Change of clopyralid concentration in recycled beef cattle compost.

Composting of beef cattle manure using sawdust or recycled compost as a bulking agent was investigated for the management of clopyralid risk, such as changes in the clopyralid concentration and the mechanism of clopyralid accumulation caused by recycled compost. These raw materials were composted with laboratory equipment, which was controlled at 60℃ after the temperature peak by autothermal composting. Clopyralid concentration did not changed during composting in the thermophilic phase; on the contrary, it increased because clopyralid accumulated in compost when recycled compost was used repeatedly as a bulking agent. The clopyralid accumulation ratio (ratio of clopyralid concentration to that in the first compost) could be explained by a model using a recurrence formula, and a correlation existed between the calculated (model) accumulation ratios and measured accumulation ratios (R2  = 0.78). Using this model, the excessive accumulation of clopyralid could be controlled when using recycled compost as a bulking agent with lower moisture content or when part of the recycled compost was replaced by another bulking agent, even if recycled compost had high moisture content. In future work, the model and its considerations should be verified in a field test.

A Bulky Three-Hindered Quadrant Bisphosphine Ligand: Synthesis and Application in Rhodium-Catalyzed Asymmetric Hydrogenation of Functionalized Alkenes.

A bulky three-hindered quadrant bisphosphine ligand, di-1-adamantylphosphino(tert-butylmethylphosphino)methane, named BulkyP*, has been synthesized via a convergent short pathway with chromatography-free procedures. The ligand is a crystalline solid and can be readily handled in air. Its rhodium(I) complex exhibits very high enantioselectivities and catalytic activities in the asymmetric hydrogenation of functionalized alkenes.

A versatile strategy for the synthesis and mechanical property manipulation of networked biodegradable polymeric materials composed of well-defined alternating hard and soft domains.

The present paper proposes a versatile strategy for the synthesis and mechanical property manipulation of networked biodegradable polymeric materials composed of well-defined alternating soft and hard domains. As an example of the strategy, we selected biodegradable poly(l-lactide) (PLLA) and poly(ε-caprolactone) (PCL) as the hard and soft components, respectively, and synthesized networked biodegradable polymeric materials composed of well-defined alternating PLLA and PCL domains with different l-lactide (LLA) unit contents via crosslinking of well-defined four-armed diblock copolymers of PLLA and PCL (4-C-L). The strategy reported here, which is also applicable to non-biodegradable polymeric materials, successfully facilitated the synthesis of the networked biodegradable materials composed of alternating hard and soft domains and their mechanical properties of the synthesized materials were largely manipulated by the LLA unit contents of the precursor four-armed diblock 4-C-L copolymers. Moreover, the crystallization behavior and thermal properties of 4-C-L copolymers before and after crosslinking were investigated and discussed.

Catalyst-Free and Regioconvergent Substitution Reactions of Bromothiophenes with a BH3-Substituted Phosphide Anion.

A range of bromothiophenes reacted with lithium boranato(tert-butyl)methylphosphide in the absence of transition-metal catalysts under mild conditions to provide the same 2,5-disubstituted and 2-monosubstituted products regardless of the substitution patterns of the starting bromothiophenes.

High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes.

Levan, a type of fructan, is an oligomer or polymer with mainly a ß(2,6)-linked fructose chain attached to sucrose. We introduced two timothy genes, PpFT1 and PpFT2, coding for two homologous sucrose:fructan 6-fructosyltransferases into sugar beet. Sugar beet produces a high concentration of sucrose, a starting substrate in fructan synthesis, in the root. Among transgenic T1 lines, we obtained sugar beet transformants that accumulated large amounts of ß(2,6)-linked levans (about 20 to 75mgg(-1) FW) in the roots. The transformed sugar beet plants possessing PpFT1 or PpFT2 produced linear levans with different degrees of polymerization (DP). Namely, the PpFT1 transformants accumulated mainly high DP levans including those with DP>40, while the PpFT2 transformants accumulated levans with DP between 3 and 40. Chromatograms showed that PpFT2 produces pure ß(2,6)-linked linear levans compared with fructans synthesized by PpFT1. These levans belong to the high DP class of plant fructans, but have much shorter DP than that of levans generally produced by microorganisms.

Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense).

Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to -3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties.

Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22 °C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP = 3-40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants.

Three-hindered quadrant phosphine ligands with an aromatic ring backbone for the rhodium-catalyzed asymmetric hydrogenation of functionalized alkenes.

The three-hindered quadrant phosphine ligands (R)-1-tert-butylmethylphosphino-2-(di-tert-butylphosphino)benzene ((R)-3H-BenzP*) and (R)-2-tert-butylmethylphosphino-3-(di-tert-butylphosphino)quinoxaline ((R)-3H-QuinoxP*) exhibited good to excellent enantioselectivities in the rhodium-catalyzed asymmetric hydrogenation of selected dehydroamino acid derivatives, enamides, and ethenephosphonates.

Rigid P-chiral phosphine ligands with tert-butylmethylphosphino groups for rhodium-catalyzed asymmetric hydrogenation of functionalized alkenes.

Both enantiomers of 2,3-bis(tert-butylmethylphosphino)quinoxaline (QuinoxP*), 1,2-bis(tert-butylmethylphosphino)benzene (BenzP*), and 1,2-bis(tert-butylmethylphosphino)-4,5-(methylenedioxy)benzene (DioxyBenzP*) were prepared in short steps from enantiopure (S)- and (R)-tert-butylmethylphosphine-boranes as the key intermediates. All of these ligands were crystalline solids and were not readily oxidized on exposure to air. Their rhodium complexes exhibited excellent enantioselectivities and high catalytic activities in the asymmetric hydrogenation of functionalized alkenes, such as dehydroamino acid derivatives and enamides. The practical utility of these catalysts was demonstrated by the efficient preparation of several chiral pharmaceutical ingredients having an amino acid or a secondary amine component. A rhodium complex of the structurally simple ligand BenzP* was used for the mechanistic study of asymmetric hydrogenation. Low-temperature NMR studies together with DFT calculations using methyl α-acetamidocinnamate as the standard model substrate revealed new aspects of the reaction pathways and the enantioselection mechanism.

S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme.

A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO3, PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 °C, being active at 0-65 °C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.

Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.).

The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of ß(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for ß(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against ß(2,1)-linked fructans, but considerably less than against ß(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy.

Enantiopure 1,2-bis(tert-butylmethylphosphino)benzene as a highly efficient ligand in rhodium-catalyzed asymmetric hydrogenation.

An electron-rich P-stereogenic bisphosphine ligand named "BenzP*" was conveniently prepared from o-dibromobenzene and enantiopure tert-butylmethylphosphine-borane. Its rhodium complex exhibited excellent enantioselectivities of up to 99.9% and high catalytic activity of up to 10,000 h(-1) TOF in asymmetric hydrogenations of various functionalized alkenes.

Hydroxynitrile lyase from Passiflora edulis: Purification, characteristics and application in asymmetric synthesis of (R)-mandelonitrile.

A hydroxynitrile lyase from leaves of Passiflora edulis (PeHNL) was purified and characterized for the first time. The enzyme is a monomer of 15kDa and 18kDa by SDS-PAGE, and gel filtration, respectively. Asymmetric synthesis of (R)-mandelonitrile from benzaldehyde and acetone cyanohydrin in a biphasic system employing the PeHNL from rinds of P. edulis was carried out. Several parameters influenced the enantiomeric purity of the product and initial velocity of the reaction. Both pH and temperature were important parameters controlling the enantiomeric purity of the product. The optimum pH and temperature were pH 4 and 10°C, respectively. At the optimum pH and temperature, the spontaneous non-enzymatic reaction yielding the racemic mandelonitrile was almost completely suppressed. The PeHNL was stable (more than 80% residual activity after incubation for 12h) in the system of methyl-t-butyl ether (MTBE), dibutyl ether (DBE), hexane (HEX), and diisopropyl ether (DIPE) while diethyl ether (DEE) and ethyl acetate (EA) were not suitable solvents. The initial velocity was markedly affected by the type of organic solvent in the biphasic system, while high enantiomeric purity was obtained when organic solvents having logP lower than 3.5 were used. The highest initial velocity of reaction and enantiomeric purity of (R)-mandelonitrile were obtained in the biphasic system of DBE with the aqueous phase content of 30% (v/v). The optimum substrate concentrations were 250mM for benzaldehyde and 900mM for acetone cyanohydrin, and the optimum enzyme concentration was 26.7units/ml. The highest enantiomeric purity of (R)-mandelonitrile was successfully obtained with conversion and enantiomeric excess of 31.6% and 98.6%, respectively. The enzyme showed considerable reusability in batch reaction with high enantiomeric purity of product. Herein, we reported the characteristics of a unique (R)-PeHNL from leaves of P. edulis. The PeHNL from rinds had been isolated for the first time and the enzyme showed great ability in transcyanation of (R)-mandelonitrile with high e.e. in DBE as the co-organic solvent in a biphasic system.

Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.).

Variation in the structures of plant fructans and their degree of polymerization (DP) can be explained as the result of diverse combinations of fructosyltransferases (FTs) with different properties. Although FT genes have been isolated in a range of plant species, sucrose:fructan 6-fructosyltransferase (6-SFT) cDNAs have only been functionally characterized in a few species such as wheat. A novel FT cDNA possessing 6-SFT activity has been identified and characterized from the temperate forage grass, timothy (Phleum pratense L.). The cDNA of an FT homolog, PpFT1, was isolated from cold-acclimated timothy. A recombinant PpFT1 protein expressed in Pichia pastoris showed 6-SFT/sucrose:sucrose 1-fructosyltransferase (1-SST) activity and produced linear beta(2,6)-linked levans from sucrose with higher DPs than present in graminans formed in vitro by wheat 6-SFT (Wft1). PpFT1 and Wft1 showed remarkably different acceptor substrate specificities: PpFT1 had high affinity for 6-kestotriose to produce levans and low affinity for 1-kestotriose, whereas Wft1 preferentially used 1-kestotriose as an acceptor. The affinity of the PpFT1 recombinant enzyme for sucrose as a substrate was lower than that of the Wft1 recombinant enzyme. It is also confirmed that timothy seedlings had elevated levels of PpFT1 transcripts during the accumulation of fructans under high sucrose and cold conditions. Our results suggest that PpFT1 is a novel cDNA with unique enzymatic properties that differ from those of previously cloned plant 6-SFTs, and is involved in the synthesis of highly polymerized levans in timothy.

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information.

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding.

Effects of cellulose supplementation on fecal consistency and fecal weight.

We investigated the effects of cellulose supplementation on fecal consistency and fecal weight. About 26 women were classified into two groups-normal defecation and constipation groups. All subjects ate the following meals during the experiment: ordinary meals (first week), experimental meals (second week), and experimental meals mixed with 4 g (third week) and 8 g (fourth week) cellulose. The experimental meal contained 16.7 g fiber. Fecal weights, fecal water content, fecal consistency, and defecation frequency were measured during the experimental period. As a result, in the normal defecation group, the mean fecal weight was 222.9 g day(-1) in the first week, and thereafter decreased. Although 20/24 g of fiber intake in the third/fourth week increased the fecal weight to over 150 g, the fecal consistency was still lower than the optimal consistency of around 300 g cm(-2). However, these changes were not observed in the constipated group.

Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in Benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia californica.

Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4'OMT transformants. HPLC/liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. japonica 4'OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production.

Sucrose metabolism of perennial ryegrass in relation to cold acclimation.

Sugar metabolism is one of the important factors involved in winter hardiness and since the discovery of sucrose biosynthesis, considerable advances have been made in understanding its regulation and crucial role. This investigation examined the changes in activities of sucrose metabolizing enzymes and sugar content during cold hardening of perennial ryegrass (Lolium perenne L.). Changes in acid invertase (AI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) along with all the three soluble sugars glucose, fructose and sucrose were measured in leaves and stem base tissue during cold acclimation. Although fructans were the predominant carbohydrate the changes in glucose, fructose and sucrose were significant. All the three soluble sugars in both leaf and stem tissues started to decrease from the first day and continued up to day 7 and thereafter started to increase until day 28. AI in the soluble fraction showed a higher activity than that in the cell wall bound fraction. In both the leaf and stem bases soluble AI activity increased during the first week and after that it started to decrease gradually. On the other hand both the SS and SPS increased gradually throughout the acclimation period. Sucrose content was negatively correlated with AI and positively correlated with SS and SPS accounting well for the relation between the substrate and enzyme activity. These results suggest that AI, SS and SPS in ryegrass are regulated by cold acclimation and play an important role in sugar accumulation and acquisition of freezing tolerance.

Screening for new hydroxynitrilases from plants.

We established a simple HPLC method to determine the activity and stereochemistry of the chiral mandelonitrile synthesized from benzaldehyde and cyanide, and applied it to screen for hydroxynitrile lyase (HNL) activity of plant origin. A total of 163 species of plants among 74 families were examined for (R)- and (S)-HNL activities using the method. We discovered that homogenate of leaves of Baliospermum montanum shows (S)-HNL activity, while leaves and seeds from Passiflora edulis, and seeds from Eriobotrya japonica, Chaenomles sinensis, Sorbus aucuparia, Prunus mume, and Prunus persica show (R)-HNL activity. Partially purified (R)-HNLs from Passiflora edulis and Eriobotrya japonica acted not only on benzaldehyde but also on aliphatic ketone. The enantiomeric excess of (R)-methylpropylketone cyanohydrin synthesized from 2-pentanone using homogenate from leaves of Passiflora edulis was 87.0%, and that of (R)-mandelonitrile synthesized by homogenate from seeds of Eriobotrya japonica was 85.0%.

A first trial in the clinical application of photodynamic therapy for the prevention of restenosis after coronary-stent placement.

BACKGROUND AND OBJECTIVES: The aim of this clinical study was to evaluate the safety of local delivery of a photosensitizer followed by photodynamic therapy (PDT), to determine its effectiveness in reducing in-stent restenosis. STUDY DESIGN/PATIENTS AND METHODS: Porfimer sodium was administered via a local delivery catheter to five coronary-stent implanted lesions followed by irradiation with a pulse laser. Coronary angiography (CAG) was performed at the baseline, after the procedure and at a 6-month follow-up. RESULTS: By the 18-month clinical follow-up, no adverse events such as photodermatosis, or myocardial ischemia had occurred. At the follow-up, no coronary embolization, dissection, or aneurysmal dilatation was observed in the CAG. In-stent diameter stenosis, late loss, and loss index were 19.16+/-8.20%, 0.37+/-0.18 mm, and 0.19+/-0.12, respectively. No in-stent restenosis was observed. CONCLUSIONS: This study suggests that PDT, with local delivery of Porfimer sodium, is safe and may be a feasible technique in preventing in-stent restenosis.