Shoshi Muto

Nagoya University, Nagoya-shi, Aichi-ken, Japan

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Publications (50)167.96 Total impact

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    ABSTRACT: The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa ( J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca2+. Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti-Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca2+, and a 40-kDa Ca2+-independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca2+-dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48 / 80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca2+ receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound.
    No preview · Article · Jul 2008 · Journal of Phycology
  • Koji Takahashi · Takashi Yuasa · Shoshi Muto
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    ABSTRACT: A 26-kDa and a 36-kDa protein that cross-reacted with anti-Ga-common and anti-Gβ antibodies, respectively, were detected in Dunaliella cells. The 26-kDa protein was solubilized from a crude membrane fraction with deoxycholate and purified to homogeneity by DE52 and hydroxylapatite chromatography and DEAE-5PW high performance liquid chromatography (HPLC). The hydroxylapatite-purified preparation had GTPγS binding and GTPase activities, but the homogeneous 26-kDa protein had none. The sequence of the 28 N-terminal amino acids of the 26-kDa protein had no homology to any GTP binding protein thus far reported.
    No preview · Article · Apr 2006 · Physiologia Plantarum
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    ABSTRACT: Plants are always exposed to the menace of oxidative stress and protect themselves by activating a variety of defense responses. However, molecular mechanisms for oxidative stress-induced gene expression are largely unknown. We here studied the roles of the oxidative stress-responsive putative voltage-dependent Ca(2+) permeable channels, NtTPC1A and NtTPC1B, and cell cycle in H(2)O(2)-induced expression of antioxidant enzymes, glutathione peroxidase (GPX) and ascorbate peroxidase (APX), in tobacco BY-2 cells. H(2)O(2)-induced [Ca(2+)](cyt) rise and expression of GPX and APX were inhibited by the cosuppression of NtTPC1A/B as well as Al ion, a specific blocker for NtTPC1s, and enhanced by overexpression of AtTPC1, suggesting that NtTPC1s are the major Ca(2+)-permeable channels activated by H(2)O(2) and that Ca(2+) influx via NtTPC1s is involved in induction of H(2)O(2)-triggered gene expression. Oxidative stress-induced signal transduction mechanisms were highly dependent on the phases of the cell cycle; H(2)O(2)-induced [Ca(2+)](cyt) rise and expression of GPX and APX as well as the level of NtTPC1s transcripts correlated with each other and were maximal at G1 phase. In contrast, the cell cycle-dependence of hypoosmotic shock-induced [Ca(2+)](cyt) rise that is known to be independent of NtTPC1s was almost reverse and maximal at S phase. These results suggest that the cell cycle-dependent regulation of oxidative stress-induced [Ca(2+)](cyt) rise and expression of NtTPC1s contribute to the cell cycle dependence of H(2)O(2)-induced expression of peroxidases. Various Ca(2+)-mediated signal transduction pathways are differentially regulated by the cell cycle.
    No preview · Article · Dec 2005 · Biochemical and Biophysical Research Communications
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    Takuya Furuichi · Izumi C Mori · Shoshi Muto
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    ABSTRACT: Protein kinases are involved in signal transduction for environmental stress responses. In response to drought and salinity, a 48-kDa protein kinase (AAPK; abscisic acid-activated protein kinase (AAPK) in guard cells is activated by abscisic acid (ABA) and phosphorylates several targets such as the carboxy-terminus of inward-rectifying K+ channel and heterogeneous mRNA binding protein to adopt to the changing environment. The AAPK expressed specifically in guard cells, and recombinant AAPK was phosphorylated only with the extract from ABA-treated guard cells but not from untreated cells. This indicates the presence of an AAPK kinase (AAPKK), which is activated by ABA and phosphorylates AAPK preceding the activation of AAPK. Both AAPK and AAPKK are involved in the protein kinase cascade for the rapid ABA-signaling.
    Full-text · Article · Sep 2005 · Zeitschrift fur Naturforschung C
  • Takuya Furuichi · Shoshi Muto
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    ABSTRACT: Using Ca2+-dependent photoprotein aequorin-transformed tobacco BY-2 cell suspensions, the sugar-induced increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) was investigated by measuring the luminescence intensity. When 0.5 M sucrose or some other sugars were fed to the cells, strong and transient luminescence was observed. Salts or sugar analogues didn't show this effect. In addition, the intensity of sucrose-induced aequorin luminescence was gradually enhanced when cells were exposed to sugar-starvation. This was observed with the concurrent expression of the sucrose/H+ co-transporter, NtSUT1A. The [Ca2+]cyt increase may initiate Ca2+-signaling leading to the expression of genes related to biosynthesis of storage carbohydrates in a sink organ. The sugar-signaling may play an important role in the conversion on nutritional stage of plant tissue, source organ to sink organ.
    No preview · Article · Sep 2005 · Zeitschrift fur Naturforschung C
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    Kawano T · Kadono T · Yang SC · Muto S
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    ABSTRACT: Cations of Al and rare earth elements (REEs) are phyto-toxic. The toxicity of such cations has been summarized as followings: (1) metal cations induce the production of superoxide (O2-) in plant cells; (2) the metal cations with higher valence induce greater oxidative burst (OXB); (3) with higher ion valence, the concentration required for maximal response is minimized; (4) the induced OXB is sensitive to inhibitors of NADPH oxidase. To date, non-redox metal cations such as those of Al, La, Ce, and Gd, have been shown to induce OXB. It has been shown that Zn2+ inhibits the REE-induced OXB. Zn is normally present in plants at high level, and its deficiency is one of the most widespread micronutrient deficiencies in plants, causing severe reductions in crop production. Increasing studies indicate that oxidative damage to plants caused by reactive oxygen species (ROS) results from a deficiency of Zn. A hypothetical model explains that the O2--generating activity of NADPH oxidase is sensitive to Zn2+, thus the REE-dependent stimulation could be inhibited or retarded by Zn2+. In this study, we tested the effect of Zn2+ supplementation on REE-induced OXB in tobacco cell culture, measured with the O2.--specific chemiluminescence (CL) of a Cypridina luciferin analog (CLA). CLA-CL specifically indicates the generation of O2- (and 1O2 with a lesser extent) but not that of other ROS. Here, the mode of Zn action against the REE-induced OXB was assessed with Lineweaver-Burk kinetics and possible eco-physiological roles for Zn in interaction with other metal cations in plants are discussed.
    Full-text · Conference Paper · Aug 2005
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    ABSTRACT: The molecular links between the cell cycle and defense responses in plants are largely unknown. Using synchronized tobacco BY-2 cells, we analyzed the cell cycle dependence of elicitor-induced defense responses. In synchronized cultured apoaequorin-expressing cells, the increase in cytosolic free Ca2+ induced by a proteinaceous elicitor, cryptogein, was greatly suppressed during the G2 and M phases in comparison with G1 or S phases. Treatment with cryptogein during the G1 or S phases also induced biphasic (rapid/transient and slow/prolonged) responses in activation of mitogen-activated protein kinases (MAPKs) and production of reactive oxygen species (ROS). In contrast, elicitor treatment during the G2 or M phases induced only a rapid and transient phase of MAPK activation and ROS production. Their slow and prolonged phases as well as expression of defense-related genes, cell cycle arrest and cell death were induced only after the cell cycle progressed to the G1 phase; removal of the elicitor before the start of the G1 phase inhibited these responses. These results suggest that although cryptogein recognition occurred at all phases of the cell cycle, the recognition during the S or G1 phases, but not at the G2 or M phases, induces the prolonged activation of MAPKs and the prolonged production of ROS, followed by cell cycle arrest, accumulation of defense-related gene transcripts and cell death. Elicitor signal transduction depends on the cell cycle and is regulated differently at each phase.
    No preview · Article · Feb 2005 · Plant and Cell Physiology
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    Tomonori Kawano · Takuya Furuichi · Shoshi Muto
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    ABSTRACT: acid (SA) that was named after Salix plant (willow), was first discovered as a major component in the extracts from willow tree bark that had been used as a natural anti-inflammatory drug from the ancient time to the 18 th century (Rainsford 1984; Weissman 1991). Acetylsalicylic acid which is widely known as aspirin is the world first synthetic drug that had been produced by Bayer Company as anti-inflammatory agent in 1897 (Weissman 1991). Since then aspirin became one of the most popular drugs among the people and has been widely used for over 100 years, and pharmacological actions of aspirin and related salicylates in animal system have been intensively studied while only little about the action of SA in plants has been elucidated. White (1979) was the first plant biologist paid attention to salicylates as disease resistance-inducing chemicals. He and his colleague demonstrated that injection of aspirin into tobacco leaves enhanced the resistance to subsequent infection by tobacco mosaic virus (TMV) (White 1979; Antoniw and White 1980). Later, it has been shown that this treatment induces the accumulation of pathogenesis-related (PR) proteins (Kessmann and Ryals 1993; Malamy et al. 1990; Métraux et al. 1990). Recent intensive studies have shown that SA biosynthesis and signaling required for plant defense should not be regarded as a linear pathway but rather as a complex network (Shah 2003). This article describes the recent progress in our understanding of SA signaling and biosynthesis leading to a controlled level of SA in plants, by covering historical milestone works. Until now, involvement of SA signaling was com-monly confirmed by the phenotypes of plants carrying the NahG transgene encoding bacterial salicylate hydroxylase which converts SA to catechol (Delaney et al. 1994; Hunt et al. 1996; Alvarez 2000). Lowered non-host resistance to a Pseudomonas syringae pv. phaseolicola strain is one of the representative NahG phenotype reported for NahG Arabidopsis plants, but a recent report presented the data questioning above effect of NahG and also reliability of NahG experiments judging the involvement of SA was very much weakened (van Wees et al. 2003). The report uncovered that the by-product of NahG action, catechol may be the actual player lowering the non-host resistance to the pathogen. The report propounded that any conclusion about SA requirement based solely on phenotypes of NahG plants should be reevaluated. Further investigations on NahG side effects are urgently required for evaluation of the earlier works using NahG plants. In contrast, the works directly blocking the SA biosynthesis for examining the SA requirement in defense response (e.g. Nawrath and Métraux 1999; Wildermuth et al. 2001) must receive
    Full-text · Article · Dec 2004 · Plant Biotechnology
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    ABSTRACT: In plant cells, Al ion plays dual roles as an inducer and an inhibitor of Ca(2+) influx depending on the concentration. Here, the effects of Al on Ca(2+) signaling were assessed in tobacco BY-2 cells expressing aequorin and a putative plant Ca(2+) channel from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). In wild-type cells (expressing only aequorin), Al treatment induced the generation of superoxide, and Ca(2+) influx was secondarily induced by superoxide. Higher Al concentrations inhibited the Al-stimulated and superoxide-mediated Ca(2+) influx, indicating that Ca(2+) channels responsive to reactive oxygen species (ROS) are blocked by high concentration of Al. H(2)O(2)-induced Ca(2+) influx was also inhibited by Al. Thus, inhibitory action of Al against ROS-induced Ca(2+) influx was confirmed. Similarly, known Ca(2+) channel blockers such as ions of La and Gd inhibited the H(2)O(2)-induced Ca(2+) influx. While La also inhibited the hypoosmotically induced Ca(2+) influx, Al showed no inhibitory effect against the hypoosmotic Ca(2+) influx. The effects of Al and La on Ca(2+) influx were also tested in the cell line overexpressing AtTPC1 and the cell line AtTPC1-dependently cosuppressing the endogenous TPC1 equivalents. Notably, responsiveness to H(2)O(2) was lost in the cosuppression cell line, thus TPC1 channels are required for ROS-responsive Ca(2+) influx. Data also suggested that hypoosmotic shock induces TPC1-independent Ca(2+) influx and Al shows no inhibitory action against the TPC1-independent event. In addition, AtTPC1 overexpression resulted in a marked increase in Al-sensitive Ca(2+) influx, indicating that TPC1 channels participate in osmotic Ca(2+) influx only when overexpressed. We concluded that members of TPC1 channel family are the only ROS-responsive Ca(2+) channels and are the possible targets of Al-dependent inhibition.
    Full-text · Article · Dec 2004 · Biochemical and Biophysical Research Communications
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    ABSTRACT: By searching a Chlamydomonas expressed sequence tag database and by comparing the retrieved data with homologous sequences from a DNA database, we identified an expressed Chlamydomonas reinhardtii putative major intrinsic protein (MIP) gene. The nucleotide sequence, consisting of 1,631 bp, contains an open reading frame coding for a 300-amino-acid protein named CrMIP1. It possesses conserved NPA motifs, but is not highly homologous to known aquaporins. CrMIP1 was expressed in Saccharomyces cerevisiae and assayed for water and glycerol transport activity. By the stopped-flow spectrophotometric assay, CrMIP1 did not enhance the osmotic water permeability of membrane vesicles of the yeast transformant. However, the transformant cells showed glycerol transport activity in the in vivo assay using [14C]glycerol. This is the first report on the isolation and functional identification of a MIP member from algae.
    Full-text · Article · Oct 2004 · Plant and Cell Physiology
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    ABSTRACT: Salicylic acid beta-glucoside (SAG) is a storage form of a defense signal against pathogens, releasing free salicylic acid (SA), to meet the requirements in plants. Since excess SA induces locally restricted cell death following oxidative burst and Ca2+ influx in plants, the effects of SAG on cell viability, Ca2+ influx, and generation of superoxide (O2*-) were examined in suspension-cultured tobacco BY-2 cells expressing aequorin. Among SA-related chemicals tested, only SAG induced the slow and long-lasting O2*- generation, although SAG was less active in acute O2*- generation, Ca2+ influx and induction of cell death. The prolonging action of SAG is likely due to gradual release of SA and the data suggested that a peroxidase-dependent reaction is involved. Notably, pretreatment with low-dose SA (50 micromu) enhanced the response to SAG by 2.5-fold. There are four possible secondary messengers in early SA signaling detectable in the BY-2 culture, namely O2*-, H2O2, Ca2+ and protein kinase (PK). If these messengers are involved in the low-dose SA-dependent priming for SAG response, they should be inducible by low-dose SA. Among the four SA-inducible signaling events, PK activation was excluded from the low-dose SA action since a much higher SA dose (> 0.4 mmu) was required for PK activation.
    Full-text · Article · Sep 2004 · Zeitschrift fur Naturforschung C
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    ABSTRACT: Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.
    No preview · Article · Jun 2004 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.
    No preview · Article · Mar 2004 · Plant and Cell Physiology
  • Takuya Furuichi · Shoshi Muto
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    ABSTRACT: . Changes in the cytosolic concentration of calcium ions ([Ca2+]i) play a key second messenger role in signal transduction. These changes are visualized by making use of either Ca2+-sensitive fluorescent dyes or the Ca2+-sensitive photoprotein, aequorin. Here we describe the advances made over the last 10 years or so, which have conclusively demonstrated a second messenger role for [Ca2+]i in a few model plant systems. Characteristic changes in [Ca2+]i have been seen to precede the responses of plant cells and whole plants to physiological stimuli. This has had a major impact on our understanding of cell signaling in plants. The next challenge will be to establish how the Ca2+ signals are encrypted and decoded in order to provide specificity, and we discuss the current understanding of how this may be achieved.
    No preview · Article · Dec 2003 · Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme
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    ABSTRACT: SUMMARYA cDNA (DtNDK1) encoding a nucleoside diphosphate (NDP) kinase with a putative mitochondrial targeting signal sequence was previously isolated from the halo-tolerant green alga Dunaliella tertiolecta. When expressed in Saccharomyces cerevisiae, the processed DtNDK1 enzyme was specifically localized to mitochondria. The present study reports several biochemical characteristics of the mitochondrial NDP kinase from D. tertiolecta. The mature protein was expressed as either N- or C-terminal hexahistidine-tagged protein and purified to homogeneity by affinity chromato-graphy. Native gel electrophoresis and sedimentation velocity in sucrose density gradients showed that the active enzyme consisted of a hexamer. The enzyme, with a pH optimum of 7, showed the highest specificity to dCDP (Km= 50 μmol/L) and the highest turnover towards the synthesis of UTP (up to 140-fold higher). The present study also provides evidence that purified DtNDK1 proteins are capable of transferring a phosphate group to another protein.
    Full-text · Article · Aug 2003 · Phycological Research
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    ABSTRACT: Trivalent cations such as those of Al, La, and Gd are phytotoxic. Our previous works showed that addition of LaCl(3) or GdCl(3) to tobacco cells triggers the generation of superoxide (O(2)*-). Here, we show that AlCl(3) at normal physiological pH (5.8) induces much greater production of O(2)*- (detected with a specific chemiluminescence probe), indicating that these trivalent cations similarly induce the oxidative bursts. It was shown that NADPH oxidase is involved in the generation of O(2)*- and the yield of O(2)*- was dose-dependent (ca. 6mM Al, optimal). Following the acute spike of O(2)*-, a gradual increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) was detected with the luminescence of recombinant aequorin over-expressed in the cytosol. Interestingly, a O(2)*- scavenger and a Ca(2+) chelator significantly lowered the level of [Ca(2+)](c) increase, indicating that the Al-induced O(2)*- stimulates the influx of Ca(2+). Compared to the induction of O(2)*- generation, the [Ca(2+)](c) elevation was shown to be maximal (340 nM) at relatively lower Al concentrations (ca. 1.25 mM). Thus, the Al concentration optimal for O(2)*- is too much (inhibitory) for [Ca(2+)](c). In addition, high concentrations of Al were shown to be inhibitory to the H(2)O(2)-induced Ca(2+) influx. This explains the ineffectiveness of high Al concentration in the oxidative burst-mediated induction of [Ca(2+)](c) increase. It is likely that Al-induced [Ca(2+)](c) elevation is manifested from the finely geared balance between the O(2)*- -mediated driving force and the channel inhibition-mediated brake. Furthermore, it is note-worthy that Al (< or =10mM) showed no inhibitory effect on the hypo-osmolarity-induced Ca(2+) influx, implying that Al may be a selective inhibitor of redox-responsive Ca(2+) channels. Possible target channels of Al actions are discussed.
    Full-text · Article · Aug 2003 · Biochemical and Biophysical Research Communications
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    ABSTRACT: A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2+-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.
    Full-text · Article · Mar 2003 · Bioscience Biotechnology and Biochemistry
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    Shoshi Muto · Tomonori Kawano
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    ABSTRACT: Until late '90s, only four enzymatic pathways have been considered to be responsible for production of reactive oxygen species (ROS) during the plant defense mechanism, namely NADPH oxidase, pH-dependent cell wall peroxidase, germin-like oxalate oxidase and amine oxidases. Among four enzymes, NADPH oxidase and the pH-dependent cell waa-bound peroxidase are considered to be the main sources of ROS in plants (Bolwell, 1995; Bolwell and Wojtaszek, 1997). H2O2 is an electron-accepting substrate for peroxidase-dependent reactions, thus peroxidases are likely considered merely as ROS detoxifying enzymes. However, it actually produces ROS at certain occasions for defenses against infection with pathogens. ROS generation by extracellularly secreted peroxidase in elicitor-treated plants has been reported (Kiba et al., 1996), although the electron-donating substrates are obscure..., (continues)
    Full-text · Chapter · Jan 2003
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    ABSTRACT: We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.
    No preview · Article · Dec 2002 · Plant and Cell Physiology
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    ABSTRACT: Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, beta-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O2*-). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc'). The O2*- generation and Asc formation were detected by O2*--specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O2*- production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H2O2, but a trace of H2O2 added to the system greatly increased the production of both O2*- and Asc*. Addition of GSH completely inhibited the PEA-dependent production of O2*- and Asc* in Hb solution. We propose that the O2*- generation and Asc* formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.
    Full-text · Article · Jul 2002 · Bioscience Biotechnology and Biochemistry