[Show abstract][Hide abstract] ABSTRACT: Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.
[Show abstract][Hide abstract] ABSTRACT: Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.
[Show abstract][Hide abstract] ABSTRACT: Our previous studies of action spectra for UV-B-induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV-B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV-B light sensing. UV-B-induced phenylalanine ammonia-lyase (PAL) activity was considerably suppressed by N-acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N-acetylserotonin-treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV-B-induced PAL activity. These results suggest the occurrence of an unidentified UV-B photoreceptor (other than UVR8, the tryptophan-based UV-B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After re-examining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV-B sensors. This article is protected by copyright. All rights reserved.
No preview · Article · Jun 2014 · Photochemistry and Photobiology
[Show abstract][Hide abstract] ABSTRACT: Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.
No preview · Article · Nov 2013 · The Journal of General and Applied Microbiology
[Show abstract][Hide abstract] ABSTRACT: We examined the effects of photoperiod, wavelength and light fluence rate on diurnal vertical migration (DVM) cycle in a coastal
raphidophyte, Chattonella antiqua. We first observed the DVM under different combinations of light–dark (LD) cycles and light spectra. Under continuous white,
UV-A or blue light, DVM followed the LD cycle established during the white light pre-conditioning, for one cycle, and then
became arrhythmic. Under red light, however, the DVM rhythms under the different LD regimes continued approximately as during
pre-conditioning. When C. antiqua cultured under continuous red light was exposed to a 2-h pulse of blue light at the beginning or end of artificial ‘night’,
the DVM was delayed or advanced, respectively. The fluence rate–response curve indicates a blue-light threshold of 10–2 μmol photons m–2 s–1 for the DVM phase shift. The equal-quantum action spectra for phase shift peaked in the UV-A/blue region (360–480 nm), which
is the part of the light spectrum most transmitted in its natural habitat. We show that C. antiqua can sense the weak blue component of sunlight throughout its depth range, allowing it to cue its DVM to the day–night cycle
regardless of weather and transparency.
Full-text · Article · May 2013 · Journal of Plankton Research
[Show abstract][Hide abstract] ABSTRACT: UVR causes erythema, which has been used as a standardized index to evaluate the risk of UVR for human skin. However, the genotoxic significance of erythema has not been elucidated clearly. Here, we characterized the wavelength dependence of the genotoxic and erythematic effects of UVR for the skin by analyzing the induction kinetics of mutation and inflammation in mouse skin using lacZ-transgenic mice and monochromatic UVR sources. We determined their action spectra and found a close correlation between erythema and an epidermis-specific antigenotoxic response, mutation induction suppression (MIS), which suppressed the mutant frequencies (MFs) to a constant plateau level only 2-3-fold higher than the background MF at the cost of apoptotic cell death, suggesting that erythema may represent the threshold beyond which the antigenotoxic but tissue-destructive MIS response commences. However, we unexpectedly found that MIS attenuates remarkably at the border wavelengths between UVA and UVB around 315 nm, elevating the MF plateaus up to levels ∼40-fold higher than the background level. Thus, these border wavelengths can bring heavier mutation loads to the skin than the otherwise more mutagenic and erythematic shorter wavelengths, suggesting that erythema-based UVR risk evaluation should be reconsidered.Journal of Investigative Dermatology advance online publication, 14 February 2013; doi:10.1038/jid.2012.504.
Preview · Article · Feb 2013 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: A cyaA-deficient Escherichia coli strain was transformed by a plasmid carrying the gene for BsPAC, a photoactivated adenylyl cyclase identified from a Beggiatoa sp., and was subjected to an antibiotic susceptibility assay and biofilm formation assay under a light or dark condition. Cells expressing BsPAC that were incubated under blue light (470 nm) were more susceptible to fosfomycin, nalidixic acid and streptomycin than were cells incubated in the dark. Cells expressing BsPAC formed more biofilms when incubated under the light than did cells cultured in the dark. We concluded from these observations that it is possible to determine the importance of cAMP in antibiotic susceptibility and biofilm formation of E. coli by photomanipulating the cellular cAMP level by the use of BsPAC. A site-directed mutant of BsPAC in which Tyr7 was replaced by Phe functioned even in the dark, indicating that Tyr7 plays an important role in photoactivation of BsPAC. Results of mutational analysis of BsPAC should contribute to an understanding of the molecular basis for photoactivation of the protein.
No preview · Article · Aug 2012 · The Journal of General and Applied Microbiology
[Show abstract][Hide abstract] ABSTRACT: 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, λpeak = 396 nm) and green-absorbing (Pg, λpeak = 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.
Preview · Article · Nov 2011 · Plant and Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: In the photoreaction of BLUF (sensor of blue light using FAD) domains, conserved Tyr/Gln nearby the FAD chromophore plays a crucial role in the formation of a red-shifted intermediate. The intermediate appears accompanying a hydrogen-bonding alteration of the Tyr/Gln. In order to reveal the hydrogen-bonding structure of the Tyr/Gln active center and FAD, FTIR spectroscopy was applied in the 4000–1800 cm–1 region, where X–H stretches appear. The present FTIR data show an unusually low O–H stretching vibration of tyrosine at 2800–2600 cm–1. This provides direct evidence of the light-induced switch of the hydrogen-bonding network, where the conserved Tyr (Tyr21 for AppA) donates an unusually strong hydrogen bond in the intermediate.Keywords: FAD; BLUF; FTIR; tyrosine; hydrogen bond
No preview · Article · Apr 2011 · Journal of Physical Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Feb 2011 · Photochemistry and Photobiology
[Show abstract][Hide abstract] ABSTRACT: The visible fluorescence spectrum of single flavoprotein at a temperature of 1.5 K has been measured by one-photon excitation. The flavoprotein studied was a photoswitchable enzyme, photoactivated adenylyl cyclase. The time course of the spectrum revealed a structural change occurring at a rate of 10(-3) s(-1) around hydrogen bonds at the flavin cofactor binding site.
No preview · Article · Feb 2011 · Physical Review Letters
[Show abstract][Hide abstract] ABSTRACT: Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PACα and PACβ), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PACαF2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PACβF2 domain, which like PACαF2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PACβF2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PACαF2 were 0.28-0.32 and 34-44 s. The remarkable differences between PACαF2 and PACβF2 may be related to the sensitivity of the photoactivation. In PACαF2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PACβF2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PACβF2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PACβF2 plays a main role in suppressing the quantum efficiency.
No preview · Article · Oct 2010 · Photochemical and Photobiological Sciences
[Show abstract][Hide abstract] ABSTRACT: Vibrational infrared absorption of a single protein molecule was detected at a few kelvins as infrared-induced recovery of visible fluorescence of a dye with which the protein was labeled. This sensitive method of detecting infrared absorption was demonstrated for a single bovine serum albumin (BSA) molecule labeled with Alexa Fluor 660 by determining the vibrational infrared absorption spectrum of the backbone vibrations of the α-helical structure in the wavelength region around 6 μm (1650 cm−1). In addition to measuring the vibrational infrared absorption spectrum, the visible fluorescence can be simultaneously used for imaging of the same dye-labeled single protein molecules.
No preview · Article · Sep 2010 · Journal of Physical Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: Petalomonas sphagnophila is a poorly studied plastid-lacking euglenid flagellate living in Sphagnum-dominated peatlands. Here we present a broad-ranging microscopic, molecular and microspectrophotometric analysis of uncultured P. sphagnophila collected from four field locations in Nova Scotia, Canada. Consistent with its morphological characteristics, 18S ribosomal DNA (rDNA) phylogenies indicate that P. sphagnophila is specifically related to Petalomonas cantuscygni, the only other Petalomonas species sequenced to date. One of the peculiar characteristics of P. sphagnophila is the presence of several green-pigmented particles approximately 5 mum in diameter in its cytoplasm, which a previously published study suggested to be cyanobacterial endosymbionts. New data presented here, however, suggest that the green intracellular body may not be a cyanobacterium but rather an uncharacterized prokaryote yet to be identified by molecular sequencing. 16S rDNA library sequencing and fluorescence in situ hybridizations show that P. sphagnophila also harbors several other endobionts, including bacteria that represent five novel genus-level groups (one firmicute and four different proteobacteria). 16S rDNA phylogenies suggest that three of these endobionts are related to obligate intracellular bacteria such as Rickettsiales and Coxiella, while the others are related to the Daphnia pathogen Spirobacillus cienkowskii or belong to the Thermoactinomycetaceae. TEM, 16S rDNA library sequencing and a battery of PCR experiments show that the presence of the five P. sphagnophila endobionts varies markedly among the four geographic collections and even among individuals collected from the same location but at different time points. Our study adds significantly to the growing evidence for complex and dynamic protist-bacterial associations in nature.
[Show abstract][Hide abstract] ABSTRACT: Using infrared high-speed video microscopy, we observed light-triggered transitory flagellar motions in flagellate reproductive cells (swarmers) of a brown alga, Scytosiphon lomentaria, under primary helical swimming conditions before and during negative phototactic orientation to unilateral actinic light. The posterior flagellum, which is autofluorescent and thought to be light-sensing, was passively dragged in the dark and exhibited one to several rapid lateral beats during orientation changes for phototactic steering. Notably, a brief cessation of anterior flagellar beating was occasionally observed concomitantly with rapid beats of the posterior flagellum. This behavior caused a pause in helical body rotation, which may contribute to the accuracy of phototactic steering. Thus, coordinated regulation of the movement of the two flagella plays a crucial role in phototactic steering.
No preview · Article · Dec 2009 · Photochemistry and Photobiology
[Show abstract][Hide abstract] ABSTRACT: Strong fluorescence emission occurred in sea urchin larvae when irradiated with blue light under a fluorescence microscope. The blue light irradiation first broke red granules in the pigment cells, releasing green fluorescent substance(s) into the cytoplasm of the pigment cells. The released and dispersed fluorescent substance(s) then made the entire pigment cell emit strong green fluorescence. With prolonged blue light irradiation, the pigment cell itself bursted, dispersing the fluorescent substance(s) into the body cavity or seawater. This resulted in “explosive emission” of the fluorescence. Cells that can emit such fluorescence first appeared at the late-blastula stage, proliferated with development, and changed into the red-pigmented cells. Similar fluorescence emission was observed in the larvae of Clypeaster japonicus, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus, of which C. japonicus larvae displayed the strongest fluorescence.
No preview · Article · Aug 2009 · ZOOLOGICAL SCIENCE
[Show abstract][Hide abstract] ABSTRACT: Apoptosis plays an important role in eliminating cells from populations when cells have been exposed to UV irradiation and damaged. Studies of cells in culture have provided some details of the mechanisms involved when stress response genes act after exposure to UV irradiation and other environmental stresses. However, little is known about the responses of intact sections of human skin growing in organ culture to UV irradiation. In the work reported here, it was found that the response of organ-cultured human skin after exposure to UV irradiation is different than the response of cultured cells. At wavelengths below 300 nm, the action spectrum obtained from organ-cultured skin samples showed a lower sensitivity than that observed at 300 nm, indicating that the overlying stratum corneum and upper epidermal cell layers had probably caused a selective absorption of incident UV radiation at some wavelengths. At 3 hours after UV irradiation, p53 was phosphorylated at Ser 15 and Ser 46, and accumulated in the cell nuclei, notably after exposure to 280-320 nm wavelengths. Accumulations of Bax, active Caspase-3 and cleaved PARP were detected in apoptotic cells at 24 hours post-exposure, along with a reduction of Bcl-2 levels, notably after exposure to 300-365 nm light. This difference in apoptotic responses may result from the characteristics of the different irradiation wavelengths used, and from details in the skin's structure. The data obtained in this study using an organ-culture system utilized direct measurements of the biological effects of different wavelengths of UV lights.
Full-text · Article · Jun 2008 · Journal of Radiation Research
[Show abstract][Hide abstract] ABSTRACT: UVA1 induces the formation of 8-hydroxy-2'-deoxyguanosines (8-OH-dGs) and cyclobutane pyrimidine dimers (CPDs) in the cellular genome. However, the relative contribution of each type of damage to the in vivo genotoxicity of UVA1 has not been clarified. We irradiated living mouse skin with 364-nm UVA1 laser light and analyzed the DNA damage formation and mutation induction in the epidermis and dermis. Although dose-dependent increases were observed for both 8-OH-dG and CPD, the mutation induction in the skin was found to result specifically from the CPD formation, based on the induced mutation spectra in the skin genome: the dominance of C --> T transition at a dipyrimidine site. Moreover, these UV-specific mutations occurred preferentially at the 5'-TCG-3' sequence, suggesting that CpG methylation and photosensitization-mediated triplet energy transfer to thymine contribute to the CPD-mediated UVA1 genotoxicity. Thus, it is the CPD formation, not the oxidative stress, that effectively brings about the genotoxicity in normal skin after UVA1 exposure. We also found differences in the responses to the UVA1 genotoxicity between the epidermis and the dermis: the mutation induction after UVA1 irradiation was suppressed in the dermis at all levels of irradiance examined, whereas it leveled off from a certain high irradiance in the epidermis.
Full-text · Article · Mar 2008 · Journal of Investigative Dermatology