PHOTOTACTIC RESPONSE OF CHLAMYDOMONAS TO FLASHES OF LIGHT –II. RESPONSE OF INDIVIDUAL CELLS

Abstract— –Video-microscope studies provide further evidence that Chlamydomonas can become oriented in response to a single short flash of light. Following a flash, 50% of the cells in a negatively phototactic population undergo a transient deflection in swimming path ('turn response'), 10% show a 'stop response', and 40% continue to swim straight ahead. The direction of turning is related to the direction of the stimulus; a majority of cells turn away from the flash source. Repetitive flashing at 60 per s elicits oriented swimming, indistinguishable from that observed with continuous light. Responses at the onset of repetitive flashing resemble single-flash responses, reinforcing the idea that response to a single flash corresponds to the initial stages of orientation to continuous light. A stop response sometimes occurs at the onset of orientation to repetitive flashing, but it is apparently not an essential component of orientation. The fact that only 60% of the cells turn or stop in response to a flash is consistent with the hypothesis that light direction is perceived by comparing light absorbed in one photoreceptive region at two instants in time (before and during the flash). The only cells to turn or to stop would be those in which the photoreceptor organelle is appropriately oriented at the instant of the flash.     

PHOTOTACTIC RESPONSE OF CHLAMYDOMON AS TO FLASHES OF LIGHT-I. RESPONSE OF CELL POPULATIONS

Abstract— Chlamydomonas reinhardi responds phototactically to a single, very short flash of blue light (6-4 μs). Net oriented response of a cell population is monitored photometrically, using the "population system" of Feinleib and Curry (1967). A single high-intensity flash elicits a small, but definite net movement away from the stimulus source. Repetitive flashing at low frequency (between 8 and 60 flashes per min) and at the same intensity elicits a prolonged response in the same direction. Net phototactic response to single or repetitive flashes varies with stimulus intensity in the same way as does response to continuous light (Feinleib and Curry, 1971b); response is positive at low intensity and negative at high intensity. These data indicate that at least some cells become oriented in response to a short flash. The occurrence of such a response has implications for the mechanism of phototactic orientation. If almost all the cells responded, one would assume that Chlamydomonas perceives light direction instantaneously by detecting an absorption gradient within the cell. Unequivocal interpretation of the short-flash response requires examination of the behavior of individual cells.     

CALCIUM CONTROL OF PHOTOTACTIC ORIENTATION IN Chlamydomonas reinhardtii: SIGN AND STRENGTH OF RESPONSE

Abstract— Chlamydomonas reinhardtii responds to a blue light stimulus by an oriented swimming (phototaxis) toward or away from the stimulus source. In this study it is established that the sign and strength of the phototactic response are a complex function of extracellular [Ca²⁺], stimulus fluence rate, time of analysis after onset of stimulation and light pretreatment. At very low extracellular [Ca²⁺] the response is weak and usually negative. At [Ca²⁺] close to the preconditioning level, phototactic response becomes stronger and positive. As [Ca²⁺] is raised further, the initial (2 s) response remains positive but the long term (20 s) becomes negative and very strong. At extremely high [Ca²⁺] the cells become immobile. This bimodal behavior suggests that two different mechanisms determine the direction of the turn. Data cannot be explained in terms of a simple model. The model which accounts for most of the details of the behavior is that of Kamiya and Witman (1984), which proposes that positive response is triggered by a transient increase in intracellular [Ca²⁺] and negative response by a decrease below unstimulated level of Ca²⁺, at least in the range of 10^-9-10^-6 M [Ca²⁺]. The strong negative orientation which follows an initial positive response above this level of [Ca²⁺], in these experiments, is best explained by an adaptation of the cells due to an increased (on average) intracellular [Ca²⁺].     

POSITIONING OF AQUATIC MICROORGANISMS IN RESPONSE TO VISIBLE LIGHT AND SIMULATED SOLAR UV-B IRRADIATION

Three species of protozoans and two crustaceans were irradiated with simulated solar ultraviolet radiation to investigate their ability to detect and avoid UV-B (320–280 nm). Horizontal and vertical movements to sheltered areas by these organisms suggest that UV-B is an important environmental factor. UV-B survival curves were determined which indicated the resistance for each organism studied. The tolerance correlated well with the positioning behavior (i.e. sensitive organisms avoided exposure by moving into sheltered areas whereas the more resistant organisms showed a less pronounced avoidance).     

DNA STRAND BREAKS IN MAMMALIAN CELLS EXPOSED TO LIGHT IN THE PRESENCE OF RIBOFLAVIN AND TRYPTOPHAN

Abstract— When mammalian cells were exposed to visible-fluorescent light or near-UV light in the medium containing riboflavin and L-tryptophan, single-strand breaks appeared in their DNA. This did not occur if either riboflavin or tryptophan was omitted from the medium. The same effect was observed when cells were added to the pre-irradiated medium, indicating that a stable photoproduct was responsible. The induced DNA lesions were shown to be equally repairable in both excision proficient and defective (xeroderma pigmentosum) human cell lines. The active photoproduct formed was shown to be hydrogen peroxide. The possible relationship between these results and the near-UV induced killing of mammalian cells is discussed.     

CIRCADIAN RHYTHM OF LOCOMOTOR BEHAVIOR IN A POPULATION OF Paramecium multimicronucleatum: ITS CHARACTERISTICS AS DERIVED FROM CIRCADIAN CHANGES IN THE SWIMMING SPEEDS AND THE FREQUENCIES OF AVOIDING RESPONSE AMONG INDIVIDUAL CELLS

Abstract The locomotor behavior of Paramecium is determined by two components: swimming speed and the frequency of avoiding responses. The circadian oscillations of these two components were examined in order to interpret characteristics of the circadian locomotor rhythm, previously found in a Paramecium population using an originally defined parameter, "traverse frequency" (Hasegawa et al ., 1978, 1982). In our present study, a modified version of the previously developed, fully computerized, close-up video/photoamplifier system was used. Results indicate that individual specimens swam fast and unidirectionally during the day, while at night, in a light–dark cycle, they swam slowly and frequently turned. This oscillatory pattern was sustained in continuous darkness, where fluctuation in the frequency of avoiding responses was a dominant characteristic. The time structure of the "random walk" of Paramecium behavior was also examined by constructing and stochastically testing histograms of the interval times between specimens consecutively traversing beneath an observation point. Statistical analyses of observation data indicated that the circadian organization of the two components by individual specimens resulted in a circadian accumulation/dispersal rhythm of the entire population. It was concluded that the circadian "traverse frequency" rhythm principally represented this circadian accumulation/dispersal rhythm.     

APPEARANCE AND DEVELOPMENT OF PHOTOSYNTHETIC ACTIVITIES IN WHEAT ETIOPLASTS GREENED UNDER CONTINUOUS OR INTERMITTENT LIGHT—EVIDENCE FOR WATER-SIDE PHOTOSYSTEM II DEFICIENCY AFTER GREENING UNDER INTERMITTENT LIGHT*

Abstract— Etiolated wheat seedlings are greened under continuous or intermittent light. Under continuous light the onset of photosystem II (PS II) activity appears after 4 h of illumination. Under intermittent light (1 ms flashes alternating with 15 min dark periods), PS II activity cannot be detected after 300–400 flashes, although the pigment composition and structural development of these plastids are similar to those observed after 4 h of continuous light. However, the appearance of PS II activity in isolated plastids can be observed in two different ways: (1) in vivo by exposing the seedlings to a short period of continuous light after the intermittent light; or (2) in vitro by addition to the isolated plastids of an electron donor for PS II, such as diphenylcarbazide. It is concluded that the intermittent light induces development of the electron transport chain from PS II to PS I, but that a deficiency occurs on the water-side of PS II.     

RAPID STOMATAL RESPONSE TO RED LIGHT IN Zea mays

Gas exchange techniques were employed to study responses of stomatal conductance to pulses of red and blue light in the grass, Zea mays. Zea mays exhibited conductance increases following brief (< 1 min) pulses of either red or blue light, in contrast to other species (e.g. Commelina communis; Assmann, 1988, Plant Physiol. 87 , 226–231) that exhibit consistent conductance responses only to pulses of blue light. Red light pulses of 450 μmol m^?2s^?1 for x min or 225 μmol m^?2s^?1 for 2x min were used to probe the fluence dependence of the red light response. The red light-stimulated conductance increase was constant for a given fluence, and increased with increasing total fluence. The conductance response to red light was larger in field grown plants (maximum growth irradiance ? 1600 μmol m-²s^?l) than in plants raised in growth chambers (maximum growth irradiance ? 150 μmol m^?2s^?1).     

QUANTITATION OF THE PHOTOMUTAGENIC RESPONSE OF Salmonella TO AN EASTERN SHALE OIL AND FLUORESCENT LIGHT

The dose-response relationship for photomutation (i.e. photosensitized mutation) by a shale-derived oil was examined. The Ames' Salmonella typhimurium tester strain TA98 was exposed to several concentrations of an Eastern shale oil and UV-visible radiation from illumination-type fluorescent lamps. Reversion to histidine prototrophy and survival were assayed following various radiation doses. Reciprocity of shale oil concentration and radiation exposure over approximately 10-fold ranges of oil concentrations and radiation doses was observed with revertant numbers per plate, percent survival, and the induced frequency of revertants (revertants per survivor). The relationship between mutation frequency and the product (shale oil concentration times radiation exposure) fit well with either a linear model or a power law model in which the frequency of induced mutations was described by the product dose raised to the 1.26th power. Similar dose-response relationships provide potential criteria for comparing potency of photomutagenic substances, comparisons that may be valuable towards assessing, and perhaps modifying, risks imposed by human exposure to synthetic fuels.     

BLUE AND ULTRAVIOLET-B LIGHT PHOTORECEPTORS IN PARSLEY CELLS

Abstract— Ultraviolet-B (UV-B) and blue light photoreceptors have been shown to regulate chalcone synthase and flavonoid synthesis in parsley cell cultures. These photoreceptors have not yet been identified. In the present work, we studied UV-B photoreception with physiological experiments involving temperature shifts and examined the possible role of flavin in blue and UV-B light photoreception. Cells irradiated with UV-B light (0.5–15 min) at 2°C have the same fluence requirement for chalcone synthase and flavonoid induction as controls irradiated at 25°C. This is indicative of a purely photochemical reaction. Cells fed with riboflavin and irradiated with 6 h of UV-containing white light synthesize higher levels of chalcone synthase and flavonoid than unfed controls. This effect did not occur with blue light. These results indicate that flavin-sensitization requires excitation of flavin and the UV-B light photoreceptor. The in vivo kinetics of flavin uptake and bleaching indicate that the added flavin may act at the surface of the plasma membrane. In view of the likely role of membrane-associated flavin in photoreception, we measured in vitro flavin binding to microsomal membranes. At least one microsomal flavin binding site was solubilized by resuspension of a microsomal pellet in buffer with high KPi and NaCl concentrations and centrifugation at 38000 g. The 38000 g insoluble fraction had much greater flavin binding and contained a receptor with an apparent K_D of about 3.6 μM and an estimated in vivo concentration of at least 6.7 × 10–⁸M. Flavin mononucleotide, roseoflavin, and flavin adenine dinucleotide can compete with riboflavin for this binding site(s), although each has lower affinity than riboflavin. Most microsomal protein was solubilized by resuspension of the microsomal pellet in non-denaturing detergents and centrifugation at 38 000 g ; however, this inhibited flavin binding, presumably because of disruption of the environment of the flavin receptor. The parsley microsomal flavin binding receptor(s) have a possible role in physiological photoreception.     

DNA SINGLE-STRAND LESIONS DUE TO ''SUNLIGHT'' AND UV LIGHT: A COMPARISON OF THEIR INDUCTION IN CHINESE HAMSTER AND HUMAN CELLS, AND THEIR FATE IN CHINESE HAMSTER CELLS

Abstract— It has been shown that the lethal properties of germicidal UV light (254 nm) and sunlight-simulating near UV light are qualitatively different (Elkind et al ., 1978). Further to compare these two radiations, the induction of single-strand DNA breaks (i.e. frank breaks plus alkali-labile lesions) was measured in two cell lines. Equal numbers of breaks in Chinese hamster cells require a dose of UV 5.5% of a near UV dose but in HeLa cells a UV dose of 7.6% of a near-UV dose is required. The rate of break production by these radiations is about 1/10-th of that due to X-rays when a comparison is made on an equal killing dose basis. The inventory of breaks in Chinese hamster cells was also followed and was found to be characteristically different for UV compared to near UV light. These data indicate that significant differences exist, at a molecular level, in the effects produced by ultraviolet and sunlight-simulating light, and further emphasize the need for caution in attempting to extrapolate from observed molecular or biological effects due to the former to those to be expected from the latter.     

FORMATION OF PHOTOPRODUCTS LETHAL FOR HUMAN CELLS IN CULTURE BY DAYLIGHT FLUORESCENT LIGHT AND BILIRUBIN LIGHT

Abstract. Irradiation of Dulbecco's modified Eagle's tissue culture medium with "Daylight,""Special Blue," or "Bilirubin" fluorescent light produces photoproducts lethal to human cells. Killing is abolished when (1) riboflavin, (2) tryptophan and tyrosine, or (3) riboflavin, tryptophan and tyrosine are deleted from medium prior to irradiation with any of the above fluorescent lamps. Toxic photoproducts are also formed when buffered salt solutions containing (a) riboflavin and tryptophan, (b) riboflavin and tyrosine, or (c) riboflavin, tryptophan and tyrosine are exposed to any of these light sources.     

PHOTOBEHAVIOR OF EUGLENA GRACILIS: ACTION SPECTRUM FOR THE STEP-DOWN PHOTOPHOBIC RESPONSE OF INDIVIDUAL CELLS

Abstract—Light-induced behavioral responses of Euglena gracilis have been investigated in single cells by means of a video system coupled to an optical microscope. Light intensity-effect curves at different wavelengths in the near UV and visible range have been determined. From these curves the action spectrum for the step-down photophobic response of Euglena has been calculated. From a comparison with the results obtained using a population method by means of a phototaxigraph, it is concluded that a single photomotile reaction is responsible for cell accumulation, brought about by trapping in the light spot and possibly by phototaxis towards scattered light from organisms already in the light field.     

INTERACTION OF 8-METHOXYPSORALEN AND NEAR-UV LIGHT CAUSES MUTATION AND CYTOTOXICITY IN MAMMALIAN CELLS

Abstract The interaction of near-UV light and a photosensitizer, 8-methoxypsoralen (8-MOP), was studied in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase system; cell survival (cloning efficiency) and mutation induction (resistance to 6-thioguanine) were quantified. Exposure of cells to either 8-MOP up to 20 μg/m l (93 μ M ) or near–UV light up to 40000 J/m² had no effect on either survival or mutation frequency. Preincubation of cells with 8–MOP from 5 to 120 min prior to irradiation with various fluences did not affect cell survival or mutation frequency. Survival decreased and mutation frequency increased linearly when either the 8-MOP concentration or fluence was increased while the other factor was held as a constant. Mutation frequency appears to show reciprocity relative to the product of 8-MOP concentration times fluence of near–UV light [(μg/m l )·(J/m²)] throughout a range apparently limited by high cell lethality. The observed pooled data on mutation, f (x), as a function of (μg/m l )·(J/m²), x , fit a linear dose–response line, f (x) = (34.2 + 0.05 x ) × 10^-6. Cell survival, however, does not appear to exhibit such reciprocity.     

EFFECT OF UVA AND BLUE LIGHT ON PORPHYRIN BIOSYNTHESIS IN EPIDERMAL CELLS*

Abstract— To study porphyrin biosynthesis in normal human keratinocytes and A431 cells derived from human epidermoid carcinoma, cultured cells were incubated with delta-aminolevulinic acid (ALA), the precursor of porphyrin synthesis, and accumulation of porphyrins was measured spectrofluorometrically. Both human keratinocytes and A431 cells accumulated porphyrins in a time-dependent and a dose-dependent fashion. Protoporphyrin was the predominant porphyrin accumulated by both cell types. Porphyrin accumulation was enhanced by Ca Mg ethylene-diaminetetraacetic acid, a ferrochelatase inhibitor, and the enhancement was reversed by the addition of iron, suggesting the utilization of iron by ferrochelatase. The effect of light on porphyrin accumulation was evaluated by exposing the ALA-loaded A431 cells to ultraviolet-A (UVA) and blue light radiation, followed by continued incubation with ALA for 2–48 h. There was an enhancement of porphyrin accumulation 2–48 h after the radiation as compared with nonirradiated controls. Consistent with this finding, ferrochelatase activity decreased in these cells at 24 h and 48 h. These data demonstrate that human keratinocytes and A431 cells are capable of porphyrin biosynthesis, and that exposure of porphyrin-containing A431 cells to light, which includes the Soret band spectrum, decreases the ferrochelatase activity, which is responsible, at least in part, for the further increase in porphyrin level.     

LIGHT INDUCED RELOCALIZATION OF SULFONATED meso-TETRAPHENYLPORPHINES IN NHIK 3025 CELLS AND EFFECTS OF DOSE FRACTIONATION

Human cervix carcinoma cells of the line NHIK 3025 were incubated for 18 h with sulfonated meso-tetraphenylporphines (TPPSn where n = 1, 2a, 2o or 4) followed by 1 h in sensitizer-free medium and then exposed to light. The fluorescing fraction of TPPS4, TPPS2o and TPPS2a has recently been shown to be located intracellularly in extracellular granules which are intracellularly localized in a similar pattern as acridine orange-stained granules, assumed to be endosomes and lysosomes (Berg, K., A. Western, J. Bommer and J. Moan. Photochem. Photobiol. 52, 481-487). Light exposure induced a relocalization of TPPS4 from its granular pattern to mainly the nuclear area while TPPS2o and TPPS2a relocalized mainly to cytoplasmic areas. After the light-induced relocalization TPPS4 became less efficient in sensitizing photoinactivation of cells as measured per fluorescing cellbound TPPS4 molecules while TPPS2a and TPPS2o became more efficient. These changes were independent of the extracellular concentration of TPPSn applied to the cells, except for cells incubated with 75 micrograms/mL TPPS4. These cells became more sensitive to light after a light exposure inactivating 20% of the cells. This increased photosensitivity seems to be related to a 2-2.5 fold increase in the amount of fluorescing cellbound TPPS4 induced by the first light exposure.     

DEVELOPMENT OF THERMOLUMINESCENCE BANDS DURING GREENING OF WHEAT LEAVES UNDER CONTINUOUS AND INTERMITTENT ILLUMINATION

Abstract— Mature wheat leaves excited by 1-min illumination at a low temperature of -60° C showed five thermoluminescence bands at -45, -10, +25, +40 and +55° C (denoted as Z_u, A, B₁ B₂ and C bands, respectively). The development of these bands during greening of etiolated wheat leaves under continuous and intermittent illumination was investigated, and the following results were obtained. (1) Etiolated leaves showed only the C band. When these leaves were greened under continuous light, the B₁ and B₂ bands appeared at 3 h and the Z_u band appeared at 10 h. The B₁ and B₂ bands were intensified during prolonged greening under continuous illumination to be the strong bands observed for mature leaves. The A band and the group of B₁ and B₂ bands were alternative: Similar experiments by excitation of thermoluminescence at -20° C showed the development of the A band instead of these B₁ and B₁ bands. (2) When the etiolated leaves were greened under intermittent illumination (1-ms light + 5-min dark), the Z_u band first appeared after 5 h of illumination (60 flashes) and was gradually intensified during further illumination with 340 flashes but, interestingly, neither the B₁ nor the B₂ band appeared even after 24–28 h of illumination with 280–340 flashes. (3) On exposure of such leaves greened under intermittent illumination to continuous light, the B₁ and B₂ bands were rapidly developed. The appearance of these bands was accompanied by the generation of the Hill activity (DCIP photoreduction with water as electron donor). (4) These results were discussed in relation to the previous study on photoactivation of the latent water-splitting system accumulated in the leaves greened under intermittent illumination. It was deduced that the structure responsible for the A band or the group of B₁ and B₂ bands is involved in the evolution of oxygen in chloroplasts, and probably involves cations of the Mn²⁺ catalyst generated by the action of light.     

CAROTENE AND CYTOCHROME c DEPENDENT MITOTIC RECOMBINATION AND KILLING IN RESPONSE TO VISIBLE LIGHT AND UV-C IN THE SMUT FUNGUS Ustilago Violacea

Abstract— The carotene (carotenoid hydrocarbons) and cytochrome c compositions for the phlph⁺ pink diploid strain of Ustilago violacea and three newly developed color variants, white (w), orange (o), and yellow (y) were quantitatively determined using high-performance liquid chromatography of lipid extracts and difference spectroscopy of alkali extracts. In addition, the effect of high-intensity incandescent and far UV (UV-C) radiation on survival and mitotic recombination in all four phlph⁺ strains were studied. The phlph⁺w and phlph⁺y strains contained relatively small amounts of cytochrome c; however, the phlph⁺y strain accumulated β-carotene while in the phlph⁺w strain only the colorless carotene phytoene was found. The phlph⁺w was very sensitive to incandescent radiation, with complete killing by 90 min of exposure. The induction of mitotic recombination in the phlph⁺w strain was inversely proportional to the level of survival, with 96% induction by 90 min of exposure. The β-carotene-accumulating phlph⁺y strain was considerably more resistant to photokilling, and exhibited induction of mitotic recombination at a lower level. Similar results were observed with these strains in response to UV-C. The phlph⁺y strain was significantly more resistant to UV-C killing than the phlph⁺w strain and it also exhibited lower induced levels of mitotic recombination. The phlph⁺o and phlph⁺ pink strains accumulated over 10 times the cytochrome c as the phlph⁺w or phlph⁺y strains. The phlph⁺o strain accumulated β-carotene at the level of the phlph* y strain, but the phlph⁺ pink strain contained only about one-tenth as much β-carotene. The phlph⁺ o and phi ph⁺ pink strains exhibited sensitivity to visible radiation that was intermediate to the phlph⁺y and phlph⁺w strains. Mitotic recombination induction by visible radiation in the orange and pink strains was slightly less than that in the phlph⁺w strain. In response to UV-C, the phlph⁺o and phlph⁺ pink strains had survival and mitotic recombination induction characteristics that were similar to the phlph⁺w strain.