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.     

Mutagenic interaction between near-(365 nm) and far-(254 nm)ultraviolet radiation in repair-proficient and excision-deficient strains of Escherichia coli.

The mutational interaction between radiation at 365 and 254 nm was studied in various strains of E. coli by a mutant assay based on reversion to amino-acid independence in full nutrient conditions. In the two repair-proficient strains (K12 AB 1157 and B/r), pre-treatment with radiation at 365 nm strongly suppressed the induction of mutations by far-UV, a phenomenon accompanied by a strong lethal interaction. The frequency of mutations induced by far-UV progressively declined with increasing dose of near-UV. Far-UV-induced mutagenesis to T5 resistance was almost unaltered by pre-treatment with near-UV. In AB 1886 uvrA there was no lethal interaction between the two wavelengths but the mutagenic interaction was synergistic. This synergism was maximal at a 365-nm dose of 8 X 10(5) J m-2. It is proposed that in the wild-type strain, cells containing potentially mutagenic lesions are selectively eliminated from the population because of abortive excision of an error-prone repair-inducing signal. In excisionless strains, 365-nm radiation may be less damaging to the error-prone than to the error-free post-replication repair system. Alternatively, mutation may be enhanced because of the occurrence of error-prone repair of 365-nm lesions by a system that is not induced in the absence of 254-nm radiation.     

Comparative ultraviolet action spectra (254-320 nm) of five "wild-type" eukaryotic microorganisms and Escherichia coli

The action spectra of five eukaryotic organisms and the prokaryote, Escherichia coli, were examined over the wavelength range, 254-320 nm. Both the repair competent and three repair defective strains (E. coli, Caenorhabditis elegans, Saccharomyces) were examined. Tetrahymena pyriformis action spectra were performed with and without the excision repair inhibitor caffeine present. Others have observed that lethality, mutation, and the production of pyrimidine dimers show much the same wavelength dependence as DNA absorption. The results presented here demonstrate several action spectra which deviate from the DNA absorption spectra. Ultraviolet sensitization ratios (repair competent/repair defective) were also examined and were shown to change over the wavelength range. These findings suggest that DNA may not be the only important chromophore leading to cell death in the uv wavelength range studied. Since uv-B is of major importance in solar uv damage, these findings may also yield important implications for solar uv studies.     

Genetic control of mitotic crossing-over in yeasts. III. Induction by 8-methoxypsoralen and long-wave UV irradiation (lambda=365 nm)

The lethal effect of 8-methoxypsoralen (8-MOP) plus 365 nm light has been studied in haploid radiosensitive strains of Saccharomyces cerevisiae. The diploid of wild type and the diploid homozygous for the rad2 mutation (this mutation blocks the excision of UV-induced pyrimidine dimers) were more resistant to the lethal effect of 8-MOP plus 365 nm light than the haploid of wild type and rad2 haploid, respectively. The diploid homozygous for rad54 mutation (the mutation blocks the repair of double-strand breaks in DNA) was more sensitive than haploid rad54. The method of repeated irradiation allowed to study the capacity of radiosensitive diploids to remove monoadducts induced by 8-MOP in DNA. This process was very effective in diploids of wild type and in the rad54 rad54 diploid, while the rad2 rad2 diploid was characterized by nearly complete absence of monoadduct excision. The study of mitotic crossing over and mitotic segregation in yeast diploids, containing a pair of complementing alleles of the ade2 gene (red/pink) has shown a very high recombinogenic effect of 8-MOP plus 365 nm light. The rad2 mutation slightly increased the frequency of mitotic segregation and mitotic crossing over. The rad54 mutation decreased the frequency of mitotic segregation and entirely suppressed mitotic crossing over. The method of repeated irradiation showed that the cross-links, but not monoadducts, are the main cause of high recombinogenic effect of 8-MOP plus 365 nm light. The possible participation of different repair systems in recombinational processes induced by 8-MOP in yeast cells is discussed.     

Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

We have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. We have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) were used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.     

Effects of 8-methoxypsoralen and ultraviolet light A on EGF receptor (HER-1) expression

Activation of psoralens by ultraviolet light irradiation at 308-400 nm (UVA) is used in the photochemical treatment of psoriasis. While the major effect of this activation is the formation of DNA adducts, it was recently demonstrated that psoralens can also bind to specific saturable high affinity cellular receptors, and that this is associated with inhibition of epidermal growth factor (EGF)-receptor binding. In view of these findings, we have examined whether 8-methoxy-psoralen (8-MOP) itself, or in combination with UVA, influences expression of the human EGF-receptor gene ("HER-1") in a human keratinocyte cell line. We have found that 8 MOP alone, and to a lesser extent UVA, induce a striking increase in cellular levels of HER-1 RNA. The combination of 8-MOP with UVA produces less induction of HER-1 RNA than that obtained with 8-MOP alone. We suggest, therefore, that this effect of 8-MOP is not due to DNA damage, but may reflect a separate effect of this compound on receptor-mediated signal transduction.     

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.     

Photomodification of blood by laser and ultraviolet radiation: a comparative study

The efficiency of in vivo blood irradiation by a laser light source (λ = 632.8 and 670 nm) and a mercury lamp (UV light, λ = 254 nm) was compared. Absorption spectra, gas content, oxyhemoglobin content, hemoglobin oxygen saturation, concentrations of lactate and glucose were studied for both irradiated and control samples. Hemoglobin was assumed to be the primary photoacceptor of light radiation for the indicated wavelengths. No substantial differences have been found between the effects of laser and non-laser irradiation. We conclude that the biological impact of the procedure is related to photoinduced changes in hemoglobin oxygen saturation.     

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%).     

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 interaction of the 7,8-dihydrodiol-9, 10-oxides of benzo(a) pyrene with bacteriophages R17 and T7

Dose-survival curves for bacteriophages R17 and T7 treated with the syn- and anti-isomers for 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in 0.02 M phosphate buffer, pH 7.0, have been determined. In both cases the anti-isomer proved to be the more toxic: mean lethal dose for R17; syn- 3 μg/ml, anti- 2 μg/ml: and for T7, syn- 3 μg/ml, anti- 0.3 μg/ml. With both reagents reaction with bacteriophage or loss by solvolysis were complete within minutes. Physico-chemical studies of the RNA failed to detect any degradation 1 and 24 h after the addition of the reagents to the bacteriophage and no change in survival of the bacteriophage occurred during this period. In experiments with bacteriophage T7 and T7-DNA reaction did not, in the first hour, introduce any significant number of alkali-labile sites in the nucleic acid. These results suggest that no reaction occurs with the phosphate groups of the nucleic acids. Following the initial loss of infectivity when bacteriophage T7 was treated with the syn-isomer there was a further, progressive loss of biological activity over 4 days which was associated with the development of alkali labile lesions. It seems probable that these latter effects are due to the loss of alkylated bases from the DNA, a process similar to the depurination reactions observed following the reaction of DNA with e.g. methylating agents.     

Survivorship, development, and DNA damage in echinoderm embryos and larvae exposed to ultraviolet radiation (290-400 nm)

Laboratory experiments utilizing ecologically relevant irradiances of ultraviolet radiation (UVR) known to occur in shallow Gulf of Maine waters were conducted on the planktonic embryos and larvae of two common benthic echinoids; the green sea urchin Strongylocentrotus droebachiensis and the sand dollar Echinarachnius parma. Significant decreases in survivorship were observed in freshly fertilized embryos of both species with greater mortality in E. parma that was associated with the absence of UVR-absorbing compounds, the mycosporine-like amino acids. Experiments on blastula, gastrula, and prism larval stages of S. droebachiensis also showed significant decreases in survivorship, delays in development, and abnormal embryos and larvae associated with exposure to UVR. Additionally, all developmental stages of S. droebachiensis experimentally exposed to UVR resulted in significant increases in DNA damage, measured as cyclobutane pyrimidine dimer photoproducts. The observed delays in early cleavage and subsequent developmental stages for S. droebachiensis are correlated with DNA damage. It is postulated that cell cycle arrest at critical checkpoints after DNA damage, mediated by a suite of cell cycle genes, is a component of the observed UVR induced developmental delays.     

Influence of urethane and of hydrostatic pressure on the growth of bacteriophages T2, T5, T6, and T7

In 0.5 per cent NaCl, nutrient broth at 35 degrees C., urethane in a concentration of 0.4 M stops the reproduction of Escherichia coli, strain B. On dilution with 20 volumes of sterile medium, growth is resumed at its former rate after a short lag. In the one-step growth of T2, 15, T6, or T7, in the same medium at the same temperature, 0.4 M urethane, when added at the time of infection, had no apparent effect on adsorption and caused no decrease in titer throughout the latent period of the control, but completely prevented a rise in titer. If diluted 1:20 with sterile medium prior to a certain critical time in the latent period, however, bacteriophage was liberated at the same time, and in the same amount as in the control. The initial stage of apparent insensitivity to the drug lasts from the time of infection until the approximate critical times of 7 minutes with T7, T2, or T6, or 13 minutes with T5. Under the conditions described, the normal latent periods were 14, 23, 30, and 44 minutes for T7, T2, T6, and T5, respectively. At the critical times referred to above, there begins a stage characterized by complete sensitivity, rather than complete insensitivity, to 0.4 M urethane, in the sense that no active phage is subsequently liberated in continued presence of the drug. The length of this completely sensitive stage, as judged by addition of the drug at successive intervals during the latent period, extends from approximately 7 until 9 minutes after infection with T7, 7 until 15 minutes with T2 or T6, or 13 until 25 minutes with T5. When the urethane is added late in this stage of T2, a decrease in initial titer takes place as judged by assays made 40 minutes after infection, the maximum effect occurring when the drug is added between 14 and 15 minutes after infection. When added subsequently to the completely sensitive stage of each type, i.e. subsequently to 9 minutes after infection with T7, 15 minutes with T2 or T6, or 25 minutes with T5, liberation of the bacteriophage takes place in presence of the drug, but the yield is reduced, the amount of reduction being greater the sooner it is added. The yield increases as addition of the drug is delayed, but it is measurably reduced when added late in the rise period. Macroscopic lysis with T7 is delayed by 0.4 M urethane, when present from the time of infection. The delay is less with increased multiplicities of infection. A similar delay occurs with T6r at a multiplicity of 4. The application of hydrostatic pressures of 7,000 to 9,000 p.s.i. early in the latent period, within 5 to 8 minutes after infection, prevents a yield in each of the four phage types, and if maintained for lengthy periods of time a reduction in initial titer occurs. If released at various times shortly after the latent period, a rise in the titer occurred after a certain interval whose length was characteristic of the phage type. The yield was less the longer the release of pressure was delayed. When the pressure was first applied late in the latent period, large amounts of phage were liberated either under pressure or explosively when pressure was released to make the assays. Hydrostatic pressure at 6,000 p.s.i. had little effect on the rate or amount of macroscopic clearing with T7 in relatively high multiplicity of infection, when applied at the start of lysis, but slowed the rate and reduced the amount of clearing when applied shortly after infection.     

Synthesis,and biological evaluation of 2-(4-aminophenyl)benzothiazole derivatives as photosensitizing agents

Photodynamic therapy (PDT) employing exogenous photosensitizers is currently being approved for treatment of basal cell carcinoma (BCC). 2-(4-Aminophenyl)benzothiazoles (6) consist of chromophoric structure and absorb light in the UVA (315–400 nm). These results encouraged us to design and synthesize a diversity of 2-phenylbenzothiazoles (6). Studies on the apoptotic mechanism involved in photosensitive effects induced by UVA-activated 6 in BCC cells are carried out in the present article. 6-UVA-treated cells displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, and activation of caspase-3. 6-UVA induced a decrease in mitochondrial membrane potential (Δψ_mt) and ATP via enhanced ROS generation and promoted phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK expression. These results suggest that 6-UVA elicits photosensitive effects in mitochondria processes which involve ERK and p38 activation, and ultimately lead to BCC cell apoptosis.     

NMR study of the drug-base overlap geometry in the dark complex of 8-methoxypsoralen and d(pApT)4

The dark binding of 8-methoxypsoralen (MOP) to d(pApT)4 was investigated by 270-MHz 1H nuclear magnetic resonance (NMR) spectra. The continuous high-field shifts of the MOP resonances by d(pApT)4 at low temperatures indicate fast exchange between free and bound drug. The limiting complexation shifts of the various MOP protons between 0.36 (CH3) and 1.20 ppm (H5) are in the range expected for an intercalation complex. The NMR line widths of the MOP ring protons vary with the square of the observed complexation shifts (maximum at H5), indicating a dominant effect of the fast exchange between free and bound drug. The corresponding kinetic parameters agree with the values previously reported for a variety of other intercalators. The observed exchange broadenings were also used as a criterion to limit the uncertainty connected with fast averaging of the signals of the drug in potential multiple binding modes: A qualitatively different pattern of broadenings (minimum at H5) is expected from fast exchange between the two binding modes related by the short 2-fold quasi-symmetry axis of MOP. The measured complexation shifts were compared to theoretical values calculated on the basis of coplanar intercalation with base pair arrangements derived from typical published intercalation site geometries. The standard deviation between observed and calculated shifts was considerably smaller for asymmetrical intercalation between the bases of the same strand (less than or equal to 0.11 ppm) than for symmetrical intercalation between the base pairs (greater than or equal to 0.28 ppm).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and phi II

To understand constraints on the evolution of bacteriophage assembly, the structures, electrophoretic mobilities (mu) and assembly pathways of the related double-stranded DNA bacteriophages T7, T3 and phi II, have been compared. The characteristics of the following T7, T3 and phi II capsids in these assembly pathways have also been compared: (1) a DNA-free procapsid (capsid I) that packages DNA during assembly; (b) a DNA packaging-associated conversion product of capsid I (capsid II). The molecular weights of the T3 and phi II genomes were 25.2 X 10(6) and 25.9 (+/- 0.2) X 10(6) (26.44 X 10(6) for T7, as previously determined), as determined by agarose gel electrophoresis of intact genomes. The radii of T7, T3 and phi II bacteriophages were indistinguishable by sieving during agarose gel electrophoresis (+/- 4%) and measurement of the bacteriophage hydration (+/- 2%) (30.1 nm for T7, as previously determined). Assuming a T = 7 icosahedral lattice for the arrangement of the major capsid subunits (p10A) of T7, T3 and phi II best explains these data and data previously obtained for T7. At pH 7.4 and an ionic strength of 1.2, the solid-support-free mu values (mu 0 values) of T7, T3 and phi II bacteriophages, obtained by extrapolation of mu during agarose gel electrophoresis to an agarose concentration of 0 and correction for electro-osmosis, were -0.71, -0.91 and -1.17(X 10(-4) cm2V-1 s-1. The mu 0 values of T7, T3 and phi II capsids I were -1.51, -1.58 and -2.07(X 10(-4] cm2V-1 s-1. For the capsids II, these mu 0 values were -0.82, -1.07 and -1.37(X 10(-4] cm2V-1 s-1. The tails of all three bacteriophages were positively charged and the capsid envelopes (heads) were negatively charged. In all cases the procapsid had a negative mu 0 value larger in magnitude than the negative mu 0 value for bacteriophage or capsid II. A trypsin-sensitive region in capsid I-associated, but not capsid II-associated, T3 p10A was observed (previously observed for T7). The largest fragment of trypsinized capsid I-associated p10A had the same molecular weight in T7 and T3, although the T3 p10A is 18% more massive than the T7 p10A. It is suggested that the trypsin-resistant region of capsid I-associated p10A determines the radius of the bacteriophage capsid.