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author:("Ito, solei")
1.  Genome-Wide Screening of Salt Tolerant Genes by Activation-Tagging Using Dedifferentiated Calli of Arabidopsis and Its Application to Finding Gene for Myo-Inositol-1-P-Synthase 
PLoS ONE  2015;10(5):e0115502.
Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis.
PMCID: PMC4433338  PMID: 25978457
2.  Tenascin C protects aorta from acute dissection in mice 
Scientific Reports  2014;4:4051.
Acute aortic dissection (AAD) is caused by the disruption of intimomedial layer of the aortic walls, which is immediately life-threatening. Although recent studies indicate the importance of proinflammatory response in pathogenesis of AAD, the mechanism to keep the destructive inflammatory response in check is unknown. Here, we report that induction of tenascin-C (TNC) is a stress-evoked protective mechanism against the acute hemodynamic and humoral stress in aorta. Periaortic application of CaCl2 caused stiffening of abdominal aorta, which augmented the hemodynamic stress and TNC induction in suprarenal aorta by angiotensin II infusion. Deletion of Tnc gene rendered mice susceptible to AAD development upon the aortic stress, which was accompanied by impaired TGFβ signaling, insufficient induction of extracellular matrix proteins and exaggerated proinflammatory response. Thus, TNC works as a stress-evoked molecular damper to maintain the aortic integrity under the acute stress.
PMCID: PMC3920275  PMID: 24514259
3.  Crystallization and preliminary X-ray analysis of geraniol dehydrogenase from Backhousia citriodora (lemon myrtle) 
A recombinant form of geraniol dehydrogenase from Backhousia citriodora has been overexpressed in Escherichia coli and purified and crystallized by the sitting-drop method using polyethylene glycol 3350 as a precipitant.
A recombinant form of geraniol dehydrogenase (EC from Backhousia citriodora was overexpressed in Escherichia coli and purified and crystallized by the sitting-drop method using polyethylene glycol 3350 as a precipitant. A data set to 2.3 Å resolution was collected from a monocrystal at 98 K using synchrotron radiation on beamline NE3A of the Photon Factory. The crystals belonged to the orthorhombic group P21212, with unit-cell parameters a = 125.00, b = 151.01, c = 51.18 Å. The asymmetric unit is expected to contain two BcGEDH molecules, with a corresponding crystal volume per protein weight of 3.1 Å3 Da−1 and a solvent content of 60.6%.
PMCID: PMC3107137  PMID: 21636906
geraniol dehydrogenase; Backhousia citriodora
4.  Crystallization and preliminary X-ray analysis of a glucansucrase from the dental caries pathogen Streptococcus mutans  
In this study, the glucansucrase from the dental caries pathogen S. mutans was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant.
Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1 Å resolution.
PMCID: PMC2935234  PMID: 20823533
glucansucrase; dental caries; Streptococcus mutans
5.  Crystallization and molecular-replacement studies of the monoclonal antibody mAbR310 specific for the (R)-HNE-modified protein 
Antigen-free Fab fragment of mAbR310, which recognizes (R)-HNE modified protein, has been crystallized. Initial phases have been obtained by molecular replacement.
4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE–histidine Michael addition-type adduct possessing three chiral centres in the cyclic hemiacetal structure. Monoclonal antibodies against HNE-modified protein have been widely used for assessing oxidative stress in vitro and in vivo. Here, the purification, crystallization and preliminary crystallographic analysis of a Fab fragment of novel monoclonal antibody R310 (mAbR310), which recognizes (R)-HNE-modified protein, are reported. The Fab fragment of mAbR310 was obtained by digestion with papain, purified and crystallized. Using hanging-drop vapour-diffusion crystallization techniques, crystals of mAbR310 Fab were obtained. The crystal belongs to the monoclinic space group C2 (unit-cell parameters a = 127.04, b = 65.31, c = 64.29 Å, β = 118.88°) and diffracted X-rays to a resolution of 1.84 Å. The asymmetric unit contains one molecule of mAbR310, with a corresponding crystal volume per protein weight of 2.51 Å3 Da−1 and a solvent content of 51.0%.
PMCID: PMC2243084  PMID: 16754982
mAbR310; monoclonal antibodies; Fab fragments
6.  Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus  
This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6222 and diffracted to a resolution of 2.4 Å.
Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6222, with unit-cell parameters a = b = 145.97, c = 118.03 Å.
PMCID: PMC1952353  PMID: 16511150
pyruvate kinase

Results 1-6 (6)