Nascent DNA Synthesis in Ultraviolet Light-Irradiated Mouse, Human, and Chinese Hamster Cells

The technique of alkaline sucrose gradient centrifugation was used to study newly synthesized DNA in control and ultraviolet light-irradiated mouse L, human HeLa, and Chinese hamster ovary cells. Nascent DNA molecular weight distributions did not appear to differ among the three cell lines for unirradiated cells. However, at short times after ultraviolet light irradiation, human HeLa cells appeared to synthesize more low molecular weight DNA than either mouse L or Chinese hamster ovary cells. Since this difference was not related to differences in either the rate of DNA synthesis or amount of ultraviolet damage in the irradiated cells it appeared to be a phenotypic characteristic of the cell lines tested. A parallel was noted for these three cell lines between an increase in the synthesis of low molecular weight DNA, detected on alkaline sucrose gradients, and cell killing as measured by the ability of irradiated cells to form colonies.     

Fluorescent-light-induced lethality and DNA repair in normal and xeroderma pigmentosum fibroblasts

Cell survival and induction of endonuclease-sensitive sites in DNA were measured in human fibroblast cells exposed to fluorescent light or germicidal ultraviolet light. Cells from a xeroderma pigmentosum patient were hypersensitive to cell killing by fluorescent light, although less so than for germicidal ultraviolet light. Xeroderma pigmentosum cells were deficient in the removal of fluorescent light-induced endonuclease sites that are probably pyrimidine dimers, and both the xeroderma pigmentosum and normal cells removed these sites with kinetics indistinguishable from those for ultraviolet light-induced sites. A comparison of fluorescent with ultraviolet light data demonstrates that there are markedly fewer pyrimidine dimers per lethal event for fluorescent than for ultraviolet light, suggesting a major role for non-dimer damage in fluorescent light lethality.     

Phytochrome-mediated flavone glycoside synthesis in cell suspension cultures of Petroselinum hortense after preirradiation with ultraviolet light

Summary Ultraviolet light was demonstrated to stimulate flavone glycoside synthesis in Petroselinum cell suspension cultures. The data presented suggest the involvement of phytochrome in this response: Flavone glycoside formation resulting from 1 h of ultraviolet irradiation was increased by subsequent continuous far-red light irradiation. However, the ultraviolet effect was reduced by a subsequent irradiation with 10 min of far-red. This far-red effect was fully reversed by a sub-sequent irradiation with 10 min of red. Red and far-red irradiations were ineffective without ultraviolet preirradiation. It is concluded that in this system ultraviolet irradiation is required in order to change the cells in such a way as to allow a physiological effectiveness of the phytochrome system.     

Photobiology of natural populations of zooxanthellae from the sea anemone Aiptasia pallida: assessment of the host's role in protection against ultraviolet radiation

Natural populations of the sea anemone Aiptasia pallida containing endosymbiotic dinoflagellates were acclimated to different irradiance regimes, with and without ultraviolet radiation (UV). They showed a compensatory response in the amount of chlorophyll and the activities of enzymes responsible for detoxifying active species of oxygen that are produced by the interaction between visible or ultraviolet radiation and photosynthetically produced oxygen. Protection from active species of oxygen is essential to prevent the photooxidation of chlorophyll and the concomitant loss of productivity. Bulk analyses of chlorophyll showed differences between the populations exposed to varying irradiance regimes, but revealed no significant independent effect of UV. However, analysis by flow cytometry of the individual cells from treated populations did detect statistically significant differences in cell size and the amount of chlorophyll fluorescence per cell, which could be attributed to treatment with ultraviolet radiation. With flow cytometry we are able to detect the population variability that is undetectable by bulk measurements which is important in assessing the effects of environmental parameters in both symbiotic and free-living microalgae. Research using simultaneous measurements by flow cytometry could add considerable insight into the population dynamics of both zooxanthellae and host cells.     

The response properties of retinula cells in the flyCalliphora erythrocephala as a function of the wavelength and polarization properties of visible and ultraviolet light

Detailed functional response properties of the class (1–6) retinula cells in the insectCalliphora erythrocephela have been determined as a function of the wavelength of monochromatic light and polarization conditions over a six log unit range of light adaptation levels. These stimuli were produced as a small parallel beam that could combine various wavelengths in both the ultraviolet and visible regions whose polarization planes could be individually controlled. The wild type and white-eyed mutant were studied and shown to have no significant differences in their response properties. It was shown that previous tests using transient stimuli, large compared to the adaptation levels produced responses of high orders of nonlinearity and rapidly changing transient properties. In contrast, the use of white-noise tests did not cause changes in the basic cell properties as established by a given light adaptation condition. These tests accurately model the visual conditions of a wide and representative range of normal behavioral conditions and the model response data thus obtained can be directly correlated to important physiological tests for the same stabilized adaptation conditions. The simultaneous application of both visible and ultraviolet monochromatic light revealed a complex response interaction which changed the self response contribution of the ultraviolet light from that due only to ultraviolet light but had no effect on the visible light response. It also produced a mutual interaction. This effect was found to be the same for all members of the (1–6) class of cells when the light was unpolarized but varied for different members of the class for polarized light depending upon the relative angles of theE-vectors for the two types of light which in turn vary for different members of the six cell classes.     

Kinetic and biophysical analysis of the m2 muscarinic receptor

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.     

X-Ray and Ultraviolet Sensitivity of Synchronized Chinese Hamster Cells at Various Stages of the Cell Cycle

Populations of Chinese hamster cells, synchronized by selecting for cells at or close to division, were exposed to 250 kvp x-rays and to ultraviolet light at different stages of the cell cycle and colony-forming ability examined thereafter. These cells were found to be most resistant to x-rays during the latter part of the DNA synthetic period (S) and to be about equally sensitive before (G₁) and after (G₂) this period. Multitarget type curves of the same slope (D_o ~ 200 rad) only approximately fitted the survival data at different stages in the cycle. The changes in response were primarily due to variations in the shoulders (or extrapolation numbers) of the curves however. The response to ultraviolet light differed from that to x-rays. Resistance was greatest in G₂ and changes in both shoulder and slope of the survival curves occurred throughout the cell cycle. The x-ray and ultraviolet responses for component stages of the cell cycle were respectively compounded into expected survival data for a log phase asynchronous population of hamster cells and found to agree well with direct experiment.     

Resistance of senescent keratinocytes to UV-induced apoptosis.

Irradiation with ultraviolet (UV) triggers programmed cell death (apoptosis) in keratinocytes. This process is believed to protect against skin carcinogenesis since the cells with damaged DNA are selectively removed, limiting the likelihood of the development of a malignant keratinocyte clone. The p53 protein is able to detect mutation-bearing DNA fragments and is thus indispensable for the UV-induced apoptosis in the epidermis. Since age is a risk factor for the development of skin tumors we investigated whether ultraviolet induces apoptosis and p53 activation in senescent keratinocytes. Cultured senescent keratinocytes were irradiated with broad-band ultraviolet, apoptosis was assessed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) technique and the p53 activation pattern was determined with Western blotting and immunofluorescent staining with a panel of anti-p53 antibodies recognising different conformational forms of the protein (PAb 122, PAb 240, DO-7). In senescent keratinocytes arrested in the G1 phase of cell cycle, ultraviolet irradiation (100-2000 J/m2) caused accumulation and nuclear translocation of p53. However, in contrast to young cells where UV induces apoptotic cell death in G1, apoptosis was not detected in senescent cells. There were subtle differences in the p53 activation pattern between senescent keratinocytes and known patterns in young keratinocytes and other cell types. In senescent keratinocytes a constitutional nuclear expression of p53 (conformational form recognized by PAb 240) was present and the p53 induction in response to ultraviolet radiation was rapid. Suppression of apoptosis in senescent keratinocytes may be an important mechanism responsible for enhanced skin carcinogenesis in old age.     

Interactions Between Light and Gas Vacuoles in Halobacterium salinarium Strain 5: Effect of Ultraviolet Light

The potential light shielding by intracellular gas vacuoles in Halobacterium salinarium strain 5 was examined by looking at the ultraviolet light inactivation curves of both wild-type cells and mutants which are defective in the production of gas vacuoles. Whereas strains defective in gas vacuole production were slightly more sensitive to ultraviolet inactivation, no significant differences in ultraviolet sensitivity were seen, indicating that these subcellular inclusion bodies are not effective as light-shielding organelles. In addition, it was shown that ultraviolet light acts as a plasmid-curing agent in Halobacterium.     

Ultraviolet light-induced stimulation of the JNK mitogen-activated protein kinase in the absence of src family tyrosine kinase activation

In T cells, the JNK mitogen-activated protein kinase is activated by simultaneous stimulation of the T-cell receptor and CD28 or by a number of stress stimuli including ultraviolet light, hydrogen peroxide, and anisomycin. Lck, a Src family kinase, is essential for T-cell receptor-mediated activation of JNK. We asked whether Lck was also involved in stress-mediated activation of JNK. JNK was activated by ultraviolet light irradiation in all of the four T-cell lines we examined, but Lck was not. Additionally, JNK activation by ultraviolet light, hydrogen peroxide, and anisomycin was completely normal in T cells lacking Lck. These data suggest that Lck is not activated by ultraviolet light irradiation, nor is it required for JNK activation in T cells by any of the stress stimuli we tested. We also examined JNK activation by ultraviolet light in mouse fibroblasts expressing no known Src kinases. The activation of JNK by ultraviolet light was completely normal in these cells. Finally, treatment of lymphoid and epithelial cells with a Src kinase family inhibitor PP2-reduced tyrosine phosphorylation of cellular proteins markedly without affecting ultraviolet light-induced activation of JNK. These results suggest that Src kinases are not essential for ultraviolet light-induced activation of JNK in a diverse variety of cell types.     

Two color light scattering identifies physical differences between lymphocyte subpopulations

Forward angle light scattering of two different wavelengths by cells in a flow cytometer was used to investigate physical differences between lymphocytes of different lineage, functional subclass and developmental stage. Correlation of the ultraviolet (UV: 351 nm and 364 nm) and 488 nm light scattering signals produced by lymphoid cells demonstrated that the two signals were not equivalent and that they placed different emphasis on the physical parameters characterizing lymphocytes. Both small T and B lymphocytes from peripheral lymphoid tissues and mitogenically activated large T and B lymphocyte blasts were discriminated by both wavelengths. Differences between the Lyt-2 negative and Lyt-2 positive T lymphocyte subsets were also apparent. Two color light scattering could also discriminate between immature thymocytes and mature peripheral T cells and between small bone marrow cells and mature peripheral B cells. In bone marrow an increase in UV light scattering coincided with the appearance of cell surface immunoglobulin on small cells. These data establish that two color light scattering is a sensitive probe for distinguishing cells of apparently similar morphology and that it can be used to study the physical changes that occur during lymphoid cell differentiation.     

A cell kinetic model to explain the time of appearance of skin reaction after X-rays or ultraviolet light irradiation.

Skin reactions to various doses of X-rays (300 and 10 kV) and ultraviolet light (u.v.) have been compared using hairless mice. Two regions of epidermis with widely differing cell kinetics and gross structure have been compared. Little evidence could be found to support the idea that the early phases of the reaction are dependent on cell cycle time. The data can be explained by a model based on the assumption that epidermis contains only a small fraction of clonogenic (stem) cells and this fraction may vary in different epidermal regions. X-rays appear to exert their greatest destructive action on these clonogenic cells while u.v. is more indiscriminate in its action, killing both clonogenic and non-clonogenic cells.     

Relative responses of an X-ray-resistant hybrid cell-line and its parent line to X-irradiation, ultraviolet light, actinomycin D and cordycepin.

Using colony formation as an assay, a rat-mouse hybrid cell-line (HD1) and one of its parent lines (H4) have been studied as to their abilities to survive exposure to ionizing radiation, ultraviolet light, and the drugs actinomycin D and cordycepin. HD1 cells are more resistant than H4 to ionizing radiation, actinomycin D and cordycepin. Both cell lines respond similarly to ultraviolet light. When both cell-lines were co-treated with actinomycin D or cordycepin, the toxic effect of ionizing radiation was enhanced, whereas that of ultraviolet light (U.V.L.) was unchanged. The data suggest that RNA synthesis is more important immediately after irradiation with X-rays than with U.V.L. and that cells resistant to the toxic effect of ionizing radiation are also resistant to the toxicity induced by inhibitors of RNA synthesis.     

A CELL KINETIC MODEL TO EXPLAIN THE TIME OF APPEARANCE OF SKIN REACTION AFTER X-RAYS OR ULTRAVIOLET LIGHT IRRADIATION

Skin reactions to various doses of X-rays (300 and 10 kV) and ultraviolet light (u.v.) have been compared using hairless mice. Two regions of epidermis with widely differing cell kinetics and gross structure have been compared. Little evidence could be found to support the idea that the early phases of the reaction are dependent on cell cycle time. The data can be explained by a model based on the assumption that epidermis contains only a small fraction of clonogenic (stem) cells and this fraction may vary in different epidermal regions. X-rays appear to exert their greatest destructive action on these clonogenic cells while u.v. is more indiscriminate in its action, killing both clonogenic and non-clonogenic cells.     

Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

Abstract Mitochondria are involved in apoptosis of mammalian cells and even single‐cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL‐ZSU‐1). The Western blot results suggested that cytochrome c in the ultraviolet‐treated SL‐1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time‐dependent manner. Flow cytometric analysis of mitochondrial membrane potential (ΔΨm) of SL‐ZSU‐1 cell treated with ultraviolet‐C (UV‐C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.     

Donor age-dependent acceleration of cellular aging by repeated ultraviolet A irradiation of human dermal fibroblasts derived from a single donor

The relationship between cellular aging and aging of entire organisms has been studied extensively. The findings are confusing, however, and no clear relationships have been demonstrated. The conflicting data may be due to individual differences among the donors of the studied cells. It is crucial to identify the changes in cellular properties that are the result of the aging process. Here, we used human dermal fibroblast cell lines established from a single donor at different ages to assess the influence of ultraviolet A (UVA) on cellular aging. These cell lines have the same genetic background and were obtained from a restricted body region. The results indicated that cellular aging was accelerated by UVA irradiation in a donor age-dependent manner. The ratio of lifespan shortening increased with donor age. Increased donor age not only decreased cell division, but also increased the growth arrest response to UVA irradiation. The characteristics of the cultured cells reflected the age-related changes in dermal fibroblasts.     

A Reporter of UV Intensity Delivered to the Cytosol during Photolytic Uncaging

Photolytic uncaging of biologically-active molecules within cells is a powerful technique. However, the delivery of uncaging light into the cytosol can vary between cell types, individual cells of the same type, and different loci within an individual cell because of optical differences in absorbance and light-scattering properties of the cytoplasm. Here, we demonstrate a simple technique for monitoring the magnitude of cytosolic ultraviolet delivery during uncaging, which also leaves a quantitative and persistent record of this within the cells. The simple method shown here provides a much needed universal monitor of the delivery of ultraviolet light to molecules within the cytosol, providing a much needed parameter for the correct interpretation of uncaging experiments.     

Differences in sensitivity between human, mouse and Chinese hamster cells to killing by monochromatic ultraviolet light

Irradiation of human (IMR-91), mouse (10T1/2) and Chinese hamster (V79) fibroblasts with monochromatic ultraviolet light (u.v.) in the far-, mid-, and near-u.v. regions resulted in cell-strain-specific changes in sensitivity as a function of the wavelength used. The data suggested cell-strain-specific action spectra for cell killing by ultraviolet light that did not correlate with the ability of examined cells to excise pyrimidine dimers.     

Effects of a novel gaseous antioxidative system containing a rosemary extract on the oxidation induced by nitrogen dioxide and ultraviolet radiation

Rosemary is commonly used as a spice and a flavoring agent in food processing. Although the antioxidative properties of its extracts have been investigated, there have been few reports on the volatile components of rosemary. We designed a novel antioxidative system which can generate the volatile constituents in the gaseous phase from a rosemary extract and evaluated the gaseous antioxidative activities against both lipid peroxidation and cell death induced by nitrogen dioxide and ultraviolet radiation. The antioxidative effects of the major volatile components on the oxidation of linoleic acid induced by azo compounds were also investigated in a solution. The volatile components in the novel antioxidative system suppressed the Jurkat cell death induced by nitrogen dioxide and the intracellular formation of reactive oxygen species in fibroblast cells induced by ultraviolet radiation. 1,8-Cineole among the volatile components exerted an antioxidative effect against the oxidation of linoleic acid in a solution induced by azo compounds and ultraviolet radiation. These data suggest that the volatile constituents of a rosemary extract had antioxidative properties and that gaseous exposure antioxidant is a promising method for promoting health.     

Alterations in the Morphology of Bacillus subtilis After Exposure to β-Lysin and Ultraviolet Light

Viable counts, turbidities, and electron micrographs of Bacillus subtilis exposed to beta-lysin and ultraviolet light (UV), singly or in combination, were compared in an attempt to relate death with changes in morphology. The decreases in survival of both the beta-lysin- and UV-treated cells were rapid and preceded decreases in turbidity, as well as the changes in morphology. No significant differences were observed in turbidity reduction or morphological alterations of control cells from those of cells exposed to UV light. These cells developed prominent subcell wall spaces during incubation in the hypertonic stabilizing medium. No observable damage in either the cell wall or the cell membrane had taken place during 4 hr, but by 20 hr extensive damage of these two structures was apparent. The control and UV-treated cells exposed to beta-lysin did not develop prominent subcell wall spaces. Within 2 hr, lesions were observable in their cell walls, and the cytoplasmic membranes were permeable to phosphotungstic acid. The damage to these structures became more extensive with time. Although the visible changes of control and UV-treated cells were evident much later than those induced by beta-lysin, the morphological alterations in all cells were similar. It appeared that beta-lysin caused an accelerated release of an autolytic enzyme which digested the cell walls.