The recovery of normal DNA replication kinetics in ultraviolet-irradiated Chinese hamster cells

The kinetics of DNA replication were analyzed in the second S phase following UV irradiation of Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by treating cells selected in mitosis with hydroxyurea for 9 h. Following UV irradiation, the cells were allowed to progress until the next mitosis; at which time they were resynchronized at the beginning of the second S phase by the same procedure. The kinetics of DNA replication were determined by measuring the proportion of DNA which achieved hybrid buoyant density on CsCl density gradients as a function of the time of incubation in the presence of 5-bromodeoxyuridine.The results of these experiments showed that even though the rate of DNA replication is substantially depressed during the first S phase following UV irradiation with a fluence of 5 J/m², the rate has recovered to the extent that it is indistinguishable from the unirradiated control by the time the cells have entered their second S phase. It was concluded from these observations that the lesions in DNA which caused the rate of DNA replication to be initially depressed during the first S phase have been either removed or modified such that they no longer are able to cause a reduction in the rate of DNA replication in the second S phase following UV irradiation.     

Induction and rejoining of gamma-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones

The induction and rejoining of gamma-ray-induced DNA strand breaks were measured in a Chinese hamster ovary cell line, AA8, and in two radiosensitive clones (EM9 and NM2) derived from it. The kinetics of recovery from sublethal damage (SLD) and potentially lethal damage (PLD) has previously been characterized in each of these lines [vanAnkeren et al., Radiat. Res., 115, 223-237 (1988)]. No significant differences were observed among the cell lines in the yields of either DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) as assayed by filter elution. Data for SSB rejoining in AA8 and NM2 cells irradiated with 7.5 Gy were fit by a biexponential process (t1/2 values of approximately 4 and 80 min). In comparison, SSB rejoining in EM9 cells was initially slower (t1/2 = 10 min) and a higher level of SSBs was unrejoined 6 h after irradiation. DSB rejoining in AA8 cells assayed at pH 9.6 was also biphasic (t1/2 values of 15 and 93 min), although when assayed at pH 7.0, most (approximately 80%) of the damage was rejoined at a constant rate (t1/2 = 45 min) during the first 2 h. EM9 cells exhibited a slower initial rate of DSB rejoining when assayed at pH 9.6 but showed no difference compared with AA8 cells in DSB rejoining when assayed at pH 7.0. These results indicate that radiosensitive EM9 cells, whose kinetics of recovery from SLD and PLD was the same as that of AA8 cells, have a defect in the fast phase of SSB rejoining but no measurable defect in DSB rejoining. Conversely, NM2 cells, which displayed a reduced shoulder width on their survival curve and decreased recovery from SLD, had no demonstrable defects in the rate or extent of rejoining of DSBs or SSBs. When compared with the SLD and PLD data reported previously, these results suggest that there is no direct correlation between either of these recovery processes and the rejoining of SSBs or DSBs as assayed here.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

Deoxyribonucleic Acid Synthesis in Ultraviolet-Light-Irradiated Chinese Hamster Cells

Two mammalian cell lines, Chinese hamster ovary (CHO) which can recover colony-forming ability between fractionated doses of ultraviolet light (UV), and Chinese hamster B-14FAF28 which cannot recover, were tested for the ability to bypass UV-induced photoproducts in DNA during postirradiation DNA synthesis. The molecular weight distributions of newly synthesized DNA in UV-irradiated populations of both cell lines showed evidence for photoproduct bypass. Hence, the bypass mechanism does not correlate with recovery after UV.     

Comparison of the effects of ultraviolet light and ethylmethanesulphonate upon the frequency of mitotic recombination in yeast

Summary Host-cell reactivation of gamma-irradiated phage T1 in strains of E. coli K-12 has been compared with HCR of UV-irradiated phage in these same strains and with the radiation sensitivities of these strains (Fig. 1–4). The pattern of the HCR of gammairradiated phage in these strains is like that of the HCR of UV-irradiated phage. HCR in strains whose genotype is uvr ⁺rec^- is like that of the wild type; whereas, HCR is minimal in strains which are uvr ^-. It is suggested that some type of gamma-ray-induced base damage in phage DNA is repaired in uvr ⁺ strains.This work was supported by the United States Atomic Energy Commission Contract No. AT(11-1)-1686. — This is report No. COO-1686-6.Supported in part by the United States Public Health Service Training Grant No. 5T1 RH-80-02(67).     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Effects of mercuric chloride on synchronized Chinese hamster ovary cells: survival and DNA replication

Chinese hamster ovary (CHO) cells in vitro were treated with HgCl2 at various stages in the cell cycle and the effects of this chemical on cell survival, DNA replication, and cell division were observed. In terms of survival the early G1 cells were the most sensitive to treatment, followed by late G1 and early S, while mid S and late S-G2 treated cells were the least sensitive. Treatment with HgCl2 also resulted in reduced rates of DNA replication and delays in cell division. The early G1 treated cells showed substantially reduced rates of DNA replication followed by 4--5 h division delay. The early S and late S-G2 treated cells had some reduction in their rates of DNA replication followed by corresponding division delay of 2.5 h in the early S treated cells and 1 h in the late S-G2 treated cells.