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Now showing items 1 - 16 of 17

  • A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis

    Iseki, Mineo   Matsunaga, Shigeru   Murakami, Akio   Ohno, Kaoru   Shiga, Kiyoshi   Yoshida, Kazuichi   Sugai, Michizo   Takahashi, Tetsuo   Hori, Terumitsu   Watanabe, Masakatsu  

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  • Action Spectrum in the Ultraviolet Region for Phototropism of Bryopsis Rhizoids

    Iseki, Mineo   Wada, Shunji  

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  • Origins of a cyanobacterial 6-phosphogluconate dehydrogenase in plastid-lacking eukaryotes.

    Maruyama, Shinichiro   Misawa, Kazuharu   Iseki, Mineo   Watanabe, Masakatsu   Nozaki, Hisayoshi  

    BACKGROUND: Plastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. Although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive.RESULTS: Here we show an extended phylogenetic analysis including novel gnd gene sequences from Excavata and Glaucophyta. Our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. The Diplonema gene was related to cytosol-type genes in red algae and Opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. Statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. In addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in Euglenida were robustly monophyletic with Stramenopiles, and this monophyletic clade was moderately separated from those of red algae. These data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses.CONCLUSION: We propose an evolutionary scenario in which plastid-lacking Excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene.
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  • Differentiation of photocycle characteristics of flavin-binding BLUF domains of alpha- and beta-subunits of photoactivated adenylyl cyclase of Euglena gracilis

    Ito, Shinji   Murakami, Akio   Iseki, Mineo   Takahashi, Tetsuo   Higashi, Shoichi   Watanabe, Masakatsu  

    Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PAC alpha and PAC beta), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PAC alpha F2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PAC beta F2 domain, which like PAC alpha F2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PAC beta F2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PAC alpha F2 were 0.28 0.32 and 34 44 s. The remarkable differences between PAC alpha F2 and PAC beta F2 may be related to the sensitivity of the photoactivation. In PAC alpha F2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PAC beta F2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PAC beta F2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PAC beta F2 plays a main role in suppressing the quantum efficiency.
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  • Photomovement in Euglena

    Haeder, Donat-P.   Iseki, Mineo  

    Motile microorganisms such as the green Euglena gracilis use a number of external stimuli to orient in their environment. They respond to light with photophobic responses, photokinesis and phototaxis, all of which can result in accumulations of the organisms in suitable habitats. The light responses operate synergistically with gravitaxis, aerotaxis and other responses. Originally the microscopically obvious stigma was thought to be the photoreceptor, but later the paraxonemal body (PAB, paraflagellar body) has been identified as the light responsive organelle, located in the trailing flagellum inside the reservoir. The stigma can aid in light direction perception by shading the PAB periodically when the cell rotates helically in lateral light, but stigmaless mutants can also orient with respect to the light direction, and negative phototaxis does not need the presence of the stigma. The PAB is composed of dichroically oriented chromoproteins which is reflected in a pronounced polarotaxis in polarized light. There was a long debate about the potential photoreceptor molecule in Euglena, including carotenoids, flavins and rhodopsins. This discussion was terminated by the unambiguous proof that the photoreceptor is a 400 kDa photoactivated adenylyl cyclase (PAC) which consists of two a-and two beta-subunits each. Each subunit possesses two BLUF (Blue Light receptor Using FAD) domains binding FAD, which harvest the light energy, and two adenylyl cyclases, which produce cAMP from ATP. The cAMP has been found to activate one of the five protein kinases found in Euglena (PK.4). This enzyme in turn is thought to phosphorylate proteins inside the flagellum which result in a change in the flagellar beating pattern and thus a course correction of the cell. The involvements of PAC and protein kinase have been confirmed by RNA interference (RNAi). PAC is responsible for step-up photophobic responses as well as positive and negative phototaxis, but not for the step-down photophobic response, even though the action spectrum of this resembles those for the other two responses. Analysis of several colorless Euglena mutants and the closely related Euglena longa (formerly Astasia longa) confirms the results. Photokinesis shows a completely different action spectrum. Some other Euglena species, such as E. sanguinea and the gliding E. mutabilis, have been investigated, again showing totally different action spectra for phototaxis and photokinesis as well as step-up and step-down photophobic responses.
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  • Possible involvement of a tetrahydrobiopterin in photoreception for UV-B-induced anthocyanin synthesis in carrot.

    Takeda, Junko   Nakata, Rieko   Ueno, Hiroshi   Murakami, Akio   Iseki, Mineo   Watanabe, Masakatsu  

    Our previous studies of action spectra for UV-B-induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV-B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV-B light sensing. UV-B-induced phenylalanine ammonia-lyase (PAL) activity was considerably suppressed by N-acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N-acetylserotonin-treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV-B-induced PAL activity. These results suggest the occurrence of an unidentified UV-B photoreceptor (other than UVR8, the tryptophan-based UV-B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After reexamining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV-B sensors. =C2=A9 2014 The American Society of Photobiology.
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  • Blue and Red Light-Induced Germination of Resting Spores in the Red-Tide Diatom Leptocylindrus danicus

    Shikata, Tomoyuki   Iseki, Mineo   Matsunaga, Shigeru   Higashi, Sho-ichi   Kamei, Yasuhiro   Watanabe, Masakatsu  

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

    Fujita, Satoshi   Iseki, Mineo   Yoshikawa, Shinya   Makino, Yumiko   Watanabe, Masakatsu   Motomura, Taizo   Kawai, Hiroshi  

    The posterior flagellum of the zoospore of the brown alga Scytosiphon lomentaria exhibits bright green autofluorescence. To identify the fluorescent flagellar substance(s), we isolated flagella from zoospores and partially purified a flavoprotein by anion-exchange and gel-filtration chromatography. Spectrofluorometric and chromatographic analyses showed that the flavoprotein had an apparent molecular mass of 41 kDa and a non-covalently bound flavin mononucleotide as a chromophore. Based on partial amino acid sequences of the protein, a cDNA of the 41-kDa flavoprotein was cloned and sequenced. The deduced amino acid sequence of the cDNA was homologous to that of the Old Yellow Enzyme family distributed in proteobacteria, yeasts and vascular plants.
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  • Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

    Ohki, Mio   Sato-Tomita, Ayana   Matsunaga, Shigeru   Iseki, Mineo   Tame, Jeremy R. H.   Shibayama, Naoya   Park, Sam-Yong  

    The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light-and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.
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  • Homologs of the sexually induced gene 1 (sig1) product constitute the stramenopile mastigonemes.

    Honda, Daiske   Shono, Takanori   Kimura, Kei   Fujita, Satoshi   Iseki, Mineo   Makino, Yumiko   Murakami, Akio  

    The tripartite tubular mastigoneme on the anterior flagellum is a morphological feature that characterizes the stramenopiles. Mastigonemes are significant and potentially informative structures not only from the viewpoint of systematics, but also of cell biology. Nevertheless, few biochemical studies have been reported on stramenopile mastigonemes. The flagella of Scytosiphon lomentaria (Phaeophyceae) were successfully isolated and analyzed using SDS-PAGE followed by protein sequencing. The partial amino acid sequence of one flagellar protein (115kDa) showed high similarity with the sexually induced gene 1 (sig1) product of centric diatoms. A polyclonal antibody against the 115-kDa protein reacted not only to the shaft of mastigonemes in Scytosiphon lomentaria, but also another distinctly different stramenopile flagellate, Sulcochrysis biplastida (Dictyochophyceae). Therefore, we propose that the 115-kDa protein (i.e. Sig1 homologs) is a constituent of the tubular shaft of the mastigoneme.
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  • Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis

    Ito, Shinji   Murakami, Akio   Sato, Kyosuke   Nishina, Yasuzo   Shiga, Kiyoshi   Takahashi, Tetsuo   Higashi, Shoichi   Iseki, Mineo  

    Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis ( Iseki et al., Nature, 2002, 415, 1047 - 1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PAC alpha and PAC beta). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD" (BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACa by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of ribo flavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28 - 0.32, and the half-life was 34 - 44 s at 25 degrees C for the recombinant PACa F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tll0078) to ca. 900 s ( AppA). The mutated recombinant Y472F and Q514G of PACa F2 and the F2 domain of the PACa homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation.
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  • Photomanipulation of antibiotic susceptibility and biofilm formation of Escherichia coli heterologously expressing photoactivated adenylyl cyclase.

    Yasukawa, Hiro   Konno, Noriko   Haneda, Yukari   Yamamori, Baku   Iseki, Mineo   Shibusawa, Mami   Ono, Yasushi   Kodaira, Ken-ichi   Funada, Hisashi   Watanabe, Masakatsu  

    A cyaA-deficient Escherichia coli strain was transformed by a plasmid carrying the gene for BsPAC, a photoactivated adenylyl cyclase identified from a Beggiatoa sp., and was subjected to an antibiotic susceptibility assay and biofilm formation assay under a light or dark condition. Cells expressing BsPAC that were incubated under blue light (470 nm) were more susceptible to fosfomycin, nalidixic acid and streptomycin than were cells incubated in the dark. Cells expressing BsPAC formed more biofilms when incubated under the light than did cells cultured in the dark. We concluded from these observations that it is possible to determine the importance of cAMP in antibiotic susceptibility and biofilm formation of E. coli by photomanipulating the cellular cAMP level by the use of BsPAC. A site-directed mutant of BsPAC in which Tyr7 was replaced by Phe functioned even in the dark, indicating that Tyr7 plays an important role in photoactivation of BsPAC. Results of mutational analysis of BsPAC should contribute to an understanding of the molecular basis for photoactivation of the protein.=20
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  • Phylogeny of primary photosynthetic eukaryotes as deduced from slowly evolving nuclear genes.

    Nozaki, Hisayoshi   Iseki, Mineo   Hasegawa, Masami   Misawa, Kazuharu   Nakada, Takashi   Sasaki, Narie   Watanabe, Masakatsu  

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  • Flagellar Motions in Phototactic Steering in a Brown Algal Swarmer

    Matsunaga, Shigeru   Uchida, Hiroko   Iseki, Mineo   Watanabe, Masakatsu   Murakami, Akio  

    Using infrared high-speed video microscopy, we observed light-triggered transitory flagellar motions in flagellate reproductive cells (swarmers) of a brown alga, Scytosiphon lomentaria, under primary helical swimming conditions before and during negative phototactic orientation to unilateral actinic light. The posterior flagellum, which is autofluorescent and thought to be light-sensing, was passively dragged in the dark and exhibited one to several rapid lateral beats during orientation changes for phototactic steering. Notably, a brief cessation of anterior flagellar beating was occasionally observed concomitantly with rapid beats of the posterior flagellum. This behavior caused a pause in helical body rotation, which may contribute to the accuracy of phototactic steering. Thus, coordinated regulation of the movement of the two flagella plays a crucial role in phototactic steering.
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  • Phylogeny of primary photosynthetic eukaryotes as deduced from slowly evolving nuclear genes (vol 24, pg 1592, 2007)

    Nozaki, Hisayoshi   Iseki, Mineo   Hasegawa, Masami   Misawa, Kazuharu   Nakada, Takashi   Sasaki, Narie   Watanabe, Masakatsu  

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  • Vibrational Microspectroscopy of Single Proteins

    Fujiyoshi, Satoru   Furuya, Yo   Iseki, Mineo   Watanabe, Masakatsu   Matsushita, Michio  

    Vibrational infrared absorption of a single protein molecule was detected at a few kelvins as infrared-induced recovery of visible fluorescence of a dye with which the protein was labeled, This sensitive method of detecting infrared absorption was demonstrated for a single bovine serum albumin (BSA) molecule labeled with Alexa Fluor 660 by determining the vibrational infrared absorption spectrum of the backbone vibrations of the a-helical structure in the wavelength region around 6 mu m (1650 cm(-1)). In addition to measuring the vibrational infrared absorption spectrum, the visible fluorescence can be simultaneously used for imaging of the same dye-labeled single protein molecules.
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