Elimination of lethal and pre-mutational DNA lesions during the photoreactivation of UV-irradiatedEscherichia coli

The comparison of the frequency oftrp ⁺ revertants ofEscherichia coli B/r Hcr⁺ thy trp after UV-irradiation on the one hand and after UV-irradiation plus photoreactivation on the other showed that both photoreversible pyrimidine dimers of the cyclobutane type and the non-photoreversible DNA lesions cause, at equal lethal effects, alsotrp ⁺ reversions with the same efficiency. If lethal, the pyrimidine dimers may thus be conceived as primary pre-mutational lesions.     

Ultraviolet-induced mutations to asparagine independence in Jensen sarcoma cells in vitro

UV-irradiation induces an exponential increase in the frequency of mutation from asparagine requirement to asparagine non-requirement in Jensen sarcoma cells grown in vitro. The corrected mutation frequency increases from the spontaneous rate of 5.1·10^?6 per cell to 1248·10^?6 per cell with a dose of 180 erg/mm² of 254 nm UV A substantial increase was oberved even without correction for survivors, and no significant difference was observed in the UV sensitivity of asparagine-requiring and non-requiring Jensen clones. When Jensen cells were plated at low densities in a feeder layer of LMTK-cells inactivated by HAT medium, an increase in the cloning ability of the former was observed as compared to appropriate controls without the feeder layer, but the increase was constant over all doses of UV tested. Revertants are stable and possess measurable asparagine synthetase.It is concluded that UV is an extremely effective mutagen in this system.     

UV-irradiation of sea urchin eggs changes the course of the Ca-activated ATPase activity

UV-irradiation of sea urchin eggs extends the time of increased Ca²⁺-ATPase activity of the first peak in the cell cycle if the eggs are irradiated before fertilization and the time where in controls the activity of the first peak declines. Irradiation after this time causes delay of the second cell cycle; it extends also the duration of increased enzyme activity of the first peak in the second cycle. The enzyme itself does not seem to be UV-sensitive as UV-irradiation of homogenates does not alter the enzymatic activity.     

The roles of translation initiation regulation in ultraviolet light-induced apoptosis

Ultraviolet light (UV) inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α). Two eIF2α kinases, PERK and GCN2, are known to phosphorylate the Serine-51 of eIF2α in response to UV-irradiation. In this report, we present evidence that phosphorylation of eIF2α plays a role in UV-induced apoptosis. Our data show that wild-type mouse embryo fibroblasts (MEF^s/s) are less sensitive to UV-induced apoptosis than MEF^A/A cells in which the phosphorylation site, Ser51, of eIF2α is replaced with a non-phosphorylatable Ala (Ser51Ala). PARP expression in MEF^A/A cells is reduced without being cleaved after UV-irradiation. In contrast, PARP is cleaved without a significant decrease in parental PARP in MEF^S/S cells after UV-irradiation. Our data also show that MEF^GCN2−/− cells, in which GCN2 is knocked out, are more sensitive to UV-irradiation, agreeing with the observation from MEF^A/A cells. However, MEF^PERK−/− cells, in which PERK is knocked out, are less sensitive to UV-irradiation. In addition, MCF-7-PERKΔC cells, which are stably transfected with a kinase domain deleted mutant of PERK (PERKΔC), are more resistant to UV-induced apoptosis than parental MCF-7 cells. Overexpression of wild-type PERK sensitizes MCF-7 cells to UV-induced apoptosis without directly inducing cell death. These results suggest that the level of eIF2α phosphorylation impacts PARP expression upon UV-irradiation. The eIF2α kinases may mediate UV-induced apoptosis via an eIF2α dependent or independent signaling pathway.     

Effect of neonatal influences on pain and audiogenic sensitivity and the content of monoamines in the adult rat brain

In adult Wistar, KM, and Wag/Rij rats, the threshold of pain sensitivity (tail-flick test) and degree of spasm attack in response to a strong sound were estimated after neonatal administration of Semaks (analog of ACTG4-10 fragment) or after placebo (administration of saline for the control of the effect of neonatal pain stimulation). These neonatal influences did no affect the rates of sensorimotor maturation at an early age (Fox tests), i.e., did not affect directly the physiological activity of rat pups at the age of up to 21 days. In all control rats injected with saline (pain stimulation), the latent periods of audiogenic attacks increased reliably, while their degree decreased. Administration of Semaks "raised" these parameters to the lvl of those in intact animals, i.e., increased the sensitivity to sound. Neonatal administration (per os) of caffeine to KM rats increased reliably the latent period of audiogenic attacks. The thresholds of pain sensitivity in the rats of all strains were significantly lower than in the intact control, just as the level of dopamine in the hippocampus of KM rats. These data are interpreted as an evidence of changes in the development of some brain systems in response to neonatal influences.     

UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2^−/− mouse embryonic fibroblasts (MEFs) while Akt1^−/− MEFs show cell cycle arrest. Here, we find that Akt1^−/− MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated β-galactosidase (SA β-gal) staining indicate that Akt1^−/− MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1^−/− MEFs suppressed SA β-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1^−/− MEFs, suggesting that UV light induces premature senescence in Akt1^−/− MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.     

Change in the binding capacity of human serum albumin following its exposure to therapeutic doses of UV radiation

UV-irradiation (254 nm) of donor blood and the blood of newborn with hemolytic disease, using the device for UV-irradiation utilized in Soviet hospitals for autotransfusion of UV-irradiated blood, produces a 1.1-2.0-fold increase in the binding capacity of serum albumin. The effect is the greater, the lower the initial level of serum albumin binding capacity.     

Substrate of a UV-induced repair system providing for W-reactivation of lambda phage]

Escherichia coli uvrA, polA and uvrD cells carrying non-UV-inducible prophage lambdac1857ind- were infected with 3H-thymidine labelled homoimmune phage lambdac1857, and the effect of UV-irradiation of super-infecting phage and lysogenic bacterial cells on the content of intracellular covalently-closed lambda DNA circles (cccDNA) and pyrimidine dimer content in lambda DNA are studied. UV-irradiation of host cells results in two-fold increase of relative content of cccDNA of UV-irradiated phage lambda in uvrD mutant, while there is no such an effect in uvrA and polA mutants. In UV-irradiated or intact uvrA lysogens cccDNA molecules, forming after the infection with UV-irradiated phage lambda, contain pyrimidine dimers, but in uvrD mutant cccDNA in free of dimers. The data indicate that the repair system induced by UV-irradiation of uvrA and polA cells acts exclusively on the DNA defects appearing after (or in the course) of phage genomes replication. UV-inducible repair system in uvrD mutant can operate also on some intermediates of abortive excision repair, possibly on long single straided excision gaps.     

Effects of UV-irradiation on the SCE frequency in human lymphocyte cultures

UV-irradiation (254 nm) was found to induce a smaller increase of SCE in human lymphocytes than in human fibroblasts and CHO cells. The UV-induced SCE frequency in human lymphocytes was not influenced by the duration between irradiation and the subsequent S-phase. UV-irradiated lymphocytes showed a slightly more than additive response to the SCE-inducing effect of HN2 and acetaldehyde in comparison with non-irradiated cells. The UV-induced SCE frequency was similar in lymphocyte cultures containing 20 and 100 microM of BrdUrd. The results suggest that human lymphocytes are relatively insensitive to the SCE-inducing effect of UV-irradiation, and that SCE-inducing damage caused by UV is not removed during the G1 phase in these cells.     

Influence of Ca2+ ions on UV-induced damage to mice peritoneal macrophage plasma membranes

It was shown that UV-irradiation caused damage to mice peritoneal macrophage plasma membranes. A decrease in extracellular Ca2+ leads to a decrease of the damaging effect. An increase in extracellular Ca2+ or adding of calcium ionophore A23187 to the medium is accompanied by an increase in a number of damaged cells. These data allow us to suppose that modification of the damaging effect of UV-irradiation by Ca2+ ions can be bound with changing of electric stability of membrane lipid matrix.     

Damaging effect of digitonin on macrophage plasma membranes exposed to UV-irradiation

It was shown that macrophage irradiation in 4.6 J/cm2 (lambda(max) = 306 nm) dose leads to small quantity of damaged cells in cell population, which doesn't change substantially during 60 min of incubation in darkness. So as detergent digitonin treatment (without irradiation) in 3 mkg/ml concentration doesn't lead to substantial cell damage. Also the result of combined influence of UV-irradiation and digitonin added after irradiation, 15 min before the damaged cells counting, has been got. It was shown that macrophage incubation for 15 minutes leads to cell damaging twice as much sum of UV (4.6 J/cm2) and digitonin (3 mkg/ml) damaging. However the level of cell damaging obtained 30 minutes later after finishing of irradiation doesn't exceed the sum of separate effects of this factors. Further increase of postradiation time leads to synergic effect again.     

Participation of the SSB protein in the repair of the DNA of Ehrlich ascites carcinoma cells following UV irradiation

Synchronous changes were detected in the SSB-protein content of the chromatin and in the rate of repair DNA synthesis at different time intervals after UV-irradiation of Ehrlich ascites tumor cells. The amount of SSB-protein in the extra-chromatin fraction was in an inverse relation to its content in the chromatin, whereas the cumulative SSB-protein content remained invariable. Similar changes in the SSB-protein content of the chromatin and in repair synthesis were also registered after the effect of various doses of UV-light. The increase of the SSB-protein content in the chromatin was not connected with the postirradiation accumulation of single-strand sites in DNA.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Effects of whole body UV-irradiation on oxygen delivery from the erythrocyte

In 16 healthy caucasian volunteers (mean age: 22.2 years) the influence of whole body UV-irradiation on the oxygen transport properties of erythrocytes was investigated. Four hours after irradiation with UV (using the minimal erythema dose, MED) no variation of haemoglobin concentration, hematocrit, mean corpuscular haemoglobin concentration, pH or standard bicarbonate could be found, whereas inorganic plasma phosphate (Pi), calcium, the intraerythrocytic 2,3-diphosphoglycerate (2,3-DPG), the activity of erythrocytic phosphofructokinase (PFK) and pyruvatekinase (PK) increased significantly. The half saturation tension of oxygen (P50-value) tended to increase. The increase of Pi causes--via a stimulation of the glycolytic pathway--an increase in 2,3-DPG concentration and thus results in a shift of the oxygen dissociation curve. It is therefore possible to enhance tissue oxygenation by whole body UV-irradiation.     

Repair defect mutants of Proteus mirabilis. II. Excision of pyrimidine dimers from the DNA of ultraviolet-irradiated P. mirabilis wildtype and UV-sensitive mutants

Summary Two photoproducts, thymine-thymine and cytosine-thymine-dimers were identified after UV-irradiation of Proteus mirabilis. It was found that 1 erg/mm² at 253 nm produced approximately 2.9×10⁶ pyrimidine dimers/thymine residues or about 8 dimers per 10⁷ nucleotides. Both photoproducts were excised at the same rate from the DNA of ultraviolet-resistant wildtype cells (PG 273, PG 758), but remained in acid precipitable DNA in ultraviolet-sensitive HC R-mutants (PG 678, PG 686).The excised dimers appeared both in the TCA-soluble cell fraction and in the medium outside the cells. EXR-mutants (PG 693, PG 699) also demonstrated excision capability. The excision ability of the REC-mutant (PG 672) could not be unambiguously demonstrated, because of high DNA-degradation. The number of excised dimers depended on the UV-dose. In contrast to HC R-mutants of Escherichia coli, HC R-mutants of P. mirabilis showed DNA-degradation at about the same rate as the wildtype strain during repair after UV-irradiation.     

Participation of the intracellular enzymes in the control of mutational processes

Summary The influence of UV-specific endonuclease and medium composition on the frequency and spectrum of genic mutations in Escherichia coli KI2 uvr ⁺ (with normal repair enzymes) and urv A6 (defective in UV-specific endonuclease) was studied. Mutations at the locus glu (gene controlling assimilation of glucose) were induced by ultra-violet irradiation and hydroxylamine treatment. To identify mutant colonies, triphenyl tetrazolium chloride (TTC) was added to the medium since it coloured the mutant colonies bright crimson and readily permitted distinction between pure mutant clones (complete mutations) and mixed clones (mosaic or sector mutations).A maximum mutation frequency after UV-irradiation was observed in E. coli uvr ⁺ cells but not in the E. coli uvr A6 strain. The curve of mutagenesis with a maximum was found in both studied strains after treatment by hydroxylamine which did not cause DNA damage recognized by UV-specific endonuclease.The highest frequency of mutations (at the point of maximum) in the series of experiments with enriched growth medium was almost 10 times higher than in the series of the experiments with poor medium.It was established that in bacteria with normal repair enzymes the frequency of complete mutations was higher than the frequency of mosaic mutations. It was also observed that the rate of UV-mutagenesis was higher in the case of E. coli uvr ⁺.The study of the distribution of mosaic mutant sectors in experiments with bacteria suspended in either a nutrient broth or a buffer during UV-irradiation revealed that the size of mutant sectors was rather variable and that the differences in the number of nucleoids per cell did not always determine the distribution of mutant sector sizes.Abbreviations HA Hydroxylamine hydrochloride - TTC Triphenyl tetrazolium chloride - TCA Trichloroacetic acid Other papers of this series are: Soyfer 1972; Soyfer et al. 1977; Soyfer and Kartel 1978     

Mechanism of the clinical effects of UV-irradiated blood: the stimulation of DNA synthetic activity of human cells in culture

Supernatants of UV-irradiated specimens of donor whole blood, leukocyte-platelet or red cell suspensions added to human embryonic cells in vitro produce a 1.4-1.6-fold increase in 3H-thymidine incorporation into human embryonic cells. Irradiation of blood plasma without the cells by the same doses as therapeutic ones is not followed by the effect indicated. Therefore the stimulation of the growth capacity of the blood after irradiation is connected with its cells. It is suggested that the effect under discussion is derived from the release of some active components from the blood cell surface (outer perimembranous layer) because of its photochemical destruction during UV-irradiation.     

Further characterization of SOS system induction in recBC mutants of Escherichia coli

Expression of several SOS functions such as induction of lambda prophage, inhibition of cell division and induction of both umuC and recA genes after UV-irradiation, nalidixic acid or mitomycin C addition was studied in an RecBC- mutant. UV-irradiation and mitomycin C induced all SOS functions studied in the RecBC- cells but at a lower level and delayed with respect to the wild-type strain. On the contrary, nalidixic acid was unable to trigger any of these SOS functions. In the RecBC- mutant, adenine only had a stimulating effect on the amplification of RecA protein synthesis following UV-irradiation. Nevertheless, in the wild-type strain the stimulating effect occurred in all SOS functions studied following UV-irradiation as well as in the amplification of RecA protein synthesis by nalidixic acid but not in the other SOS functions triggered by this compound. Furthermore, adenine produced a decrease in the mitomycin C-mediated induction of all SOS functions studied in both RecBC- and wild-type strains.     

Sensitivity of Vibrio cholerae to the lethal and mutagenic effects of UV-irradiation mediated by the presence of plasmids

The effect of UV-irradiation on Vibrio cholerae cells and its changes mediated by the plasmid R245 have been studied. Vibrio cholerae strains 569B and RV31 have been shown to be considerably more sensitive to lethal effect of UV-irradiation as compared with Escherichia coli and Salmonella typhimurium cells. Highly toxigenic strain 569B and practically atoxigenic strain RV31 have the same UV-sensitivity. Lethal effect of UV-irradiation on Vibrio cholerae cells is increased when the irradiated cells are plated on enriched media. UV-induction of mutations was not registered in plasmidless strains of Vibrio cholerae. Plasmid R245 increases UV-resistance of vibrio cells and makes them UV-mutable.     

Problems in the estimation of mutation rates and in the detection of induced mutations in man

The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stiationary phase haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the “petite” recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of “petites” in stationary phase cells (increase of the frequency of “petites” during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.