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PURPOSE The aim of this study was to investigate topographic "hot spots" on the anterior corneal surface before Descemet stripping automated endothelial keratoplasty (DSAEK) and their effects on postoperative visual acuity and hyperopic shift. METHODS Twenty-seven eyes of 27 patients with bullous keratopathy, who underwent DSAEK were studied. We defined a hot spot as a focal area with relatively high refractive power on the anterior corneal surface in eyes with bullous keratopathy. Refractive spherical equivalent, keratometric value, and corneal topography were retrospectively evaluated using anterior segment optical coherence tomography (AS-OCT). RESULTS Hot spots were identified in 11 eyes (42.3%) before DSAEK and disappeared in 9 eyes of these eyes (81.8%) at 6 months after DSAEK. AS-OCT revealed focal epithelial thickening in the same areas as the hot spots. There was no significant difference in the postoperative visual acuity between eyes with and without hot spots (P > 0.05). The keratometric value of the anterior corneal surface significantly flattened from 45.7 ± 2.7 diopters (D) before DSAEK to 44.2 ± 2.7 D 1 month after DSAEK in eyes with hot spots (P = 0.01), whereas in eyes without hot spots, there were no significant differences in the keratometric values before and after DSAEK. At 6 months, the refractive change was +1.1 ± 1.3 D in eyes with hot spots and -0.2 ± 0.6 D in eyes without hot spots (P = 0.034). CONCLUSIONS In eyes with focal epithelial thickening, topographic hot spots on the anterior corneal surface were observed using AS-OCT. The hot spots disappeared after DSAEK and had no influence on the postoperative visual acuity.

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source of these amino acids. Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of BCAAs. The metabolic control of BCAA biosynthesis involves allosteric regulation of ALS by the end-products of the pathway, i.e., valine, leucine and isoleucine. ALS holoenzyme seems to consist of two large catalytic subunits and two small regulatory subunits. In a previous study, using homologous recombination dependent gene targeting we created rice plants in which W548Land S627I mutations were induced into the endogenous gene encoding the ALS catalytic subunit. These two amino acid substitutions conferred hypertolerance to the ALS-inhibiting herbicide bispyripac-sodium. In this study, we revealed that feedback regulation by valine and leucine was reduced by these two amino acid substitutions. Furthermore, in leaves and seeds of ALS mutants with W548Land/or S627I substitution, a 2- to 3-fold increase in BCAAs was detected. Our results suggest that the ALS catalytic subunit is also involved in feedback regulation of ALS, and that judicious modification of the regulatory and catalytic subunits of ALS-coding genes by gene targeting can lead to the efficient accumulation of BCAA in plants.

The selective detection of methanol by photoluminescence under environmental conditions has been a great challenge for materials science. Herein, a reversible, turn-on-type photoluminescence triggered by methanol vapor in square-planar palladium(II) and platinum(II) complexes, newly prepared from [MCl2(1,3-bis(diphenylphosphino)propane)] and l-cysteine, is reported. Both the “turn-on” and “turn-off” states of the complexes were crystallographically characterized, which revealed the presence of intermolecular OH···O and CH···π interactions between methanol and the complex molecules in the turn-on state. These interactions prevent the vibrational quenching of the luminescence, leading to the turn-on-type luminescence in this system.

Pyribencarb is a novel benzylcarbamate-type fungicide having excellent activity against a wide range of plant pathogenic fungi, especially gray mold diseases caused by Botrytis cinerea. The primary target site of pyribencarb is the Qo site of cytochrome b in the electron transfer system of the respiratory chain. The inhibitory effects of pyribencarb to cytochrome b enzyme on the transfer system of a QoI-resistant strain of B. cinerea are stronger than those of other QoI fungicides. Pyribencarb exhibited adequate control in greenhouse trials when QoI-resistant strains were inoculated. We have developed a practical method for evaluating the sensitivity of B. cinerea to pyribencarb. The final concentrations of pyribencarb in the PDA medium were set at 1 and 100mg a.i. per liter with 1mM salicylhydroxamic acid (SHAM). The isolates that were completely inhibited at 1mg a.i. per liter of pyribencarb were “sensitive strains,” and the isolates that were inhibited by more than 80% at 100mg a.i. per liter of pyribencarb were “weakly resistant strains.” In our assays so far, no strains with mycelial growth inhibition rates of less than 80% at 100mg per liter of pyribencarb have ever been found. In the event that such strains should be found in the future, it will be necessary to monitor their sensitivity to pyribencarb using an in vivo assay.

The action mechanism of fenoxasulfone was studied by examining the inhibitory effects of this herbicide on the biosynthesis of very-long-chain fatty acids (VLCFAs). Fenoxasulfone treatment decreased VLCFAs, such as C20:0, C20:1, C22:0, C24:0, C24:1 and C26:0 fatty acids, in barnyard millet cultured cells, and increased long-chain-fatty acids and medium-chain-fatty acids, such as C18:0 and C15:0, which are precursors of VLCFAs. Fenoxasulfone potently inhibited activities of VLCFA elongase (VLCFAE) in the microsomal fraction of etiolated barnyard millet seedlings, which catalyzed the elongation steps from C22:0 to C24:0 and C24:0 to C26:0, respectively. These results strongly suggested that fenoxasulfone is a potent inhibitor of plant VLCFAEs and should be categorized within the K3 group of the Herbicide Resistance Action Committee. VLCFAE activity of recombinant Fatty Acid Elongation 1 (FAE1) of Arabidopsis that catalyzes the elongation step from C18:1 to C20:1, was inhibited by fenoxasulfone in a time-dependent manner, which has been shown in the inhibition of VLCFAEs by other well-known VLCFAE-inhibiting herbicides. On the other hand, VLCFAE activity of the microsomal fraction of etiolated barnyard millet seedlings, which catalyzes the elongation step from C24:0 to C26:0, was inhibited by fenoxasulfone in a time-independent manner. This time-independent inhibition raised a new inhibition mechanism of VLCFAE by fenoxasulfone likely with pyroxasulfone, which is classified in the same chemical class as fenoxasulfone.

Pyroxasulfone has been shown to be a potent inhibitor of very-long-chain fatty acid elongase (VLCFAE) of plants; therefore, it is categorized within the K3 group of herbicides. In this paper, we studied the properties of pyroxasulfone as a VLCFAE inhibitor in more detail by examination of its inhibitory effects on VLCFAEs from some plants. Pyroxasulfone inhibited microsomal VLCFAE activities of rice, Italian ryegrass, barnyard millet, wheat, corn and soybean with time-independent reversible inhibition. There were differences in the inhibitory potencies against VLCFAEs between pyroxasulfone-susceptible plants (rice, Italian ryegrass and barnyard millet) and pyroxasulfone-tolerant plants (wheat, corn and soybean). This result confirmed that the difference in the sensitivities of VLCFAEs to pyroxasulfone was one of the factors involved in the selectivity of pyroxasulfone between crops and weeds. We succeeded in preparing recombinant VLCFAEs of Arabidopsis (FAE1) and rice (Q6F365) using yeast and rice cultured cells and constructed each assay system. VLCFAE activities of FAE1 and Q6F365 were potently inhibited by pyroxasulfone; however, a critical difference was found in the inhibition manner between FAE1 and Q6F365. Pyroxasulfone inhibited FAE1 in a time-dependent manner, whereas it inhibited Q6F365 in a time-independent manner. The time-independent reversible inhibition of rice Q6F365 and microsomal VLCFAEs of plants proposes a new inhibition mechanism of VLCFAEs by pyroxasulfone.

A temperature-dependent gas-surface kinetic model for methane oxidation over palladium is proposed. Thermodynamic data for the surface species (O, H, OH, H 2 O, and CO) are derived from statistical mechanic analysis using literature heats of desorption and vibrational frequencies. The rate parameters in the model also satisfy thermo-kinetic constraints. The hydrogen oxidation submodel is validated against literature stagnation flow reactor experiments at 1300 K and 13 Pa. The current model is further tested against catalytic methane ignition in a laminar flow reactor at atmospheric pressure, and with time-resolved measurements of the size distribution of palladium nanoparticles generated in situ from an aerosol containing palladium acetate. The improved gas-surface model predicts closely the experimental data. The role of palladium nanoparticles in enhancing methane ignition is attributed to heat release due to catalytic methane oxidation over distributed nanoparticle surfaces, leading to a temperature rise and thus an accelerated gas-phase chain-branching process.

Pyribencarb is a novel benzylcarbamate-type fungicide, which is active against a wide range of plant pathogenic fungi. In this paper, the inhibitory effects of this fungicide on the electron transport system of fungi, plants, rat and carp were examined to elucidate its mode of action and selectivity. Pyribencarb potently inhibited succinate-cytochrome c reductase (SCR) activities of Botrytis cinerea (cucumber gray mold), Corynespora cassiicola (leaf spot) and decylubiquinol-cytochrome c reductase (UCR) activity of B. cinerea. Pyribencarb inhibited the UCR of B. cinerea in an uncompetitive manner with respect to decylubiquinol, which was the same as strobilurin fungicides, and the substrate-dependent inhibition constant was found from calculation to be 13 nM. These results suggested that the target site of pyribencarb is cytochrome b of complex III in the electron transport system of the respiratory chain. On the other hand, the inhibitory potency of pyribencarb on SCR activities of plants, rats and carp was relatively weak compared with that of strobilurin fungicides, indicating that pyribencarb is a Qo inhibitor of cytochrome b, whose properties are superior to well-known Qo inhibitor fungicides in terms of target. The binding site of pyribencarb on cytochrome b was assumed to be a little different from that of strobilurin fungicides, because pyribencarb inhibited SCRs of strobilurin fungicide-resistant strains of B. cinerea and C. cassiicola with relatively low concentrations. The binding site was also discussed through comparison of amino acid sequences of plants, rats, carp, yeast and fungi, including B. cinerea, which was elucidated in this paper.

Previously, we showed that four mutated acetolactate synthase (ALS) genes derived from rice and Arabidopsis (W548L/S627IOsALS, S627IOsALS, W574L/S653IAtALS and S653IAtALS) confer high levels of resistance to pyrimidinylcarboxylate type ALS inhibitors (Kawai et al. 2008). Mutated ALS genes of rice were obtained from rice cells cultured in the presence of an ALS-inhibitor. The mutated ALS genes of Arabidopsis, which have the same amino acid substitutions as those of rice, have been generated artificially. Here, we demonstrate that these mutated genes function as effective selectable markers for transformation of Arabidopsis. Specifically, we studied expression of the mutated ALSs in Arabidopsis and their effect on the sensitivity of transgenic Arabidopsis plants to the ALS inhibitors. Our results show that the degree of resistance to the ALS inhibitors of transformants expressing Arabidopsis mutated ALSs was greater than those of transformants expressing rice mutated ALSs. The amino acid sequences of ALSs derived from monocotyledonous plants and those derived from dicotyledonous plants were clearly divided into two clusters in a phylogenetic tree. Based on these results, it would be preferable to use rice and Arabidopsis mutated ALS genes for generating monocotyledonous and dicotyledonous transgenic plants, respectively. Moreover, our findings are particularly useful when generating transgenic plants with a known ALS nucleotide sequence. In such cases, their own ALS gene carrying these mutations could be used as a selectable marker because amino acid residues at the point of mutation are conserved among plant species.

The mechanism of action of pyroxasulfone was studied by examination of the inhibitory effects of this herbicide on the biosynthesis of very-long-chain fatty acids (VLCFAs) both in vivo and in vitro. Pyroxasulfone treatment drastically reduced the biosynthesis of VLCFAs and caused a buildup of fatty acid precursors in cultured rice cells. Pyroxasulfone specifically inhibited the elongation steps from C18:0 to C20:0, C20:0 to C22:0, C22:0 to C24:0, C24:0 to C26:0 and C26:0 to C28:0, catalyzed by VLCFA elongases (VLCFAEs) in plants including rice. These results suggested that pyroxasulfone is a potent inhibitor of VLCFA biosynthesis, and should be categorized within the K3 group of herbicides. Twenty putative VLCFAEs of rice were identified by blastp search with the amino acid sequences of Arabidopsis VLCFAEs. Oligo microarray and real time RT-PCR analysis revealed that Q5Z6S3 and Q8H7Z0, which were identified by their Uniplot ID number, might play important roles during the biosynthesis of C28:0 and C30:0 VLCFAs in shoot formation, and biosynthesis of C20:1 and C22:1 VLCFAs in cell proliferation, respectively. These VLCFAEs are likely targets for pyroxasulfone.

Herbicide-resistant turfgrass can be an efficient tool that will allow easier turf maintenance. Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways leading to the branched-chain amino acids, and amino acid substitutions in ALS have been known to confer resistance to ALS-inhibiting herbicides. A two-point mutated rice ALS gene [OsALS (dm)] has been shown to confer strong resistance to bispyribac-sodium (BS), an ALS-inhibiting herbicide. In this study, we introduced into turf-type tall fescue (Festuca arundinacea Schreb.) the OsALS (dm) gene by using Agrobacterium-mediated transformation for conferring herbicide resistance. Stable integration of the transgene was confirmed by Southern blot analysis. Transgenic and wild-type plants were sprayed on the leaves with herbicide containing BS; approximately half of the transgenic plants were unaffected by the treatment and showed resistance to the herbicide, whereas the wild-type plants died. ALS activity in the leaf tissue of transgenic-resistant plants incubated with BS was almost equivalent to that in wild-type plants without BS and was higher than in wild-type plants incubated with BS. These indicate that the transgenic-resistant plants actively produced OsALS (dm) protein under herbicide treatment. This is the first report of herbicide-resistant transgenic tall fescue after introduction of a mutated ALS gene.

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T(1) soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3′-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

Summary We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.

A low power (lower than 50mW) and high resolution (332ppi) VGA LCD with integrated 6-bit digital data drivers has been developed using CLC (CW-Laser Lateral Crystallization) technology. Logic operations at 3V and low power analog buffer circuits are realized by improved uniformity of TFT characteristics. Integration of digital drivers with line-at-a-time driving scheme is achieved by fine lithography technology (2μm pitch wiring).

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum . A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp :: zhd101 gene ( gfzhd101 ), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (a w ) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an a w of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.

A two-point mutated gene of acetolactate synthase (ALS) was isolated from rice cells, which was cultured together with an ALS-inhibiting herbicide, bispyribac-sodium (BS). The mutations involved residues of tryptophan at position 548 to leucine (W548L) and serine at position 627 to isoleucine (S627I). The ALS expressed in Escherichia coli from this gene showed resistance to multiple herbicides including pyrimidinylcarboxylate (PC), sulfonylurea and imidazolinone herbicides, and showed stronger resistance to PC herbicides than to other herbicides. BS, a PC herbicide, had almost no effect on the enzyme even at 100 μM, which is an approximately 10,000-fold higher concentration than the concentration required for 50% inhibition of the wild-type. The resistance level of W548L/S627I mutating ALS to BS was stronger than the additive effect predicted from the degree of resistance of each single amino acid mutated ALS. Transformed rice cells carrying this gene and a regenerated rice plant expressed resistance to BS, suggesting that this gene is useful as a selectable marker for introducing foreign traits into rice when used with PC herbicides.

We performed functional analysis of transgenic plants expressing the W548L/S627I mutated ALS gene from rice as well as studies on inheritance of the mutated gene, phenotype and fertility of transgenic plants to ensure availability of the mutated gene as a selectable marker for plant genetic transformation. Expression levels of the ALS (endogenous+mutated ALS) gene of transgenic rice plants were correlated with the resistance of transgenic plants to bispyribac-sodium (BS). The BS-resistant trait of a transgenic plant was stably inherited by the progeny in a Mendelian manner. A homozygote of transgenic plants harboring the mutated gene was normal in its growth and fertility compared with the wild type. These results ensured that the W548L/S627I mutated gene from rice can be efficiently used as a selectable marker for genetic transformation of rice in combination with BS.

Centrifugal pump performance of electric submersible pump (ESP) is analyzed experimentally during emulsification process in methane-water pipe flows. The ESP is installed in a horizontal pipe section of a flow loop. The flow loop is placed vertically with the total length of 10.4 m, which is composed of the stainless steel pipeline of 2.1 cm inner diameter and the upflow test section of 5 cm inner diameter. The system temperature is kept isothermally at 4 °C and the initial gas-phase volume fraction is fixed at 0.016 while the pump speed and the flow rate are varied up to 2500 rpm and 50 L/min, respectively. The flow regime of the initial gas-liquid flows is determined to be turbulent bubbly flows. Transformation of vapor methane into solid methane hydrate is made by elevating the system pressure up to the super cooling condition through water injection. The detail emulsification process is characterized with use of a high speed camera and in-situ particle size analyzer, both of which are located at the pump downstream. The particle size analyzer employs the focused beam reflectance measurement (FBRM) technique. It is found that the extent of phase transformation is limited by the pump speed as it determines the smallest size of bubbles generated in the flow line. Emulsion is appeared to be white-opaque, which is made by clouds of microbubbles encapsulated by methane hydrate. The opaque particles can be detected by FBRM and exhibit uniform size distributions, indicating that the particles are emulsified homogeneously in the entire flow domain through fragmentation and coagulation kinetics. The majority of methane hydrate particles are appeared to be transportable by the bulk fluid though some particle deposition on the viewing glass window can be occurred. Higher water power is given by the ESP under the emulsified flows than that of water-only flows, indicating that the apparent fluid viscosity can be increased by methane hydrate particles emulsified in water solution.

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the necrotrophic cereal pathogen Fusarium graminearum. This mycotoxin is detoxified by ZHD101, a lactonohydrolase from Clonostachys rosea, or EGFP:ZHD101, its fusion to the C-terminus of an enhanced green fluorescence protein. We previously showed that egfp:zhd101 is efficiently expressed in T(0) leaves of rice. In this study, we assessed the feasibility of in planta detoxification of the mycotoxin using progeny. When protein extract from T(1) leaves was incubated with ZEN, the amount of the toxin decreased significantly as measured by HPLC. ZEN degradation activity was also detected in vivo in transgenic T(2) seeds. These results suggest that zhd101 can be exploited as an efficient and cost-effective system for protection of important cereals that are more susceptible to the pathogen (e.g., wheat and maize) from contamination with the estrogenic mycotoxin.

Seeds of rice (Oryza sativa L. var. Koshihikari) were soaked in water for 2 d. Thereafter, the embryo containing an apical meristem was inoculated with Agrobacterium tumefaciens by piercing a site of the husk overlying the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were then grown to maturation (T0 plants) and allowed to pollinate naturally to set seeds (T1 plants) in pots under nonsterile conditions. To examine the transformation by various means, three different strains of A. tumefaciens were used for transformation: an M-21 mutant, which is an avirulent mutant with a Tn5 insertion in the iaaM gene, and two LBA4404 strains each with a different binary vector. Five different lines of evidence were demonstrated the transformation: the altered phenotype and its inheritance by the next generation, histochemical detection of beta-glucuronidase, resistance to hygromycin B, detection of the transgene by PCR and rescue of a plasmid consisting of the integrated T-DNA and the flanking rice genome DNA. Transformation efficiency of T1 plants was estimated to be 40% and 43% by PCR and a histochemical assay of beta-glucuronidase, respectively.

Potentialities of rapid thermal annealing to enhance the photoluminescence emission of Si nanocrystals in SiO2 have been investigated. Ion implantation was used to synthesize specimens of SiO2 containing excess Si with different concentrations. Si precipitation to form nanocrystals in implanted samples takes place with a conventional furnace anneal. The photoluminescence intensity and the peak energy of emission from Si nanocrystals depend on implanted ion fluence. Moreover, the luminescence intensity is strongly enhanced with a rapid thermal anneal prior to a conventional furnace anneal. The luminescence intensity, however, decreases with a rapid thermal anneal following a conventional furnace anneal. It is found that the order of heat treatment is an important factor in intensities of the luminescence. Moreover, the luminescence peak energy is found to be dependent, but a little, on thermal history of specimens. Based on our experimental results, we discuss about the mechanism of an enhancement of the photoluminescence, together with the mechanism of photoemission from encapsulated Si nanocrystals produced in a SiO2 matrix by ion implantation and annealing.

We have established a simple diagnosis method for rice blast fungus resistant to MBI-D. This involves the preparation of PCR templates directly from the lesions in combination with primer-introduced restriction enzyme analysis PCR (PIRA-PCR).

The objective of this study was to investigate the properties of granules and tablets prepared by a novel Fluidized Hot-Melt Granulation (FHMG) technique. Macrogol 6000 (polyethylene glycol 6000, PEG 6000), macrogol 20000 (polyethylene glycol 20000, PEG 20000), and glyceryl monostearate (GMS) were used as binders with different levels of viscosity and water solubility. The properties of both granules and tablets were compared with those obtained using the Standard Tablet Formulation (STF, lactose/corn starch/hydroxypropylcellulose/ magnesium stearate: 66/30/3.5/0.5) for fluidized-bed granulation, which is widely used for wet granulation. To obtain suitable flowability as granules for tabletting, the content of the melting material should be approximately 10 w/w%. The rate of increase in the mean diameter of the granules during FHMG was affected by both the melting temperature and the viscosity of the melting material used in the granules. The compression properties of granules prepared by FHMG were also investigated, demonstrating that these granules had a high pressure transmittance. The hardness and the disintegration time of tablets obtained from granules prepared by FHMG were influenced by the properties of the melting material, such as its compaction behavior, solubility, and wettability. No significant differences of hardness were observed when compared to STF tablets. Tablets prepared from FHMG granules disintegrated within 15 min, whereas the STF tablets showed faster disintegration. It was also demonstrated that the hardness and disintegration time of tablets prepared from FHMG granules were not affected by the tablet porosity. Therefore, tablets with a constant quality may be obtainable under a wide range of compression forces. The results of this study suggested that FHMG is a useful method of preparing granules for tableting without using any solvents or water.

N-Isobutyl-N-(4-substituted benzyl)-4-halo-2-pentenamide 類はヒエに対し顕著な光要求性の除草活性を示す. ワタの幼植物およびそれより調製した酵素標品, ならびに培養した単細胞藻類を用いたアッセー系により, 殺草のメカニズムの解明を試み, 一連の供試化合物は明らかに protoporphyrinogen oxidase (PPO) 阻害活性を示したが, 植物毒性の強度変化は, むしろクロロフィル生合成阻害活性の強度変化によく対応した. したがって, 本系列の化合物の植物毒性には, PPOの阻害以外にクロロフィル生合成のほかの段階の阻害が重なっていることが示唆された.

We have measured the implantation dose dependence as well as the oxidation effect of the photoluminescence behaviour of Si nanocrystals in SiO2 layers fabricated by ion implantation and a subsequent annealing step. After annealing at high temperature, a characteristic photoluminescence band, peaking just below the 1.7 eV was observed. The peak energy and the intensity of the photoluminescence were found to be affected by the dose of implanted Si ions, but to be independent of annealing time and excitation photon energy. We also present experimental results of an oxidation induced continuous peak energy shift of the photoluminescence peak up to around 1.8 eV. This peak energy, however, was found to return to its previous position with re-annealing. These results indicate that whilst the excitation photons are absorbed by Si nanocrystals, the emission is not simply due to electron–hole recombination inside the Si nanocrystals, but is related to the presence of defects, most likely located at the interface between the Si nanocrystals and the SiO2, for which the characteristic energy levels are affected by cluster–cluster interactions or the roughness of the interface.

We report recent results showing broad-band excitation of erbium ions implanted into thin films of silica containing silicon nanocrystals. Evidence for the existence of nanocrystals is presented in the form of HR-TEM images of crystalline regions of the thin films. Indirect excitation of the rare-earth ions is mediated by the nanocrystals, which are either grown in during plasma enhanced CVD of the films, or are formed by implantation of thermally grown SiO 2 layers with Si + ions. We demonstrate efficient flashlamp pumping of the erbium 1.535 μm PL band and discuss the device implications of this material.

眼外傷後に30～35Δの内斜視を発症した7才女児の症例に対し、斜視角の軽減を目的としてプリズム漸減法を行なった。途中2回の斜視角のもどりが生じたが、眼鏡箔を併用することによって斜視角の軽減を得た。その結果、斜視角は発症時の1/3の10Δと安定し、1眼の5mmの内直筋の後転で良好な眼位を得ることが出来た。両眼視機能は良好に保たれていた。

A possible mechanism for the photoemission from Si nanocrystals in an amorphous SiO2 matrix fabricated by ion implantation is reported. We have measured the implantation dose and the temperature dependence as well as the oxidation effect of the photoluminescence behaviour of Si nanocrystals in SiO2 layers fabricated by ion implantation and a subsequent annealing step. After annealing, a photoluminescence band, peaking just below 1.7 eV was observed. The peak energy of the photoluminescence was found to be affected by the dose of implanted Si ions and the temperature during ion implantation, but to be independent of annealing time and excitation photon energy. We also present experimental results of an oxidation-induced continuous peak energy shift of the photoluminescence peak up to around 1.8 eV. This peak energy, however, was found to return to its previous position with re-annealing. These results indicate that whilst the excitation photons are absorbed by Si nanocrystals, the emission is not simply due to electron-hole recombination inside the Si nanocrystals, but is related to the presence of defects, most likely located at the interface between the Si nanocrystals and the SiO2, for which the characteristic energy levels are affected by cluster-cluster interactions or the roughness of the interface.

Si nanoclusters are formed in a SiO 2 matrix by ion implantation and annealing, and a possible mechanism for the light emission from Si implanted SiO 2 layers is reported. We have measured dose (concentration of excess Si atoms), annealing time and excitation energy dependence of the photoluminescence. After annealing, a photoluminescence band around 1.7 eV has been observed. The peak energy of the photoluminescence is found to be independent of annealing time and excitation energy, while the intensity of the luminescence increases as the annealing time and excitation energy increase. Moreover, we found that the peak energy of the luminescence is strongly affected by dose of implanted Si ions especially in the high dose range. These results indicate that the emission of photon is not simply due to direct electron–hole recombination inside Si nanoclusters, but is related to defects probably at the interface between Si nanoclusters and SiO 2 , for which the energy state is affected by Si cluster–cluster interaction. It seems that Si nanoclusters react via a thin oxide interface and the local concentrations of Si nanoclusters plays an important role in the peak energy of the photoluminescence.

A novel method for the fabrication of luminescent Si nanoclusters in an amorphous SiO 2 matrix by ion implantation and annealing, and the detailed mechanisms for the photoluminescence from implanted Si nanoclusters are reported. We have measured dose (concentration of excess Si atoms), annealing time and excitation energy dependence of the photoluminescence of Si nanoclusters in SiO 2 layers. The samples were fabricated by Si ion implantation into SiO 2 and subsequent high temperature annealing. After annealing, a photoluminescence band below 1.7 eV has been observed. The peak energy of the photoluminescence is found to be independent of annealing time and excitation energy, while the intensity of the luminescence increases as the annealing time and excitation energy increase. Moreover, we found that the peak energy of the luminescence is strongly affected by dose of implanted Si ions especially in the high dose range. These results indicate that the photons are absorbed by Si nanoclusters, for which the band-gap energy is modified by the quantum confinement effects, and the emission is not simply due to direct electron-hole recombination inside Si nanoclusters, but is related to defects probably at the interface between Si nanoclusters and SiO 2 , for which the energy state is affected by Si cluster–cluster interactions. It seems that Si nanoclusters react via a thin oxide interface and the local concentrations of Si nanoclusters play an important role in the peak energy of the photoluminescence.

A method for the fabrication of luminescent Si nanoclusters in an amorphous SiO 2 matrix by ion implantation and annealing, and the detailed mechanisms for the photoluminescence are reported. We have measured the implanted ion dose, annealing time and excitation energy dependence of the photoluminescence from implanted layers. The samples were fabricated by Si ion implantation into SiO 2 and subsequent high-temperature annealing. After annealing, a photoluminescence band below 1.7 eV has been observed. The peak energy of the photoluminescence is found to be independent of annealing time and excitation energy, while the intensity of the luminescence increases as the annealing time and excitation energy increase. Moreover, we found that the peak energy of the luminescence is strongly affected by the dose of implanted Si ions especially in the high dose range. These results indicate that the photons are absorbed by Si nanoclusters, for which the band-gap energy is modified by the quantum confinement effects, and the emission is not simply due to direct electron–hole recombination inside Si nanoclusters, but is related to defects probably at the interface between Si nanoclusters and SiO 2 , for which the energy state is affected by Si cluster–cluster interactions. It seems that Si nanoclusters react via a thin oxide interface and the local concentrations of Si nanoclusters play an important role in the peak energy of the photoluminescence.

A method for the fabrication of luminescent Si nanoclusters in an amorphous SiO2 matrix by ion implantation is reported. We have measured the dose (concentration of excess Si atoms) and annealing time dependence of the photoluminescence of Si nanoclusters in SiO2 layers at room temperature. The samples were fabricated by ion implantation and subsequent annealing. After annealing, a photoluminescence band below 1.7 eV has been observed. The peak energy of the photoluminescence is found to be almost independent of annealing time, while the intensity of the luminescence increases as the annealing time increases. Moreover, we found that the peak energy of the luminescence is strongly affected by the dose of implanted Si ions, especially in the high-dose range. We also show direct evidence of widening of the band-gap energy of Si particles of a few nanometers in size by employing photoacoustic spectroscopy. These results indicate that the photons are absorbed by Si nanoclusters, for which the band-gap energy i...

The activity of a number of O-(4,6-dimethoxypyrimidin-2-yl)salicylic acids and their thio analogs inhibiting acetolactate synthase (ALS) preparation was measured. The effects of substituents on the salicylic-benzene ring on the inhibitory activity were analyzed quantitatively with physicochemical substituent parameters. For 6-substituted (thio)salicylic acids, the activity was shown to vary parabolically with the 'intramolecular' steric parameter (E g ). In addition, the higher steric dimension of substituents in terms of the STERIMOL width or length parameter lowered the activity. The field-inductive electron-withdrawing property of the 6-substituents in terms of the Swain-Lupton-Hansch F was favorable for the activity of salicylic acid series. In 5-substituted salicylic acids, the activity was increased by electron-donating substituents with smaller size. The relationships between ALS inhibitory and herbicidal activities were also analyzed with some weed species. Both pre- and post-emergence activities against barnyard grass, Echinochloa crus-galli, were linearly related to the ALS inhibitory activity after allowing for the hydrophobic factor that may contribute to the transport processes. Those against two broad-leaved weed species, Polygonum convolvulus and Abutilon theophrasti were linearly related to the in-vitro activity with no significant participation of the hydrophobic factor.

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.

Aino virus (JaNAr 28 strain), a possible agent of a congenital anomaly of calves, was inoculated into the yolk sac of chick embryos at a dose of 10(2), 10(3), 10(4) or 10(5) TCID50 0.2 ml-1 at four, six or eight days of incubation. At 21 days of incubation all the unhatched embryos or hatched chickens in the experimental and control groups were sacrificed for pathological examination. The incidence of hydranencephaly, cerebellar hypoplasia or agenesis and arthrogryposis or scoliosis was highest (85 per cent in the chicks inoculated with 10(3) TCID50 0.2 ml-1 at eight days of incubation. The lesions were very similar to those found in congenital abnormalities in calves suspected of a natural infection with Aino virus.

Three feline coronavirus (FCoV) isolates KUK-H, M91-266, and M91-267 were examined to elucidate their biological and antigenic properties as well as disease potential in cats. Immune stainings of virus-infected cells by using FCoV type-specific monoclonal antibodies indicated that their antigenic specificity was serotype II. However, antigenic variations among these serotype II FCoVs were detected by neutralization assay with hyperimmune antisera against FCoVs and canine coronaviruses, and with experimentally infected cat sera; there were two subtypes in serotype II FCoVs. The isolates efficiently grew in fcwf-4 cell culture showing lytic CPE enough to form distinct plaques: when measured 48 hr after infection, plaque sizes of both M91-266 and M91-267 were approximately 1 mm in diameter, and a mixture of small (less than 1 mm in diameter) and large (approximately 3 mm in diameter) plaques were produced in the case of KUK-H. Strains KUK-H, M91-266 and M91-267 produced feline infectious peritonitis (FIP) in 50%, 67% and 89% of experimentally inoculated kittens, respectively. Furthermore, 80% of the kittens inoculated with the small plaque former of KUK-H developed FIP accompanied by more prominent clinical signs as well as pathological changes when compared with 28.6% of kittens inoculated with the large plaque former. These results suggest that serotype II FIPVs producing smaller size of plaques are more virulent than those producing larger size of plaques.