Action spectrum for photoperiodic control of thyroid-stimulating hormone in Japanese quail (Coturnix japonica).

At higher latitudes, vertebrates exhibit a seasonal cycle of reproduction in response to changes in day-length, referred to as photoperiodism. Extended day-length induces thyroid-stimulating hormone in the pars tuberalis of the pituitary gland. This hormone triggers the local activation of thyroid hormone in the mediobasal hypothalamus and eventually induces gonadal development. In avian species, light information associated with day-length is detected through photoreceptors located in deep-brain regions. Within these regions, the expressions of multiple photoreceptive molecules, opsins, have been observed. However, even though the Japanese quail is an excellent model for photoperiodism because of its robust and significant seasonal responses in reproduction, a comprehensive understanding of photoreceptors in the quail brain remains undeveloped. In this study, we initially analyzed an action spectrum using photoperiodically induced expression of the beta subunit genes of thyroid-stimulating hormone in quail. Among seven wavelengths examined, we detected maximum sensitivity of the action spectrum at 500 nm. The low value for goodness of fit in the alignment with a template of retinal1-based photopigment, assuming a spectrum associated with a single opsin, proposed the possible involvement of multiple opsins rather than a single opsin. Analysis of gene expression in the septal region and hypothalamus, regions hypothesized to be photosensitive in quail, revealed mRNA expression of a mammal-like melanopsin in the infundibular nucleus within the mediobasal hypothalamus. However, no significant diurnal changes were observed for genes in the infundibular nucleus. Xenopus-like melanopsin, a further isoform of melanopsin in birds, was detected in neither the septal region nor the infundibular nucleus. These results suggest that the mammal-like melanopsin expressed in the infundibular nucleus within the mediobasal hypothalamus could be candidate deep-brain photoreceptive molecule in Japanese quail. Investigation of the functional involvement of mammal-like melanopsin-expressing cells in photoperiodism will be required for further conclusions.

Somatic cell mutations caused by 365 nm LED-UVA due to DNA double-strand breaks through oxidative damage.

Evidence is accumulating indicating that UVA (320-400 nm ultraviolet light) plays an important role in photo-carcinogenesis. UVA is thought to produce reactive oxygen species in irradiated cells through photo-activation of inherent photosensitizers, and was recently reported to cause DNA double-strand breaks (DSBs) in exposed cells. We have investigated the involvement of UVA in mutations and DNA damage in somatic cells using Drosophila melanogaster larvae. Using the Okazaki Large Spectrograph, we previously observed that longer wavelength UVA (>330 nm) was more mutagenic in post-replication repair-deficient D. melanogaster (mei-41) than in the nucleotide excision repair-deficient strain (mei-9). LED-light has recently been developed as a high-dose-rate UVA source. LED-UVA light (365 nm) was also more mutagenic in mei-41 than in mei-9. The mei-41 gene was shown to be an orthologue of the human ATR gene, which is involved in the repair of DSBs through phosphorylation of histone H2AX. In order to estimate the extent to which oxidative damage contributes to mutation, we established a new D. melanogaster strain (urate-null mutant) that is sensitive to oxidative damage and has a marker to detect somatic cell mutations. When somatic cell mutations were examined using this strain, LED-UVA was mutagenic in the urate-null strain at doses that were non-mutagenic in the urate-positive strain. In an effort to investigate the generation of DSBs, we examined the presence of phosphorylated histone H2AvD (H2AX D. melanogaster homologue). At high doses of LED-UVA (>800 kJ m(-2)), levels of phosphorylated H2AvD (γ-H2AvD) increased significantly in the urate-null strain. Moreover, the level of γ-H2AvD increased in the excision repair-deficient strain but not in the ATR-deficient strain following UVA-irradiation. These results supported the notion that the generation of γ-H2AvD was mediated by the function of the mei-41 gene. It was reported that ATR functions on DSB repair in D. melanogaster. Taken together, we propose a possible pathway for UVA-induced mutation, whereby DNA double-strand breaks resulting from oxidative stress might be responsible for UVA-induced mutation in somatic cells of D. melanogaster larvae.

Novel photosensory two-component system (PixA-NixB-NixC) involved in the regulation of positive and negative phototaxis of cyanobacterium Synechocystis sp. PCC 6803.

Two wild-type substrains of a motile cyanobacterium Synechocystis sp. PCC 6803 show positive phototaxis toward a light source (PCC-P) and negative phototaxis away from light (PCC-N). In this study, we found that a novel two-component system of photoresponse is involved in the phototactic regulation. Inactivation of slr1212 (pixA), which encodes a photoreceptor histidine kinase, reverted the positive phototaxis of PCC-P to negative phototaxis, and inactivation of the downstream slr1213 (nixB) and slr1214 (nixC), which encode AraC-like transcription factor-type and PatA-type response regulators, respectively, reverted the negative phototaxis of PCC-N to positive phototaxis. Opposite effects of pixA and nixBC disruption implies an unexpected signal transduction pathway in the switching of positive and negative phototaxis. The blue/green-type cyanobacteriochrome GAF domain of PixA was expressed in Synechocystis and phycocyanobilin-producing Escherichia coli. The holoprotein covalently bound a chromophore phycoviolobilin and showed reversible photoconversion between the violet- (Pv, λ(peak) = 396 nm) and green-absorbing (Pg, λ(peak) = 533 nm) forms, although the protein from E. coli partially bound a precursor phycocyanobilin. These results were discussed with regard to an idea that PixA serves as a violet light receptor for switching of positive and negative phototaxis by transcriptional and functional regulation.

Blue and red light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus.

Photophysiological and pharmacological approaches were used to examine light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus. The equal-quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light-induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca(2+) influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein-tyrosine-phosphatase, protein kinase C and actin polymerization and translation.

Distinct responses of chloroplasts to blue and green laser microbeam irradiations in the centric diatom Pleurosira laevis.

The centric diatom Pleurosira laevis is a large unicellular alga, in which ca 200 chloroplasts migrate toward the nuclear cytoplasm through the transvacuolar cytoplasmic strands in response to blue-light irradiation and, on the contrary, toward the cortical cytoplasm in response to green-light irradiation. We analyzed these light-induced chloroplast migrations using a scanning laser microbeam provided by a confocal microscope for intracellular irradiation. Spot irradiation of a blue laser microbeam induced rapid assemblage of chroloplasts into the nuclear cytoplasm regardless of the spot position and spot number. On the other hand, one or two spots of green laser microbeam induced chloroplast accumulation at the spots, although increasing spot numbers suppressed chloroplast accumulation at each spot. In our experimental condition, ca 1 min of blue-light irradiation was sufficient to stimulate movement, whereas green-light irradiation required uninterrupted and longer irradiation time (ca 15 min). Chloroplast assemblage induced by blue-light required extracellular Ca2+, and was inhibited by Ca2+ channel antagonists. Furthermore, higher efficiencies of chloroplast migration were obtained when a single beam spot was fragmented and scattered over wider area of plasma membrane. These observations suggested that blue-light induced a response at the plasma membrane, which subsequently activated Ca2+ permeable channels. This sequence of physiological events is identical to what was previously observed with chloroplast movement in response to mechanical stimulation. Furthermore, experiments with the cytoskeleton-disrupting agents, colchicine and cytochalasin D, indicated that blue-light-induced chloroplast movement required microtubules whereas the green-light-induced response to beam spot required actin filaments.

Action spectrum for expression of the high intensity light-inducible Lhc-like gene Lhl4 in the green alga Chlamydomonas reinhardtii.

Lhl4 encodes a distant relative of light-harvesting Chl-a/b proteins in the green alga Chlamydomonas reinhardtii. Lhl4 mRNA markedly accumulated within 30 min after illumination and in proportion to the light intensity up to a fluence rate much higher than that required for photosynthesis. The high intensity light (HL)-induced accumulation of Lhl4 mRNA required continuous illumination, and the mRNA level rapidly decreased when the cells were placed in the dark. HL only slightly stabilized the mRNA, suggesting that the HL-induced expression of the Lhl4 gene is primarily regulated at the level of transcription. Blue light was more effective for inducing Lhl4 gene expression than green or red light, and far-red light had no effect. The action spectrum for Lhl4 gene expression was examined at wavelengths between 325 and 775 nm using the Okazaki Large Spectrograph. The obtained spectrum showed a distinct peak in the blue region (450 nm) and a shoulder in the UV-A region (375 nm). The curve in the spectrum rose steeply in the short wavelength UV region. In addition, we observed two minor peaks in the green (575 nm) and the red (675 nm) regions. The action spectrum suggests that a blue/UV-A light photoreceptor with a flavin-based chromophore participates in the HL response of Lhl4 gene expression. However, the hypersensitivity to near UV-B light suggests the involvement of an unidentified UV light perception system in the expression of the Lhl4 gene.

Action spectra of apoptosis induction and reproductive cell death in L5178Y cells in the UV-B region.

We investigated the action spectra for the induction of apoptosis and reproductive cell death in mouse lymphoma L5178Y cells using a high-performance spectroirradiator, the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki. L5178Y cells were exposed to monochromatic light at different wavelengths in the UV-B and UV-A regions. The frequencies of apoptosis induction and reproductive cell death were determined by counting cells with chromatin condensation and by a semi-solidified agarose colony formation assay, respectively. The measured action spectra for the two end-points were similar. The sensitivity decreased steeply with an increase of wavelength in the UV-B region, but showed no further decrement in the UV-A region. The action spectra were slightly steeper than that for the minimum erythematic dose (MED), and were similar to the light-absorption spectrum of DNA in the UV-B region. On the other hand, in the UV-A region, the spectra for both endpoints were close to the MED, but not to DNA absorption spectra. The difference between the measured spectra and that for MED may have been caused by the absorption of the light by the skin. Differences in the time course and morphological difference of apoptosis were found between the UV-B and UV-A region. These results suggest that although DNA damage induced by UV-B light can trigger apoptosis, or lead to reproductive cell death, other damage (membrane, protein and so on) may trigger those effects in the UV-A region.