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author:("Saito, chieo")
1.  Qualitative difference between “bulb” membranes and other vacuolar membranes 
Plant Signaling & Behavior  2011;6(12):1914-1917.
“Bulb” is a mobile and complex structure appearing in vacuolar membrane of plant cell. We recently reported new fluorescent marker lines for bulbs and bulb-less mutants. We tried multicolor visualization of vacuolar membrane to show distinct segregation of bulb-positive protein (γTIP or AtVAM3) and bulb-negative protein (AtRab75). Unexpectedly, GFP-AtRab75 resulted to localize in bulb under the condition of co-expression with TagRFP-AtVAM3. The signal intensities of GFP-AtRab75 and TagRFP-AtVAM3 were quantified and compared. The result indicates that TagRFP-AtVAM3 is concentrated in bulb than GFP-AtRab75.
doi:10.4161/psb.6.12.18061
PMCID: PMC3337177  PMID: 22105033
AtRab75; AtVam3; plant growth; Rab-GTPase; SNARE; vacuolar membranes; “bulb”
2.  c-JUN N-TERMINAL KINASE MODULATES OXIDANT STRESS AND PEROXYNITRITE FORMATION INDEPENDENT OF INDUCIBLE NITRIC OXIDE SYNTHASE IN ACETAMINOPHEN HEPATOTOXICITY 
Acetaminophen (APAP) overdose, which causes liver injury in animals and humans, activates c-jun N-terminal kinase (JNK). Although it was shown that the JNK inhibitor SP600125 effectively reduced APAP hepatotoxicity, the mechanisms of protection remain unclear. C57Bl/6 mice were treated with 10 mg/kg SP600125 or vehicle (8% dimethylsulfoxide) 1h before 600 mg/kg APAP administration. APAP time-dependently induced JNK activation (detected by JNK phosphorylation). SP600125, but not the vehicle, reduced JNK activation, attenuated mitochondrial Bax translocation and prevented the mitochondrial release of apoptosis-inducing factor at 4–12 h. Nuclear DNA fragmentation, nitrotyrosine staining, tissue GSSG levels and liver injury (plasma ALT release and necrosis) were partially attenuated by the vehicle (−65%) and completely eliminated by SP600125 (−98%) at 6 and 12h. Furthermore, SP600125 attenuated the increase of inducible nitric oxide synthase (iNOS) mRNA and protein. However, APAP did not enhance plasma nitrite+nitrate levels (NO formation); SP600125 had no effect on this parameter. The iNOS inhibitor L-NIL did not reduce NO formation or injury after APAP but prevented NO formation caused by endotoxin. Since SP600125 completely eliminated the increase in hepatic GSSG levels, an indicator of mitochondrial oxidant stress, it is concluded that the inhibition of peroxynitrite was mainly caused by reduced superoxide formation. Our data suggest that the JNK inhibitor SP600125 protects against APAP-induced liver injury in part by attenuation of mitochondrial Bax translocation but mainly by preventing mitochondrial oxidant stress and peroxynitrite formation and thereby preventing the mitochondrial permeability transition pore opening, a key event in APAP-induced cell necrosis.
doi:10.1016/j.taap.2010.04.015
PMCID: PMC2885557  PMID: 20423716
Acetaminophen; Hepatotoxicity; Mitochondria; JNK; SP600125; Bax; Oxidant Stress
3.  MECHANISM OF PROTECTION BY METALLOTHIONEIN AGAINST ACETAMINOPHEN HEPATOTOXICITY 
Toxicology and applied pharmacology  2009;242(2):182-190.
Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the US. Metallothionein (MT) expression attenuates APAP-induced liver injury. However, the mechanism of this protection remains incompletely understood. To address this issue, C57BL/6 mice were treated with 100 µmol/kg ZnCl2 for 3 days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. Liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining), MT expression, hepatic glutathione (GSH) and glutathione disulfide (GSSG) levels were determined after 6 h. APAP alone caused severe liver injury with oxidant stress (increased GSSG levels), peroxynitrite formation and DNA fragmentation, all of which were attenuated by zinc-induced MT expression. In contrast, MT knockout mice were not protected by zinc. Hydrogen peroxide-induced cell injury in primary hepatocytes was dependent only on the intracellular GSH levels but not on MT expression. Thus, the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species. Zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI). However, MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins, which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage.
doi:10.1016/j.taap.2009.10.006
PMCID: PMC2789886  PMID: 19835899
Acetaminophen; hepatotoxicity; metallothionein; oxidant stress; liver failure; covalent binding
4.  NOVEL MECHANISMS OF PROTECTION AGAINST ACETAMINOPHEN HEPATOTOXICITY IN MICE BY GLUTATHIONE AND N-ACETYLCYSTEINE 
Hepatology (Baltimore, Md.)  2010;51(1):246-254.
Acetaminophen (APAP) overdose is a major cause of acute liver failure. The glutathione (GSH) precursor N-acetylcysteine (NAC) is used to treat patients with APAP overdose for up to 48 h. Although it is well established that early treatment with NAC can improve the scavenging of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), protective mechanisms at later times remain unclear. To address this issue, fasted C3Heb/FeJ mice were treated with 300 mg/kg APAP and then received intravenously 0.65 mmol/kg GSH or NAC at 1.5 h after APAP. The animals were sacrificed at 6 h. APAP alone caused severe liver injury with peroxynitrite formation and DNA fragmentation, all of which was attenuated by both treatments. However, GSH (−82%) was more effective than NAC (−46%) in preventing liver injury. Using nuclear magnetic resonance spectroscopy to measure tissue ATP levels and the substrate flux through the mitochondrial Krebs cycle, it was observed that the reduced liver injury correlated with accelerated recovery of mitochondrial GSH content, maintenance of ATP levels and an increased substrate supply for the mitochondrial Krebs cycle compared to APAP alone. NAC treatment was less effective in recovering ATP and mitochondrial GSH levels and showed reduced substrate flux through the Krebs cycle compared to GSH. However, increasing the dose of NAC improved the protective effect similar to GSH suggesting that the amino acids not used for GSH synthesis were used as mitochondrial energy substrates.
Conclusion
Delayed treatment with GSH and NAC protect against APAP overdose by dual mechanisms, i.e. by enhancing hepatic and mitochondrial GSH levels (scavenging of reactive oxygen and peroxynitrite) and by supporting the mitochondrial energy metabolism.
doi:10.1002/hep.23267
PMCID: PMC2977522  PMID: 19821517
Drug-induced liver injury; oncotic necrosis; oxidant stress; acute liver failure; mitochondrial bioenergetics
5.  Application of Lifeact Reveals F-Actin Dynamics in Arabidopsis thaliana and the Liverwort, Marchantia polymorpha 
Plant and Cell Physiology  2009;50(6):1041-1048.
Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact–Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact–Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.
doi:10.1093/pcp/pcp055
PMCID: PMC2694730  PMID: 19369273
Actin; Arabidopsis thaliana; Lifeact; Liverwort; Marchantia polymorpha

Results 1-5 (5)