Hula-twist is a volume-conserving photoisomerization reaction mechanism postulated in 1985 to account for the rapid photoisomerization of the retinyl chromophore in rhodopsin. The requisite stereochemical consequence of simultaneous isomerization of a double bond and an adjacent single bond has recently been demonstrated in isomerization of pre-vitamin D in an organic glass and by many other examples of organic systems already reported in the literature This paper reports the consequence in applying the mechanism to the primary photochemical process of several photosensitive biopigments: bilirubin, photoactive yellow protein, bacteriorhodopsin and rhodopsin. It is shown that the anchored nature of the chromophores must first be taken into consideration.

Photoperiodism is an important adaptive phenomenon in various physiological parameters including reproduction to cope with seasonal changes. Involvement of extraretinal photoreceptors in the photoperiodism in non-mammalian vertebrates has been well established. In addition, circadian clock system is known to be involved in the photoperiodic time measurement. The pathway consists of light-input system, time measurement system (circadian clock), gonadotropin releasing hormone (GnRH) production in the hypothalamus, luteinizing hormone (LH) and follicle stimulating hormone (FSH) production in the pituitary, and final gonadal development. Recently, several laboratories reported photopigments newly cloned in the pineal, eyes and deep brain in addition to already known visual pigments in the retina. These are pinopsin, parapinopsin, VA-opsin, melanopsin, etc. All these photopigments belong to the opsin family having retinal as the chromophore. However, the function of these photopigments remains unknown. I reviewed the studies on the location of the photopigments by immunocytochemistry. I also discussed the results on the action spectra for induction of gonadal development in relation with the location of the photoreceptors. Various physiologically active substances distribute in the vertebrate brain. Such substances are GnRH, GnIH, neuropeptide Y, vasoactive intestinal peptide, c-Fos, galanin, neurosteroids, etc. I summarized the immunhistochemical studies on the distribution and the photoperiodic changes of these substances and discussed the route from the deep brain photoreceptor to GnRH cells.

Circadian rhythms can be seen in a variety of physiological functions in insects. Light is a powerful zeitgeber not only synchronizing but also modulating the rhythm to adjust insect's temporal structure to seasonal changes in the environmental cycle. There are two general effects of the length of light phase within 24 hr light cycles on the circadian rhythms, i.e., the modulation of free-running period and the waveform. Since the photoperiodic modulation of the free-running period is induced even in the clock mutant flies, per$\^$s/, the free-running period is not fully determined genetically. In crickets, the ratio of activity (a) and rest phase (p) under the constant darkness (DD) is clearly dependent on the photoperiod under which they have been kept. When experienced the longer photoperiod it becomes smaller. The magnitude of change in a/p-ratio is dependent on the number of cycles they experienced. The neuronal activity of the optic lobe in DD shows the a/p-ratio changing with the preceding photoperiod. These data suggest that a single circadian pacemaker stores and maintains the photoperiodic information and that there is a system that accumulates the effects of single photoperiod to cause greater effects.

A comparison of the functional components underlying photoperiodism and circadian rhythmicity in the same species is an interesting issue in the context of unravelling clock mechanisms. In the present study, covering or surgical removal of the compound eyes was performed to localize photoreceptors for photoperiodism to control reproductive diapause and for entrainment of circadian locomotor rhythms in the blow fly Protophormia terraenovae. Intact flies showed a long-day photoperiodic response. When the compound eyes were covered by silver paint, diapause incidence increased under diapause-averting conditions of a long-day photoperiod and constant light, as if flies were kept under constant darkness. Covering of a medial region of the head capsule or solvent painting of the compound eyes gave no significant effects. When the compound eyes were removed, flies did not distinguish the photoperiod, whereas removal of antennal lobes or ocelli did not affect the photoperiodism. Intact flies showed a freerunning rhythm under constant darkness. The rhythm entrained to light-dark (LD) cycles with light of high and low intensity. When the compound eyes and ocelli were surgically removed, the rhythm entrained to LD cycles with light of high intensity but freeran under LD cycles with light of low intensity. The results suggest the retinal pathways are involved in photoperiodism and that flies use both retinal and extraretinal pathways for rhythm entrainment. Under dim light-LD cycles, the retinal pathways mainly mediate rhythm entrainment. Retinal photoreceptors seem to be used both for photoperiodism and entrainment of the rhythm.

We have cloned a cDNA for an opsin (Boceropsin) from the silkworm larval brain which was suggested to contain the photoperiodic receptor. Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins, and are presumed to be photoperiodic receptor. RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain, but not in the subesophageal (Sg) and thoracic ganglion. Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of the Bombyx larva. Boceropsin was considered not to be involved in the circadian photoreception, because carotenoids are not indispensable for the photoreception and formation of circadian rhythms in the silkworm.

Pineal organs of poikilotherm vertebrates transform the environmental light information into a humoral message and a neuronal activity. The former is melatonin, and the latter is modulation of the impulse in ganglion cells. The ganglion cells are physiologically classified into luminosity (achromatic) type and chromatic one, as the neural activity is modulated in two ways. We attempted to classify the pineal ganglion cells with morphological characteristics by means of the three- dimensional reconstruction method. In the pineal ganglion cells of river lamprey, there are two different features, oval and spherical. For comparison of their projection region in the brain, the tracing investigation was also carried out. The application of the neural tracer near mesencephalic tegmentum showed that only oval-shaped ganglion cells were labeled in the pineal organ. These results suggest that the oval-shaped ganglion cell is functionally different from the spherical one.

The chicken pineal gland has been used for studies on the circadian clock, because it retains an intracellular phototransduction pathway regulating the phase of the intrinsic clock oscillator. Previously, we identified chicken clock genes expressed in the gland (cPer2, cPer3, cBmal1, cBmal2, cCry1, cCry2, and cClock), and showed that a cBMALl/2-cCLOCK heteromer acts as a regulator transactivating cPer2 gene through the CACGTG E-box element found in its promoter. Notably, mRNA expression of cPer2 gene is up-regulated by light as well as is driven by the circadian clock, implying that light-dependent clock resetting may involve the up-regulation of cPer2 gene. To explore the mechanism of light-dependent gene expression unidentified in animals, we first focused on pinopsin gene whose mRNA level is also up-regulated by light. A pinopsin promoter was isolated and analyzed by transcriptional assays using cultured chicken pineal cells, resulting in identification of an 18-bp light-responsive element that includes a CACGTG E-box sequence. We also investigated a role of mitogen-activated protein kinase (MAPK) in the clock resetting, especially in the E-box-dependent transcriptional regulation, because MAPK is phospholylated (activated) in a circadian manner and is rapidly dephosphorylated by light in the gland. Both pulldown analysis and kinase assay revealed that MAPK directly associates with BMAL1 to phosphorylate it at several Ser/Thr residues. Transcriptional analyses implied that the MAPK-mediated phosphorylation may negatively regulate the BMAL-CLOCK-dependent transactivation through the E-box. These results suggest that the CACGTG E-box serves not only as a clock-controlled element but also as a light-responsive element.

Studies till date suggest the existence of a fetal biological clock in suprachiasmatic nuclei entrained by the circadian signal from mother. Melatonin from maternal pineal gland reaches to the fetus by crossing every biological barrier including placenta, hence fetuses were exposed to similar melatonin variation as their mother. Experimental modulations of maternal pineal gland activity of pregnant females either by exposing the them to different photoperiodic schedules or by exogenous melatonin treatments till the date of parturition, regulated the fetal plasma level of melatonin, thereby the prenatal (fetal) growth and development. This clearly suggests the maternal transport of melatonin to their fetus through placenta since fetal retina-hypothalamic tract was incomplete. An extension of experimental schedules till 60 days of post-partum period regulated the neonatal pineal gland activity and gonadal maturation along with their plasma levels of melatonin and sex steroids suggesting clearly the phenomenon of maternal transfer of melatonin to their young ones during the post-natal period, when the neonates were solely dependent on the mother's milk for their nutrition and energetic demands. On the basis of above observations we may suggest that the maternal pineal gland activity regulate the prenatal development by passing its melatonin to fetus via placenta and post-natal growth and sexual maturation by passing maternal melatonin to neonates via milk. Hence, the photoperiod perceived by mother is translated into the maternal plasma level of melatonin which not only regulates the prenatal but also the post-natal growth and sexual maturation of neonates.

In vertebrate rhodopsins the glutamic acid at position 113 serves as a counterion to stabilize the protonated retinylidene Schiff base linkage and to shift the spectrum to the visible region. Invertebrate rhodopsins and retinochrome have the amino acid residue different from glutamic acid or asparatic acid at this position and therefore, these pigments may have a counterion at different position. We first investigated the counterion in retinochrome by site specific mutagenesis. The results showed that the counterion is the glutamic acid at position 181, where almost of all the pigments including vertebrate and invertebrate rhodopsins in the rhodopsin family have a glutamic acid or an aspartic acid. In vertebrate rhodopsins, however, Glu 181 does not act as a counterion, and the red-sensitive cone pigments have a histidine at this position, which serves as a chloride-binding site for red-shift of the absorption spectrum. These findings suggested that the role of Glu181 as a counterion may be weakened by the newly acquired counterion at position 113. Taken together with our recent studies on an invertebrate-type rhodopsin, the rhodopsin diversity was discussed from viewpoint of counterion.

Ascidians are lower chordates, and their tadpole-like larvae share a basic body plan with vertebrates. To study photoreceptive systems in ascidians, we have isolated and characterized cDNA clones for three opsins, five G protein ${\alpha}$ subunits (G${\alpha}$), catalytic and regulatory subunits of cGMP phosphodiesterase (PDE), and arrestin from the ascidian Ciona intestinalis tadpole larva. Ci-opsin1 and Ci-opsin2 are vertebrate-type opsins, while Ci-opsin3 is a retinal photoisomerase similar to retinochrome and mammalian RGR. Both Ci-opsin1 and arrestin are specifically localized in the photoreceptor cells of the ocellus, whereas Ci -opsin2 is not expressed in the photoreceptors, but is co-localized in another population of neurons in the brain with PDE (Ci-PDE9 and Ci-PDE$\delta$). Ci-opsin3 is present in the entire region of the brain. Though five different cDNAs encoding Ga have been cloned, no transducin-type G protein has been found yet. Interestingly, one of G${\alpha}$i isoform is conspicuously expressed in the entire region of the brain. The Ci-opsin3 gene expression was observed in a broad area of the brain vesicle as well as in the visceral ganglion. Genes encoding ascidian homologs of CRALBP and ${\beta}$-CD, whose function is required for the mammalian visual cycle, are co-expressed with Ci-opsin3 in the brain vesicle and visceral ganglion. Localization of Ci-opsin3, CRALBP, and ${\beta}$-CD in a broad area of the brain suggests that the brain of the ascidian larva has a visual cycle system similar to that of the vertebrate RPE. Based on these data, we discuss the evolution of vertebrate visual systems.

In photoreceptor cells, light activates visual pigments consisting of a chromophore (retinal) and a protein moiety (opsin). Activated visual pigments trigger an enzymatic cascade, called phototransduction cascade, in which more than ten phototransduction proteins are participating. Two types of vertebrate photoreceptor cells, rods and cones, play roles in twilight and daylight vision, respectively. Cones are further classified into several subtypes based on their morphology and spectral sensitivity. Though the diversities of vertebrate photoreceptor cells are crucial for color discrimination and detection of light over a wider range of intensities, the molecular mechanism to characterize the photoreceptor types remains unclear. We investigated the amino acid sequences of about 50 vertebrate opsins, and found that these sequences can be classified into five fundamental subfamilies. Clear relationships were found between these subfamilies and their characteristic spectral sensitivities. In addition to opsins, we studied other phototransduction proteins. The amino acid sequences of phototransduction proteins can be classified into a few subfamilies. Even though their spectral sensitivity is considerably different, cones fundamentally share the phototransduction protein isoforms which are different from those found in rods. It is suggested that the difference in phototransduction proteins between rods and cones is responsible for their sensitivity to light. Isoforms and their selective expression may characterize individual photoreceptor cells, thus providing us with physiological functions such as color vision and daylight/twilight visions.

In the vertebrate retina, rods mediate twilight vision and cones daylight vision. Rods have been purified easily from the retina, and thus the phototransduction mechanism in rods is now well documented. However, it has not been possible to purify cones in large quantities, and therefore, the knowledge on the mechanism in cones is limited. Here we report purification of carp (Cyprinus carpio) cones with a stepwise Percoll gradient. Using purified cells, we compared the phototransduction mechanism between rods and cones. The results showed that both transducin activation and phosphodiesterase activation are less effective, and visual pigment phosphorylation is faster in cones. These differences explain lower light-sensitivity and briefer photoresponse time course in cones.

Raft is a distinctive membrane domain enriched in a certain class of lipids, cholesterol, and proteins observed on the plasma membrane. Growing evidence has revealed that such membrane domains play key roles in signal transduction, fertilization, development, transmitter release, and so on. Recently, we have isolated raft-like detergent-resistant membrane (DRM) fraction from bovine photoreceptor rod outer segments. Transducin and its effecter, cGMP-phosphodiesterase, elicited stimulus-dependent translocation between detergent-soluble membrane and DRM. This suggested potential importance of such distinct membrane domains in vertebrate phototransduction. Here, we will discuss physiological meaning of the translocation of major components of cGMP cascade to raft-like membrane in phototransduction. We would like to propose a hypothesis that raft-like membrane domains on the disk membrane are the place where cGMP cascade system could be quenched.

We propose a novel mechanism (Twist Sharing Mechanism) for the cis-trans photoisomerization of rhodopsin, based on the molecular dynamics (MD) simulation study. New things devised in our simulations are (1) the adoption of Mt. Fuji potentials in the excited state for twisting of the three bonds C9=C10, C11=C12 and C13=14 which are modeled using the detailed ab initio quantum chemical calculations and (2) to use the rhodopsin structure which was resolved recently by the X-ray crystallographic study. As a result, we found the followings: Due to the intramolecular steric hindrance between 20-methyl and 10-H in the retinal chromophore, the C12-C13 and C10-C11 bonds are considerably twisted counterclockwise in rhodopsin, allowing only counterclockwise rotation of the C11 =C12 in the excited state. The movement of 19-methyl in rhodopsin is blocked by the surrounding three amino acids, Thr 118, Met 207 and Tyr 268, prohibiting the rotation of C9=C10. As a result only all-trans form of the chromophore is obtainable as a photoproduct. At the 90$^{\circ}$ twisting of C11=C12 in the course of photoisomerization, twisting energies of the other bonds amount to about 20 kcal/mol. If the transition state for the thermal isomerization is assumed to be similar to this structure, the activation energy for the thermal isomerization around C11=C12'in rhodopsin is elevated by about 20 kcal/mol and the thermal isomerization rate is decelerated by 10$\^$-14/ times than that of the retinal chromophore in solution, protecting photosignal from the thermal noise.

The reaction center Dl protein of photosystem II is the target of photodamage by excess illumination. The Dl protein is damaged by reactive oxygen species generated by photochemical reactions and then degraded by specific proteolytic enzymes. We found that the Dl protein also cross-links with the surrounding polypeptides, such as D2 and CP43 in isolated thylakoids or photosystem II-enriched membranes from spinach under the illumination with strong visible light. The cross-linking was observed in spinach leaf discs as well when they were illuminated at higher temperature (40°C). It was also shown that the cross-linked products are digested efficiently by a protease(s) in the stroma. Thus the cross-linking/digestion processes of the Dl protein seem to comprise a new pathway in the turnover of the photodamaged Dl protein. It should be noted, however, that the cross-linked products of the Dl protein and CP43 induced by endogenous cationic radicals in the donor-side photoinhibition are resistant to proteolytic digestion. Accumulation of these cross-linked products in the thylakoids may lead to the decay of the function of chloroplasts and finally to the death of plant cells. Thus, we suggest that the quality control of photosystem II, especially removal of the cross-linked products of the Dl protein, is crucial for the survival of chloroplasts under the light stress.

In chilling-sensitive plants, the donor side of Photosystem II is inhibited by the chilling treatment in the dark, while the acceptor side of Photosystem I is inhibited by the chilling under the moderate light. Since the addition of inhibitors of electron transfer from Photosystem II protects Photosystem I from chilling induced photoinhibition of Photosystem I, inhibition or down-regulation of Photosystem II activity in vivo may also protect Photosystem I from photoinhibition. It was revealed that dark-chilling pretreatment actually protected Photosystem I from photoinhibition. The results imply that down-regulation of Photosystem II under stress conditions may have a role to protect Photosystem I from photoinhibition.

This report concerns a putative role of mycosporine-like amino acids (MAA) as natural scavengers of singlet oxygen ($^1$O$_2$) in marine organisms. MAA prepared from the ascidian Lissoclinum patella were found to protect biological systems against detrimental effects of the type II photosensitization in vitro. L. patella MAA were resolved into five components, and the relative $^1$0$_2$ quenching efficiencies were measured for three major components in aqueous media. It turned out that they were all effective in scavenging $^1$0$_2$, to different degrees albeit. The results suggest that physiological relevance of MAA in marine organisms may be found in a 'built-in' defense against photooxidative effects of sunlight.

Zinc chlorin 1 possessing tertiary 3$^1$_hydroxy and 13$^1$-oxo groups was synthesized as a model for the antenna chlorophylls of photosynthetic green bacteria. Self-aggregation of 1 in nonpolar organic solvents was examined and compared to 2 and 3 possessing a secondary and primary 3$^1$_hydroxy group, respectively. Zinc chlorin 1 self-aggregated in I％(v/v) CH$_2$Cl$_2$-hexane to form oligomers and showed a red-shifted Qy maximum at 704 nm compared to the monomer (648 nm in CH$_2$CI2$_2$). This red-shift is larger than that of 3$^1$S-2 (648 to 697 nm) and comparable to that of3$^1$R-2 (648 to 705 nm), but smaller than that of 1 (648 to 740 nm), indicating that while a single 3$^1$-methyl group (primary to secondary OH) suppressed tight and/or extended aggregation, the additional 3$^1$-methyl group (secondary to tertiary OH) did not further suppress aggregation. The relative stability of the aggregates was in the order 3> 3$^1$R-2∼ 1 > 3$^1$S-2 as determined by visible spectral analyses. Molecular modeling calculations on oligomers of zinc chlorins 1, 3$^1$ R-2 and 3 gave similar well-ordered energy-minimized structures, while 3 stacked more tightly than 3$^1$ R- 2 and 1. In contrast, 3$^1$S-2 gave a relatively disordered (twisted) structure. The calculated oligomeric structures could explain the visible spectral data of 1-3 in nonpolar organic solvents. Moreover, self- aggregation of synthetic zinc 13$^1$_oxo-hlorins 4-6 possessing a 2-hydroxyethyl, 3-hydroxypropyl and 3- hydroxy-I-propenyl group at the 3-position in nonpolar organic solvents was discussed.

A cyanobacteria-like organism Aaryochloris marina performs oxygenic photosynthesis with near far-red light by the use of chlorophyll d. Reaction center chlorophyll (Chl) of Photosystem (PS) II of A. marina was studied by analysis of millisecond-delayed fluorescence. Delayed fluorescence is emitted by Chi d indicating efficient energy transfer between antenna Chi d molecules and the unknown primary electron donor of PS II. P740 a reaction center Chl of PS I of A. marina is shown to give a dimer type cation, and triplet state with a D value of 245xlO$\^$-4/ cm$\^$-l/ in contrast to the 280-290 xlO$\^$-4/cm$\^$-l/ values of P700 suggesting triplet spins interacting at a 5% larger distance in P740 than in P700.

Chlorophyll (Chi) d was assigned to an antenna pigment of red algae in 1943, but its presence and function in red algae have not been necessarily clear for a long time. In 1996, it was shown that Chi d functioned as a major antenna pigment in a peculiar oxygenic photosynthetic prokaryote, Acaryochloris marina, isolated as a symbiont of a colonial ascidian from coral reefs. This finding evoked the necessity for reexamination of the presence and function of Chi d in red algae. We found Chi d in methanol-extract from several marine red algae, and the relative content was high in one species, Ahnfeltiopsis flabelliformis. Absorption and fluorescence spectra, HPLC analysis, and NMR and mass spectroscopy characterized Chi d extracted from the red algal thalli, and those were essentially identical to those of Chi d isolated from A. marina. However, micro-spectrophotometric analysis suggested that Chi d was not an actual constituent of the red algae but came from epiphyte(s) attached to surface of red algal thalli.

Fluorescence rise and decay curves of carotenoids were measured in solutions and in pigment protein complexes with a femtosecond time-resolved fluorescence spectroscopy. For linear carotenoids, the S$_2$ lifetimes showed the maximum value around n = 9-10. The conjugation of a keto-carbonyl group shortened the S$_2$lifetime and prolonged the S$_1$lifetime. The excitation relaxation dynamics within carotenoids and the excitation energy transfer kinetics from carotenoids to chlorophylls are discussed as a function of molecular structure of carotenoids.

33kDa extrinsic protein, an important protein in oxygenic photosynthesis, was known to have no fixed configuration in solution. At 20$\^{C}$ and pH 6, 33kDa extrinsic protein showed changes of free energy of -14.6 kJ/mor$\^$-1/ and of standard volume of -120mL/mol, respectively, with increase of hydrostatic pressure, comparatively lower than for most proteins. NBS modification of Trp241 in 33kDa extrinsic protein dramatically changes the secondary protein structure, its affinity to photosystem II as well as photosynthetic oxygen evolution. The relationship between structural change and transport of oxygen, water and proton is deserved a further study.

Blue light activates proton pump, and creates electrical gradient across the plasma membrane and drives $K^{+}$ uptake in stomatal guard cells. In this presentation, we provide evidence for regulatory mechanisms of the pump and the identification of blue light receptor. The pump is shown to be the plasma membrane H$^{+}$- ATPase and is activated through phosphorylation of the C-terminus. Phosphorylation occurred and 14-3-3 protein bound to the phosphorylation site. The binding of 14-3-3 protein was required for the H$^{+}$-ATPase activation. We also found that phot1 phot2 double mutant does not respond to blue light but other mutants respond to blue light by stomatal opening. However, all these mutants are capable of stomatal opening in the presence of fusicoccin, an activator of the H$^{+}$-ATPase. These results suggest that both photl and phot2 act as blue light receptors in guard cells.d cells.

Phytochrome A (phyA) and phytochrome B (phyB) perceive light and adapt to fluctuating circumstances by different manners in terms of effective wavelengths, required fluence and photoreversibility. Action spectra for induction of seed germination and inhibition of hypocotyl elongation using phytochrome mutants of Arabidopsis showed major difference. PhyA is the principal photoreceptor for the very low fluence responses and the far-red light-induced high irradiance responses, while phyB controls low fluence response in a red/far-red reversible mode. The structural requirement of their bilin chromophores for photosensory specificity of phyA and phyB was investigated by reconstituting holophytochromes through feeding various synthetic bilins to the following chromophore-deficient mutants: hy1, hyl/phyA and hyl/phyB mutants of Arabidopsis. We found that the vinyl side-chain of the D-ring in phytochromobilin interacts with phyA apoprotein. This interaction plays a direct role in mediating the specific photosensory function of phyA. The ethyl side-chain of the D-ring in phycocyanobilin fails to interact with phyA apoprotein, therefore, phyA specific photosensory function is not observed. In contrast, both phytochromobilin and phycocyanobilin interact with phyB apoprotein and induce phyB specific photosensory functions. Structural requirements of the apoproteins and the chromophores for the specific photoperception of phyA and phyB are discussed.

Light is one of the most important environmental factors that influence plant growth and development. It does not function independently but exerts its role through coordinated interactions with intrinsic developmental programs, such as hormonal regulation. One typical example is hypocotyl growth in which light signals are modulated through growth hormones. However, the underlying molecular mechanisms are largely unknown. We demonstrated that brassinosteroids play an important role in the light signal transduction in etiolated hypocotyl growth. A light-responsive Ras-like G-protein, Pra2 from pea, physically and functionally interacts with a cytochrome P450 that specifically catalyzes C-2 hydroxylation in brassinosteroid biosynthesis. The cytochrome P450 expression, along with Pra2, is induced in the dark and predominantly localized in the rapidly elongating zone of etiolated pea epicotyls. Transgenic plants with a reduced level of Pra2 exhibit a dark-specific dwarfism, which is completely rescued by brassinosteroid application. On the contrary, overexpression of the cytochrome P450 results in enhanced hypocotyl growth even in the light, which phenocopies the etiolated hypocotyl growth. It is therefore envisioned that Pra2 is a molecular switch that mediates the crosstalk between light and brassinosteroids in the etiolation process.

Euglena gracilis abruptly changes its swimming direction after a sudden increase or decrease in incident light intensity, that is, step-up or step-down photophobic responses, resulting in photoavoidance or photoaccumulation, respectively. To identify the photoreceptor molecules for these UV-A/blue-light type photobehaviors, we purified a flavoprotein from isolated putative photosencory organelles (PFBs) of Euglena. The purified flavoprotein, which noncovalently bound flavin adenine dinucleotide (FAD), seemed to be a heterotetramer of alpha- and beta-subunits. Predicted amino acid sequences of each of the subunits were similar to each other and contained two FAD-binding domains each followed by an adenylyl cyclase catalytic domain. The purified flavoprotein actually showed adenylyl cyclase activity, being drastically elevated by blue-light irradiation. Suppression of gene expression of the flavoprotein (Photoactivated Adenylyl Cyclase, PAC) by RNA interference (RNAi) caused loss of the step-up photophobic response, demonstrating that PAC actually mediates photoavoidance of Euglena.

Phoborhodopsin (pR or sensory rhodopsin II, sRII; the absorption maximum of ∼ 500 nm) is a retinoid protein and works as a photoreceptor of the negative phototaxis of Halobacterium salinarum. pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. These sensory proteins form a complex with a cognate transducer protein in the membrane, and this complex transmits the light-signal to the cytoplasm to evoke avoidance reaction from blue-green light. Recently, the functional expression in Escherichia coli membrane of ppR was achieved, which can afford a large amount of the protein and enables mutant studies to clarify the role of various amino acid residues. A truncated transducer which can bind to ppR is also expressed in Escherichia. coli membrane. In this article, we will review properties of ppR mainly using observations of our laboratory; which contains photochemistry (photocycle), light-driven proton uptake, release and transport, F -helix titling during photocycle and association of the transducer.

Archaeal rhodopsins possess retinal molecule as their chromophores, and their light-energy and light-signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of protein then leads to functional processes, proton pump in bacteriorhodopsin (bR) and transducer activation in phoborhodopsin (pR). It is known that sensory rhodopsins can pump protons in the absence of their transducers. Thus, there should be common and specific features in their protein structural changes for function. In this paper, our r ecent studies on pR from Natronobacterium pharaonis (ppR) by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy are compared with those of bR. In particular, protein structural changes upon retinal photoisomerization are studied. Comparative investigation of ppR and bR revealed the similar structures of the polyene chain of the chromophore and water-containing hydrogen-bonding network, whereas the structural changes upon photoisomerization were more extended in ppR than in bR. Extended protein structural changes were clearly shown by the assignment of the C=O stretch of Asnl05. FTIR studies of a ppR mutant with the same retinal binding site as in bR revealed that the Schiff base region is important to determine their colors.

We demonstrate here a dynamic structure of bacteriorhodopsin (bR) as revealed by $^{13}$ C NMR studies on [3_$^{13}$ C]_,[1-$^{13}$ C]Ala- and/or Val-labeled wild type and a variety of site-directed mutants at ambient temperature. For this purpose, well-resolved (up to twelve) I$^{13}$ C NMR peaks were assigned with reference to the displacement of peaks due to the conformation-dependent I$^{13}$ C chemical shifts and reduced peak-intensities due to site-directed mutations. Revealed bR structure was not rigid as anticipated from 2D crystals of hexagonal array but a dynamically heterogeneous, undergoing a variety of local fluctuations depending upon specific site with frequency range of 10$^2$ -10$^{8}$ Hz. In particular, dynamics- dependent suppression of peaks turned out to be very sensitive to the motion of 10$^{-4}$ s and 10$^{-5}$ s interfered with frequency of magic angle spinning and proton decoupling, respectively. It is also noteworthy that such dynamic feature is strongly dependent upon the manner of 2D crystalline packing: $^{13}$ C NMR peaks of monomeric bR yielded either highly broadened or completely suppressed signals, depending upon the type of $^{13}$ C-labeled amino-acid residues.

Ultraviolet resonance Raman (UVRR) spectroscopy was used to elucidate the dynamic change of the protein structure of bacteriorhodopsin (BR) during the photocycle. The photointermediates minus light- adapted (LA) BR difference spectra show Trp difference signals, which are assigned to Trp189 or Trp182 on helix F by using the mutants, W182F and W189F. The Difference signals of Trp 182 indicates an increase in hydrogen bonding strength at the indole nitrogen and a large change in the side chain conformation (X$\^$2,1/ torsion angle) in the M$_1$ \longrightarrow M$_2$ transition. On the other hand, Trp189 shows an increased hydrophobic interaction. These results suggest that the tilt of helix F occurs in the M$_1$\longrightarrow M$_2$ transition. In the M$_2$ \longrightarrow N transition, the hydrophobic interaction of Trp182 decreases drastically, The decrease in hydrophobic interaction of Trp182 in the N state suggests an invasion of water molecules that promote the proton transfer from Asp96 to the Schiff base. Structural reorganization of the protein after the tilt of helix F may be important for efficient reprotonation of the Schiff base.

A state of bacteriorhodopsin at high temperature was studied by various spectral measurements. The stability measurements indicated that the onset temperature of the denaturation was 70$^{\circ}C$ in the dark and 60$^{\circ}C$ under illumination. The reactivity of hydroxylamine with the Schiff's base also significantly increased in the temperature range between 60 and 70$^{\circ}C$. A spectral band at about 470 nm appeared in the temperature range higher than 60$^{\circ}C$. The circular dichroism spectra in the visible region started to change from a bilobed exiton type to a positive band at about 60$^{\circ}C$, suggesting that the two-dimensional configuration of bacteriorhodopsin molecules changed from crystalline to amorphous. All the measurements suggested a new state between 60 and 70$^{\circ}C$ in which bacteriorhodopsin is stable only in the dark.

Coherent oscillations in is fluorescence dynamics of W.-t. PYP and its site-directed mutants have been observed. Two oscillatory modes coupled with the ultrafast fluorescence due to the twisting of the excited chromophore were identified, a high ftequency mode (∼135 cm$\^$-1/) with ∼550 is damping time and a low frequency overdamped mode (-45 cm$\^$-1/) with ∼250 is damping time, respectively. Both modes disappear in the fluorescence dynamics of denatured PYP emphasizing the important role of the protein nanospace as the environment for photoreaction. The qualitative picture of fluorescence dynamics in site-directed mutants was rather similar to that in W.-t. PYP, i.e., similar oscillatory modes (∼130-140 cm$\^$-1/ and ∼40-70 cm$\^$-1/) have been observed. This indicates that the vibrational modes and electron-vibration couplings do not change dramatically due to the mutation though the damping time of low frequency mode a little decreases as the protein nanospace structure becomes looser and more disordered by mutation. On the other hand, in the case of some PYP analogues, the qualitative picture of fluorescence dynamics changes, showing the familiar picture of solvation effect whereas the oscillations are almost damped. Comparative analyses of these observations are presented.

The chromophore/protein interactions in the photocycle intermediates of photoactive yel- low protein (PYP) were probed by site-directed mutagenesis. The absorption spectra of L- intermediates produced from E46Q, T50V, and R52Q mutants were calculated using the absorption spectra of dark states and difference absorption spectra between L-intermediates and dark states, and compared with that of PYP$\_$L/. The absorption spectrum of R52Q$\_$L/ agreed with that of PYP$\_$L/, but those of E46Q$\_$L/ and T50V$\_$L/ were red-shifted. The effect of these mutations on the absorption spectrum for L-intermediate was comparable to that for the dark state, suggesting that the interaction around the phe-nolic oxygen of the chromophore is conserved in PYP$\_$L/ unlike the crystal structure. On the other hand, we have reported that the absorption spectra of Y 42F$\_$M/, T50V $\_$M/, and R52Q$\_$M/ agreed with that of PYP$\_$M/, but that of E46Q$\_$M/ was red-shifted, suggesting that the hydrogen bond of the chromophore with Glu46 is conserved but that with Tyr42 is broken in PYP$\_$M/. These results suggest that the chromophore inter-acts with Glu46 throughout the photocycle, but never directly interacts with Arg52. This model con- flicts with some of the structural model of PYP intermediates proposed based on the high-resolution X -ray crystallography.

The last step in the photocycle of photoactive yellow protein (PYP) is a spontaneous recovery of the dark state from the active state in which the p-coumaric acid chromophore is thermally isomerized, concomitantly with the deprotona- tion of the chtomophore and the refolding of the protein moicty. For the purpose of understanding the mechanism of the thermal back-isomerization, we have investigated the rate-determining step by analyzing mutant PYPs of Met100, which was previously shown to play a major role in facilitating the reaction (1). The mutation to Lys, Leu, Ala, or Glu decelerated the dark state recovery by 1 to 3 three orders of magnitude. By evaluating temperature-dependence and pH-dependence of the kinetics of the dark state recovery, it was found that the retardation by mutations resulted from elevation of the activation enthalpy ( H$\^$┿/) and that the pKa of the chromophore, which was affected by the mutation, is in a linier correlation with the amplitude of the rate constants. It was, therefore, deduced from the correlation that the free energy for crossing the activated state in the dark recovery process is proportional to the free energy for the deprotonation of the chromophore, identifying the rate-determining step as the deprotonation of the chromophore. (1) Devanathan, S. Genick, U. K. Canestrelli, I. L. Meyer, T. E. Cusanovich, M. A. Getzoff, E. D. Tollin, G., Biochemistry 1998, 37, 11563 - 11568

PYP consists of a water-soluble apoprotein and 4-hydroxycinnamyl chromophore bound to Cys69 via thiolester linkage, Upon absorption of a photon, the photocycle is initiated, leading to formation of several photo-intermediates. Among them, M intermediate is important to understand the signal transduction mechanism of PYP, because it is a putative signaling state. As well known, the dynamics of a protein is closely correlated with the occurrence of its function. Here we report the results of IO ns molecular dynamics (MD) simulation for the M intermediate in aqueous solution and discuss the characteristic feature of this state from a viewpoint of structural fluctuation.

A photo-labile compound which is bioinactive but, upon irradiation with light, yields bioactive species is called as "caged compound". Photolysis of caged compounds generating bioactive species, has become a general method to produce a desired amounts of bioactive species in the specific time interval at the desired place or area of the target biological systems. For this purpose, we designed and synthesized caged hydroxyl radical., "Photo-Fenton Reagent" NP-IIl. NP-IIl has a strong absorption maximum at 377 nm and yields hydroxyl radicals upon UV light irradiation. The antioxidant activity of the ${\alpha}$ -lipoic acid and other naturally occurring compounds has been examined by using NP-IIl as a molecular probe. For example, upon photoirradiation of NP-lII with BSA or apolipoprotein of human low density (LDL), the significant oxidative modifications were observed in both cases. The oxidation was completely suppressed in the presence of ${\alpha}$-lipoic acid, which clearly demonstrates the strong hydroxyl radical scavenging activity of ${\alpha}$-lipoic acid. Other applications of NP-lII will also be described

A strict anaerobe, Prevotella melaninogenica is highly sensitive to oxidative stress. Oxidative stress such as exposure to oxygen or addition of hydrogen peroxide, increased 8-hydroxydeoxyguanosine (80HdG), a typical of oxidative DNA damage, and decreased the bacterial cell survival rate. We could detect the generation of reactive oxygen species in P. melaninogenica after exposure to oxygen. UVA irradiation also increased 80HdG in the bacterium. On the other hand, such oxidative stress did not increase 80HdG in a facultative anaerobe. These findings suggest that P. melaninogenica is a suitable material to study the biological effects of oxidative stress, to evaluate antioxidants, and to study the effects of oxygen or reactive oxygen species on molecular evolution.

Oxidative stress evoked hy Ultraviolet (UV) exposure has been suggested to be involved in UV-induced skin carcinogenesis. In this study, the role of oxidative stress in UV-carcinogenesis was evaluated by applying N-Acetylcysteine (NAC) in animal model of hairless-mouse. NAC is known to be a precursor of glutathione, which was converted to glutathione in cytoplasm, acting as an intracellular free radical scavenger. The glutathione levels in hairless mouse skin after one time application of NAC increased significantly. With and without the pre-treatment of NAC, hairless-mice were exposed to UVB three times a week, at total dose 274.4 kJ in 80 times, and the timing of tumor-development, incidence of skin tumor and the histopathology of tumors were observed. 8-hydroxy-2'-deoxyguanosine (8-0HdG), a typical form of oxidative damage in DNA has been also investigated in the course of experiment. The decrease of 8-0HdG formation of UVB- exposed skin compared to controls was observed in the early stage of experiment in the NAC-treated mice. In addition, initial tumor development delayed significantly in NAC-treated group. Finally the number of the tumor developed in the NAC-treated mice was fewer though not significant. These results suggest that antioxidants may have inhibitory effect in the initial step of UVB-induced carcinogenesis of hairless mice.

Solar UV light is a well-known carcinogen. UVA radiation is probably carcinogenic to humans. In addition, recent investigations point to the importance of UVA irradiation in the photoaging. We investigated the mechanism of sequence- specific DNA damage using $\^$32/P-Iabeled DNA fragments in relation to carcinogenesis and aging. Furthermore, we investigated whether UVA accelerates the telomere shortening in human WI-38 fibroblasts. The exposure of double- stranded DNA fragments to 365 nm light in the presence of endogenous sensitizers produced sequence-specific cleavage at the 5' site of 5'-GG-3' and 5'-GGG-3' sequences. In addition, HPLC analysis revealed that sensitizers plus 365 nm light increased the 8-oxodG content of double-stranded DNA. We discuss the mechanisms of guanine-specific DNA damagecaused by excited photosensitizers in relation to carcinogenesis and aging.

Direct, continuous, and accurate measurements of solar ultraviolet irradiance (290-400 nm: UVR) have been carried out since 1990, by using both band-spectral ultraviolet-B (290-320 nm: UV-B) and ultraviolet-A (320-400 nm: UV-A) radiometers at Tokai University in Hiratsuka, Japan (35$^{\circ}$N, 139$^{\circ}$E). From our observations, the following findings are provided: 1) an increasing trend in solar UV -B from Oct. 1990 to Sept. 2000; 2) a regional comparison of solar UVR in Japan; 3) the distinct characteristics of UV-B and UV-A irradiance, such as diffuse property, daily and seasonal variation; and 4) human body protection against solar UVR. An increasing 10-year trend in global solar UV - B in Hiratsuka corresponded to a decrease in the total ozone amount measured at Tsukuba (36$^{\circ}$N, 140$^{\circ}$E), giving supportive evidence for a direct link between these two parameters. Furthermore, a strong correlation was found between solar UV-B and total ozone amount from results of UVR measurements at four Tokai University monitoring stations dispersed throughout Japan. Additional results revealed different diffuse properties in global solar UV and in global solar total (300-3000 nm: Total) irradiances. For example, in the global UVR, the diffuse component was dominant: about 80 % independent of weather, with more than 60 % of global UV-B, and more than 50% of global UV-A with even a cloudless clear sky. On the other hand, the portion of the diffuse in the global total irradiance was very low, less than 10 % on a cloudless clear day. Daily and seasonal variations of UV -B and UV -A irradiances were found to be quite different, because of the marked dependence of UV -B irradiance on the atmospheric ozone amount. Moreover, UV -B irradiance showed large daily and seasonal variations: the ratio between maximum and minimum irradiances was more than 5. In contrast, the variation in UV-A was small: the ratio between maximum and minimum was less than 2. Three important facts are proposed concerning solar UVR protection of the human body: 1) the personal minimal erythema dose (MED); 2) gender based difference in MED values; and 3) proper colors for UVR protective clothing.

UV-B radiation gives harmful effects on plants, such as production of several types of DNA lesions, and growth inhibition. On the other hand, plants have some protective mechanisms, including filtering effect due to accumulation of phenolic compounds in epidermal cells and reactivation of DNA lesions, which are enhanced by UV-B irradiation. We have investigated the mechanism of UV-B effects on plants using cucumber seedlings as plant materials. Cucumber plants were cultivated in an artificially lit growth chamber. Supplemental UV-B irradiation, of which intensity was almost equal to the level of natural sunlight, retarded the growth of first leaves. The growth retardation must result trom the inhibition of cell division and/or cell growth. Microscopical observation of leaf epidermis suggested that the growth retardation might be mainly caused by cell growth inhibition. The retardation was, however, restored within 2 or 3 days after the termination of UV-B irradiation. It is known that UV-B irradiation lowers the activity of photo system II (PS II). In the present experimental conditions, however, UV-B irradiation has little effect on PS II activity as estimated by chlorophyll fluorescence. The stomatal conductance, a major factor determining photosynthetic rate, of first leaves increased during the growth. The increase of stomatal conductance was suppressed by UV-B irradiation and restored by termination of the irradiation. It has not been clear, however, what mechanisms are involved in the suppression of increase of stomatal conductance.

Rice is widely cultivated in various regions throughout Asia. Over a five-year period, we investigated the effects of supplemental UVB radiation on the growth and yield of Japanese rice cultivars in the field. The findings of that study indicated that supplemental UVB radiation has inhibitory effects on the growth and grain development. Furthermore, we investigated the sensitivity to UVB radiation of rice cultivars of 5 Asian rice ecotypes, and found that rice cultivars vary widely in UVB sensitivity. The aim of our study is improving UVB resistance in plants by bioengineering or breeding programs. In order to make it, there is need to find the molecular origin of the sensitivity to UVB. Cyclobutane pyrimidine dimer (CPD) is major UV-induced DNA lesions. Plants possess two mechanisms to cope with such DNA damage. The first is the accumulation of UV-absorbing compounds. Our previous data showed that the steady-state CPD levels in leaves of rice grown under chronic radiation in any culture were not so greatly influenced by the increased UV-absorbing compounds content, although there was a significant positive correlation between the CPD levels induced by challenge UVB exposure and the UV-absorbing compounds content. The other is the repair of DNA damage. Photorepair is the major pathway in plants for repairing CPD. We found that the sensitivity to UVB could seriously correlate with the low ability in CPD photorepair in rice plants. These results suggest that photo lyase might be an excellent candidate for restoration by way of selective breeding or engineering in rice.

This paper is a part of the research into effects of increased UV-B (Ultraviolet-B, 280-320nm) upon the ecosystem of field wheat. Based on a 3-year project with intensified UV-B influencing the crop, observation and calculation were made of such factors and parameters as the morphology of a single wheat plant including its leaf area, leaf base angle and proportion of spacing between joints. The results show that the enhanced UV-B signifantly decreased wheat's SSLA, and the increased percentage of the lower LAI is associated with the change in leaf base angle and proportion of spacing between joints.

Space is full of energetic events emitting high-energy radiations which may be fatal to all living things unless protected. The present paper briefly describes high-energy photons and particles incident on Earth surface and their common properties toward living things. Role of radiation played in evolution of life and earth environment will be presented.

Sugar-linked porphyrin and chlorin compounds have been synthesized. Phototoxicity of these compounds against the HeLa cell line was also examined. For the porphyrin derivatives, the higher activity was observed for a derivative having four OH-protected sugar moieties. For the chlorin derivatives, OH-unprotected free-base derivatives were generally effective. Singlet oxygen producing ability were examined to evaluate the activity on photodynamic therapy of the compounds.

Pyropheophorbide and pheophorbide-photosensitizers as chlorin analogues are promising new compounds for PDT because the chlorin analogues are activated with much longer red light at > 670nm and produce less long-term normal tissue phototoxicity than Photofrin. The various chlorin derivatives can be obtained by moditying peripheral substituted group among which meso-H, vinyl group and exocyclic ring are the most active positions. These characteristics prompted us to introduce various groups for constructing modified pyropheophorbide and pheophorbide a compounds. A stereospecific introduction of various double bonds at 3-position was performed to methylpheophorbide a to give a long hydrophobic moiety and cyclic derivatives. Chlorin-C$_{60}$ dyad and chlorin- $C_{60}$-porphyrin triad also were easily prepared by the reaction of terminal aldehyde of methyl pyropheophorbide a. For the reaction on meso $\delta$-position bromination and Vismeier formylation can occur. N,N-dimethylaminoacrolein also reacted on $\delta$-position and was cyclized to isobacteriochlorin, but other modification has not been succeeded. Exocyclic keto function was also modified to give purpurin derivatives, bicyclic and spiro compounds. In this presentation we report a series of modified pyropheophorbide and pheophorbide a derivatives.s.

There are 3 UV DNA repair endonuclease activities in mammalian cells that cleave UV -irradiated DNA. Interestingly, mammalian UV endonuclease III with MW of 26.7kD has a lyase activity on AP sites. It also cleaves the phosphodiester bond within a cyclobutane pyrimidine dimer. Genomic analysis of human repair endonuclease III gene revealed that this gene has 100% sequence identity with ribosomal protein S3 (rpS3). Therefore, rpS3 seems to function both in translation and in DNA repair. This gene of about 6.1 kb contains 6 introns and 7 exons, and the first and fifth introns of human rpS3 gene contain functional U15 small nucleolar (sno) RNAs which appear to be involved in ribosome assembly. It is to be noted that the column profile of the endonuclease activity of rpS3 appears to be altered in Xeroderma Pigmentosum (XP) group D cells compared to normal cells indicating that this protein is involved in XP disease as well. XP is a human disease characterized by high sensitivity of skin by UV- or sun-light irradiation and by high frequency of developing skin cancers. We also report here that rpS3 protein is involved in other cellular functions.

Many of the adverse effects of solar UV exposure appear to be directly attributable to damage to epidermal DNA. In particular, cyclobutane pyrimidine dimers (CPD) may initiate mutagenic changes as well as induce signal transduction responses that lead to a loss of skin immune surveillance and micro-destruction of skin structure. Our approach is to reverse the DNA damage using prokaryotic DNA repair enzymes delivered into skin using specially engineered liposomes. T4 endonuclease V encapsulated in liposomes (T4N5 liposome lotion) enhanced DNA repair by shifting repair of CPD from the nucleotide excision to the base excision repair pathway. Following topical application to humans, increased repair limited UV-induction of cytokines, many of which are immunosuppressive. In a recent clinical study, topical treatment of UV-irradiated human skin with T4N5 liposome lotion reduced the suppression of the nickel sulfate contact hypersensitivity response. Similarly, the photoreactivating enzyme enhances repair by directly reversing CPDs after absorbing activating light. Here also treatment of UV-irradiated human skin with photoreactivating enzyme in liposomes and photoreactivating light restored the response to the contact allergen nickel sulfate. These findings confirm in humans the observation in mice that UV induced suppression of contact hypersensitivity is caused in part by CPDs. We have tested the ability of T4N5 liposome lotion to prevent UV-induced skin cancer in patients with xeroderma pigmentosum (XP), who have an elevated incidence of skin cancer resulting from a genetic defect in DNA repair. Daily use of the lotion for one year in a group of 20 XP patients reduced the average number of actinic keratoses by 68% and basal cell cancers by 30% compared to 9 patients in the placebo control group. Delivery of DNA repair enzymes to skin is a promising new approach to photoprotection.

The functional disruption of Langerhans cells (LC) by UVB radiation is involved in antigen-specific immunosuppression of contact hypersensitivity. We tested whether UVB radiation inhibits the endocytotic activity of LC, which leads to impaired subsequent migration and maturation. Human monocyte-derived LC that took up lucifer yellow (L Y) or FITC-dextran (Fd) exclusively migrated in response to 6Ckine and matured. Exposing LC to 10-40 mJ/cm$^2$ of UVB radiation reduced their endocytotic activity in fluid phase pinocytosis (measured by uptake of LY) and in receptor-mediated endocytosis (measured by uptake of Fd). Membrane ruffling and CD32 expression were also suppressed by UVB radiation. UVB-irradiated, endocytosing LC had less movement towards 6Ckine, expressed less CD54 and CD86, and had less effective stimulatory activity in allo-MLR than nonirradiated, endocytosing LC. Endocytosis up-regulated TNF-$\alpha$ production by LC, but prior UVB radiation inhibited this enhancement. The finding that impaired endocytosis of LC by UVB radiation inhibits subsequent migration and maturation was also confirmed in murine epidermal cells obtained from unirradiated and 2OmJ/cm$^2$ of UVB-irradiated skin.

While ultraviolet radiation alters various cutaneous cell functions, little is known about photo-immunological and photobiological effects of infrared radiation (IR) on the skin except its local thermal effects. The fIrst part of this study demonstrated that single exposure of mouse skin to near IR (0.7 - 1.3 $\mu$m) reversibly suppressed the proliferating activity of the epidermis, the density of Langerhans cells, and the ability of skin to induce contact hypersensitivity reaction. The second part demonstrated that the rate of wound closure was significantly accelerated by repeated exposures in animal models. The production of transforming growth factor-$\beta$l and matrix metalloproteinase-2, which are responsible for the wound healing processes, was significantly upregulated by irradiation, as shown by enzyme immunoassay, zymography, and reverse transcription polymerase chain reaction. Thermal controls were negative. The results suggest that near-IR irradiation can modulate the epidermal proliferation and part of the skin immune system, and stimulate the wound healing processes, presumably by non-thermal effects.

UVR-induced immunosuppression contributes to skin cancer. The aim was to construct accurate dose response curves for primary and secondary contact sensitivity for solar-simulated UVR (ssUVR; 290-400nm), UVA and UVB as the role of UVA in immunosuppression is controversial. We used a xenon arc source. The mice were immobilised, enabling accurate dosing. C57BL/6 mice were immunosuppressed at half the dose of ssUVR required to cause sunburn but not by higher doses (up to the sunburn dose). Thus, ssUVR causes systemic immunosuppression only over a narrow, low dose range. UVA caused suppression at low but not high doses whereas UVB induced immunosuppression at all doses tested. 8 weeks later the mice were resensitised to assess tolerance. Mice exposed to the minimum immunosuppressive dose of ssUVR prior to primary sensitisation were tolerant to re-sensitisation. However, at higher doses of ssUVR, these mice were protected from tolerance. Interestingly, while low doses of UV A caused immunosuppression, even lower doses enhanced the response to the second sensitisation. Higher doses of UVA had no affect. UVB induced tolerance in a dose related manner. Thus, ssUVR only induces immunosuppression and tolerance over a narrow dose range. Both UVA and UVB are immunosuppressive at this dose, while higher doses of UVA protect from the suppressive effects of UVB. Surprisingly very low doses of UVA enhanced memory development. Thus UVR has complex effects on the immune system depending on dose and spectrum.

Epidermal cells produce a panel of antioxidants as well as cytokines after UVB irradiation, which counteract reactive oxygen species, however, how these antioxidants might regulate melanogenesis is unclear. An important constituent of the cellular antioxidant buffering system which controls the redox state of proteins is thioredoxin (TRX), a 13-kD protein that catalyzes thiol-disulfide exchange reactions, regulates activation of transcription factors, and possesses several other biological functions similar to cytokines. TRX suppressed the UVB-induced production and secretion of $\alpha$-melanocyte stimulating hormone ($\alpha$-MSH) and of adrenocorticotropic hormone (ACTH), and also suppressed proopiomelanocortin (POMC) mRNA expression by normal human keratinocyte (KC)s. Further, L-cysteine, N-acetyl-cysteine, $\alpha$-tocopheryl ferulate showed suppressive effect on UVB-induced POMC mRNA expression. However, TRX released from UVB-irradiated KCs stimulated melanogenesis by up-regulating MSH receptor expression and its binding activity in melanocyte (MC)s. UVB-induced KC derived cytokines such as IL1, IL6, and ET1 upregulated MSH-receptor binding ability as well as MCl-R mRNA expression in cultured normal human MCs. MCl-R has a tendency to be upregulated by UVB-induced KC-derived cytokines as well as by direct UVB irradiation. These results suggest that antioxidants such as TRX suppresses UVB induction of POMC, but in the case of MCl-R, this gene can be mainly in the trend of upregulation by UVB-induced KC-derived factors including TRX.

Nitric oxide (NO) is a newly described transmitter involved with cell to cell communication that is generated in biologic tissues by specific types of nitric oxide synthase (NOS), which metabolize L-arginine and molecular oxygen to citrulline and nitric oxide. In the skin. NO has been reported to play an important role in such diseases as psoriasis, atopic dermatitis, and contact dermatitis, as well as act as an important modulator in UVB-induced erythema. Ultraviolet B irradiation to the skin evokes an increase in NO production in the epidermis through two pathways; induction of inducible NOS, mediated by inflammatory cytokines, and elevation of constitutive neuronal NOS activity. In a cell culture system, it has been demonstrated that NO functions as a melanogen after being produced in keratinocytes in response to UVB-irradiation. NO-stimulated melanogenesis in melanocytes is mediated by the cGMP/PKG pathway. In this study, up-regulation of tyrosinase gene expression by NO-stimulation and the involvement of NO in UVB-induced pigmentation were examined. In NO-induced melanogenesis, protein synthesis and tyrosinase activity increased along with an up-regulation of tyrosinase gene expression. In an animal model, UVB-induced pigmentation in skin was suppressed by sequential daily treatments with a specific inhibitor of NOS. Thus, NO plays an important role in UVB-induced pigmentation, where its function as a melanogen is considered to be one of the mechanisms. Together with its role in the development of erythema, NO contributes to the total protective response of skin against UVB-irradiation.

Cutaneous hyperpigmentation is a well-known consequence of both acute and chronic X-irradiation, although the molecular mechanisms involved are not well understood. Recently, protein kinase C-$\beta$ (PKC-$\beta$) was shown to activate tyrosinase, a key and the rate-limiting enzyme in melanogenesis [1]. In this study, we have investigated its role in mediating ionizing radiation-induced pigmentation by exposing cultured human melanocytes to X-irradiation. Increased tyrosinase activity after the 4 Gys exposure was observed within 48 hrs and total melanin content doubled after 7 days. Interestingly, tyrosinase mRNA level was not affected by X-irradiation. However, there was a 2-3 fold increase in PKC-$\beta$ mRNA after 48 hours of irradiation, coinciding with the increase in tyrosinase activity. This induction was not due to non-specific heat generated during the irradiation because when melanocytes were incubated at 4$0^{\circ}C$, there was no induction of PKC-$\beta$ mRNA. Taken together, these data suggest that X-irradiation induces cutaneous hyperpigmentation, at least in part, by up-regulating the level of PKC-$\beta$.

Skin pigmentation, also known as complexion coloration, results from the biosynthesis of melanin within the melanocytes of the Stratum basalum and the subsequent transfer, translocation, and degradation of this pigment to, in, and by the neighboring keratinocytes respectively, Melanins are produced and retained in melanosomes synthesized in the cell body that are translocated along the dendrites using microtubules via motor proteins. Melanosomes are eventually captured and retained at the tips of dendrites by attachment to the peripherally localized actin. Melanosomes reaching the dendritic tips are transferred to keratinocytes, primarily via phagocytosis of released melanosomes by keratinocytes. Molecules responsible for cell/cell recognition and interaction that regulate transfer are being identified. Some of these putative mediators appear to be affected by ultraviolet radiation. After the keratinocytes receive melanosomes, the granules are distributed individually or as clusters in dark versus light skin respectively. These melanosomes are then aggregated over the nucleus for photoprotection ofkeratinocyte DNA and eventually degraded.

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation. PI staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes after UVB irradiation. The level of p53 and Bax revealed a dose-dependent increase with increasing dose of UVB, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved trom a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of Bcl-2. We next examined the downstream targets of apoptosis. Our results showed that a precursor form of caspase-3 disappeared with increasing doses of UVB. We also observed cleavage of poly(ADP-ribose) polymerase (PARP) after UVB irradiation. In addition, UVB irradiation resulted in a remarkable activation of c-Jun N-terminal kinase (JNK). These results indicate that UVB may induce apoptosis via JNK activation in human melanocytes.

Carcinogenesis can be theoretically divided to intiation step and promotion step. Intiation associates with genetic alterations including p53 tumor suppressor gene and ras oncogene. Promotion involves in clonal expansion of of an initiated cell by epigenetic mechanism, mainly through signal transduction and gene expression. Ultraviolet light (UV) acts as both initiator and promoter. Initiation is closely related with DNA damage induced by UV, including cyclobutane pyrimidine dimers, (6-4) photoproducts and 8-hydroxy-2'-deoxyguanosine. Cyclobutane pyrimidine dimers and (6-4) photoproducts are directly induced by UV, while 8-hydroxy-2'-deoxyguanosine is induced indirectly by the reactive oxygen species. Because initiation is an irreversal genetic event, while promotion is a reversal and epigenetic event, to know the molecular mechanisms of tumor promotion in UV-carcinogenesis is crucial to develop preventive medicine and suppress UV-carcinogenesis. Because ROS is also involved in signal transduction of the cell, anti-oxidant could be the good candidate of anti-promoting agent. Here, we describe the suppressive effect of UV-carcinogenesis by various anti-oxidant including olive oil. In addition, we discuss about the mechanism of UVB-induced expression of cyclooxygenase-2, which might be a representative molecule involved in promotion of UV-carcinogenesis.

Bcl-2 is a member of large bcl-2 family and protects cells from apoptosis. Using bcl-2-expressing adenovirus vector (Ad-bc1-2), we investigated the effect of bc1-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bc1-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (lx10$^{6}$ ) were transfected at Ixl0$^{8}$ PFU/ml. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on bcl-2-expression level. Following UVB irradiation caspase 8, 3, 9 activities were stimulated in NMK cells, while in bc1-2-transfected cells, only caspase 8, but not caspase 3 or 9 activities were stimulated. In order to investigate the effect of bc1-2 in vivo, topical application of Ad-bc1-2 on tape-stripped mouse skin was performed. Following the application, Bc1-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bc1-2 at 1st day following the application of lxl0$^{9}$ PFU in 200ml. The introduced Bc1-2 remained at least for 6 days. UVB irradiation (1250 J/m$^2$) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Bc1-2-transfected mice skin were resistant to UVB-induced apoptosis. Topical application of empty adenovirus vector alone had no effect on Bc1-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.

There are two distinct UV-responsive signaling pathways in UV-irradiated mammalian cells, i.e., the DNA damage-dependent and -independent pathways. The former occurs in nucleus and results in growth arrest and apoptosis via post-translational modification of p53. The latter is initiated by oxidative stress and/or by damages in cell membrane or cytoplasm, which activate signaling cascade through intracellular molecules including mitogen activated protein kinases (MAPK). In normal human fibroblastic cells, all of MAPK family members, extracellular signal-related kinases (ERK), c-Jun N-terminal kinases (JNK) and p38, were rapidly phosphorylated following UV-irradiation. ERK phosphorylation was suppressed by an inhibitor of receptor tyrosine kinases (RTK). As ERK usually responds to mitogenic stimuli from RTK ligands, UV-induced ERK phosphorylation may be linked to the proliferation of survived cells. In contrast, phosphorylation of JNK and p38, as well as apoptosis, were modulated by the level of UV-generated oxidative stress Therefore, JNK and p38 may take part in oxidative stress-mediated apoptosis. Phosphorylation of p53 at Ser and Thr residues are essential for stabilization and activation of p53. Among several sites reported, we confirmed phosphorylation at Ser-15 and Ser-392 after UV-irradiation. Both of these were inhibited by a phosphoinositide 3-kinase inhibitor, presumably due to the shutdown of signals from DNA damage to p53. Phosphorylation at Ser-392 was also sensitive to an antioxidant and a p38 inhibitor, suggesting that Ser-392 of p53 is one of the possible points where DNA damage-dependent and -independent apoptic signals merge. Thus, MAPK pathway links UV-induced intracellular signals to the nuclear responses and modifies DNA damage-dependent cellular outcome, resulting in the determination of cell death.

Hydroa vacciniforme (HV) is a disease of Epstein-Barr virus (EBV)-associated NK/T cell lymphoproliferative disorder, and patients with severe HV-like eruptions have a high risk to progress to various EBV-related hematological complications. Photosensitivity in HV might be a 'photo-Koebner' phenomenon mediated by infiltration of EBV-infected NK/T cells.

Sunscreens have been advocated as an important means of preventing skin cancer. UV-induced immunosuppression is important for skin cancer development, yet the effectiveness of sunscreens in protecting the human immune system from UV radiation is unclear. The only currently accepted method of sunscreen rating is the Sun Protection Factor (SPF) based on prevention of erythema. We developed an in vivo non-invasive method for evaluating protection of the human immune system from UV radiation based on recall contact sensitivity to nickel, a common allergen. Using this system we showed that broad-spectrum sunscreens provide greater protection to the immune system than sunscreens which protect from UVB only. UVA was found to be immunosuppressive. We developed this technique to enable the study of solar simulated UV radiation dose responses and determined Immune Protection Factors (IPFs) for six commercially available sunscreens based on limits of protection from the dose response data. We found that the IPF did not correlate with the SPF and that protection from erythema therefore cannot be used to predict protection of the immune system. However, IPF was significantly correlated to the UVA protective capability of the sunscreens, indicating that sunscreen protection from UVA is important for prevention of immunosuppression. We recommend that sunscreens should be rated against their immune protective capability to provide a better indication of their ability to protect against skin cancer.

In Drosophila melanogaster, it is known that the circadian clock consists of an autoregulatory feedback loop, which includes so-called clock genes, such as per, tim, dClk and cyc and produces periodical expression of per. It is recently suggested, however, that the circadian oscillation without the rhythmical expression of per is involved in the regulation of circadian locomotor rhythms. In the present study, we examined the existence and the property of the possible per-less oscillation using arrhythmic clock mutant flies carrying per$^{01}$, tim$^{01}$, dClk$^{Jrk}$ or cyc$^{01}$. When temperature cycles consisting of 25$^{\circ}$Ｃ and 30$^{\circ}$Ｃ with varying periods (T = 8~32 hr) were given, they showed rhythms synchronizing with the given cycle under constant darkness (DD). per$^{01}$ and tim$^{01}$ flies always showed a peak around 7 hr after the onset of thermophase irrespective of Ts of temperature cycles, while dClk$^{Jrk}$ and cyc$^{01}$ flies did not. In addition, several days were necessary to establish a clear temperature entrainment in per$^{01}$ and tim$^{01}$ flies, when they were transferred from a constant temperature to a temperature cycle under DD. These results suggest that per$^{01}$ and tim$^{01}$ flies have a temperature-entrainable weak oscillatory mechanism. The fact that dClk$^{Jrk}$ and cyc$^{01}$ flies did not show any sign of the endogenous oscillation suggests that the per-less oscillatory mechanism may require CLK and CYC.

Pigment-dispersing factor (PDF) is an octadecapeptide distributed in the optic lobe and the brain in a variety of insect species. There are lines of evidence suggesting possible involvement of PDF in the insect circadian system. However, its physiological roles in the circadian time keeping mechanism have not been clearly defined. In this study, we have examined the phase shifting effects of Gryllus-PDF on the circadian locomotor rhythm in the cricket Gryllus bimaculatus of which circadian clock is located in the optic lobe. Phase shifts in the circadian activity rhythm were measured following microinjection of 22nl of vehicle (Ringer's solution) or O.lmM PDF into the optic lobe through the compound eye at various circadian times. The results showed that PDF induced phase shifts of the circadian clock in a phase-dependent manner, suggesting that it may play a role as an input signal for the circadian clock.

Light is a major environmental signal for entrainment of the circadian clock, but little is known about the phototransduction pathway triggered by light-activation of photoreceptive molecule(s) responsible for the phase shift of the clock in vertebrates. The chicken pineal gland and retina contain the autonomous circadian oscillators together with the photic entrainment pathway, and hence they provide useful experimental model for the clock system. We previously demonstrated the expression and light-dependent activation of rod-type transducin $\alpha$-subunit (Gtl$\alpha$) in the chicken pineal gland. It is unlikely, however, that the pineal Gt$_1$$\alpha$ plays a major role in the photic entrainment, because the light-induced phase shift is unaffected by bloking the signaling function of Gt$_1$$\alpha$. Here, we show the expression of G 11 $\alpha$, an $\alpha$-subunit of another heterotrimeric G-protein, in the chicken pineal gland and retina by cDNA cloning, Northern blot and Western blot analyses. GIl$\alpha$-immunoreactivity was colocalized with pinopsin in the chicken pineal cells and it was found predominantly at the outer segments of photoreceptor cells in the retinal sections, suggesting functional coupling of G11 $\alpha$ with opsins in the both the tissues. By coimmunoprecipitation experiments using the retina, we showed the light- and GTP-dependent interaction between rhodopsin and G11 $\alpha$. Upon ectopic expression of a Gq/ 11-coupled receptor in cultured pineal cells, pharmacological (non-photic) activation of endogenous G11 induced phase-dependent phase shifts of the melatonin rhythm in a manner very similar to the effect of light. These results suggested opsin-G11 pathway contributing to the photic entrainment of the circadian clock.

The avian pineal as well as the retina has been known to contain several types of photoreceptors with different visual pigments such as rhodopsin, iodopsin and the pineal specific opsin, pinopsin. These organs are also known to have circadian clock to regulate melatonin production. Exposure of animals to light causes a decline of the melatonin level and the phase shifts of melatonin rhythms in the pineal and retina. Therefore, the circadian clock system of these organs seem to consist of three elements, i.e., light input, oscillator and melatonin output systems. In birds, it was suggested that rhodopsin might be involved in the entrainment of pineal melatonin rhythms from the action spectrum experiment for controlling NAT activity rhythms. However, there are much more pinopsin-immunoreactive (Pino-IR) cells than rhodopsin (Rho-IR) and iodopsin (Iodo-IR) cells in the avian pineal. We found that Pino-IR cells appeared earlier embryonic stages than Rho-IR and Iodo-IR cells. So, we tried to identify the visual pigments involved in the circadian melatonin rhythms in the pineal and retina. Organ cultured pineals were exposed to monochromatic light to find out which opsin participates in regulation of melatonin rhythms. The action spectra showed a peak at 475nm, suggesting that pinopsin is the major photopigment to regulate melatonin production in birds.

Djungarian hamsters show distinct seasonal rhythms in several physiological parameters. One of them is daily torpor that occurs in winter with decreased body temperature (about 1O-20$^{\circ}$C) during daytime. Daily torpor is induced by short-day photoperiod, food restriction and castration. But the mechanism to induce daily torpor has not been clarified. In the present study, we tried to clarify the process of daily torpor induction in detail. Adult male hamsters were kept in long photoperiod and high temperature (LP-HT) before the experiment and, thereafter, the animals were transferred to short photoperiod and low temperature (SP-LT), and they were kept in this condition for about six months. The daily rhythms of locomotor activity and body temperature were recorded every three-minutes by using the Minimitter telemetry system. Locomotor activity and body temperature showed very closely synchronized rhythms. All animals under LP-HT showed daily rhythms with higher locomotor activity and body temperature in nighttime than in daytime. Under SP-LT, there were two types of animals with and without showing daily torpor. Thus, they have individual differences in the response to SP -LT.

In birds, the photoperiodic seasonal breeding involves encephalic photoreception at the initial step of triggering the well-known endocrinal cascade. Especially in Japanese quail (Coturnix coturnixjaponica), the reproductive neuroendocrine function responds to a single long day, and hypothalamic regions are known to be important for the reproductive response. However, little is known about where and how the light and time signals are integrated to detect daylength information and transduced to the endocrinal responses. To gain insights into this issue, we are interested in the c-Fos expression in the hypothalamus of the Japanese quail. Meddle and Follett (1997) previously identified two hypothalamic regions where c-Fos-like immunoreactivities were induced in response to a long day by using an antibody to carboxyl terminal region of human c-Fos (Lys$^{347}$ -Leu$^{367}$ ). In the present study, we used a different anti-c-Fos antibody recognizing a region from Lys$^{128}$ to Ala$^{152}$ of human c-Fos, and found in long-day- stimulated quails many c-Fos-like immunoreactive nuclei localizing within two regions, nucleus anterior medialis hypothalami and nucleus periventricularis hypothalami, which are distinct from those identified in the previous study. Then, we focused on the difference in the cross-reactivities of the antibodies used, and determined the whole coding sequence of quail c-Fos to compare the antigenic sequences of the two antibodies with the amino acid sequence of quail c-Fos. We found that the antibody we used would recognize quail c-Fos more specifically than that used in the previous study.

We investigated the interaction of serotonin and galanin (GA) by a double immunostaining method in the Japanese quail. Serotonin-immunoreactive (IR) cells were located in the paraventricular organ (PVO) and infundibular nucleus (IF). The number of the cells under short-day photoperiod (SD) was less in the dark phase than in the light phase. GA-IR cells were found in the PVO, IF and median eminence. The number of GA-IR cells in SD was significantly greater than that in long-day photoperiod (LD). Numerous GA- IR varicose fibers ran along serotonin- IR cell bodies and nerve fibers in the PVO and IF of the same sections. Very few serotonin-IR fibers ran along GA-IR cell bodies and GA-IR nerve fibers in the ventral part of the IF. The present results suggest that the possibility of functional interaction takes place between serotonin- and GA- IR neurons in the PVO and IF.

The presence of retinals (retinal and 3,4-didehydroretinal) has been known in the eggs of wide range of oviparous vertebrates, but the biological significance of the egg retinals has yet to be clarified. We here show that retinals are the major components of retinoids in the eggs of all species of chordate animals we examined. The egg retinals were commonly bound to egg yolk proteins, the storage proteins, via a Schiff base linkage. The Schiff base linkage, which protects the reactive aldehyde group, would negate the toxicity of aldehyde, and enable to accumulate much amount of retinals. The retinals in chordate eggs are considered to be the precursor of functional retinoids, such as photoreceptive pigment chromophores and retinoic acid, during development. The results of the present research strongly suggest that retinals in the eggs of oviparous chordates are the common and essential mode of retinoid storage.

The absorption wavelength of the protonated retinal Schiff base can be controlled by the surrounding environment. An external anion is related to fine adjustment of the absorption wavelength. The addition of anion to retinochrome solution caused blue shift in spectra. The increase of the shift was dependent on the ion concentration. The large shift value was obtained as 20 nm at the saturated concentration of nitrate. The shift intensity for the nitrate addition exceeded that of chloride. Seemingly, it depends on the ionic strength or lyotropic character of the anion. However, neither of sulphate nor gluconate ion showed remarkable blue shift. These phenomena were accounted for with (1) delocalization of the positive charge in the conjugated polyene system, (2) ionic bonding strength between the counter ion (glutamate) and the proton, and/or (3) interaction of the added anion with the proton on Schiff base.

A vertebrate retina is an organ belonging to the central nerve system (CNS), and is usually difficult to regenerate except at an embryonic stage in life. However, certain species of urodele amphibians, such as newts and salamanders, possess the ability to regenerate a functional retina from retinal pigment epithelial (RPE) cells even as adults. After surgical removal of neural retinas from adult newt eyes, the remaining RPE cells lose their pigment granules, transdifferentiate into retinal progenitor cells, which further differentiate into various retinal neurons, and then finally reform a functional neural network. To understand the molecular mechanisms of CNS regeneration, we attempted to investigate the genes expressing in regenerating newt retina. mRNAs were isolated from regenerating retinas at 18-19 days after the surgical removal of the normal retina, and a cDNA library (regenerating retinal cDNA library) were constructed. Our EST analysis of 112 clones in the regenerating cDNA library revealed that about 70% clones are closely related to the genes previously identified. About 40% clones are housekeeping genes, and about 15% clones encode proteins related to the regulation of gene expression and to the proliferation of the cells. Sequences similar to neural retina- and RPE-specific genes were not detected at all. These results led us to suppose that the regenerating retinal cells are in a state considerably different from those of neither neural retina nor RPE cells.

To investigate the visual and extra-ocular photoreception, we cloned the opsin genes in ayu (Plecoglossus allivelis). Amplified fragments encoding exon-4 (-5) of opsin cDNAs were cloned from the retina and brains of ayu, and sequenced. One clone was identified as rod (AYU-Rh), two as green cone (AYU-GI, -G2), one as red cone (A YU-R), two as ultraviolet cone (AYU-UVl, UV2), one as VA (AYU-VA), and one as extra-ocular rod (AYU-ExoRh) opsins. 335 amino acids sequence deduced from the full-length cDNA of AYU-Rh showed high identity with that of other fish. Southern blotting analysis indicated that ayu possess two 'rhodopsin' genes, one is visual rhodopsin and the other is non-visual extra-ocular rhodopsin. In situ hybridization showed that the mRNA of AYU-Rh was localized only in rod cells in the retina. On the other hands, AYU-ExoRh was expressed only in the pineal. We cloned two isoforms (AYU-VAM and -VAL) of VA opsin from ayu. The deduced amino acid sequences of these variants were identical to each other within the first 342 residues, but they showed divergence in the C-terminal sequence. AYU- VAL corresponded to the long isoform found in other fish, and AYU-VAM was identified as a new type of VA opsin variant. Pal-VAM is a new probably functional non-visual photoreceptive molecule in fish.

The anterior brain vesicle of ascidian larvae contains two distinct pigment cells. Ultrastructure of these pigment cells has been shown that the anterior pigment cell is an otolith for perception of gravity and the posterior pigment cell is an ocellus for light reception. The larva has remarkably simple central nervous system (CNS) composed of about 330 cells. We focused to study neural networks of visual systems. In the present paper, we report the whole structure of the photoreceptors of the ascidian larva visualized by an antibody against arrestin. Visual arrestin is the key protein for the termination of phototransduction and one of the abundant proteins in photoreceptors. Recently, we cloned an arrestin homologue gene, Ci-arr and the expression of Ci-arr was found to be restricted to the photoreceptors in the ocellus. To study the whole structure of the photoreceptors in the larva, we prepared an antibody against Ci-Arr. It is found that anti Ci-Arr antibody specifically stains the photoreceptors, including the cell bodies, the axons, and the nerve terminals. The photoreceptor cell bodies lies in row outside the pigment cup which penetrate the pigment cell and is continuous with the outer segments of the photoreceptor cell, inside the concavity of the pigments. The axons form bundle into a single tract. The tract extends toward the midline, where the nerve terminals diverge and seem to form synapses

We have recently found that a detergent-resistant raft like membrane (DRM) can be prepared from bovine rod outer segment membranes as a low-density buoyant fraction in sucrose density gradient ultracentrifugation. G protein (transducin) and its effector enzyme (phosphodiesterase: PDE) drastically change their affinities to DRM in the process of phototransduction. We report here that the recruitment of transducin and/or $^2$PDE to DRM has close relationship with their states in signal transduction. Active T$\alpha$/PDE-complex has a high affinity to DRM, whereas inactive transducin, or inactive PDE are excluded from DRM. Active T$\alpha$/PDE-complex seems to bind to a GTPase activating protein (GRS9) in multi- protein complexes localized on DRM. Physiological significance of the multi-protein complex on the raft-like membrane in vertebrate phototransduction would be discussed.

We have cloned a hydromedusan opsin cDNA and showed that the deduced amino acid sequence of the cytoplasmic loop between helices 5 and 6 (loop 5-6) was clearly different from that reported so far. The amino acid sequence of the loop 5-6 is important on determination of the specificity for the coupled G- protein. To clarify which class of G-protein mediates the phototransduction system in the ocellus of the hydromedusan, we investigated G-proteins expressed in the ocellus. By PCR against the cDNA of the ocellus with primers designed according to the conserved amino acid sequence in G-protein a subunit, we obtained three kinds of cDNA fragments. Based on the sequence similarities, ttwo of them (JGI and JG3) were classified as $G_{i}$ and $G_{q}$, respectively. The other one (JG2) was a new subtype within $G_{*}$ class. Electron microscopic immunocytochemistry with the antiserum against the C-terminal sequence of $G_{q}$ or $G_{t}$ revealed the presence. of the both classes in the ocellus. The similarity of the C-terminal sequence of the JG2 with that of bovine $G_{t}$ suggests that the anti- $G_{t}$ antiserum would bind to JG2. These results suggest the possibility that the hydromedusan rhodopsin decides the specificity for the coupled G-protein by the other domain than the loop 5-6.oop 5-6.5-6.

S-modulin in frog or its bovine homologue, recoverin, is a 26 kDa EF-hand $Ca^{2+}$-binding protein found in rod photoreceptors. The $Ca^{2+}$ -bound form of S-modulin binds to rhodopsin kinase (Rk) and inhibits its activity. Through this regulation, S-modulin is believed to modulate the light-sensitivity of a rod. In the present study, we tried to identify the interaction site of the $Ca^{2+}$ -bound form of S-modulin to Rk. First, we mapped roughly the interaction regions by using partial peptides of S-modulin. The result suggested that a specific region near the amino terminus is the interaction site of S- modulin. We then identified the essential amino acid residues in this region by using S-modulin mutant proteins: four amino acid residues were suggested to interact with Rk. These residues are located in a small closed pocket in the $Ca^{2+}$-free, inactive form of S-modulin, but exposed to the surface of the molecules in the $Ca^{2+}$ -bound, active form of S-modulin. Two additional amino acid residues were found to be crucial for the $Ca^{2+}$ -dependent conformational changes of S-modulin. The present study firstly identified the functional site of S-modulin, a member of a neuronal calcium sensor protein family.in family..

The inverted retina of the Onchidium dorsal eye (DE) is composed only of ciliary-type photoreceptor cells (CC's). The outer segment (OS) of the CC is a concentric lamellar structure consisting of many modified ciliary membranes and stains positively with anti-$\beta$-tubulin antibody. Near the base of the OS there are about 30 basal bodies each connecting individually to a cilium. The cilia are rod-shaped at the base, progressing upwards to a flattened sheet-like shape with increasing surface area. Three-dimensional analysis on serial sections demonstrates the ladle-shape of a modified cilium. Many modified cilia wrap around each other like the leaves of a cabbage. Nine pairs of microtubules (MT's) are located regularly in a ring at the base of the cilium, gradually losing their regular arrangement towards the periphery, where they separate into two subgroups that are contained within two swollen portions of a modified cilium. Within the CC of the Onchidium DE, MT's in the modified cilium exist as two poles extending longitudinally in a thin expanded ciliary membrane. This arrangement may support the photoreceptive OS and serve to maintain its structural integrity.

Planarians show negative phototaxis and have extensive regenerative ability, including the ability to regenerate the brain. Recently the process of regeneration of the planarian brain has been divided into three steps based on the expression of neural markers. In this study, we have analyzed the process of recovery of the light response during head regeneration. Although morphological observations indicated that regeneration of the eyes and optic nerves appeared to be completed by the fourth day, the recovery of the evasion behavior against light was not recovered within 4 days after amputation. Functional recovery of the evasion behavior could be detected starting 5 days after amputation and then gradually recovered. We previously identified genes which are specifically expressed in the brain after the recovery of morphological structures. This characteristic suggested that these genes may be involved in functional recovery of the brain. To investigate the function of these genes, we performed gene knockout analysis using the RNA interference method. The results clearly indicated that these genes are involved in the functional recovery of the visual system.

The intermediate M420 formed in a solution of firefly luciferin in deoxygenated dimethyl-sulfoxide added potassium f-butoxide was observed to emit yellow-green and red light by filter-photometry. By H-NMR, M420 was found to be deprotonated at the site where luciferin reacts with oxygen.

The structural stability of bacteriorhodopsin (bR) solubilized by Triton X-100 (TX-100) was studied by measuring the denaturation kinetics in the dark and under illumination, and compared with the structural stability of bR solubilized by octyl-${\beta}$-glucoside (OG). In the dark, bR solubilized by TX- 100 was more stable than bR solubilized by OG. Under illumination, bR solubilized by TX-100 showed light-induced denaturation in the same manner as bR solubilized by OG. These results in the dark well correlated with the experimental results of the visible CD band. Although solubilized bR in the TX-100 concentration range of 2-50 mM showed almost identical positive CD band and did not denature in the dark at 35$^{\circ}$C, the kinetic constant of the photobleaching increased with the increase of TX-100 concentration. These results suggested that photo-intermediates of solubilized bR are destabilized by TX-100 micelles.

Irreversible photobleaching of bacteriorhodopsin (bR), namely denaturation induced by illumination of visible light, was investigated by absorption kinetic measurements. The denaturation kinetics revealed that light illumination significantly enhanced the structural decay of bR. The kinetic analyses showed that the molecular structure of bR denatures according to a single-exponential decay, whereas irreversible photobleaching has two decay components. The decay constant of the slow component of photobleaching is almost same as that in the dark. An Arrhenius plot of the denaturation kinetic constants for the fast and slow components showed similar activation energies of approximately 19 kcal/mol.

Recent denaturation experiments of bacteriorhodopsin (bR) in the dark and under illumination at high temperatures revealed that irreversible thermal bleaching occurs above ~ 70°C and the preceding reversible structural changes in the dark above 60°C are closely related to irreversible photobleaching observed in the same temperature range (Yokoyama et al. (2002). J Biochem. 131,785). In this study, structural properties of bacteriorhodopsin (bR) at high temperatures were extensively probed by hydroxylamine reactivity with the Schiff base in the dark and hydrogen-deuterium (H-D) exchange in the peptide groups. In the Arrhenius plot from kinetics measurements of the hydroxylamine reaction, a good linear relationship between the reaction time constant and the inverse of the absolute temperature was observed below 60°C, while significant increase started above 60°C, suggesting that remarkable increase in water accessibility of the Schiff base in the temperature region. FT-IR spectroscopic studies on the H-D exchange suggested increase in the deuterium exchanges rate of the peptide hydrogen in the same temperature region.

pharaonis phoborhodopsin (ppR), a photophobic sensor of haloalkaliphilic bacteria, Natronobacterium phar-aonis, has retinal as a chromophore covalently bound to Lys in G-helix via a protonated Schiff base (PSB), as is the same as bacteriorhodopsin (bR). For ppR, the corresponding counter-ion is Asp residue (Asp75) located in C-helix. Here we investigated the influence of the protonated state of this counter-ion and its location on the chromophore configuration. Under alkaline condition, the chromophore configuration of D75E mutant was analyzed by HPLC. D75E had a much larger content of 13-cis isomer: the ratio of 13-cis to all-trans was 6:4 while the wild-type had this ratio of 1 :9. On the other hand, under acidic condition where Glu was associated, D75E had no 13-cis retinal isomer. Mutants whose Asp75 was replaced by neutral amino acids (D75N and D75Q) did not contain 13-cis retinal. Furthermore, retinal isomer compositions and the change in the visible ab- sorption spectra (indicating the dissociation state of Glu75) were measured under varying pH, and these were almost the same dependencies. These results indicate that an important factor determining the 13-cis isomer content is the presence of negative charge of the counter-ion against PSB, but not the size of this residue. Com- parison between the wild-type and D75E in alkaline solutions indicates the influence of the location of the counter-ion.

Spectroscopic titration of D 193N and D 193E mutants of pharaonis phoborhodopsin (ppR) were performed to evaluate the pK$_{a}$ of the Schiff base Asp 193 corresponds to Glu204 of bacteriorhodopsin (bR). The pK$_{a}$ of the Schiff base (SBH$^{+}$) of D193N was 10.1~10.0 (at XH$^{+}$) and 11.4~11.6 (at X) depending on the protonation state of a certain residue (designated by X) and independent on CI$^{[-10]}$ , while those of the wild-type and D193E were> 12. pK$_{a}$ of XH$^{+}$ were; 11.8~11.2 at the state of SB, 10.5 at SBH$^{+}$ state in the presence of CI$^{[-10]}$ , and 9.6 at SBH$^{+}$ without CI$^{[-10]}$ These imply the presence of a long-range interaction in the extracellular channel.r channel.

Bacteriorhodopsin (bR) and halorhodopsin (hR), which exist in the membrane of Halobacterium salinarum, are light-driven ion pumps. In spite of high similarity of primary and tertiary structures between bR and hR, these membrane proteins transport different ions, proton and chloride, in the opposite direction. From alignment of the amino acid sequences, Thr-89 of bR is homologous to Ser-l15 of hR from Halobacterium salinarum (shR). X-ray structure of shR has revealed that OH group of this residue directly interacts with CI$\^$-/ Thus, Ser-lI5 of shR is expected to play an important role in CI$\^$-/ binding and transport. In this study, we expressed wild type hR from Natronobacterium pharaonis (PhR) and Sl30A, which corresponds to Ser-l15 of shR, in E. coli in order to clarify binding affinity of chloride ion and photocycle reactions. From the titration with CI$\^$-/, affinity of Sl30A became quite lower than that of WT (WT 6 mM, Sl30A 89 mM). Furthermore, from the flash photolysis with pulse laser of λ$\_$max/ at 532 nm, the reaction rate of SI30A from 0 intermediate to hR ground state was found to become apparently slower than that of WT. The singular value decomposition (SVD) and global fitting analyses of the photocycles were performed to identify all photointermediates and determine the reaction rates.

X-ray structures of pharaonis phoborhodopsin (ppR) show the different direction of the side chain of Arg72 from that of the corresponding residue (Arg82) of bacteriorhodopsin, BR. For BR, this residue is considered to play an important role in the proton pumping. In order to investigate the role of Arg72 in ppR, we constructed Arg72 mutants of R72A, R72K and R72Q, and measured the photocycle and proton pumping activities. The pH-titration curves on the absorption maximum of the mutants were shifted to alkaline in comparison of that of the wild-type. This may imply the increase of pKa of D75, suggesting the presence of the (probably electric) interaction between D75 and Arg72. Rate constants of the M-decay were 3-7 times faster than that of the wild-type, and the time for the completion of the photocycling was also reduced. Using Sn0$_2$ electrode, the rate of transmembrane proton transport was measured upon illumination. The photo-induced proton pumping activities were estimated after the corrections that are the percentages of the associated form of D75 (which has no pumping activity) and the photocycling rates. R72A and R72Q showed the reduced activity while R72K did not reduce the activity.

In halobacterial membrane, pharaonis phoborhodopsin (or pharaonis sensory rhdopsin II, psRII) forms a complex with its transducer pHtrII. Flash-photolyis of D75N mutant did not yield M-intermediate but an O-like intermediate is observed. We examined the interaction between D75N of ppR and t-Htr (truncated pHtrII). These formed a complex in the presence of n-dodecyl-$\beta$-D-maltoside, and the association accelerated the decay of the 0 of D75N from 15 to 56 s$\^$-1/. From the decay time constants under varying ratios of D75N and t-Htr, n, the molar ratio of D75N/t-Htr in the complex, and K$\_$D/, the dissociation constant, were estimated. The value of n was unity and K$\_$D/ was estimated to 146 nM. This K$\_$D/ value can be considered as the association between the photo-intermediate and t-Htr, which is deduced by the method of estimation. Previously we (Photochem. Photobiol. 74, 489-494 (2001)) reported K$\_$D/ of 15 $\mu$M for the interaction between the wild-type and t-Htr by means of the change of M-decay rates. Therefore, this value should be the K$\_$D/ value for the interaction between M of the wild-type and t-Htr.

All-optical switching has been demonstrated in bacteriorhodopsin (bR) based on nonlinear intensity induced excited state absorption. The transmission of a cw probe laser beam at 410 nm corresponding to the peak absorption of M state through a bR film is switched by a pulsed pump laser beam at 570 nm that corresponds to the maximum initial 8 state absorption. The switching characteristics have been analyzed using the rate equation approach considering all the six intermediate states (B, K, L, M, N and 0) in the bR photocycle. The switching characteristics are shown to be sensitive to life time of the M state, absorption cross-section of the 8 state at probe wavelength ($\sigma$ $\_$Bp/) and peak pump intensity. It has been shown that the probe laser beam can be completely switched off (100 % modulation) by the pump laser beam at relatively low pump powers, for $\sigma$$\_$Bp/ = O. The switching characteristics have been used to design all-optical NOT, OR, AND and the universal NOR and NAND logic gates for optical computing with two pulsed pump laser beams.

Archaerhodopsin (aR), a light-driven proton pump found in HaIonubrum sp. aus-l, was crystallized into an octahedral crystal belonging to the space group P4$_3$2$_1$2. It is shown that aR is composed of7 helical segments and an anti-para1leI ${\beta}$ sheet. The main-chain sIrudure of aR is nearly identical to that of bacteriorhodopsin, but a significant structural difference is observed in the protein surface, especially at lipid binding sites.

Bacillus Anthracis is the causative agent of anthrax. The major virulence factors are a poly-D glutamic acid capsule and three-protein component exotoxin, which is collectively known as anthrax toxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin and edema toxin), causing different pathogenic responses in animals and cultured cells. However, it remains to be elucidated for pathogenic mechanism of anthrax toxin. In this study, we constructed toxin component in bacterial overexpression system and purified the native toxin from Bacillus anthracis delta sterne F32 using FPLC system. Recombinant toxin showed high homogeneity and rapid purification processes. Also, this recombinant toxin was comparable to B. anthracis native toxin in terms of cytotoxic effects on cultured cell lines.

In many plant cells, the positions of chloroplasts change in response to changes in light conditions. In the epidermal cells of the aquatic angiosperm Vallisneria gigantea, the avoidance response of chloroplasts is induced specifically by irradiation with blue light of high intensity. Possible roles of actin cytoskeleton in the blue-light-induced avoidance response of chloroplasts were investigated by partial irradiation and phalloidin staining. We showed that the blue-light-dependent redistribution of chloroplasts was induced only in the limited area, where exposed to blue light, even in individual cells. In addition. in the exposed area, the configuration of actin filaments strikingly changed compared with that before the irradiation. Short and thick bundles of actin filaments surrounding the chloroplasts changed to much longer and thinner bundles with a more stretched array. In contrast, in the unexposed area, neither the distribution of chloroplasts nor the configuration of actin filaments exhibited any changes. Cytochalasin D and latrunculin B inhibited the avoidance response of chloroplasts concomitantly with the fragmentation of actin filaments. These results indicate that the reorganization of actin filaments plays a crucial role in the induction of avoidance response of chloroplasts.

There is a difference in the inhibitory effects to supplemental UVB (wavelengths 280 to 320 nm) among Japanese rice (Oryza sativa L.), the cultivar Norin I is less resistant while the cultivar Sasanishiki is resistant. UVB induces photodamage in DNA. Cyclobutane pyrimidine dimer (CPD) is a major UV-induced DNA lesion. Photorepair, which is mediated by photolyase, is the major pathway in plants for repairing CPD. We have analyzed CPD induction and repair in Sasanishiki and its close relative Norin I using alkaline agarose gel electrophoresis. Norin I is deficient in CPD photoreactivation and excision, thus UV sensitivity correlates with deficient dimer repair [I]. The photorepair deficiency in Norin I results from a functionally altered photolyase with a photoflash analysis [2]. In this paper, we examined the UVB-sensitivity of several other UV-sensitive and -resistant cultivars and found that the CPD photolyase activity was deficient in UV-sensitive ones. It was also evident that there was a variation in the deduced amino acid sequences of CPD photolyases of the UV-sensitive and -resistant cultivars, whereas each deduced amino acid sequence of the UV-sensitive cultivars and of the UV-resistant ones was the same. These results suggest that the difference in the CPD photolyases of UV-sensitive and -resistant rice might be due to the structural alteration of CPD photolyase.

We examined the effects of supplementary ultraviolet-B (UV-B) radiation on the changes in synthesis and degradation of ribulose-I, 5-biphosphate carboxylase /oxygenase (Rubisco) and light-harvesting chlorophyll a/b binding protein of PSII (LHCII), as well as mRNA levels for small and large subunits of Rubisco (rbcS and rbcL, respectively) and LHCII (cab) with leaf age in UV-sensitive rice (Norin I) and UV-resistant rice (Sasanishiki). Both Rubisco and LHCII were actively synthesized until the leaf had fully expanded, and then decreased with leaf age. Synthesis of Rubisco, but not LHCII, was significantly suppressed by UV-B in Norin 1. The degradation of Rubisco was enhanced by UV-B around the time of the leaf maturation in the two cultivars. The levels of rbcS and rbcL were reduced by UV-B at the early leaf stages after emergence in both cultivars. The level of cab was first present at the highest level in the two cultivars, but drastically decreased due to UV-B treatment immediately after leaf emergence in Norin 1. It was proved that synthesis and degradation of Rubisco and LHCII greatly changed with leaf age: Rubisco synthesis was significantly suppressed by supplementary UV-B radiation at the transcription step during the early leaf stages. It was also suggested that the difference between the two rice cultivars in sensitivity to UV-B in the synthesis of Rubisco might be due to the specific suppression not only after transcription but also at transcription.

Blue light (BL) induces stomatal opening through activation of H$^{+}$ pump, which creates electrical gradient across the plasma membrane for $K^{+}$ uptake into guard cells. The pump is the plasma membrane H$^{+}$ -ATPase and is activated via phosphorylation of the C-terminus with concomitant binding of the 14-3-3 protein. The opening is initiated by the perception of BL through phototropin (phot), which are recently identified as BL receptors in stomatal guard cells. In this study, we provide the biochemical evidence for phots as BL receptors in stomatal guard cells. vfphot was phosphorylated reversibly by BL, and phosphorylation levels of vfphot increased earlier than those of the plasma membrane W-ATPase. BL-dependent phosphorylations of vfphot and H$^{+}$-ATPase showed similar fluence dependency. Staurosporin, an inhibitor of serine/threonine protein kinase, and diphenyleneiodonium chloride (DPI), an inhibitor of flavoprotein, inhibited BL-dependent phosphorylations of vfphot and H$^{+}$ -ATPase. These results indicate that vfphot acts as a BL-receptor mediating stomatal opening.l opening.

In broom sorghum, Sorghum bicolor Moench, UV causes anthocyanin synthesis having action peaks in UVA and UVB regions. We previously reported that UV induces anthocyanin synthesis through UVB photoreceptor and phytochrome activated by UV. Furthermore, UVA and UVB amplify phytochrome-induced anthocyanin synthesis (PIAS). Our action- spectroscopic research indicated that a UV -receptor for amplification of PIAS is likely to be the same or same type of UVB photoreceptor for induction of anthocyanin synthesis. UVA-amplification of PIAS can be explained by the action of a cryptic red light signal (CRS), an amplification factor for PIAS produced by a distinct phytochrome-species activated by UVA. We suggest that UVA photoreceptors are not involved in anthocyanin synthesis in the broom sorghum.

Bacteriochlorophyll(BChl)-c containing species of green sulfur photosynthetic bacterium Chlorobium (ChI.) vibrioforme, which has BChl-d mainly, was detected. We obtained colonies on agar plates by spreading the liquid culture of ChI. vibrioforme f. sp. thiosulfatophilum strain NCIB 8327 which contained the high ratio of BChl-c/BChl-d, and transferred each colony into a new liquid medium. These cultures after growing were found to be classified into two categories. One possessed BChl-d as a light-harvesting pigment and the other did BChl-c. No colonies examined here contained both BChls-d and c. Therefore, the presence of both BChls-d and c in our cultures of ChI. vibrioforme was ascribed to the coexistence of two different cells which had BChl-d and c as the chlorosomal pigment, respectively. The change of pigment composition observed in our liquid cultures can be thus explained by the difference of growth rates between two kinds of cells.

Eight intact bacteriochlorophyll (BChl)-d homologs and isomers were isolated from a strain of green sulfur bacterium Chlorobium vibrioforme. All the molecular structures of the BChl-d components were fully determined by a combination of mass spectrometry and $^1$H-NMR spectroscopy. The aggregation behavior of the isomerically pure BChls-d in hydrophobic organic solvents was examined with respect to the stereoisomeric configuration at the C3$^1$ position as well as the bulkiness of the C8 and C12 side-chains by using electronic- absorption spectroscopy.

Synthetic zinc chlorins possessing a hydrophylic polyoxyethylene chain at the 17 -position were prepared. An amphiphilic zinc chlorin possessing a single chIorin moiety showed absorption maxima at 675 nm in an aqueous medium, indicating that the zinc chIorin did not form large aggregates but a dimeric structure. In contrast, amphiphilic zinc chlorin dyads in which two zinc chlorin moieties were connected with a hydrophilic polyoxyethylene linkage showed red-shifted absorption band around 720-740 nm in an aqueous medium. The result indicated that the amphiphilic zinc chlorin dyad self-aggregated to form chlorosome-like oligomer.

We examined the bacteriochlorophyill/bacteriopheophytin ratios in several species of purple bacteria containing only LHI. The pigment ratios depended greatly on species. Further, Rhodospirillum rubrum showed wide variation when grown under different light intensity, and Rhodobium marinum showed significant variation from culture to culture even under the same light conditions. The protein ratios of a/RC and $\beta$/RC estimated by SDS-PAGE of chromatophores of Rsp. rubrum and Rbi. marinum exhibited the ratio of $\beta$/$\alpha$ > 1. These findings gave us the novel idea that there are two types of LHl; one is a C-shaped open antenna composed by $\alpha$$\beta$ units surrounding a RC, and another is a small closed ring antenna composed by $\alpha$$\beta$ units located peripherally in a variable ratio to the core complex like LH2.

We prepared 3-(1-hydroxyethyl)-bacteriopyrochlorophy11-a (3) possessing magnesium atom and phytyl ester from modification of natural bacteriochlorophyll(BChl)-a. A dichloromethane solution of (3$^1$R) and (3$^1$S)-3 was diluted with 100~1000 fold volume of cyclohexane to give new species absorbing near-infrared lights. The resulting Q, maximum of (3$^1$R)-3 was 860 nm and red-shifted by 2150 $cm^{-1}$ / from the monomeric. In the nonpolar organic solvent, epimeric (3$^1$S)-3 showed a 1ess red-shifted peak at 798 nm as well as a residual monomeric band. Such visible spectra indicated that 3 diastereose1ectively aggregated in cyclohexane to afford oligomers possessing a simi1ar supramolecular structure with chlorosomal aggregates of natural BChl-d, 7,8-dehydro-form of 3.

Regioselective 3$^1$-$^{18}$ O-labelling of chlorophyll derivatives possessing a 3-formyl group such as methyl (pyro) pheophorbide-d (3, 4) was carried out efficiently by a simple one-step procedure; by stirring a homogeneous solution of tetrahydrofuran and H$_2$$^{18}$ O containing a small amount of trifuluoroacetic acid.

A green mutant of Rhodobacter sphaeroides 60I was acquired by chemical induction. The blue-shifted of the carotenoid absorption was found in the Light-harvesting complex II (LH2) of the mutant. With the excitation at different wavelength, we observed that the evolution of excited-state dynamics in LH2 of Rhodobacter sphaeroides 60I. The dynamical traces demonstrate a dominant absorption followed concomitantly by an ultrafast transmission increase and decay with 818nm excitation.

Since chlorophyll a and bacteriochlorophyll a are asymmetric molecules, an external ligand can coordinate to the central Mg atom either from the chiorin macrocycle side where the C13$^2$-methoxycarbonyl moiety protrudes (denoting as the 'back' side) or frome the other side (the 'face' side). We investigated which side of the macrocycle is favored for the ligand coordination, by survey of the highly resolved crystal structures of various photosynthetic proteins and theoretical model calculations. It is found that chlorophyll a as well as bacteriochlorophyll a and b in the photosynthetic proteins mostly bind their ligands on the 'back' sides. This finding was confirmed by the theoretical calculations for methyl chlorophyllide a and methyl bacteriochlorophyllide a as models: the 'back' type ligand-(bacterio )chlorophyll complex was more stable than the 'face' type one. The calculations predicted influence of the Cl3$^2$-stereochemistry on the choice of the side of the ligand coordination, which is discussed in relation to the presence of the Cl3$^2$-epimer of chlorophyll a in photosystem I reaction center [I].

Photoadaptation of Chlorobium (Cb.) phaeobacteroides was investigated under dim and strong light intensity. Absorption spectra of these whole cellIs were different each other. The Soret band intensity and the Qy bandwidth of BChl e in c디l grown under dim light intensity were smaller and more broadened than those under strong light intensity. From HPLC analysis of the pigments, total carotenoid (Car) / bacterochorolphyll (BChl) e ratio of cell increased wi1h increase of light intensities. But camposition of BChl e hamologs almost unchanged. Cb. phaeobacteroides contains 11 kinds of Car including isorenieratene and beta-isorenieratene as major Car. The campositions of Car were different for cells grown under dim and strong light intensities. In conclusion, Cb. phaeobacteroides changes total amount and canposition of Car to adapt various light intensities, while homolog canposition of BChle unchange.

Chlorophyll (ChI) a' is the Cl3$^2$-epimer of ChI a which is the constituent of P700, the primary electron donor of Photosystem (PS) I, of a thrmophilic cyanobacterium, Synechococcus elongatus, whose structure was recently determined by X-ray crystallography. To determine whether PS I of diverse oxygenic photosynthetic organisms universally contain one molecule of ChI a ’, pigment compositions of thylakoid membranes and PS I complexes isolated from cyanobacteria, green algae, red algae and higher plants were determined by reversed-phase HPLC. The results show that involvement of one ChI a'molecule in PS I is the universal feature for Chi a-based PS I of oxygenic photosynthetic organisms.

In order to investigate effect of the carotenoids (Car) on aggregation of Bacterochlorophyll (BChl) in chlorosome, we studied the spectral difference in aggregates of BChl e formed in the absence and presence of a few kinds of Car in dimethyl sulfoxide (DMSO) -water solution. The absorption spectra of aggregates made of only BChl e and those made of a mixture of BChl e and Car were almost the same. However, the kinetics and circular dichroism (CD) spectra of aggregate of these were markedly different by kind of Car. Specifically, the rate of aggregation for a mixture of BChl e and isorenietene that contains phenyl as end groupe was faster than that for only BChl e. CD spectra of aggregates made of a mixture of BChl e and isorenietene dramatically changed compared to that made of only BChl e. We propose that BChl might form several kinds of rod-like supramolecular structures to in the presence of some kind of Car in chlorosome.

Lhcb genes encoding light-harvesting chlorophyll-a/b binding (LHC) proteins of photosystem (PS) II were comprehensively characterized using the expressed sequence tag (EST) databases in the green alga, Chlamydomonas reinhardtii. The gene family was composed of eight Lhcb genes including four new genes, which were isolated and sequenced. The effects of light intensity on the levels of mRNAs accumulation of multiple Lhcb genes were studied under various conditions. The results indicate that Lhcb genes are coordinately regulated in response to light conditions, and repressed when the input light energy exceeded the requirement for $CO_2$ assimilation. The effects of high light on the expression of the Lhcb genes observed in the presence of an electron transport inhibitor, DCMU, and in mutants deficient in photosynthetic reaction centers suggest the presence of two alternative mechanisms for regulating the genes expression under high-light conditions.

Role of cl$^{[-10]}$ in photosynthetic oxygen-evolving complex was studied by light-induced Fourier transform infrared (FTIR) spectroscopy. cl$^{[-10]}$ depletion resulted in the suppression of amide I and amide II IR modes upon S$_1$ to S$_2$ transition. Br$^{[-10]}$ , 1$^{[-10]}$ and N0$_3$$^{[-10]}$ substituted FTIR difference spectra were very similar to that in cl$^{[-10]}$ reconstitution. F$^{[-10]}$ and $CH_3$COO$^{[-10]}$ substituted spectra were largely distorted. We succeeded in detecting the structural change of N0$_3$ $^{[-10]}$ in the cl$^{[-10]}$ site upon the S$_1$ to S$_2$ transition from $^{14}$ N0$_3$$^{[-10]}$ /$^{15}$ N0$_3$$^{[-10]}$ difference spectrum.

The major electron transfer cofactors in photosystem II have been studied by pulsed EPR, pulsed electron electron double resonance (PELDOR) and laser excited spin polarized electron spin echo envelope modulation (ESEEM) methods, in non-oriented and oriented photosystem II membranes. Distances between radical pairs were determined trom the observed dipole interaction constants to be 27.3 A for P680-QA, 30 A, etc. with the error within 1 A. Angles between the distance vector and membrane normal was determined by orientation dependence of oriented membranes with the accuracy of 5˚ The results were compared with the recent structural data by X-ray analysis.

When illuminated with strong visible light, the reaction center Dl protein of photo system II is photodamage and degraded. Reactive oxygen species and endogenous cationic radicals generated by photochemical reactions are the cause of the damage to the Dl protein. Recently we found that the photodamaged Dl protein cross-links with the surrounding polypeptides such as D2 and CP43 in photosystem II. As the cross-linking reaction is dependent on the presence of oxygen, reactive oxygen species are suggested to be involved. Among the reactive oxygen species examined, ？ OH was most effective in the formation of the cross-linked products. These results indicate that the cross-linking is mostly due to ？ OH generated at photosystem II. The cross-linking site of the Dl protein is not known. As several tyrosine residues exist at the D­E loop of the Dl protein, there is a possibility that di-Tyr is formed between the D­E loop of the Dl protein and surrounding polypeptides during the strong illumination. Therefore, we examined the formation of di-Tyr using the monoclonal antibody against di-Tyr under excess illumination of the photosystem II membranes. The results obtained here suggest that no di-Tyr is formed during the excess illumination of photosystem II.

Direct EPR evidence of the photo-generation of superoxide radicals ( $O_2$$^{-.}$) was obtained by using spin trapping techniques in spinach photosystem II (PSII) membranes. $O_2$$^{-.}$ was detected by following the formation of 5-diethoxyphosphoryl-5-methyl-1 -pyrroline-N-oxide (DEPMPO) superoxide adducts, DEPMPO-OOH. The significant increase of the EPR signal amplitude of DEPMPO-OOH in N$H_2O$H-, CaC $l_2$- and NaCl-treated PSII membranes showed that the oxygen-evolving system has a close relation to the $O_2$$^{-.}$ production. PSII membranes with inactivated donor side could not prevent the $O_2$$^{-.}$ production efficiently. Treatments on PSII donor side also influence the maximum level and the kinetics of Chlorophyll (Chi) a fluorescence. Results suggested that manganese cluster and extrinsic proteins might affect Chi a fluorescence in ways different from that happens at the acceptor side of PSII.SII.SII.

Two ferredoxin (Fd) fractions, namely, Fd-A and Fd-B were isolated from Heliobacillus mobilis cells, and purified by ammonium sulfate fractionation, DEAE, gel-permeation and Phenyl-Superose column chromatographies under anaerobic conditions. Their absorption spectra were typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. Their N-terminal amino acid sequences were determined, which showed that both of them contain a [4Fe-4S] cluster binding motif. Fd-B was sensitive to oxygen, and itsA$_{385}$ value decreased by about 50% in 2 h at 4$^{\circ}C$ under aerobic conditions. In contrast, $A_{385}$ of Fd-A was essentially unchanged up to 24 h under the same conditions.

Effects of illumination with xenon or sulfur lamp on growth of tomato seedlings were studied. Results showed no obvious differences in net photosynthetic rate and apparent quantum efficiency. In comparison with xenon lamp, illumination with sulfur lamp slightly reduced ChI a and ChI b content in leaves, relatively shortened epicotyl and hypocotyl length, but significantly increased anthocyanin content of leaves and promoted root growth of tomato seedling plants.

The emission of microwave sulfur lamp is mainly composed of visible light. This lamp producing little infrared radiation, has high light efficiency, long lifespan and less power consumption. In comparison with xenon lamp, growing under sulfur lamp apparently postponed jointing, increased the number of tillers, accelerated root growth of wheat. Relatively, xenon lamp evidently promoted heading and grain maturation. The development characteristics of wheat plant under sulfur lamp were more similar with that in natural condition.

The seedling height, leaf width and leaf length of pepper increased in plants grown from seeds irradiated with the low dose of 4 Gy. The $O_2$ evolution in the 4 Gy irradiation group was 1.5 times greater than the control. Pmax was decreased with increasing illumination time by 20% in the control, while hardly decreased in the 4 Gy irradiation group. Fv/Fm was decreased with increasing illumination time by 50% after 4 hours, while Fv/Fm in the 4 Gy irradiation group was decreased by 37% of inhibition, indicating that the low dose $\gamma$ radiation increased resistance of plants to photoinhibition.

We collected seeds of Solidago virgaurea plants growing at different altitudes on the Mt Hakusan area in Japan and cultivated them in a naturally-lit green house. Three-week-old seedlings were irradiated with supplemental UV-B for 12 h each day for 1 and 2 weeks. After a week of itradiation the seedlings of the population collected from the higher altitude at Oh-nanjiho (ON) had accumulated more anthocyanins than those from the lower altitude at Bettoh-deai (BD). Levels of anthocyanins in the ON seedlings were highly correlated with the dose of UV-B radiation and the correlation was also observed after 2 weeks. The growth of the third leaves was retarded by UV-B radiation in both populations. The extent of growth retardation in the third leaves was correlated with the dose of UV -B radiation in both populations. However, no significant difference in the extent of leaf area growth was observed between the ON and BD populations. The increase in plant fresh weight was extensively inhibited in the ON seedlings after 1 week of UV-B radiation. The inhibition was recovered to those in the BD population by 2 weeks irradiation. These results indicate that these populations respond differentially to supplementary UV -B radiation during the first week. Because flavonoids such as anthocyanins play an important role in protection against UV-B radiation in many plants, populations growing at higher altitude may be better able to adapt to increased global levels of UV-B radiation.

To study photocatalytic mechanism of metal doped $Ti0_2$, we investigated photodecomposition effect, photocurrent effect and antibacterial effect. When aluminium content was 2 wt %, photodecomposition effect was better than the others. Silver doped thin films had high photocurrent efficiency and antibacterial effect. This reactions were caused by dissolved oxygen in solution and oxygen adsorbed on surface of thin films.

In order to know the characteristics of circadian rhythms in core temperature in tropical inhabitants, we measured rectal temperatures every 10 min for 24 hrs in 6 Vietnamese, 20 - 22 yrs (5 males and I females) under natural conditions. Average light intensity was 16000 lx. Ambient temperatures ranged from 33 to 36 oC. These data obtained were compared with those in Japanese setters and the Polish inhabitants. The participants were sitting mostly during wakefulness and lying in bed during sleep. The results obtained are summarized as follows: I) The average maximum value was 37.7 oC, which was significantly higher than in the Japanese and Polish as well. 2) The average minimum value was 36.4 oC, which was also lower. 3) A range of oscillation was 1.3 oC, which was clearly greater than in the people living in the temperate areas. The higher maximum value of core temperature, which was actively regulated under warm temperature, seemed of adaptive significance in order to reduce water consumption. A greater rage of oscillation in tropical Vietnamese people might have ecological significance for efficient acclimatization in the environment with strong light intensity and high ambient temperature, suggesting that the setpoint of core temperature could show a greater range of oscillation.

In order to prevent side reactions due to free radical formation occurring in the reactions of singlet oxygen generated in solution phse, it was required that singlet oxygen is generated in gas phase from an apparatus of a solid-gas system. We have accomplished to construct an apparatus generating singlet oxygen in solid-gas system, which is composed of a flexible optical fibre tube connected by a dye-sensitizer probe containing rose bengal dye on polymer or inorganic material. Through the optical fibre tube visible light from a laser and an oxygen stream are passed into the sensitizer probe where singlet oxygen is generated. The determination of singlet oxygen was carried out by two methods. One involves the detection of the luminescence of singlet oxygen at 1268 mn and the other involves the chemiluminescence reaction of a dihydroisobenzofuran with singlet oxygen emitting luminescence at 456 nm.

Several peroxovanadium(V) complexes with an organic chelate ligand decompose spontaneously, depending on the nature of the chelate ligand. The self-decomposition reactions of the dinuclear peroxovanadium(V) complex with 2-oxo-l,3-diaminopropane-N,N,N',N'-tetraacetate (dpot) and the peroxovanadium(V) complexes with N-carboxymethylhistidinate (cmhist) and histamine-N,N-diacetate (histada) accompany the reduction of vanadium(V) to vanadium(IV). This implies that the peroxide anion acts as a reducing agent and thus the peroxide is oxidized in the decomposition process of the peroxovanadium(V) complexes. The oxidized dioxygen species have been characterized spectrophotometrically. Superoxide anion has been detected in 2-3 % yields using the reduction of cytochrome c method and chemiluminescence method utilized MCLA as a fluorescer. Singlet oxygen has also been detected in higher yields on the basis of chemiluminescence of tryptophan.

Juvenile visceral steatosis (JVS) mouse is an animal model of the systemic camitine deficiency. JVS mice first develop fatty liver following cardiac hypertrophy. hyperammonemia, etc. To clarify the relationship between fatty liver and other symptoms. lipid hydroperoxides levels of peripheral oragans in JVS mice at 1 month were determined by the use of phosphine derivatives. We also report here a new method to quantitate the lipid components level in fatty liver of JVS mice.

We measured previously the lipid hydroperoxides level in the brain and peripheral organs such as heart, liver, lung and kidney of senescence acceIerated-prone (SAMP8) and -resistant(SAMR1) mice at 3,6 and 9 months of age. It was found that the lipid hydroperoxide leve1s in the brain did not show any age-dependent change, and that they Were significantly higher in SAMP8 than in SAMR1 over the defined periods. In contrast, the lipid hydroperoxide leve1s in the peripheral organs, including liver, Were increased with aging in both substrain, and they were significantly higher in SAMP8 than in SAMR1 at 3 and 6 months of age. In addition, the lipid hydroperoxide levels in the peripheral organs were higher than those in the brain in both substrains. To elucidate the difference of lipid hydroperoxide levels between the brain and the peripheral organs, we further carried out lipid component analysis in the brain and liver, one of the peripheral organs, of SAMP8 and SAMR1 at 6 months of age.

Phellinus linteus mycelia have many pharmacological effects, although their pharmacological efficacy principles have not been known yet. In the course of screening for biological activity of the extracts of Phellinus linteus mycelia, we found strong antioxidative activity in some fraction of water-insoluble. Therefore, we tried to isolate the active principle(s) from the extract. The isolation of the active compound was guided by superoxide anion radical scavenging activity. As a result, caffeic acid was isolated as an active compound. The IC$\_$50/ of the compound was 3.05 $\mu$g/ml (16.9$\mu$M).

Recently, much attention has been paid to the physiological functions of flavonoids associated with their antioxidant properties. However, there was a lack of information on the molecular mechanism at which flavonoids play the antioxidative role. We have already studied on the oxidation of quercetin with hydrogen peroxide and sodium hypochlorite in alcoholic aqueous solution and determined the oxidation products. Through the structural analysis of the oxidation products, it was clarified that the hydroxyl group at C-3 in the C ring plays the important role in the antioxidative action of quercetin. Successively, rutin and (+)-catechin were oxidized with sodium hypochlorite and their mono- and di-chlorinated derivatives were obtained. These facts indicate that these flavonoids can directly scavenge hypochlorous acid and the active site in this scavenging reaction is not the hydroxyl group at C-3.

We studied iron release from ferritin by irradiating the visible light, and then followed ferritin-mediated lipid peroxidation in the rod outer segment (ROS) fraction of the porcine retina. In the presence of several phosphorus compounds such as ADP and ATP, iron release from ferritin at pH 7.0 could be induced by irradiation of the visible light to the reaction mixtures. Furthermore, iron release from ferritin in the presence of ADP depended on the incubation time and the visible light irradiation. Moreover, we investigated lipid peroxidation level in the ROS fraction by two independent assay systems including the thiobarbituric acid (TBA) and ferrous oxidation/xylenol orange (FOX) methods. The visible light induced ferritin-mediated lipid peroxidation in the ROS fraction in time- and irradiance-dependent manners. In the dark condition, iron release and lipid peroxidation were not observed. Iron release from ferritin by irradiating the visible light may play an important role in the etiology of phototoxic injuries in vivo.

The changes in expression of copper-zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn-SOD) and glutathione peroxidase (GPX) in light-damaged rat retinas were examined. Sprague-Dawley rats (male, 6-weeks-old) were maintained on a cyclic photoperiod (12 hours light and 12 hours darkness) for 2 weeks. The illumination intensity during the light period was 80 lux. To induce light damage to the retina, a high-intensity illumination (3000-lux) was applied to the animals for 24 hours. After light exposure, the animals were returned to cyclic lighting. Eyes were enucleated 12 and 24 hours after light exposure started or 1,3, and 7 days after light exposure ended. Eyes were fixed and embedded in paraffin wax. Tissues were cut into 4${\mu}{\textrm}{m}$-thick sections. Sections were immunostained using antibody against CuZn-SOD, Mn-SOD, GPX and 8-hydroxy-deoxyguanocine (8-OHdG) as oxidative stress marker. 8-OHdG was observed in the outer nuclear layer (ONL) and retinal pigment epithelium (RPE) during light exposure. In light-damaged retinas CuZn-SOD labeling was up regulated in the ONL and RPE. Mn-SOD labeling was up regulated in rod inner segments (RIS) during light exposure and that in the RPE was up regulated after exposure. GPX labeling was observed in rod outer segments (ROS) during light exposure. GPX labeling was also observed in the RPE during and after light exposure. All three enzymes were observed in the outer retina, which suffered light damage, but occurred in defferent layers except within the RPE, in which case all three were expressed. These enzymes may play complementary roles as protective factors in light-damaged retinas.

In the present study, we investigated the actions of sphingosine-I-phosphate (SPP) in Mel-Ab melanocytes. We observed the cytoprotective effect of SPP on UVB-induced cell death. Following exposure of cells to UVB, a significant protective effect was seen in cultures pretreated with SPP. Since SPP is well known as a mitogenic agent, it is possible that the mitogenic effect of SPP may contribute to cell survival. Surprisingly, we found that SPP inhibited DNA-synthesis significantly. We were next interested in the regulation of the extracellular signal-regulated protein kinase (ERK) and Akt pathways by SPP. We clearly observed that SPP potently stimulated the phosphorylation of both ERK and Akt against UVB-induced cell death. Based on these results, we conclude that SPP may show its cytoprotective effect through ERK and Akt activation.

Since solar radiation contains wavelength essential for photosynthesis accompanying with near-UV light, UV-B effects on biological parameters and acclimation mechanisms are influenced by photosynthetically active radiation (PAR). Therefore, to elucidate near-UV shielding mechanism in higher plants, we cultivated cauliflower under usual solar radiation and increased UV-B from fluorescent lamps, two- or three-fold excess over continuously estimated UV-B dose in PAR during daytime, using computer regulated systems. Increased UV-B radiation had little effect on growth expressed as fresh weigh and leaf area. Water soluble low molecular weight compounds showing absorption in near UV region were enhanced according to the irradiated UV-B dose. One of compounds in cauliflower leaves was identified as chlorogenic acid. This was found to have no near-UV photosenSitizerable activity and is known to have an ability to scavenge a wide species of active oxygen. Another pro-oxidant compound that generates superoxide anion radical under near-UV irradiation was not induced by increased UV-B during cultivation, and identified as lumazine, a degradation product from folic acid.

Cyanobacteria are the dominant micro flora in rice-fields, contributing significantly to fertility as a natural biofertilizer. Recent studies show a continuous depletion of the stratospheric ozone layer, and the consequent increase in solar UV-B (280-315 nm) radiation reaching the Earth's surface. UV-B radiation causes reduction in growth, survival, protein content, heterocyst frequency and fixation of carbon and nitrogen in many cyanobacteria. UV -B induced bleaching of pigments, disassembly of phycobilisomal complexes, thymine dimer formation and alterations in membrane permeability have also been encounterd in a number of cyanobacteria. However, certain cyanobacteria produce photoprotective compounds such as water soluble colorless mycosporine-like amino acids (MAAs) and the lipid soluble yellow-brown colored sheath pigment, scytonemin, to counteract the damaging effects of UV-B. Cyanobacteria, such as Anabaena sp., Nostoc commune, Scytonema sp. and Lyngbya sp. were isolated from rice fields and other habitats in India and screened for the presence of photoprotective compounds. A circadian induction of the synthesis of MAAs by UV -B was noted in a number of cyanobacteria. Polychromatic action spectra for the induction of MAAs in Anabaena sp. and Nostoc commune also show the induction to be UV-B dependent peaking at 290 nm. Another photoprotective compound, scytonemin, with an absorption maximum at 386 nm (also absorbs at 300, 278, 252 and 212 nm), was detected in many cyanobacteria. In conclusion, a particular cyanobacterium having photoprotective compounds may be a potent candidate as biofertilizer for crop plants.

Seedlings of spinach (Spinacia oleracea L. "King of Denmark" and "Minsterland") were grown with or without supplemental UV-B under field conditions for five or nine days when their fifth or sixth leaves were expanding. Except two leaves which were just expanded and expanding, all other leaves of each seedling were removed before tested. One of these two leaves was exposed to UV-B irradiation and the other leaf was covered with lumiror film (no transmission below 320 nm) to prevent it from UV-B exposure. l,l-diphenyl-2-picrylhydrazyl scavenging activities (antioxidative activity) in leaves without covers were increased according to UV-B doses they were exposed to. And removal of UV-B exposure with lumiror decreased foliage antioxidative activities. This increase or decrease of activities in leaves did not dependent on the UV-B doses their paired leaves were exposed to in both two cultivars. The results demonstrated that spinach foliage antioxidants induced by UV-B exposure were not transported into another leaf.

The compositional changes in mycosporine-like amino acids (MAAs) were investigated in the marine dinoflagellate Scrippsiella sweeneyae exposed to four different spectral compositions and five relative intensities of UV-B (280-320 nm) to UV-A (320-400 nm) + photosynthetically available radiation (PAR: 400-700 nm). Neither dose nor wavelengths of UVR significantly affected the growth rates. UVR caused a significantly increase in cell volume. Cell volume in the >280nm treatment was more than two times greater at 6.8 % of UVR intensity. Production of UVR induced MAAs was dependent on the dose of UVR. However. the induction of MAAs was related to the cell growth. Greater induction of MAAs was observed at shorter wavelengths. The composition of MAAs varied with increasing light intensity of UVR.

DNA samples extracted from Japanese cypress leaf tissues contain isopropyl alcohol-precipitable, high molecular weight compounds, which interfere ELISA for cyclobutane pyrimidine dimers (CPD). Removal of the compounds is achieved by DEAE ion-exchange column chromatography and improves the ELISA responses of the DNA. When extracting DNA repeatedly from the same leaf tissues, the DNA samples show CPD responses which increase with the order in sequential extraction, and hence for a reliable detennination of DNA lesion a thorough extraction of DNA is required. Clearing these two problems it was demonstrated that CPD level was slightly higher in the leaves of trees growing under full sunlight than in those growing under UBV -cut sunlight.

A new concept of "photo" -antisense method has been evaluated, where the inhibition of gene expression by the conventional antisense method is enhanced by photochemical binding between antisense oligonucleotides conjugated with photo-reactive compound and target mRNA or DNA. Fluorescein labeled oligodeoxyribonucleotides (F-DNA) was delivered to cell nuclei in the encapsulated form in multilamellar lecithin liposomes with neutral charge. F-DNA was previously shown to photo-bind to the complementary stranded DNA, and the delivery system using neutral liposome to be effective in normal human keratinocytes. In the present study, we used human kidney cancer G401.2/6TG.1 cell line to be advantageous in reproducible experiments. p53 was adopted as a target gene since antisense sequence information has been accumulated. The nuclear localization ofF-DNA was identified by comparing the fluorescence ofF-DNA with that of Hoechst 33258 under fluorescence microscope. After 7hr incubation to accumulate p53 protein induced by UV -B, p53 protein was quantified by Western blot. After 2hrs from F-DNA application, about 30% of cell population incorporated F-DNA in their nuclei with some morphological change possibly due to liposomal toxicity. Irradiation of visible light longer than 400nm from solar simulator at this time enhanced the inhibitory action of antisense F-DNA. The present results suggest that photo-antisense method is promising to control gene expression in time and space dependent manner. Further improvement of F-DNA delivery to cancer cells in the stability and toxicity is in progress. progress.

It is important to determine the action spectrum of UV-B radiation contained in the sunlight to estimate the risk of skin cancer. We have investigated action spectra for induction of apoptosis and reproductive cell death in L5178Y cells using the Okazaki Large Spectrograph at NIBB. L5178Y cells were exposed to light at different wavelengths in UV-B or UV-A region. Frequencies of apoptosis induction and reproductive cell death were determined by counting cells with chromatin condensation, and by the colony formation assay, respectively. The measured sensitivity spectra for the two end-points were in very good agreement. Sensitivity decreased steeply with increase of wavelength in UV-B region and remains nearly constant in UV-A region. The action spectra were also slightly steeper than that for the minimum erythematic dose (MED), but very similar to the light absorption spectrum of DNA in UV-B region. On the other hand, the spectra for both endpoints were similar to MED spectrum but not DNA spectrum in the UV-A region. Also different time-course and morphological difference of apoptosis were found between UV-B (long time, fragmentation) and UV-A (short time, shrinkage) region. These results suggest that DNA damage induced by UV-B light triggers apoptosis and reproductive cell death, but other damaged targets (membrane, protein and so on) trigger these effects in UV-A region.

Sunlight causes various types of adverse skin changes on the sun-exposed areas of the skin, in which the most hazardous one is the induction of malignant skin tumours. FT -IR spectra were obtained from specimens excised from normal skin, BCCs, SCCs, MMs, nevi, lesions of solar keratosis and Bowen's disease. Tissue samples from freshly frozen specimens were cut into 2 sections in strictly sequential order to be stained with H & E for histopathological analysis, and then to be air-dried on CaF$_2$ slide glasses for further spectral data acquisition from defined area of interest. Intra- and inter-sample variations were estimated within grouped lesion categories according to each skin component. Mean spectra for each type of tissue pathology in the 800-1800 $cm^{-1}$ / region was interpreted using the classical group frequency approach that showed the most visible differences in spectra of benign, premalignant and malignant changes directly related to protein conformation and nucleic acid bases. The relative intensity of the nucleic acid peak was increased with progression to malignancy. In addition, PCA was able to evaluate and maximise the differences in the spectra by reducing the number of variables characterizing each patient and pathology category. This type of approach to non-destructively estimate the complexity of IR-spectra of inhomogeneous samples such as skin demonstrates the advantage of FT -IR microspectroscopy to be able to observe diseased states (benign, premalignant, malignant) and distinguish them from normal against a huge background of inter- and intra-subject variability.

Absorption spectra of biological specimens in the soft X-ray region have been presented with special reference to the XANES (X-ray absorption Near Edge Structure) of constituent elements. Absorption spectrum in this wavelength region is characterized by the absorption edges from which elemental content could be derived. In addition, XANES has a characteristic profile for chemical environment around the element such as chemical bond. Using the specific absorption peak we can assign not only the chemical bond but also molecules having such a chemical bond. In the present paper, absorption spectrum of DNA was measured in the wavelength range from 1.5nm to 5nm. Spectrum of Chinese Hamster Ovary (CHO) cells was compared with the DNA spectrum. XANES were distinct at the K absorption edges of major elements, C, N and O. In the spectrum of the cells prominent peaks at the L absorption edge of minor element Ca were also detectable. XANES profiles in small local areas in a cell could also be measured in combination with X-ray microscopy. These give information about local chemical environment in a cell. XANES at the phosphorus K absorption edge in a human HeLa cell was successfully obtained corresponding to a sharp and intensive XANES peak of DNA.

RIKEN Structural Genomics Beamlines have been constructed for the crystallographic analysis in the structural genomics research at synchrotron radiation facility SPring-8. Synchrotron radiation accelerates the crystallographic analysis of protein structure. The target of the research and development is focused on the automatic beamline operation to maximize beamline efficiency. We are developing the sample management system, which is composed of the sample auto-changer and the database system, for high-throughput data collection. The sample management system and the beamline operating system make it possible to execute automatic data collection without any operators. The beamlines will be ready for user operation in autumn 2002. The concept of automatic beamline operation and the present status of RIKEN Structural Genomics Beamlines will be presented.

An experiment has been conducted to measure the impact of UV radiation sensitivity on dermatophytes (Microsporum boullardii) by different UV radiation exposure time interval (1 min, 2 min 5 min, 10 min and 20 min) in degradation of keratin (Feather) in growth promoting substances of protein, cysteine, cystine and methionine from 7 to 28 days of incubation period. Mutant strain caused maximum weight loss with 1 minutes of UV radiation exposure at 21 day and mutant strain became immune in sensitivity at 14 days for decomposition of feathers. Maximum protein caused at 21st days with 20 minutes U.V radiation exposure and immune sensitivity had deducted with other UV radiation exposure time. On 28 days, mutant strains became immune with all exposure times, Whereas maximum methionine caused at 21st days with 20 minutes UV radiation exposure. Maximum cysteine caused at $14^{th}$ day with 5 minutes UV radiation exposure and mutant strain showed immune response at all time periods. Cystine production was also followed by cysteine at 21 day and also showed complete immune response with 1 and 2 minutes UV radiation exposure at7 and 14 days. Thus mutant strain of Microspornm boullardii can be used as a biotechnological tool for production of growth promoting substances.

It has been well documented that dermal irradiation by ultraviolet A (UVA) locally decreases the number of Langerhans cells and suppresses contact hypersensitivity of the skin. We found that topical irradiation of UVA to the eye systemically decreased the number of Langerhans cells (LC) in the dorsalskin and lymph nodes and elicited lymphocyte apoptosis in the latter tissues but not in the thymus. Optic nerve resection, but not ciliary ganglionectomy, eliminated the UVA-induced decrease in dermal Langerhans cells by a mechanism that was partially inhibited by hypophysectomy. The immunosuppressive effect of UVA was not observed in knockout mice lacking inducible-type of nitric oxide synthase (iNOS). These results suggested that topical irradiation of UVA to the eye induced immunosuppression via NO-dependet neuronal pathways.

Till date the phenomenon of maternal transfer of photic information was reported to regulate the fetal/neonatal growth, however its influence on neonatal immune system is still an enigma. In the present study, we observed an increase in maternal plasma melatonin level under short day length (SOL) condition with a consequent decrease in TLC and LC in their respective neonates. However, a significant decrease in maternal plasma melatonin level was noted under constant darkness (DD) with an increase in TLC and LC of their neonates. The blastogenic response (BGR) to Con A of splenocytes exhibited a significant increase in neonates of SDL females and a significant decrease in the neonates of DD females. Hence, it appears that the increase in maternal plasma melatonin under SOL condition transmitted information to decrease the immune status. Continuous exposure of females to darkness (DD) negatively regulated the maternal pineal gland activity thereby decreasing their plasma melatonin level. This information was transmitted for elevation of immune status in neonates, so that they exhibit better growth and sexual maturation. Therefore, we may suggest that the maternal photic information transmitted either prenatally through placenta or postnatally via the milk regulate the hormonal profile of Melatonin to regulate the immune status of neonates in order to influence their growth and sexual maturation.

In zoological research, penetration of light has been reported of the frontal bones of fish, birds, and reptiles, suggesting the existence of physiologically direct photic routes to frontal lobes and/or deep parts of the brain. We studied the influences of frontal exposure to photodiode light on frontal alpha wave and peripheral NK cells. Repetitive exposure of the subject's forehead to a red light diode (660nm) significantly increased the effective amplitude of the frontal alpha waves (using a mean frequency with a range of +1.0 Hz), peripheral NK activity, and the level of CD57-CD16+. Frontal alpha wave activity and the level of CD57-CD16+ increased, suggesting the possibility of a non-invasive procedure for the activation of the frontal lobe and the increase of NK cells. This light is considered to penetrate the frontal bones of humans directly, and to act on the frontal lobe and/or other immunological regulatory centers in the brain, resulting in some neuro-immunological changes.

UV-induced DNA damage causes cell killing and mutations leading to carcinogenesis. In normal human cells, UV damage such as cyclobutane pyrimidine dimers (CPDs) and primidine-prymidone (6-4) photoproducts are mainly repaired by nucleotide excision repair mechanism. The molecular processes have been well characterized recently. To know the influence of mitochondrial genome on the nucleotide excision repair mechanism against CPDs, we comparatively examined the production of CPDs by UVC irradiation and their repair kinetics in human cells completely lacking mitochondrial DNA (mtDNA) and the parental HeLa S cells. Whole DNA extracted from the cells exposed to UVC was treated with T4-endonuclease V to break the phosphodiester bond adjacent to CPDs. The DNA was electrophoresed in a denaturing agarose gel, which was visualized by ethidium bromide staining. The relative amount of CPDs was determined by image analysis using NIH Image software. MtDNA- less (rho-O) cells were apparently more sensitive to UVC than HeLa S cells, while the level of induction of CPDs in rho-O and HeLa cells was comparable. The repair of CPDs was less efficient in rho-O cells compared with HeLa cells. The residual amount of CPDs after 24-h repair was larger in rho-O cells than in HeLa cells where more than 90 % of CPDs were repaired by then. The non-repaired CPDs would lead to apoptosis in rho-O cells. These results suggest that mitochondrial genome may contribute to some ATP-dependent steps in nucletide excision repair by supplying sufficient ATP which is generated through a respiratory chain in mitochondria.

UV irradiation activates various intracellular signaling pathways causing cell death in a DNA damage-dependent and an independent manner. As DNA photoproducts, major forms of DNA damage, are maximally formed by UV light at 260-nm, short wavelength UV (UVC) is more harmful than middle wavelength UV (UVB). However, the differences or similarities in responses of DNA damage-independent intracellular signaling molecules to UVB and UVC are not elucidated. We examined activation of signaling molecules towards apoptosis in normal human fibroblastic cells after irradiation with UVB or UVC at a dose generating the equal amount of DNA photoproducts. Both UVB and UVC induced transient phosphorylation of ERK and sustained phosphorylation of p38. Phosphorylation of p53 at Ser15 and at Ser392 residues were also observed, which were inhibited by a phosphoinositide 3-kinase inhibitor, wortmannin. In contrast, an antioxidant N-acetyl-cysteine and a p38 inhibitor SB203580 suppressed only Ser392 phosphorylation, suggesting that UV-induced oxidative stress and p38 activation were involved in the phosphorylation of this site. The apoptic signals such as mitochondrial cytochrome C release and annexin V binding were then observed. Overall, no difference was found in chronological responses of p53, MAPK, and apoptosis between UVB-irradiated and UVC-irradiated cells. These results suggested that DNA damage-independent intracellular signaling molecules similarly responded to UVB and UVC when the equal level of DNA photoproducts were generated.

Cells receive signals for survival as well as death, and the balance between the two ultimately determines the fate of the cells. UV-triggered apoptotic signaling has been well documented, whereas UV-induced survival effects have received little attention. We have reported previously that UVB irradiation prevented apoptosis, which was partly dependent on activation of the phosphatidylinositol 3-kinase (PI3-kinase)/ Akt pathway. In this study, anti-apoptotic effects of UV with different wavelength ranges, UVA, UVB and UVC, were examined. NIH3T3 cells showed apoptotic cell death by detachment from the extracellular matrix under serum-free conditions, which was prevented by all wavelengths. However, the effect of UVA was less than those of UVB and UVC. Reduction of mitochondrial transmembrane potential and activation of caspase-9 and -3 were suppressed by all three wavelengths of UV, showing wavelength-dependent effects as mentioned above. The PI3-kinase inhibitor wortmannin partially inhibittrl the UVB and UVC-induced suppression of apoptosis, but not the inhibitoty effect of UVA. The Akt phosphotylation by UVB and UVC was completely inhibittrl by addition of wortmannin, but that by UVA was not P38 MAP kinase inhibitor SB203580 partially inhibited the UVB and UVC-induced suppression of apoptosis and Akt phosphotylation, and completely inhibited UVA-induced those. These results suggested the existence of two different survival pathways leading to suppression of apoptosis, one for UVA that is independent of the PI3-kinase/Akt pathway and dependent on p38 MAP kinase, and the other for UVB and UVC that is dependent on both pathways.

DNA photodamage mediated by photosensitizers are believed to play an important role in solar UVA carcinogenesis. We investigated the relationship between the DNA-damaging abilities of photoexcited xanthone analogues (as photosensitizers) and their highest occupied molecular orbital (HOMO) energies. DNA damage was examined using /sup 32/P-labeled DNA fragments obtained from the p53 tumor suppressor gene. These compounds induced DNA photodamage in a similar manner, and the extents of DNA damage were following order: xanthone> thioxanthone > acridone. Photoexcited xanthone caused nucleobase oxidation specifically at 5'-G of GG sequence in double-stranded DNA. An oxidative product of 2'-deoxyguanosine, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was detected, and the amount was decreased by DNA denaturation. These findings suggest that photoexcited xanthone generates 8-OHdG at 5'-G of GG in double-stranded DNA through electron transfer. The calculated HOMO energies of these photosensitizers decreased in the following order: xanthone> thioxanthone > acridone. This study has demonstrated that DNA-damaging abilities of these photosensitizers increased exponentially with an increase in their HOMO energies.

Erythrodermic actinic reticuloid (EAR) is a photosensitive disorder characterized by dense lymphocytic infiltration in the sun-exposed areas and an increased number of atypical lymphocytes in the peripheral blood. We have reported 2 patients with EAR with circulating atypical lymphocytes and photosensitivity to both ultraviolet (UV) B and A. Although no clonal proliferation of T-cells was observed in the peripheral blood, CD8+ cells were increased in number in an oligoclonal fashion. A number of proliferating CD8+ cells were small, but most cells expressed bcl-2. These findings suggest EAR is a photosensitivity dermatitis characterized by the overspill of oligoclonal CD8+ lymphocytes responsive to UV irradiation.

Fluorouracil (FU) is well known to induce discoid lupus-like eruption at the sun exposure sites in Japan. It means the associations of UVB with drug induced DLE. It is still obscure which cytokines are involved in the development of DLE. To address the issue, we established a murine model of FU and UVB-induced discoid lupus and could show the Th1 dominant cytokine profiles in DLE model of TCR $\alpha$ chain KO mice treated with FU and UVB.

We experienced 29 cases of tentative photo allergic disorders from April 2000 to March 2002. For those 29 patients, photopatch tests have been done to explore possible causative photoallergens. We performed photopatch tests with photo-related allergens and possible causative products that the patients brought to us. After applications for 48hrs, one test site was irradiated with half of MRD or 6.0 J/cm$^2$ of UVA. Readings were evaluated according to the reading criteria of the ICDRG. Photoallergens were suggested in 20 cases (69.0%). Among them, we could find out the causative products in 7 cases (24.1%). Photopatch tests, performed by an appropriate method, are useful in some cases not only for diagnosis of photocontact dermatitis but also diagnosis of systemic photoallergic disorders.

In vitro and in vivo studies have reported the induction of matrix metaloproteinase (MMP)-1 in the fibroblasts by ultraviolet (UV) A irradiation. We constructed the skin equivalent model using HaCaT cells as keratinocytes and human neonatal dennal fibroblasts as fibroblasts in the present study. The induction of MMP-l in the fibroblasts was confirmed immunohistochemically 6 hours after UVA irradiation using this model. This model was simply composed of human keratinocytes and fibroblasts. To our knowledge, there have been a few papers concerning the skin equivalent model in the field of photobiology. The effect of UVA exposure to fibroblasts through keratinocytes was examined using this model. The cross-talk can be examined between keratinocytes and fibroblasts. This model can be a useful tool in the field of photobiology.

Three patients suffering from widespread erythrodermic cutaneous T-cell lymphoma were treated with low-dose UVAI phototherapy with a single dose of 20 - 30J/$cm^2$, resulting in cumulative doses of 520 - 1890 J/$cm^2$. Two patients showed complete responses, while other had a partial improvement.

Photodynamic therapy using topical 5-ALA has been used for non-melanoma skin cancers. Recently, the therapeutic method using incoherent light brought beneficial result in the treatment for mycosis fungoides. We used ALA-PDT for two Japanese patients suffering from lymphadenosis benigna cutis. In both cases, lesions were markedly faded and histologically, the number of infiltrated cells also decreased. We suggest that ALA-PDT can be used as an effective and safe modality in the treatment of benign cutaneous lymphoma.

In this study, we investigated apoptotic cell death induced by photodynamic therapy using 5-aminolaevulinic acid (ALA-PDT) in human promyelocytic leukemia cells (HL-60). ALA-PDT induced apoptosis in HL-60 cells as confirmed by DNA agarose gel electrophoresis and nuclear staining with Hoechst 33342. The apoptotic cell death was inhibited by addition of broad-spectrum caspase inhibitor Z-Asp-CH$_2$-DCB, indicating that the apoptotic cell death was induced in a caspase-dependent manner. Actually, western blotting analysis revealed that caspase-3 was processed as early as 1.5 h after ALA-PDT. Cytoplasmic cytochrome c released from mitochondria was detected by western blotting. However, inhibitor of caspase-9, a cysteine protease located in the downstream of cytochrome c release, was not able to reduce the apoptotic cell death. Therefore, the mitochondrial apoptotic pathway was not involved in the ALA-PDT-induced apoptosis. On the other hand, it was found that ALA-PDT-induced apoptosis was clearly inhibited by pretreatment of caspase-8 inhibitor. These data suggest that caspase-8-mediated apoptotic pathway is important in ALA-PDT-induced cell death.

S-aminolevulinic acid (ALA) is a new kind drug used in photodynamic therapy. ALA-PDT have successfully used in superficial malignancies and some skin diseases. Here the effects of ALA-PDT were studied on leukemia cells and hepatoma cells to explore the application on different kind cancers. It was found from the fluorescence emission spectra, that after ALA incubation the sensitizer - protoporphyrin IX (PpIX) was endogenously produced in both leukemia and hepatoma cells. The fluorescence images showed that the PpIX distribute in cytoplasm. However the efficiency of ALA photodynamic inactivation to two cell lines was different. The leukemia cells were more sensitive for ALA-PDT than hepatoma cells, revealing that the ALA-PDT effect is cell line dependent. However by using ALA-Hexyl ester (He-ALA) instead of ALA, the cell photo-inactivation was improved. The PDT efficiency of He-ALA was 10 times high than that of ALA, showing He-ALA is a very promising drug in ALA-PDT.

Photodynamic therapy (PDT) is a cancer treatment modality which utilizes the cytotoxicity of the active singlet oxygen derived from irradiation of a tumor accumulated photosensitizer. As the oxygen in the singlet state radiates an emission of 1270nm wavelength when it decays to the triplet state, detection of the emission helps us to understand the mechanism of PDT or to evaluate photosensitizers. We detected the 1270nm emission from photosensitizers Photofrin and ATX-SI0 in vitro and in vivo by means of high sensitive NIR detectors. We obtained the maximum amount of singlet oxygen at irradiation wavelength of 665-670nm from a HeLa tumor in a nude mouse which is injected with ATX-S10.

Photodynamic therapy (PDT) is a treatment modality based on photochemical reaction and the resultant cytotoxic reactive oxygen species. The platelet thrombus formation leading to stasis observed in vivo during PDT is called vascular shut down (VSD) effect. To investigate the mechanism of the VSD effect, we observed Human Umblical Vein Endothelial Cell (HUVEC) injury induced by photochemical reaction. We observed cell retraction and blebbing after PDT. It seems that the injury was not fetal and only morphological change. Then, the cytoplasm was stained by Calcein-AM and subendothelial area was evaluated from fluorescence microscopy. The rate of subendothelial area after PDT increased significantly. Second, we investigated interaction between neutrophils and HUVEC. Human promyelocytic leukemia cells (HL-60) were differentiated into neutrophil by incubation with all-trans retinoic acid. Calcein-AM labeled neutrophil adhesion to HUVEC was evaluated from fluorescence microscopy. PDT-induced neutrophil adhesion to HUVEC depended more on the exposure of subendothlial area than on neutrophil activation. This result suggests that there is a certain interaction between neutrophil and HUVEC during PDT.

5-aminolevulinic acid (ALA) has been used to stimulate endogenous protoporphyrin IX (PpIX) in tumor and then initiate PDT. Recently, ALA-Hexyl ester (He-ALA) was found much effective than ALA on producing PpIX in cancer cells. To clarify the transportation mechanism of ALA and He-ALA, the detection of them is the important step. ALA and its derivatives all don't emit fluorescence, so the Raman spectroscopy was used here for the direct detection of ALA and He-ALA. The results showed that ALA and He-ALA have the common strong Raman peaks at 2930, 2950 CM$\^$-1/, due to the CH$_2$ vibration. The peak 3050 CM$\^$-1/ appeared in ALA spectrum can be attributed to OH vibration, while the peaks of 2860, 2900 CM$\^$-1/ in He-ALA spectrum were assigned as the modes of CH$_3$. This Raman spectral characteristic is consistence with the structure difference of He-ALA and ALA. Thus, Raman spectroscopy provides a new way to detect and distinguish ALA and He-ALA, and could be explored further in biology system.

Hypericin was found to exhibit the highest antitumoral activity in treating EAT by photodynamic therapy (PDT): Hypericin>HPde>PII>TPPS$_4$>ALA. Moreover, 25% of mice after Hyp-based PDT survived 4 months, if compare with control group. Antitumor activity of these photosensitizers was in rather clear correlation with accumulation potential.

Photodynamic therapy (PDT) is a medical treatment using laser and photosensitizing drug taken up to destroy cancer cells. Singlet oxygen ($^1$0$_2$) generation is strongly related to this treatment. We have built a direct detection system monitoring feeble luminescence, in the near IR region, from $^1$0$_2$, We have comparatively studied the photophysical and photochemical properties in solution of a newly developed drug ATX-S10 and Photofrin already investigated clinically. We demonstrdted that ATX-S10 was capable of efficiently yielding $^1$0$_2$, which may lead to highly efficient PDT treatment. Successive laser excitation photobleached ATX -S10 readily in a dose-dependent manner. This result shows that ATX -S10 is useful in setting up suitable medical treatment conditions to minimize side effects.

We present a case of The Miller Fisher Syndrome (MFS), showing a remission during a recently developed noninvasive therapy. Two weeks after an appearance of cough and fever, a 35 years old Japanese male developed diplopia, ataxia and numbness of his fingers and toes. He was diagnosed as MFS, and a fixed dose of prednisolone acetate (60mg/day) was administered for 3 months, but little improvement was observed. In addition to this administration, we tried 20 minutes of Photic Feedback (PFB) treatment daily for 40 days. The PFB system detects brain waves from the subject's forehead, and extracts alpha waves by the band-pass filter with a center frequency set at 10.0Hz. It also simultaneously modulates the augmentation of a red light-emitting diode, corresponding with the amplitudes of the extracted alpha waves. In this treatment, this adjusted photic stimulation was given to the subject's closed eyes, resulting in the effective alpha enhancement by photic driving response. The numbness increased during each of PFB treatment, but the symptoms started to improve gradually after 10 days. Other symptoms disappeared after 40 days. CD20 levels increased with this treatment. This case suggests that the PFB treatment may speed the natural remission of MFS. This treatment may be worth considering in patients who suffer polyneuropathy.

A halophilic archaeon, Halobacterium salinarum, exhibits phototactic behaviors, by which the organism is guided to red-orange light and evades shorter wavelengths of light. The phototaxis is mediated by two retinal proteins, sensory rhodopsin I and II (SRI and SRII), whose structures are analogous to the cognate protein bacteriorhodopsin, a light-driven proton pump. SRI mediates both attractant and repellent swimming behaviors to orange light and near- UV light, respectively. The two different signaling through the single photoreceptor have been ascribed to the presence of two active structures of SRI (S$\_$373/ and P$\_$520), which are produced upon orange light illumination of SRI and upon subsequent near-UV illumination of S$\_$373/, respectively. In the present study, we have measured the difference FTIR spectra of S$\_$373/ and P$\_$520/ states. In P$\_$520/, the isomeric structure of the chromophore is assignable to all-trans, and the Schiff base of the chromophore is protonated with concomitant deprotonation of Asp76, a combination which allows for the formation of a salt bridge between them. It was suggested that the way of interaction between the Schiff base and the counterion, which is different among SRI$\_$587/, S$\_$373/ and P$\_$520/ and which has been shown to drive the conformational changes in the cognate protein, bacteriorhodopsin, is the key to controlling conformational changes for the attractant and the repellent signaling by SRI.