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Tatsuomi Matsuoka - One of the best experts on this subject based on the ideXlab platform.
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blepharismins produced by the protozoan Blepharisma japonicum form ion permeable channels in planar lipid bilayer membranes
FEBS Letters, 2001Co-Authors: Yoshinori Muto, Akemi Kida, Tatsuomi Matsuoka, Yukio Okano, Yutaka KirinoAbstract:Blepharismins are polycyclic quinones found in the pigment granules of the ciliated protozoan, Blepharisma. Exposure to purified blepharismins results in lethal damage to several other ciliates. We here report that, at cytotoxic concentrations, blepharismins formed cation-selective channels in planar phospholipid bilayer membranes. The channels formed in a diphytanoylphosphatidylcholine bilayer had a K + /Cl 3 permeability ratio of 6.6:1. Single channel recordings revealed the conductance to be quite heterogeneous, ranging from 0.2 to 2.8 nS in solutions containing 0.1 M KCl, possibly reflecting different states of aggregation of blepharismin. Our observations suggest that channel formation is a cytotoxic mechanism of blepharismin's action against predatory protozoa. fl 2001 Fed- eration of European Biochemical Societies. Published by Else- vier Science B.V. All rights reserved.
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photosensory transduction in unicellular eukaryote Blepharisma
Journal of Experimental Zoology, 2001Co-Authors: Tatsuomi Matsuoka, Hiyoshizo KotsukiAbstract:In the ciliated protozoan Blepharisma, step-up photophobic response is mediated by a novel type of photosensory complex of pink-colored pigment “blepharismins” and 200-kDa membrane protein contained in the pigment granules located just beneath the plasma membrane. We found that the fluorescence intensity of isolated blepharismins decreased prominently with a decrease of H+ concentration in the surrounding medium. In the present study, therefore, we utilized the endogenous pigment blepharismins as the pH indicator. Light stimulation evoked a sudden decrease in fluorescence intensity in a photosensitive anterior portion of the cell, suggesting that a drop in H+ concentration occurred in the anterior region. The result indicates that the photosignal is transduced into cytoplasmic signaling of H+ translocation across the outer membrane surrounding the pigment granules, so that cytosolic H+ concentration in the vicinity of plasma membrane might be increased. J. Exp. Zool. 289:467–471, 2001. © 2001 Wiley-Liss, Inc.
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high yielding tfoh catalyzed condensation of phenols with aromatic aldehydes at high pressure a model synthesis of the benzylidene biphenol key skeleton of blepharismins
Tetrahedron Letters, 2001Co-Authors: Takeshi Ohishi, Tatsuomi Matsuoka, Tomoyuki Kojima, Motoo Shiro, Hiyoshizo KotsukiAbstract:Abstract An approach to the benzylidene biphenol key component of blepharismins, photoreceptor pigments isolated from Blepharisma japonicum , is reported. This method relies on an efficient TfOH-catalyzed condensation of phenols with aromatic aldehydes in EtOH as a solvent at 3 kbar pressure.
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analyses of structure of photoreceptor organelle and blepharismin associated protein in unicellular eukaryote Blepharisma
Photochemistry and Photobiology, 2000Co-Authors: Tatsuomi Matsuoka, Hiyoshizo Kotsuki, Masaki Ishida, Daisuke Tokumori, Motoaki Matsushita, Satoshi Kimura, Takao Itoh, Giovanni CheccucciAbstract:In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as ‘‘blepharismins‘‘ (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules’ three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyBlepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment‐200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Ble
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Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma
Journal of Photochemistry and Photobiology B-biology, 2000Co-Authors: Tatsuomi Matsuoka, Naoko Moriyama, Akemi Kida, Tomohiko Suzuki, Kazuo Okuda, Hiyoshizo KotsukiAbstract:Abstract The blepharismin–200 kD protein complex of the ciliated protozoan Blepharisma is a novel type of photosensor responsible for the step-up photophobic response of the cell. In immunoblotting assays, the 200 kD protein is weakly cross-reacted with anti-inositol triphosphate receptor antibody (anti-IP 3 R antibody). Indirect immunofluorescence assays show that the pigment granules in which the blepharismin–200 kD protein complex is localized are labelled by anti-IP 3 R antibody. When the anti-IP 3 R antibody or antisense oligonucleotide for IP 3 receptor is introduced into the living cells of Blepharisma , both the photosensitivity of the cells and content of blepharismin–200 kD protein are reduced. The results suggest that the photoreceptor 200 kD protein is possibly an IP 3 receptor-like protein.
Francesco Lenci - One of the best experts on this subject based on the ideXlab platform.
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picosecond transient circular dichroism of the photoreceptor protein of the light adapted form of Blepharisma japonicum
Chemical Physics Letters, 2009Co-Authors: F Hache, Giovanni Checcucci, Pascal Plaza, Monique M Martin, Maithu Khuc, Johanna Brazard, Francesco LenciAbstract:We present a picosecond transient circular dichroism study of OBIP, the putative photoreceptor protein involved in the photophobic response of Blepharisma japonicum. The probe wavelength was chosen at 230 nm. The results are compared to those of the isolated chromophore, OxyBP, in solution. The CD changes in OBIP and OxyBP do not show the same dynamics: OBIP’s signal relaxes in a few ps whereas no such decay is obtained for OxyBP. This observation brings support to the formerly evoked existence of a fast photoinduced reaction in the chromoprotein, and demonstrates the implication of local geometrical changes that accompany this process. 2009 Published by Elsevier B.V.
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fast excited state reaction in the photoreceptor pigment protein complex of the ciliate Blepharisma japonicum
Femtochemistry and Femtobiology#R##N#Ultrafast Events in Molecular Science VIth International Conference on Femtochemistry Maison de la Chimie Paris F, 2004Co-Authors: Mathilde Mahet, Giovanni Checcucci, Pascal Plaza, Monique M Martin, Francesco LenciAbstract:ABSTRACT The primary phototransduction steps of the protozoan Blepharisma japonicum 's light perception were studied by subpicosecond transient absorption spectroscopy. The free chromophore of the light-adapted cell, oxyblepharismin, was characterized in solution and compared to the pigment-protein complex. Although showing similar steady-state and transient spectra, oxyblepharismin and its protein complex display noticeably different excited-state dynamics. In the chromoprotein, a pronounced fast biexponential decay (5 ps and 60 ps) is observed, which is not found in the corresponding kinetics of the isolated pigment. This early behavior could be the signature of a specific primary phototransduction process.
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electron transfer fluorescence quenching of Blepharisma japonicum photoreceptor pigments
Photochemistry and Photobiology, 1998Co-Authors: Nicola Angelini, Giovanni Checcucci, Pill Soon Song, Annamaria Quaranta, Francesco LenciAbstract:Abstract— The hypericin analogs blepharismin (BP), oxyblepharismin (OxyBP) and stentorin (ST), the photosensing chromophores responsible for photomotile reactions in the ciliates Blepharisma japonicum (red and blue cells) and Stentor coeruleus, represent a new class of photoreceptor pigments whose chemical structures have recently been determined. In the case of ST it has been shown that the first excited singlet state can be deactivated by donation of an electron to an appropriate acceptor molecule (e.g. a quinone molecule). This charge transfer can be considered a possible mechanism for the primary photoprocess for the photomotile responses in S. coeruleus. To determine whether an electron transfer process also occurs in the deactivation of excited blepharismin, we studied the fluorescence quenching of OxyBP in dimethyl-sulfoxide (DMSO) and in ethanol using electron acceptors with different reduction potentials. Under our experimental conditions ground state and excited state complexes (like fluorescent exciplexes) are not formed between the fluorophore and the quenchers. In DMSO the bimolecular quenching constant values (kq) calculated on the basis of the best fitting procedures clearly show that the quenching efficiency decreases with the quencher negative reduction potential, E0. The kq (M-1 s-1) and E0 (V) values are, respectively, 7.8 times 109 and -0.134 for 1,4-benzoquinone, 8.9 times 109 and -0.309 for 1,4-naphthoquinone, 2.4 times 109 and -0.8 for nitrobenzene, 0.009 times 109 and -1.022 for azobenzene and 0 and -1.448 for benzophenone. These findings point to the conclusion that upon formation of the encounter complex between OxyBP and the quencher, an electron is released from excited OxyBP to the quencher, similar to what happens in ST. It is suggested that in the pigment granules such a light-induced charge transfer from excited blepharismin to a suitable electron acceptor triggers sensory transduction processes in B. japonicum.
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sensory perception and transduction of uv b radiation by the ciliate Blepharisma japonicum
Biochimica et Biophysica Acta, 1997Co-Authors: Francesco Lenci, Francesco Ghetti, Domenico Gioffrè, Giovanni Checcucci, Antonella SgarbossaAbstract:A key question to answer studying the biological effects of ultraviolet radiation on planktonic micro-organisms is whether they can perceive UV-B radiation as a sensory signal, likewise they do with visible light. We have faced this problem performing an individual-cell analysis of Blepharisma japonicum photomotile responses to UV-B stimuli. Our results on spectral responsiveness and on the effects of a photoresponse inhibitor indicate that B. japonicum is capable to perceive and transduce UV-B radiation as an environmental sensory stimulus, which it escapes from gathering in shadowed areas. Similar UV-B avoidance motile reactions could serve as a behavioural defence mechanism contributing to avoid harmful overexposure to UV-B.
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photosensory transduction in ciliates role of intracellular ph and comparison between stentor coeruleus and Blepharisma japonicum
Journal of Photochemistry and Photobiology B-biology, 1993Co-Authors: Hanna Fabczak, Francesco Ghetti, Pill Soon Song, Stanislaw Fabczak, Giovanni Checcucci, Francesco LenciAbstract:To test the hypothesis that light signal transduction in the unicellular ciliates Stentor coeruleus and Blepharisma japonicum involves a change in intracellular pH as an initial signal following photoexcitation, we studied the dependence of the photophobic responses of the cells to changes in extracellular pH and to reagents that specifically affect intracellular pH. The extracellular pH can modify not only the intracellular pH, but can even reverse the sign of the pH gradient across the cell membrane. Thus, as predicted by the hypothesis, low extracellular pH reversibly inhibited the photophobic response of the ciliates. The intracellular pH-modulating reagents tested included ammonium chloride, a membrane-permeable weak acid that lowers the intracellular pH, and the protonophores carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)-phenyl-hydrazone (FCCP), which collapse the pH gradient across the cell membrane. The low pH and protonophore treatments caused a gradual inhibition of the photophobic responses in both ciliates. The observed reduction of the responsiveness of the cells to visible light can be attributed to the alteration of the intracellular pH, which is suggested to play a specific role in the photosensory transduction in both Stentor coeruleus and Blepharisma japonicum.
Hanna Fabczak - One of the best experts on this subject based on the ideXlab platform.
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signal recognition in lower organisms light induced control of cell movement in the ciliates Blepharisma and stentor
2015Co-Authors: Hanna FabczakAbstract:Unicellular organisms, such as related ciliates Blepharisma and Stentor, show a number of light-induced motile responses to spatial and temporal variations in the photic environment. Thanks to these photosensory capabilities, in unevenly lighted areas both species accumulate in places of optimal light intensity (photodispersal) most suitable for their growth, survival, and development. Both microorganisms lack clearly specified sense organs, as are found in multicellular organisms, however they bear conspicuous subpellicular granules (photoreceptive units) able to perceive the quantity and quality of light. The absorption of photons by cells is thereby converted to internal biophysical/biochemical signals, resulting in the modification of ciliary beating and hence the pattern of motile behavior. Blepharisma and Stentor have been found quite suitable for the study of cellular photoreception employing different biophysical or biochemical experimental methods as well as behavioral observations. In particular, electrophysiological studies using intracellular microelectrodes have improved the understanding of photoelectrical and electromotor coupling at the cellular level. The aim of this article is not only to provide an account of acquired knowledge on the photosensory phenomena in unicellular organisms, but also to show that the study of protozoa and their sensory properties may also hold a key to discovery of as-yet unresolved molecular mysteries in the future.
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effect of phosducin silencing on the photokinetic motile response of Blepharisma japonicum
Photochemical and Photobiological Sciences, 2011Co-Authors: Katarzyna Sobierajska, Ewa Joachimiak, Cezary Bregier, Stanislaw Fabczak, Hanna FabczakAbstract:The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of βγ-dimer of G-proteins (Gβγ) with β-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gβ (∼36 kDa) was reduced, whereas the level of Gβ localized to the membrane (∼32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.
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A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus
Photochemical & Photobiological Sciences, 2008Co-Authors: Hanna Fabczak, Katarzyna Sobierajska, Stanislaw FabczakAbstract:Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at p I s of 4.5 and 7.0 for Blepharisma , and three spots at p I s of 4.4, 5.0 and 6.0 for Stentor , indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.
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phosducin interacts with the g protein betagamma dimer of ciliate protozoan Blepharisma japonicum upon illumination
The Journal of Experimental Biology, 2007Co-Authors: Katarzyna Sobierajska, Hanna Fabczak, Stanislaw FabczakAbstract:Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the betagamma-subunits of G-protein (Gbetagamma) in the ciliate Blepharisma japonicum. Immunological techniques revealed that illumination of cells resulted in a decrease in phosphorylation levels of Pdc and its colocalization with Gbetagamma. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of Gbetagamma binding by this molecule. Possible formation of the Pdc-Gbetagamma complex in cells exposed to light was corroborated by FRET between these proteins. Treatment of cells with okadaic acid, an inhibitor of phosphatase activity, entirely prevented Pdc dephosphorylation by light, colocalization of this phosphoprotein with Gbetagamma and generation of the Pdc-Gbetagamma complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc-Gbetagamma complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of Gbeta. Moreover, a 33 kDa immunoanalog of 14-3-3 protein was identified and we showed that this protein is bound by phosphorylated Pdc in a cell adapted to darkness. The results of this study provide additional detailed characterization of the functional properties of the ciliate Pdc. The likely functional role of Pdc in Blepharisma is discussed.
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4213 The Journal of Experimental Biology 210, 4213-4223 Published by The Company of Biologists 2007
2007Co-Authors: Katarzyna Sobierajska, Hanna Fabczak, Stanislaw FabczakAbstract:doi:10.1242/jeb.005132 Phosducin interacts with the G-protein ��-dimer of ciliate protozoan Blepharisma japonicum upon illuminatio
Giovanni Checcucci - One of the best experts on this subject based on the ideXlab platform.
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Main photophysical properties of oxyblepharismin.
Biophysical chemistry, 2017Co-Authors: Barbara Storti, Francesco Ghetti, Giovanni Checcucci, Riccardo Nifosì, Ranieri BizzarriAbstract:Oxyblepharismin is the photo-oxidized form of blepharismin, the chromophore responsible for the photophobic response of heterotrich ciliate Blepharisma japonicum, and represents a nice model for the study of photo-transduction. In this work, we focused on the photophysical characterization of OxyBP, in view of highlighting the main features related to excitation and emission. By a combined experimental and computational approach we identified the main features of absorption and fluorescence emission of the molecule in solvents of different properties, identifying the nature of transitions as well as the possible heterogeneity at ground/excited state. The thorough photophysical characterization of OxyBP is meant to provide the starting point for the elucidation of the photo-transduction pathway in vivo.
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picosecond transient circular dichroism of the photoreceptor protein of the light adapted form of Blepharisma japonicum
Chemical Physics Letters, 2009Co-Authors: F Hache, Giovanni Checcucci, Pascal Plaza, Monique M Martin, Maithu Khuc, Johanna Brazard, Francesco LenciAbstract:We present a picosecond transient circular dichroism study of OBIP, the putative photoreceptor protein involved in the photophobic response of Blepharisma japonicum. The probe wavelength was chosen at 230 nm. The results are compared to those of the isolated chromophore, OxyBP, in solution. The CD changes in OBIP and OxyBP do not show the same dynamics: OBIP’s signal relaxes in a few ps whereas no such decay is obtained for OxyBP. This observation brings support to the formerly evoked existence of a fast photoinduced reaction in the chromoprotein, and demonstrates the implication of local geometrical changes that accompany this process. 2009 Published by Elsevier B.V.
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fast excited state reaction in the photoreceptor pigment protein complex of the ciliate Blepharisma japonicum
Femtochemistry and Femtobiology#R##N#Ultrafast Events in Molecular Science VIth International Conference on Femtochemistry Maison de la Chimie Paris F, 2004Co-Authors: Mathilde Mahet, Giovanni Checcucci, Pascal Plaza, Monique M Martin, Francesco LenciAbstract:ABSTRACT The primary phototransduction steps of the protozoan Blepharisma japonicum 's light perception were studied by subpicosecond transient absorption spectroscopy. The free chromophore of the light-adapted cell, oxyblepharismin, was characterized in solution and compared to the pigment-protein complex. Although showing similar steady-state and transient spectra, oxyblepharismin and its protein complex display noticeably different excited-state dynamics. In the chromoprotein, a pronounced fast biexponential decay (5 ps and 60 ps) is observed, which is not found in the corresponding kinetics of the isolated pigment. This early behavior could be the signature of a specific primary phototransduction process.
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analyses of structure of photoreceptor organelle and blepharismin associated protein in unicellular eukaryote Blepharisma
Photochemistry and Photobiology, 2000Co-Authors: Tatsuomi Matsuoka, Hiyoshizo Kotsuki, Masaki Ishida, Daisuke Tokumori, Motoaki Matsushita, Satoshi Kimura, Takao Itoh, Giovanni CheccucciAbstract:In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as ‘‘blepharismins‘‘ (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules’ three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyBlepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment‐200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Ble
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electron transfer fluorescence quenching of Blepharisma japonicum photoreceptor pigments
Photochemistry and Photobiology, 1998Co-Authors: Nicola Angelini, Giovanni Checcucci, Pill Soon Song, Annamaria Quaranta, Francesco LenciAbstract:Abstract— The hypericin analogs blepharismin (BP), oxyblepharismin (OxyBP) and stentorin (ST), the photosensing chromophores responsible for photomotile reactions in the ciliates Blepharisma japonicum (red and blue cells) and Stentor coeruleus, represent a new class of photoreceptor pigments whose chemical structures have recently been determined. In the case of ST it has been shown that the first excited singlet state can be deactivated by donation of an electron to an appropriate acceptor molecule (e.g. a quinone molecule). This charge transfer can be considered a possible mechanism for the primary photoprocess for the photomotile responses in S. coeruleus. To determine whether an electron transfer process also occurs in the deactivation of excited blepharismin, we studied the fluorescence quenching of OxyBP in dimethyl-sulfoxide (DMSO) and in ethanol using electron acceptors with different reduction potentials. Under our experimental conditions ground state and excited state complexes (like fluorescent exciplexes) are not formed between the fluorophore and the quenchers. In DMSO the bimolecular quenching constant values (kq) calculated on the basis of the best fitting procedures clearly show that the quenching efficiency decreases with the quencher negative reduction potential, E0. The kq (M-1 s-1) and E0 (V) values are, respectively, 7.8 times 109 and -0.134 for 1,4-benzoquinone, 8.9 times 109 and -0.309 for 1,4-naphthoquinone, 2.4 times 109 and -0.8 for nitrobenzene, 0.009 times 109 and -1.022 for azobenzene and 0 and -1.448 for benzophenone. These findings point to the conclusion that upon formation of the encounter complex between OxyBP and the quencher, an electron is released from excited OxyBP to the quencher, similar to what happens in ST. It is suggested that in the pigment granules such a light-induced charge transfer from excited blepharismin to a suitable electron acceptor triggers sensory transduction processes in B. japonicum.
Stanislaw Fabczak - One of the best experts on this subject based on the ideXlab platform.
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effect of phosducin silencing on the photokinetic motile response of Blepharisma japonicum
Photochemical and Photobiological Sciences, 2011Co-Authors: Katarzyna Sobierajska, Ewa Joachimiak, Cezary Bregier, Stanislaw Fabczak, Hanna FabczakAbstract:The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of βγ-dimer of G-proteins (Gβγ) with β-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gβ (∼36 kDa) was reduced, whereas the level of Gβ localized to the membrane (∼32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.
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A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus
Photochemical & Photobiological Sciences, 2008Co-Authors: Hanna Fabczak, Katarzyna Sobierajska, Stanislaw FabczakAbstract:Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at p I s of 4.5 and 7.0 for Blepharisma , and three spots at p I s of 4.4, 5.0 and 6.0 for Stentor , indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.
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phosducin interacts with the g protein betagamma dimer of ciliate protozoan Blepharisma japonicum upon illumination
The Journal of Experimental Biology, 2007Co-Authors: Katarzyna Sobierajska, Hanna Fabczak, Stanislaw FabczakAbstract:Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the betagamma-subunits of G-protein (Gbetagamma) in the ciliate Blepharisma japonicum. Immunological techniques revealed that illumination of cells resulted in a decrease in phosphorylation levels of Pdc and its colocalization with Gbetagamma. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of Gbetagamma binding by this molecule. Possible formation of the Pdc-Gbetagamma complex in cells exposed to light was corroborated by FRET between these proteins. Treatment of cells with okadaic acid, an inhibitor of phosphatase activity, entirely prevented Pdc dephosphorylation by light, colocalization of this phosphoprotein with Gbetagamma and generation of the Pdc-Gbetagamma complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc-Gbetagamma complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of Gbeta. Moreover, a 33 kDa immunoanalog of 14-3-3 protein was identified and we showed that this protein is bound by phosphorylated Pdc in a cell adapted to darkness. The results of this study provide additional detailed characterization of the functional properties of the ciliate Pdc. The likely functional role of Pdc in Blepharisma is discussed.
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4213 The Journal of Experimental Biology 210, 4213-4223 Published by The Company of Biologists 2007
2007Co-Authors: Katarzyna Sobierajska, Hanna Fabczak, Stanislaw FabczakAbstract:doi:10.1242/jeb.005132 Phosducin interacts with the G-protein ��-dimer of ciliate protozoan Blepharisma japonicum upon illuminatio
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photosensory transduction in unicellular eukaryotes a comparison between related ciliates Blepharisma japonicum and stentor coeruleus and photoreceptor cells of higher organisms
Journal of Photochemistry and Photobiology B-biology, 2006Co-Authors: Katarzyna Sobierajska, Hanna Fabczak, Stanislaw FabczakAbstract:Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.