Effects of abiotic stressors on synthesis of the mycosporine-like amino acid shinorine in the Cyanobacterium Anabaena variabilis PCC 7937

In the present investigation we show that the cyanobacterium Anabaena variabilis PCC 7937 produces a single mycosporine-like amino acid (MAA), shinorine (retention time = 2.3 min and absorption maximum at 334 nm) when isolated and purified by HPLC. Although there was significant induction of MAA synthesis from its initial value under 395 or 320 nm cutoff filters, MAA induction was significantly more pronounced in samples covered with 295 nm cutoff filters after 72 h of exposure. Heat as a stress factor had no effect on MAA induction with or without UV radiation. In contrast, salt and ammonium treatment had synergistic effects with UV stress. MAA synthesis was also induced by salt and ammonium in a concentration-dependent manner without UV stress in samples covered with 395 nm cutoff filters. The results indicate that MAAs may have other functions in addition to photoprotection in this organism.     

Wavelength dependence of mycosporine-like amino acid synthesis in Gyrodinium dorsum

The synthesis or accumulation of mycosporine-like amino acids (MAAs) is an important UV tolerance mechanism in aquatic organisms. To investigate the wavelength dependence of MAA synthesis in the marine dinoflagellate Gyrodinium dorsum, the organism was exposed to polychromatic radiation (PAR and UV) from a solar simulator for up to 72 h. Different irradiance spectra were produced by inserting various cut-off filters between lamp and samples. A polychromatic action spectrum for the synthesis of MAA synthesis was constructed. PAR and long wavelength UV-A radiation showed almost no effect while the most effective wavelength range was around 310 nm. Shorter wavelengths where less effective in the induction of MAA synthesis. Wavelengths below 300 nm damaged the organisms severely as indicated by a decrease in chlorophyll a absorption.     

Polychromatic action spectrum for the induction of a mycosporine-like amino acid in a rice-field cyanobacterium, Anabaena sp

A polychromatic action spectrum for the induction of an ultraviolet-absorbing/screening mycosporine-like amino acid (MAA) has been determined in a filamentous and heterocystous nitrogen-fixing rice-field cyanobacterium, Anabaena sp. High-performance liquid chromatographic (HPLC) studies revealed the presence of only one type of MAA, which was identified as shinorine, a bisubstituted MAA containing both glycine and serine groups having a retention time at 2.8 min and an absorption maximum at 334 nm. Exposure of cultures to simulated solar radiation in combination with various cut-off filters (WG 280, 295, 305, 320, 335, 345, GG 400, 420, 455, 475, OG 515, 530, 570, RG 645, 665 and a broad-band filter, UG 11) clearly revealed that the induction of the MAA takes place only in the UV range. Photosynthetic active radiation (PAR) had no significant impact on MAA induction. The ratio of the absorption at 334 nm (shinorine) to 665 nm (chlorophyll a) and the action spectrum also showed the induction of MAA to be UV dependent peaking in the UV-B range at around 290 nm. The results indicate that the studied cyanobacterium, Anabaena sp. may protect itself from deleterious short wavelength solar radiation by its ability to synthesize a mycosporine-like amino acid in response to UV-B radiation and thereby screen the negative effects of UV-B.     

MULTIPLE EFFECTS OF UV RADIATION (265–330 NM) ON FUNGAL SPORE EMERGENCE

We have investigated the influence of narrow-band UV radiation, 265–330 nm. on germination of spores of the fungus Cladosporium cucumerinum Ellis and Arth., using a Xe arc lamp and filters. Reciprocity of time and dose rate was demonstrated when fungal spores were subjected to UV radiation at 325 nm but failed to hold at 265 nm. Based on these findings, data on fluence response, and partial action spectra, we propose that there are two biologically active sites in this organism that are affected by radiation between 265 and 330 nm and that might be influenced by changes in the stratospheric ozone layer: a short-wave-sensitive site (265–295 nm) and a long-wave-sensitive site (300–330 nm). Data obtained with narrow-band interference filters confirmed previous reports of damage to nucleic acid from UV at 265–295 nm and in addition demonstrated significant inhibition by UV at 300–320 nm. Further studies of the 300 330 nm portion of the spectrum, using combinations of plastic and glass filters, showed that the influence of UV radiation in this region was primarily to produce a non-photoreactivable delay in germ-tube emergence.     

A monochromatic action spectrum for the photoinduction of the UV-absorbing mycosporine-like amino acid shinorine in the red alga Chondrus crispus

To determine the action spectrum for photoinduction of the ultraviolet (UV)-absorbing mycosporine-like amino acid shinorine, specimens of the marine red alga Chondrus crispus were irradiated with monochromatic light of various wavelengths using the Okazaki large spectrograph at the National Institute for Basic Biology, Okazaki, Japan. Fluence response curves were determined for the wavelengths between 280 and 750 nm, by irradiating the algae with monochromatic light for 10 h, followed by 4 h of 25 micromol m(-2) s(-1) photosynthetically active radiation and 10 h darkness. Samples were taken after the second exposure interval. A linear correlation between fluence rate and accumulated shinorine concentration was detected for wavelengths between 350 and 490 nm in the fluence rate range of 20-30 micromol m(-2) s(-1), whereas there was no induction above 490 nm. Below 350 nm a decline in shinorine concentration could be observed at fluence rates above 30 micromol m(-2) s(-1), probably due to an inhibition of photosynthetic activity and a subsequent impairment of shinorine biosynthesis. The constructed action spectrum indicated that the photoreceptors mediating shinorine photoinduction might be an unidentified UV-A-type photoreceptor with absorption peaks at 320, 340 and 400 nm.     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

The Photoreceptor for Phototaxis in the Photosynthetic Flagellate Euglena gracilis

The unicellular flagellate Euglena gracilis shows positive phototaxis at low fluence rates (≤10 W m ²) and negative phototaxis at high fluence rates (≥100 W m ²). Currently, retinal or flavins/pterins are discussed as chromo-phores of the photoreceptor. When grown in the presence of 4 mM nicotine, a retinal inhibitor, for several generations, the cells still showed both responses, indicating that retinal is unlikely to be the chromophoric group of the photoreceptor responsible for phototaxis. The native flavin(s) can be substituted by growing the cells in roseo-flavin dissolved in the medium. The absorption spectrum of roseoflavin extends well beyond the action spectrum for phototaxis (up to 600 nm). Excitation at wavelengths >550 nm does not cause phototactic orientation in control cells but causes both positive and negative phototaxis in roseoflavin-grown cells, indicating an uptake and assembly of the chromophore in the photoreceptor complex. The white mutant strain 1224-5/1f, induced by streptomycin treatment, lacks flavins as indicated by fluorescence spectroscopy. The phototaxis-deficient pheno-type cannot be complemented by the addition of external riboflavin. Fluorescence spectra of intact paraxonemal bodies (PAB) indicate that both pterins and flavins are involved in photoperception and that the excitation energy is efficiently funneled from the pterins to the flavins. This energy transfer is disrupted by solubilization of the PAB. In intact PAB flavins are not accessible to reducing or oxidizing substances, indicating that they are located inside the structure, while pterins are accessible, so that their localization can be assumed to be on the surface. The results described above are discussed with regard to the potential involvement of flavins and pterins as well as retinal in photoperception.     

COMPARISON OF UV ACTION SPECTRA FOR LETHALITY AND MUTATION IN Salmonella typhimurium USING A BROAD BAND SOURCE AND MONOCHROMATIC RADIATIONS

Abstract— The UV-B region (280–320 nm) is thought to be primarily responsible for the mutagenic, lethal, and carcinogenic effects of solar radiation. We have conducted UV-B action spectroscopy for mutagenesis and survival of Ames' Salmonella typhimurium strain TA98 ( uvrB , pKM101) using both monochromatic radiation from a dye laser and broader bandwidth radiation emitted from FS-20 sunlamps. A series of optical filters having different transmission cut-offs together with the sunlamp source provided bandwidths having successively less short wavelength components from which a "broad band" action spectrum was deduced. The two sets of action spectra differed both qualitatively and quantitatively: in comparison to the monochromatic action spectra, the "broad band" spectra showed up to a 200-fold reduced efficiency for both mutation induction and lethality by UV-B wavelengths. These results suggest a large protective effect of the background UV-A and/or visible radiations which were present during the broad spectrum irradiations and which are also present in solar radiation. Additional experiments show that to the extent tested this protective effect is not due to photo-reactivation or irradiance (dose rate) effects     

WAVELENGTH DEPENDENT FORMATION OF THYMINE DIMERS AND (6-4)PHOTOPRODUCTS IN DNA BY MONOCHROMATIC ULTRAVIOLET LIGHT RANGING FROM 150 TO 365 nm

We investigated the wavelength dependence of cyclobutane thymine dimer and (6-4)photoproduct induction by monochromatic UV in the region extending from 150 to 365 nm, using an enzyme-linked immunosorbent assay with two monoclonal antibodies. Calf thymus DNA solution was irradiated with 254-365 nm monochromatic UV from a spectrograph, or with 220-300 nm monochromatic UV from synchrotron radiation. Thymine dimers and (6-4)photoproducts were fluence-dependently induced by every UV below 220 nm extending to 150 nm under dry condition. We detected the efficient formation of both types of damage in the shorter UV region, as well as at 260 nm, which had been believed to be the most efficient wavelength for the formation of UV lesions. The action spectra for the induction of thymine dimers and (6-4)photoproducts were similar from 180 to 300 nm, whereas the action spectrum values for thymine dimer induction were about 9- and 1.4-fold or more higher than the values for (6-4)photoproduct induction below 160 nm and above 313 nm, respectively.     

ULTRAVIOLET ACTION SPECTRA FOR PHOTOBIOLOGICAL EFFECTS IN CULTURED HUMAN LENS EPITHELIAL CELLS

Abstract— The action spectrum for cell killing by UV radiation in human lens epithelial (HLE) cells is not known. Here we report the action spectrum in the 297–365 nm region in cultured HLE cells with an extended lifespan (HLE B-3 cells) and define their usefulness as a model system for photobiological studies. Cells were irradiated with monochromatic radiation at 297, 302, 313, 325, 334 and 365 nm. Cell survival was determined using a clonogenic assay. Analysis of survival curves showed that radiation at 297 nm was six times more effective in cell killing than 302 nm radiation; 297 nm radiation was more than 260, 590, 1400 and 3000 times as effective in cell killing as 313, 325, 334 and 365 nm radiation, respectively. The action spectrum was similar in shape to that for other human epithelial cell lines and rabbit lens epithelial cells. The effect of UV radiation on crystallin synthesis was also determined at different wavelengths. To determine whether exposure to UV radiation affects the synthesis of β-crystallin, cells were exposed to sublethal fluences of UV radiation at 302 and 313 nm, labeled with [³⁵S]methionine and the newly synthesized βY-crystallin was analyzed by immunoprecipitation and western blotting using an antibody to β-crystallin. The results show a decrease in crystallin synthesis in HLE cells irradiated at 302 and 313 nm at fluences causing low cytotoxicity. The effect of radiation on membrane perturbation was determined by measuring enhancement of synthesis of prostaglandin E₂ (PGE₂). Synthesis of PGE₂ occurs at all UV wavelengths tested in the 297–365 nm region. The slope of the PGE₂ response curves was higher than that of cell killing curves in cultured HLE cells. These data show that cultured HLE cells with extended lifespan are a suitable system for investigating photobiological responses of cells to UV radiation.     

Mycosporine-like amino acids in the marine dinoflagellate Gyrodinium dorsum: induction by ultraviolet irradiation

Cultures of the marine dinoflagellate Gyrodinium dorsum have been exposed to polychromatic radiation (photosynthetically active radiation and UV) from a solar simulator for up to 72 h. Different irradiance spectra in the ultraviolet are produced by inserting cut-off filters between lamp and samples. The mycosporine-like amino acid (MAA) content and composition are investigated by spectroscopic and chromatographic analysis. The study reveals that G. dorsum contains a complex mixture of several aminocyclohexenimine-MAAs and one aminocyclohexenone-MAA. UV irradiation around 320 nm induces an increase in the concentration of all MAAs in the samples. In contrast, exposure to short-wavelength UV-B radiation results in decreased overall MAA production. Furthermore, there is a spectral shift in the absorption of the MAA mixture towards shorter wavelengths, indicating that short-wavelength UV-B induces an altered MAA composition. The amount of MAAs is normalized to the chlorophyll a concentration.     

Photosensitizing properties of biopterin and its photoproducts using 2'-deoxyguanosine 5'-monophosphate as an oxidizable target

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through photosensitized reactions. Biopterin (Bip) and its photoproducts 6-formylpterin (Fop) and 6-carboxypterin (Cap) accumulate in the skin of human beings suffering from vitiligo, a depigmentation disorder where the protection against UV radiation fails because of the lack of melanin. This study was aimed to evaluate the photosensitizing properties of oxidized pterins present in the skin and to elucidate the mechanisms involved in the photosensitized oxidation of purine nucleotides by pterins in vitro. For this purpose, steady-state and time-resolved experiments in acidic (pH 5.0-5.8) aqueous solution were performed using Bip, Fop and Cap as photosensitizers and the nucleotide 2'-deoxyguanosine 5'-monophosphate (dGMP) as an oxidizable target. The three pterin derivatives are able to photosensitize dGMP, being Fop the most efficient sensitizer. The reactions proceed through two competing pathways: (1) electron transfer from dGMP to triplet excited-state of pterins (type I mechanism) and (2) reaction of dGMP with (1)O(2) produced by pterins (type II mechanism). Kinetic analysis revealed that the electron transfer pathway is the main mechanism and the interaction of dGMP with the triplet excited-state of pterins and the formation of the corresponding dGMP radicals were demonstrated by laser flash photolysis experiments. The biological implications of the results obtained are also discussed.     

ACTION SPECTRA FOR UV INDUCTION AND PHOTOREVERSAL OF A SWITCH IN THE DEVELOPMENTAL PROGRAM OF THE EGG OF AN INSECT (SMITTIA)*

Abstract— An action spectrum was established for the induction by ultraviolet (UV) radiation of the aberrant body segment pattern 'double abdomen' in the egg of the Chironomid midge Smittia. The action spectrum shows a peak at 285 nm wavelength, a shoulder at 265 nm, a slight increase from 245 to 240 nm, and a steep decline towards 300 nm. Corrections for wavelength-dependent shielding within the egg result in transformation of the shoulder at 265 nm into a minor peak. These results are compatible with the assumption that a nucleic acid-protein complex is involved in the initial photoreaction. This assumption is supported by the fact that the UV induction of the aberrant body segment pattern 'double abdomen' in the egg of Smittia is photoreversible. Wavelengths effective in this photoreversal range from 310 to 460 nm, with a peak at 440 nm. These spectral characteristics agree with action spectra for photoreactivation in other systems. Indirect photoreactivation does not occur in the egg of Smittia under the conditions of the experiments. Photoreversal with 440 nm radiation at high dose rate is temperature-dependent. The results support the assumption that the molecular basis for photoreversal in the egg of Smittia is similar to the predominant molecular mechanism of direct photoreactivation in other systems. Targets possibly involved in the UV induction of the 'double abdomen' are discussed.     

ACTION SPECTRA FOR THE trans TO cis PHOTOISOMERISATION OF UROCANIC ACID in vitro and IN MOUSE SKIN

Urocanic acid (UCA) is a major UV chromophore in the upper layers of the skin where it is found predominantly as the trans isomer. UV irradiation induces photoisomerisation of trans-UCA to cis-UCA which has been shown to mimic some of the immunosuppressive properties of UV exposure. We examined the wavelength dependence for trans-UCA to cis-UCA photoisomerisation in vitro and in mouse skin in vivo over the spectral range270–340 nm. The resulting action spectra were very similar with maximal effectiveness at300–315 nm and equal activity at 270 nm and325–330 nm, demonstrating that UVA-II radiation (320–340nm) is efficient at UCA photoisomerisation. These action spectra differed markedly from the trans-UCA absorption spectrum in vitro and also the reported action spectrum for UV suppression of contact hypersensitivity in mice. These findings suggest that the relationship between cis-UCA formation in skin and UV-induced immunosuppression may be complex.     

An estimation of biological hazards due to solar radiation

A spectrum evaluator based on four different dosimeter materials has been employed to estimate the spectral irradiances of solar radiation for exposed humans. The result is used to calculate the biologically effective irradiance using the erythemal action spectrum and a fish melanoma action spectrum. Measurements are made in winter at a sub-tropical site on the chest and shoulder of subjects during normal daily activities. Up to 95% of the total UV exposure received is in the UV-A waveband (320-400 nm). The UV-A waveband is found to contribute approximately 14% of the erythemal UV and 93% of the biologically effective UV for fish melanoma. Extrapolation to humans suggests that exposure to the UV-A band will contribute to photodamage in human skin during exposure to solar radiation.     

SINGLE-STRAND BREAKS INDUCED IN BACILLUS SUBTILIS DNA BY ULTRAVIOLET LIGHT: ACTION SPECTRUM and PROPERTIES

Abstract— The induction of single-strand breaks (alkali-labile bonds plus frank breaks) in the DNA of Bacillus subtilis irradiated in vivo by monochromatic UV light at wavelengths from 254 to 434 nm was measured. The spectrum consists of a major far-UV (below 320 nm) component and a minor near-UV shoulder. A mutant deficient in DNA polymerase I accumulates breaks caused by near-UV (above 320 nm) wavelengths faster than the wild-type strain proficient in polymerase I. Measurable breaks in extracted DNA are induced at a higher frequency than those induced in vivo. Anoxia, glycerol, and diazobicyclo (2.2.2.) octane inhibit break formation in extracted DNA. Alkali-labile bonds induced by 365-nm UV radiation are largely (78%) covalent bond chain breaks, the remainder consists of true alkali-labile bonds, probably apurinic and apyrimidinic sites.     

A NOVEL EFFECT OF UV-B IN A HIGHER PLANT (SORGHUM VULGARE)

Abstract— Two non-photosynthetic photoreceptors (phytochrome and the usual blue/UV light photoreceptor) were previously found to be involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings (Drumm and Mohr, 1978). The decisive point is that phytochrome can act only after a blue/UV light effect has occurred. On the other hand, the expression of the blue/UV light effect is controlled by phytochrome ('obligatory sequential action'). A strong positive interaction between the blue/UV-A and the UV-B part of the spectrum was found, in addition to the above sequential action: an inductive effect of blue or UV-A light can only express itself fully if short wavelength UV (approximately 300–320nm. UV-B range) is also given, either after the blue/UV-A light or simultaneously. Since even small amounts of the UV-B are strongly effective it is probable that this effect plays a role under natural conditions and may not be considered as a mere laboratory artifact.     

Ultraviolet wavelength dependence of photomorphological and photosynthetic responses in Brassica napus and Arabidopsis thaliana

Among the photomorphological responses in plants induced by ultraviolet-B radiation (UVB; 290 nm-320 nm) are leaf asymmetry, leaf thickening and cotyledon curling. We constructed an action spectrum of cotyledon curling in light-grown Brassica napus to characterize the UVB photoreceptor that initiates this response. Cotyledon curling was also characterized in Arabidopsis thaliana. Peak efficiency for this response occurred between 285 and 290 nm. Additionally, UVB-induced changes in epidermal cells from A. thaliana cotyledons were assessed because they are the likely site of UVB photoreception that leads to curling. Investigation of cellular structure, chlorophyll a fluorescence and chlorophyll concentration indicated that cotyledon curling is not concomitant with gross cellular damage or inhibition of photosynthesis, which only occurred in response to wavelengths <280 nm. Many UVB effects are apparently an indirect consequence of UVB radiation, dependent on UVB-mediated increases in reactive oxygen species (ROS) that either act as a signal in the UVB transduction pathway or cause oxidative damage. The cotyledon curling response was impeded by ascorbate and cystine, ROS scavengers and was promoted by H(2)O(2), a ROS. We suggest that following absorption by a UVB chromophore, ROS are generated via photosensitization, ultimately leading to cotyledon curling.     

ULTRAVIOLET RADIATION IN ANTARCTICA: INHIBITION OF PRIMARY PRODUCTION

With the seasonal formation of the ozone hole over Antarctica, there is much concern regarding the effects of increased solar UV-B radiation (280–320 nm) on the marine ecosystem in the Southern Ocean. In situ incubations of natural phytoplankton assemblages in antarctic waters indicate that under normal ozone conditions UV-B radiation is responsible for a loss of approximately 4.9% of primary production in the euphotic zone, whereas UV radiation with wavelengths between 320 and 360 nm causes a loss of approximately 6.2%. When combined with data on the action spectrum for photoinhibition by UV radiation, our data suggest that the enhanced fluence of UV-B radiation under a well-developed ozone hole (150 Dobson units) would decrease daily primary productivity by an additional amount of 3.8%. Calculations that take into consideration the extent and duration of low stratospheric ozone concentrations during September to November indicate that the decrease in total annual primary production in antarctic waters due to enhanced UV-B radiation would be 0.20%.     

Outdoor ultraviolet polychromatic action spectra for growth responses of Bellis perennis and Cynosurus cristatus

Polychromatic ultraviolet (UV) action spectra for various growth responses of the dicotyledon Bellis perennis L. (daisy) and the grass Cynosurus cristatus L. (crested dog's-tail) have been measured. The plants were grown in the natural environment and ambient daylight was supplemented with five different UV irradiances centred at eight different wavelengths (313, 318, 320, 322, 339, 348, 356 and 377 nm). Destructive growth analysis was performed on B. perennis and C. cristatus after 300 and 122 days respectively. Dose response curves were created to construct action spectra for individual responses. Different spectral responses were observed in these two plant types. B. perennis exhibited a substantial action maximum at 313 nm for the inhibition of aerial, root and total dry weight; a similar action maximum at 313 nm for the inhibition of leaf expansion was observed. Longer wavelengths were relatively ineffective on these growth parameters, with the exception of a small but statistically significant (P < 0.05) response to 320 nm radiation. By contrast, C. cristatus showed negligible response to 313 nm radiation, for inhibition of aerial, root and total dry weight but substantial responses to longer wavelengths, especially at 339 and 348 nm. These action spectra add weight to suggestions in the literature that UV-A has a role to play in responses in this region of the spectrum. The possible implications of these observations are discussed.