Hiroko Sato

Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

Establishment of an efficient in vitro culture and particle bombardment‐mediated transformation systems in Miscanthus sinensis Anderss., a potential bioenergy crop

Plants belonging to the genus Miscanthus are considered promising bioenergy crops. Here, we report the establishment of tissue culture system through particle bombardment‐mediated transformation in Miscanthus sinensis Anderss. Callus was induced efficiently from mature seeds in a medium containing a combination of a relatively high level of 2,4‐dichlorophenoxyacetic acid (5 mg L−1) and a relatively low level of 6‐benzyladenine (BA) (0.01 mg L−1). Callus induction potential of 18 accessions of M. sinensis, which were collected from various sites in Japan, was compared. Significant correlation was detected between compact (embryogenic) callus induction frequency and average annual air temperature in collection sites. An accession from Tanegashima Island showed the highest production of compact callus. We found that a higher level of BA causes callus browning; the 2 mg L−1 BA is the optimal concentration for regeneration. Both compact and friable calli were suitable for particle bombardment transformation. Through selection under the presence of 50 mg L−1 hygromycin for 3 weeks and further selection under the presence of 150 mg L−1 for 1 month, hygromycin‐resistant calli survived, of which 72.2% had been entirely transformed. Plants were regenerated from calli in the presence of hygromycin; transcripts of the hpt and gfp genes, which were cobombarded to the calli, were detected in the regenerated plants. This is the first report on the establishment of the in vitro culture of M. sinensis using mature seeds, the variation of callus formation among accessions collected from various sites in Japan, and particle bombardment‐mediated transformation in the genus Miscanthus.

Enhanced growth of human vascular endothelial cells on negative ion (Ag-)-implanted hydrophobic surfaces

Silver negative ions (Ag-) were implanted to an insulator, polystyrene, in a relatively low ion energy ranging from 5 to 30 keV, and in a dose ranging from 10(14) to 6 x 10(16) ions. cm-2. Surfaces of Ag--implanted polystyrene were studied by means of secondary ion mass spectrometry, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and micro-Raman spectroscopy, and contact angle measurement. As a result of Ag- implantation, the polystyrene surfaces underwent degradation, thereby becoming more hydrophilic with increasing dose and ion energy except an ion energy of 30 keV. The Ag- implantation in polystyrene led to enhanced growth of human vascular endothelial cells, which grew to more extent with increased hydrophilicity of Ag--implanted surfaces except an ion energy of 30 keV. Polystyrene surfaces on which Ag- were implanted up to an ion energy of 30 keV caused the same hydrophobic level as polystyrene surface itself. Nevertheless, the Ag--implanted polystyrene showed relatively good biocompatibility different from polystyrene. Such an improvement in cell adhesion may be related to the formation of a graphite-like structure on polystyrene surfaces by a Ag--implanted process. Moreover, upon plating in a high cell density, human vascular endothelial cells survived even on the polystyrene region of Ag--implanted polystyrene for longer than 1.5 months, while the cells did not grow on untreated polystyrene in the same culture conditions.

Neuron cell positioning on polystyrene in culture by silver-negative ion implantation and region control of neural outgrowth

Abstract A new method to control the position of neuron cell attachment and extension region of neural outgrowth has been developed by using a pattering ion implantation with silver-negative ions into polystyrene dishes. This technique offers a promising method to form an artificially designed neural network in cell culture in vitro. Silver-negative ions were implanted into non-treated polystyrene dishes (NTPS) at conditions of 20 keV and 3×10 15 ions / cm 2 through a pattering mask, which had as many as 67 slits of 60 μm in width and 4 mm in length with a spacing of 60 μm . For cell culture in vitro, nerve cells of PC-12h (rat adrenal phechromocytoma) were used because they respond to a nerve growth factor (NGF). In the first 2 days in culture without NGF, we observed a selective cell attachment only to the ion-implanted region in patterning Ag − implanted polystyrene sample (p-Ag/NTPS). In another 2 days in culture with NGF, the nerve cells expanded neurites only over the ion-implanted region. For collagen-coated p-Ag/NTPS sample of which collagen was coated after the ion implantation (Collagen/p-Ag/NTPS), most nerve cells were also attached on the ion-implanted region. However, neurites expanded in both ion-implanted and unimplanted regions. The contact angle of NTPS decreased after the ion implantation from 86° to 74°. The region selectivity of neuron attachment and neurite extension is considered to be due to contact angle lowering by the ion implantation as radiation effect on the surface.

Neuron attachment properties of carbon negative-ion implanted bioabsorbable polymer of poly-lactic acid

Abstract Modification of a bioabsorbable polymer of poly-lactic acid (PLA) by negative carbon ion implantation was investigated with resect to radiation effects on surface physical properties and nerve-cell attachment properties. Carbon negative ions were implanted to PLA at energy of 5–30 keV with a dose of 1014–1016 ions/cm2. Most C-implanted PLA samples showed contact angles near 80° and almost same as that of unimplanted PLA, although a few samples at 5 keV and less 3×1014 ions/cm2 had contact angles larger than 90°. The attachment properties of nerve cells of PC-12h (rat adrenal phechromocytoma) in vitro were studied. PC-12h cells attached on the unimplanted region in C-implanted PLA samples at 5 and 10 keV. On the contrary, the nerve cells attached on only implanted region for the C-implanted PLA sample at 30 keV and 1×1015 ions/cm2.

Kinetic study on the initial stage of the fibrinogen-fibrin conversion by thrombin (I) application of mathematical treatment to turbidimetrical method

Kinetics on the initial reaction of the fibrinogen-fibrin conversion catalyzed by thrombin was studied by turbidimetrical measurement. The turbidity change caused by the conversion reaction was analyzed by a mathematical treatment, assuming that the dimer formation of fibrin monomers was the rate-determining reaction in the stage of polymerization. The obtained relations were investigated under experimental conditions at neutral pH and at low ionic strength, in which fast polymerization rate of fibrin monomers can be expected in comparison with the rate of enzyme reaction. The experimental results analyzed from turbidity-time curves were generally in agreement with theoretical prediction. On the other hand, the plots relating to the enzyme concentration did not lead to anticipated linear relationship. Hence, it was concluded that every fibrin monomer did not always polymerize via dimer formation, but that dimers of fibrin monomers behaved as nuclei for polymerization.

Complex coacervation in sulfated polyvinyl alcohol- aminoacetalyzed polyvinyl alcohol system: II. Formation of coacervate droplets

SummaryConditions for complex coacervation in aqueous solution to occur were quantitatively investigated by using a new type of polyclectrolyte salt prepared from an equivalent mixture of partly sulfated polyvinyl alcohol (polyanion) and partly aminoacetalyzed polyvinyl alcohol (polycation). The effects of the chain length of poly electrolytes, the thermodynamic interaction between water and polymer, the temperature, the electrostatic interaction, and the number of charges of polyelectrolyte chain on the complex coacervation were discussed on the basis of the free energy equation proposed by us.ZusammenfassungBedingungen für die komplexe Koacervation in wäßriger Lösung wurden quantitativ untersucht unter Verwendung eines neuen Types von Polyelektrolytsalz, das aus einer äquivalenten Mischung von teilweise sulfatiertem Polyvinylalkohol (Polyanion) und teilweise aminoacetalysiertem Polyvinylalkohol (Polykation) präpariert wurde. Die Auswirkungen der Kettenlängen der Polyelektrolyten, die thermodynamische Wechselwirkung zwischen Wasser und Polymer, die Temperatur, die elektrostatische Wechselwirkung und die Ladungszahl der Polyelektrolytkette auf die komplexe Koacervation werden auf Grundlage der Gleichung für die Freie Energie, wie sie von uns vorgeschlagen wird, diskutiert.

Kinetic study on the effects of acidic polysaccharides on the interaction of fibrinogen and thrombin

SummaryRetardation effect of acidic polysaccharides on the interactions of fibrinogen with thrombin was kinetically investigated by measuring the turbidity as a measure of the concentration of fibrin polymer formed in the system. To this aim, the fibrinogenfibrin conversion curve was analyzed kinetically based on the elementary reactions, and the slope and the induction period of the conversion curve were expressed as functions of the concentrations of fibrinogen, thrombin, and polysaccharide. The strong retardation effect of dextran sulfate and heparin was quantitatively interpreted. Seven kinds of polysaccharides were examined.

Extracellular matrix absorption properties of negative ion-implanted polystyrene, polydimethylsiloxane and poly-lactic acid

Abstract Absorption properties of extracellular matrix (ECM) were investigated for carbon negative-ion-implanted polymers of polystyrene (PS), polydimethylsiloxane (PDMS) and poly-lactic acid (PLA) to estimate cell attachment properties of the modified surface without cell culture. Carbon negative ions were implanted to these polymers at 10 keV with a dose of 10 14 –10 16 ions/cm 2 . Implanted samples were dipped in a culture medium including serum for 2 h at 37 °C. After the samples were rinsed with phosphate-buffered saline (PBS) and with pure water and then dried, nitrogen atoms absorbed on the surface were investigated by XPS. The molecular structure of many kinds of extracellular matrix proteins contains nitrogen atoms; polymers of PS, PDMS and PLA do not include nitrogen. Therefore, we can evaluate the absorption properties of ECM by the nitrogen atoms absorbed on the surface. The nitrogen content increased after ion implantation for these polymers: from 5.5 to 8% for PS; from 1 to 5% for PDMS; and from 6 to 8.3% PLA. The contact angle for C-implanted PS and PDMS decreased with increasing ion dose, and corresponded well with the absorption properties of the extracellular matrix. However, the contact angle for PLA did not significantly change after ion implantation. Thus, the absorption properties of ECM represent another measure for the estimation of cell-attachment properties.

Kinetic study on the initial stage of the fibrinogen-fibrin conversion by thrombin. (III) Effects of competitive inhibitors.

By using the turbidimetrical procedure, the inhibition constants, Kip, of competitive inhibitors of thrombin were estimated in systems of fibrinogen-thrombin-inhibitor. In the presence of a competitive inhibitor such as benzamidine, p-aminobenzamidine, tosylarginine methyl ester, MD-805, and antithrombin III-heparin, the turbidity change of reaction mixture was traced on the initial stage of the fibrinogen-fibrin conversion catalyzed by thrombin. The formation rate of the smallest polymer as detectable by turbidimetry was measured from evaluating the induction period of turbidity-time curves. The Kip values thus obtained were a little less than or agreed well with the inhibition constants, Ki, reported on systems of synthetic substrate-thrombin-inhibitor.