two systems of reparation take part in the removal of photoproducts induced by 8-MOP in DNA of yeast cells.     

Methylene blue plus light mediates 8-hydroxyguanine formation in DNA

Exposure to methylene blue (MB) plus light mediates formation of large levels of 8-hydroxyguanine in DNA. The amount of 8-hydroxy-2'-deoxyguanosine (8-OHdG) present in DNA increased as the amount of MB concentration increased throughout the 2 to 200 microM range studied and was dependent on light exposure. As the time of light exposure increased so did the 8-OHdG content to levels of about 750 8-OHdG/10(5) deoxyguanosine after 15 min of light exposure when MB was at 20 microM. Even though previous research has demonstrated that hydroxyl free radicals formed from a variety of sources mediate 8-OHdG formation in DNA, inclusion of mannitol, superoxide dismutase, catalase, and desferal in the MB plus light experiments demonstrated that these scavengers of oxygen free radical intermediates or precursors caused either no change or an increase in the 8-OHdG content of DNA exposed to MB plus light. These results appear to rule out the direct role of oxygen free radical intermediates in the primary events involved in the MB plus light mediated formation of 8-OHdG in DNA. Oxygen was essential to cause MB plus light mediated 8-OHdG formation in DNA. It was noted that when the reaction was carried out where the deuterium oxide content had been increased to 100%, the amount of 8-OHdG formed in DNA increased about threefold over that observed when comparable reactions were carried out in pure H2O. Use of the singlet oxygen scavenger 2,5-dimethylfuran has yielded variable results on the MB plus light mediated formation of 8-OHdG in DNA. The data taken collectively clearly indicate that MB plus light mediates 8-OHdG formation in DNA. The D2O data and the requirement for oxygen suggest that singlet oxygen may be an intermediate.     

Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation

Quantitation of UV-induced DNA damages in nanogram quantities of non-radiactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.     

DNA damage by 8-methoxypsoralen plus near ultraviolet light (PUVA) and its repair in Escherichia coli: genetic analysis

Mutants of Escherichia coli, hyper-resistant and sensitive to 8-methoxypsoralen plus near ultraviolet light (PUVA) have been isolated and studied. Results show that a mutation, located at 57.2 min on the linkage map of E. coli, is responsible for the hyper-resistant phenotype. It is also responsible for the synthesis of a 55-kdal protein in high concentrations. In a wild-type cell the synthesis of this enzyme is inducible by mitomycin C. There are indications that the mutation may have occurred in a regulatory gene, puvR, and as a result the operon, including a putative puvA gene (the structural gene for the synthesis of the 55-kdal protein), is expressed constitutively. A model for the control of the PUV operon is proposed.     

Repair of plasmid DNA treated with 8-methoxypsoralen and long-wave UV light (lambda=365 nm) in wild type and mutant rad2 cells of Saccharomyces cerevisiae

The method of repeated irradiation has been used to study excision of 8-MOP monoadducts from plasmid and chromosomal DNA in cells of wild type and rad2 mutant of Saccharomyces cerevisiae. The measurement of kinetics of monoadduct removal from chromosomal DNA in intact and competent yeast cells showed that monoadducts were excised in both types of cells with normal repair, but this process was blocked in intact and competent cells of the rad2 mutant. The survival of pYF91 plasmid treated in vitro with 8-MOP plus near UV-light has been studied in the cells of the wild type and in incision-defective rad2 mutant by the measurement of cell transformation frequency. Episomic pYF91 plasmid used in these experiments contained the yeast nuclear LEU2 gene, a portion of 2 mkm DNA and DNA of bacterial plasmid pBR322 with resistance to ampicillin. The pYF91 plasmid was treated with 8-MOP plus near UV-light in vitro, then unbound 8-MOP was removed by dialysis. This DNA was used for transformation. The transformed yeast cells were irradiated repeatedly. The quantitative alteration of the yield of transformants, depending on the time of keeping these yeast cells in complete liquid medium at 30 degrees C, prior to repeated irradiation, allowed to measure the kinetics of monoadduct excision from plasmid DNA. It was shown that monoadducts were removed equally effectively from plasmid DNA introduced into cells of the wild type and rad2 mutant. Possibly, the repair system of both these strains provides excision of monoadducts from plasmid DNA, but this process is blocked in the rad2 mutant, relatively to monoadduct excision from chromosomal DNA.     

The random (non-specific) forward mutational response of gene loci in Aspergillus conidia after photosensitisation to near ultraviolet light (365 nm) by 8-methoxypsoralen

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Evidence is presented that a treatment with 8-methoxypsoralen plus near UV light generally produces a random (non-specific) mutational effect on different gene loci in Aspergillus conidia.     

Light effects in yeast: relation between the respiratory deficiency and light sensitivity in yeast

Visible light of 5,000 lux intensity has been shown to photokill yeast cells at 12 degrees C. In the present report some of isogenic respiratory deficient mit- and nuclear mutants were compared for their sensitivity to light. No close correlation between the cytochromes spectra and light resistance was observed. Although, the nuclear and rho- mutants which lack cytochromes a + a3 and b are as a rule light resistant. Photokilling effect in yeast seems to be dependent both on the sufficiency of respiratory chain and on protein synthesis probably on cytoplasmic level.     

Mutagenic DNA repair in Escherichia coli. XVI. Mutagenesis by ultraviolet light plus delayed photoreversal in recA strains

Mutagenesis was demonstrable after delayed photoreversal of UV-irradiated strains carrying a recA deletion indicating that RecA protein is not essential for the misincorporation process that is revealed by delayed photoreversal. Moreover, the data suggest that RecA protein actually depresses misincorporation to varying extents depending on the recA allele. No delayed photoreversal was demonstrable in reA1 or recA56 bacteria unless the lexA102(ind-) allele was also present. It is suggested that the level of these RecA proteins may be lower in the lexA102(ind-) strains thus minimising their depressive effect. Delayed photoreversal mutagenesis in strains carrying the recA441 allele was not affected by either adenine or guanosine plus cytidine, substances which affect the proteolytic activity of RecA441 protein.     

DNA repair and the genetic effects of thymidylate stress in yeast

Folate antagonists, such as aminopterin, methotrexate and various sulfonamides, block de novo thymidylate biosynthesis in Saccharomyces cerevisiae. The resulting starvation for thymine nucleotides is lethal and recombinagenic in RAD wild-type strains. In this paper we report our studies of these effects in repair-deficient yeast. Antifolate treatment of various rad mutants revealed that repair defects influence the killing and recombination caused by thymidylate deprivation. Compared to a RAD wild-type strain, diploids homozygous for rad3, rad6 or rad18 were more resistant to cell killing. Thus, contrary to findings with conventional DNA-damaging agents, the lethal effects of thymidylate starvation appear to be ameliorated by certain DNA repair deficiencies. On the other hand, a rad50 strain was extremely sensitive to the antifolates. Within this series of diploids, increasing sensitivity to thymidylate starvation was accompanied by an increase in recombination frequencies. The degrees of lethality and recombination, induced by thymidylate depletion, were correlated with the severity of DNA-strand breakage in the RAD and rad50 strains. Experiments with diploids homozygous for rad52, rad54 or rad57 suggested that aborted recombination events, provoked by thymidylate deprivation, caused chromosome loss. Furthermore, the repair defects in these mutants indicated that double-strand breaks are among the lethal lesions induced by thymine nucleotide starvation. Finally, we discuss the possibility that the recombinagenicity of thymidylate stress may account for one type of acquired resistance to methotrexate in mammalian cells.     

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue greater than azure B greater than azure A greater than toluidine blue greater than thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap alpha-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascorbate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.     

Isolation and Identification of Xanthotoxin (8-Methoxypsoralen) and Bergapten (5-Methoxypsoralen) from Celery Infected with Sclerotinia sclerotiorum

Methods for the production, isolation, and identification of xanthotoxin and bergapten from celery diseased by Sclerotinia sclerotiorum (Libert) de Bary were investigated. The only conditions under which this mold was capable of producing xanthotoxin and bergapten occurred when the mold was actively growing on fresh (metabolizing) celery. Neither compound was found in uninfected celery, in the mold growing on nutrient media, on nutrient media fortified with 30% filter-sterilized celery juice, or on nonmetabolizing celery. Maximal xanthotoxin production of 320 mug per g of dry rotted celery occurred at 20 C, although mycelial growth increased until 30 C. Neither xanthotoxin nor bergapten was found when the mold grew on 11 agricultural commodities other than celery.     

甲氧补骨脂素对大鼠成骨细胞生物活性影响的实验研究

为探讨甲氧补骨脂素对体外培养新生大鼠颅骨成骨细胞增殖与分化作用的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中以不同浓度加入甲氧补骨脂素,MTT法检测加药后不同时间细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量;用放射免疫法测定细胞内骨钙素含量。结果显示:与对照组相比,甲氧补骨脂素组在24 h和36 h时促进体外大鼠成骨细胞增殖的作用更明显;在24、48 h和72 h时均能提高成骨细胞碱性磷酸酶活性(ALP)和骨钙素(BGP)的分泌。甲氧补骨脂素对体外培养的大鼠成骨细胞的增殖与分化均有明显的促进作用。