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Many observations are reported that organic mercury compounds are involved in increasing intracellular Ca2+ levels. However, the issue of which substances on the cell membrane participate in the Ca2+ uptake that is induced by ethyl mercury is unclear. The findings of this study suggest that the P2X receptor participates in this process. The uptake of Ca2+ by C8-B4 cells was induced in the presence of ethyl mercury. Ca channels in the cell membrane were not affected in this process. In contrast, pretreatment with suramin, an antagonist of the P2X receptor, inhibited the Ca2+ uptake induced by ethyl mercury, and also brilliant blue G, a nonselective antagonist of P2×4, P2×5, and P2×7 receptors. In addition, A438079 and A740003, selective antagonists of P2×7 receptor, reduced Ca2+ uptake, while 5-BDBD, a selective antagonist of P2×4 receptor, did not. Furthermore, the mRNAs of both the P2×4 and P2×7 receptors were expressed in the presence of ethyl mercury, but the P2×5 receptor mRNA was not. These findings ...

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.

Among commonly applied molecular markers, simple sequence repeats (SSRs, or microsatellites) possess advantages such as a high level of polymorphism and codominant pattern of inheritance at individual loci. To facilitate systematic and rapid genetic mapping in soybean, we designed a genotyping panel comprised 304 SSR markers selected for allelic diversity and chromosomal location so as to provide wide coverage. Most primer pairs for the markers in the panel were redesigned to yield amplicons of 80–600 bp in multiplex polymerase chain reaction (PCR) and fluorescence-based sequencer analysis, and they were labelled with one of four different fluorescent dyes. Multiplex PCR with sets of six to eight primer pairs per reaction generated allelic data for 283 of the 304 SSR loci in three different mapping populations, with the loci mapping to the same positions as previously determined. Four SSRs on each chromosome were analysed for allelic diversity in 87 diverse soybean germplasms with four-plex PCR. These 80 loci showed an average allele number and polymorphic information content value of 14.8 and 0.78, respectively. The high level of polymorphism, ease of analysis, and high accuracy of the SSR genotyping panel should render it widely applicable to soybean genetics and breeding.

Saponins are a diverse group of secondary metabolites widely distributed in plants. Some saponins in soybean seeds have medicinal properties, but the terminal acetylated sugar at the C-22 position of group A saponins in seed hypocotyls causes a bitter and astringent taste. We used cultivated and wild soybean accessions classified into four different group A saponin phenotypes, including non-acetylated A0-αg and deacetyl-Af, as parents to obtain four F2 populations and a population of recombinant inbred lines to test their genetic relationship. The gene controlling the phenotype of the group A saponins in each line was mapped near the simple sequence repeat marker Satt336 on soybean chromosome 7 (linkage group M). An allelism test of the A0-αg and deacetyl-Af variants revealed no segregation of Aa and Ab phenotypes in the progeny, although the genes controlling the two phenotypes have been assigned to two different loci, Sg-1 and Sg-2. These results suggest that the four group A saponin phenotypes are controlled by multiple alleles at the single locus Sg-1.

A series of cis-1,2-diaminocyclohexane derivatives possessing a 6-6 fused ring for the S1 moiety were synthesized as novel factor Xa (fXa) inhibitors. The synthesis, structure-activity relationship (SAR), and physicochemical properties are reported herein, together with the discovery of compound 45c, which has potent anti-fXa activity, good physicochemical properties and pharmacokinetic (PK) profiles, including a reduced negative food effect.

Pod dehiscence (shattering) is a major cause of yield loss in mechanical harvesting of soybeans. To develop useful selection markers, we conducted a high-resolution mapping of a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1. The progeny of a residual heterozygous line, which was a recombinant inbred line segregating only for the genomic region around qPDH1, was screened for flanking markers to obtain various recombinants in the vicinity of the QTL. Analysis of the relationship between degree of pod dehiscence and graphical genotype of these lines confined the location of qPDH1 to a 134-kb region on chromosome 16 (formerly linkage group J), where ten putative genes were predicted to be present. None of these genes showed significant sequence homology with the Arabidopsis genes that have previously been reported to be associated with pod dehiscence, suggesting the presence of a novel gene and mechanism underlying pod dehiscence in soybean. Sequencing analysis of the parental shattering-resistant and -susceptible cultivars for the candidate genes revealed a high-frequency nucleotide polymorphism in this genomic region between the cultivars. Three markers were developed using insertion/deletion variations in the region. Polymorphism at these marker loci was basically conserved between diverse shattering-resistant and -susceptible cultivars/lines, suggesting the versatility and usefulness of these markers for marker-assisted selection.

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.

Photosensitivity plays an essential role in the response of plants to their changing environments throughout their life cycle. In soybean [Glycine max (L.) Merrill], several associations between photosensitivity and maturity loci are known, but only limited information at the molecular level is available. The FT3 locus is one of the quantitative trait loci (QTL) for flowering time that corresponds to the maturity locus E3. To identify the gene responsible for this QTL, a map-based cloning strategy was undertaken. One phytochrome A gene (GmPhyA3) was considered a strong candidate for the FT3 locus. Allelism tests and gene sequence comparisons showed that alleles of Misuzudaizu (FT3/FT3; JP28856) and Harosoy (E3/E3; PI548573) were identical. The GmPhyA3 alleles of Moshidou Gong 503 (ft3/ft3; JP27603) and L62-667 (e3/e3; PI547716) showed weak or complete loss of function, respectively. High red/far-red (R/FR) long-day conditions enhanced the effects of the E3/FT3 alleles in various genetic backgrounds. Moreover, a mutant line harboring the nonfunctional GmPhyA3 flowered earlier than the original Bay (E3/E3; PI553043) under similar conditions. These results suggest that the variation in phytochrome A may contribute to the complex systems of soybean flowering response and geographic adaptation.

A well-saturated molecular linkage map is a prerequisite for modern plant breeding. Several genetic maps have been developed for soybean with various types of molecular markers. Simple sequence repeats (SSRs) are single-locus markers with high allelic variation and are widely applicable to different genotypes. We have now mapped 1810 SSR or sequence-tagged site markers in one or more of three recombinant inbred populations of soybean (the US cultivar ‘Jack’ × the Japanese cultivar ‘Fukuyutaka’, the Chinese cultivar ‘Peking’ × the Japanese cultivar ‘Akita’, and the Japanese cultivar ‘Misuzudaizu’ × the Chinese breeding line ‘Moshidou Gong 503’) and have aligned these markers with the 20 consensus linkage groups (LGs). The total length of the integrated linkage map was 2442.9 cM, and the average number of molecular markers was 90.5 (range of 70–114) for the 20 LGs. We examined allelic diversity for 1238 of the SSR markers among 23 soybean cultivars or lines and a wild accession. The number of alleles per locus ranged from 2 to 7, with an average of 2.8. Our high-density linkage map should facilitate ongoing and future genomic research such as analysis of quantitative trait loci and positional cloning in addition to marker-assisted selection in soybean breeding.

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmPDIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon–intron structure across plant species. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmPDIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the α′-subunit of β-conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or β-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.

We analyzed length polymorphisms of 377 simple sequence repeat (SSR) markers in 87 cultivated or wild soybeans, including Japanese elite cultivars and irreplaceable genetic resources. The average number of SSR markers in the 20 linkage groups was 18.9, with a range of 13 to 25. All SSR markers showed polymorphism on agarose gel electrophoresis, which revealed 1380 alleles for all accessions. The number of alleles at each locus ranged from 2 to 10, with an average of 3.7, and the polymorphic information content of each marker ranged from 0.02 to 0.86, with a mean of 0.44. Cluster analysis separated the accessions into three clusters. Most Japanese cultivars were found in cluster 1, which was further divided into three subclusters that corresponded to the geographic origin of the cultivars. Most foreign cultivars and all wild accessions were present in clusters 2 and 3, respectively. Our data on the polymorphism of these SSR loci should facilitate the genetic mapping of agronomically important traits and the choice of appropriate markers for marker-assisted selection in soybean breeding programs in Japan.

誘導結合型プラズマ質量分析法（ICP-MS），誘導結合型プラズマ発光分析法（ICP-AES）を利用した下水汚泥中多元素定量法を開発した．試料の溶液化はマイクロ波酸分解法又はアルカリ融解法で行い，元素測定にはICP-MS及びICP-AESを用いた．分析値の評価には，Institute for Reference Materials and Measurements（ヨーロッパ共同体標準物質計測研究所，IRMM）より頒布されている下水汚泥標準物質（BCR-144R）を使用した．アルカリ融解法では，Zn，Cu，Pb，Ni，Co，Cdで保証値より定量値が低くなった．この濃度低下はZn，Pb，CoとCdでは揮発，Cu及びNiではPtるつぼへの合金形成が主な要因であることが分かった．また，Nb，Ta，Hfの溶解にはマイクロ波酸分解法及びアルカリ融解法双方において溶解液へのフッ化水素酸の添加が必要であった．そこで，徳島市で採取した下水汚泥実試料をマイクロ波酸分解/ICP-MS，ICP-AES法により分析した結果，計44元素の定量が可能であった．

The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression. We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co-immunoprecipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin. In addition, GmPDIM associated with the alpha' subunit of beta-conglycinin, a seed-storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone.

Anthranilate synthase (AS) is a key metabolic enzyme of the tryptophan (Trp) biosynthetic pathway in plants and microbes, and its activity is regulated by feedback inhibition by Trp. A rice gene ( OASA1D ) for a modified α-subunit of anthranilate synthase that is resistant to such feedback inhibition was introduced into azuki bean [ Vigna angularis (Willd.) Ohwi & Ohashi] by Agrobacterium -mediated transformation followed by selection with hygromycin B. Five independent azuki bean lines expressing OASA1D in which the transgene was fixed in the T 2 generation were obtained. The free Trp level in seeds of these lines (1.3–3.3 mg/g of dry weight) was increased compared with that in wild-type seeds (0.2 mg/g). In contrast, the amount of protein-bound (fixed) Trp in seeds was not affected by expression of the transgene. The AS activity in the transgenic seeds showed a reduced sensitivity to feedback inhibition by Trp or the Trp analog 5-methyltryptophan, indicating that the rice OASA1D subunit was able to interact functionally with the β-subunit of azuki bean AS. These results reveal that transformation with OASA1D is a feasible approach to increase Trp synthesis and accumulation in mature seeds of grain legumes.

In Romania, the communist planned economy system was in place for 42 years but ended due to a revolution in 1989. An environmental administration was started 1990. In order to assist with environment related activities, one of the authors worked in Bucharest for two years from March 2002 to March 2004 as a JICA (Japan International Cooperation Activity) member. One of the most important problems regarding environmental protection in Romania is waste management. In Japan, the regulations and penalties system of waste management have been used for a long time. However, there are many serious problems with the system, such as a decrease in waste disposal facilities and illegally disposed waste. The authors studied a new plan that uses a certification system in order to promote recycling industries. This system is both efficient and inexpensive so it is possible that this method of waste management could be transmitted to Romania.

In an attempt to increase the efficiency of soybean transformation by particle bombardment, we examined the effects of bombardment parameters such as gold particle size, target distance, acceleration pressure, amount of DNA per bombardment, and number of bombardments. Transgene delivery to embryogenic tissue grown in suspension culture was evaluated by monitoring the transient expression of a gene for a modified form of jellyfish green fluorescent protein [sGFP(S65T)]. Optimal transient expression of sGFP(S65T) was obtained when the tissue was bombarded twice at an acceleration pressure of 7.6 MPa (1,100 psi) and a distance of 6 cm with gold particles that were 0.6 μm in diameter and coated with 0.8 μg of DNA. Application of these optimized conditions proved effective for the generation of stable transgenic soybean plants. Stable transgene integration in the transformants was confirmed by Southern blot analysis. The average transformation efficiency achieved with the optimized protocol was siginificantly higher than that with the conventional protocol.

Isotype switching of immunoglobulin (Ig)-secreting cells is regulated by a set of cytokines. In the present study, we studied the relation between the number of peripheral blood mononuclear cells spontaneously secreting IgG, IgM, IgA, and their subclasses and the disease severities in autoimmune thyroid diseases. Ig-secreting cells were measured by enzyme-linked immunospot (ELISPOT) assay in 99 euthyroid patients with Graves' disease (GD) or Hashimoto's disease (HD) and 13 normal subjects. The number of IgG3-secreting cells was significantly higher in patients with intractable GD who had been undergoing treatment with antithyroid drugs for more than 5 years but who did not go into remission than in patients with GD in remission. This number correlated significantly with the serum level of thyrotropin receptor antibody (TRAb) in all patients with GD. These data suggest that the number of IgG3-secreting cells whose isotype switching is stimulated by interleukin (IL)-10 and IL-4 may be related to the disease severity of GD and to the level of TRAb after long-term treatment with antithyroid drugs.

In the present research, amenity effect of ornamental foliage plants, imitation plants and photograph on human psychology were analyzed by means of electroencephalogram (EEG) and semantic differential technique. The experiments were carried out in a room in Ehime University in May of 2000. The following five objects with ornamental foliage plants, imitation plants or photograph in the room were evaluated by seventeen students (nine males and eight females) . Electroencephalogram of each subject was measured in the room. 1) Without plants (Control) ; 2) photograph of six ornamental foliage plants of Epipremunum aureum (Photograph) ; 3) six ornamental foliage plants (Plant) ; 4) mixture of three ornamental foliage plants and three imitation plants (Mixture) ; 5) six imitation plants (Imitation) . Ratio of alpha wave to beta wave was calculated as an index expressing the degree of amenity. As for semantic differential technique, factor analysis was performed based on twenty-five SD scales. Higher ratio of alpha wave to beta wave was observed in “Plant” for male subjects, indicating the amenity effect of ornamental foliage plants on human psychology. Three factors were extracted by the factor analysis. The first factor was concerned with comfortable psychology and the second factor was concerned with space arround the subject. The first factor score of “Plant” was higher than those of the other objects in female subjects but not in male subjects. The second factor score of “Photograph” was higher than that of “Mixture.” These results indicated the amenity effect on human psychology by plants.

In the present study, amenity effect on human psychology as affected by ornamental foliage plants used as a screen was analyzed by means of electroencephalogram (EEG) and semantic differential technique.The experiments were carried out in a room at Ehime University in November and December of 1997. The following three conditions concerning the arrangement of ornamental foliage plants or screens around the subject were rated by eleven students.(a) a screen on each side of the subject(b) a screen on the one side and three ornamental foliage plants on the other side of the subject(c) three ornamental foliage plants on each side of the subjectElectroencephalogram of each student were measured in the room. Ratio of alpha wave to beta wave was calculated as an index expressing the degree of amenity. As for semantic differential technique, factor analysis was performed based on nineteen SD scales.Higher ratio of alpha wave to beta wave was observed in the condition (c). Two factors were extracted by the factor analysis. The factor scores of the condition (b) and the condition (c) were higher than the factor score of the condition (a). These results indicated the amenity effect on human psychology by plants.

In the present study, amenity effect of ornamental foliage plants on human psychology was analyzed by means of brain waves and semantic differential technique. The experiments were carried out in a room in Ehime University in January and February of 1997. Four objects with or without ornamental foliage plants in the room were rated by ten students (six males and four females). Brain waves of each student were measured in the room. Ratio of alpha wave to beta wave was calculated as an index expressing the degree of amenity. As for semantic differential technique, factor analysis was performed based on twenty-three SD scales.Higher ratio of alpha wave to beta wave was observed in the objects with ornamental foliage plants, indicating the amenity effect of ornamental foliage plants on human psychology. Two factors were extracted by the factor analysis. The objects with ornamental foliage plants were evaluated higher in the first factor than the objects without plants. As for the arrangement of ornamental foliage plants, the object with ornamental foliage plants placed on a straight line along the window was evaluated higher in the second factor than the object with plants interspersed in the room.

The desired 5,6-dihydro-4H-pyrrolo[3,4-d]thiazole (VII) is readily prepared via a Mitsunobu reaction of diol (IV) with sulfonamide (V).

Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar-dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1(a) allele encodes the xylosyltransferase UGT73F4, whereas Sg-1(b) encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1(a) and Gly-138 in Sg-1(b) proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-1(0) is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.

3-Dimethoxymethyl-2-(N-cyanoimino)thiazolidine 1e reacts with secondary amines to afford 2,4-diamino-s-triazines 4 unexpectedly, while the triazine 5 bearing different amino groups at 2 and 4 positions is selectively obtained by the reaction with a mixture of two kinds of amines.

Airborne particulate matter (PM) collected in Bucharest (Romania) and Tokushima (Japan) was comprehensively characterized through multielemental analysis by ICP-MS to the PM samples. Prior to an elemental characterization of the sample, a multielemental determination method composed of acid digestion (HClO(4)/HNO(3)/HF or H(2)O(2)/HNO(3)/HF) and inductively coupled plasma mass spectrometry/inductively coupled plasma atomic emission spectrometry (ICP-MS/ICP-AES) was established to analyze a certified reference material of vehicle exhaust matter (NIES No. 8) for verifying the method. As the results, 39 elements in the certified reference material could be determined. They were a good agreement with the certified and reference values for HClO(4)/HNO(3)/HF acid digestion. Chalcophile elements, such as cadmium, antimony, and lead in Bucharest showed higher enrichment factors than those in Tokushima.

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an alpha subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.

Previously, we reported that racemic cis-piperidine diamine derivatives (±)-1 showed high anti-fXa, anticoagulation activity and oral activities. To confirm the active enantiomer, (3R,4S)-piperidine diamine derivatives (-)-1A and (-)-1B were synthesized enantioselectively from Boc-D-serine. These synthetic routes and intermediates could be utilized for the optimization of these derivatives. From the activities of (-)-1A and (-)-1B, (3R,4S)-isomer was confirmed to be the active enantiomer.

We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real-time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real-time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR-negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real-time PCR is suitable for rapid diagnosis as a first screening test to determine first-line antibacterial agents to be used against these infectious diseases.

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmPDIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon-intron structure across plant species. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmPDIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the alpha'-subunit of beta-conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or beta-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.

The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression. We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co-immunoprecipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin. In addition, GmPDIM associated with the alpha' subunit of beta-conglycinin, a seed-storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone.

Previously, we identified cyclohexane diamine derivative 1 as orally bioavailable factor Xa inhibitor. We have investigated two racemic cis-piperidine diamine derivatives 2 and 3 based on 1. Compounds 2a-e showed higher fXa inhibitory activity, anticoagulant activity, and aqueous solubility than 3a-e having same substituent. Compounds 2a, 2c, 2e, and 2g-m having sp2 nitrogen, especially amide and urea derivatives, showed potent anticoagulant activity. Compounds 2h and 2k showed high oral activities in rats.

Serine protease factor xa (fXa) inhibitor 1 showed good ex vivo anti-fXa activity upon oral administration in rats. However, it has been revealed that 1 had low metabolic stability against human liver microsomes. To improve the metabolic stability, we attempted to modify the S1 and S4 ligands of 1. These modifications resulted in compound 34b, which exhibited selective anti-fXa activity and excellent anti-coagulation activity.

A simultaneous high-performance liquid chromatographic method for the determination of ephedrine, pseudoephedrine, norephedrine and methylephedrine (ephedrine alkaloids) in Kampo medicines which contain Ephedrae Herba was established. The analysis can be accomplished within 25 min with a Wakosil-II 5C18 HG column by isocratic elution using a mixture of water, acetonitrile and sodium dodecyl sulfate (65:35:0.4) as the mobile phase at a flow-rate of 1.0 ml min(-1), and detection at 210 nm. The detection limits of ephedrine alkaloids are 0.37-1.06 microM per injection (5 microl). This method was applied to analyze the quantities in eight Kampo decoctions; Mao-to, Makyo-yokukan-to, Makyo-kanseki-to, Yokuinin-to, Sho-seiryu-to, Keima-kakuhan-to, Kakkon-to and Kakkon-to-ka-senkyu-sin'i. The concentration (per Ephedrae Herba gram) of ephedrine alkaloids was higher in the Makyo-kanseki-to decoction than in the others. Calcium sulfate from Gypsum Fibrosum raised ephedrine alkaloids dissolution in the Makyo-kanseki-to decoction.

Previously, we reported that racemic cis-piperidine diamine derivatives (±)-1 showed high anti-fXa, anticoagulation activity and oral activities. To confirm the active enantiomer, (3R,4S)-piperidine diamine derivatives (-)-1A and (-)-1B were synthesized enantioselectively from Boc-D-serine. These synthetic routes and intermediates could be utilized for the optimization of these derivatives. From the activities of (-)-1A and (-)-1B, (3R,4S)-isomer was confirmed to be the active enantiomer.

3-Dimethoxymethyl-2-(N-cyanoimino)thiazolidine 1e reacts with secondary amines to afford 2,4-diamino-s-triazines 4 unexpectedly, while the triazine 5 bearing different amino groups at 2 and 4 positions is selectively obtained by the reaction with a mixture of two kinds of amines.

3-Alkyl-2-(N-cyanoimino)thiazolidines were methylated mainly at the nitrile nitrogen atom with trimethyloxonium tetrafluoroborate (Me 3 O.BF 4 ). The reaction of the methylated products, the nitrilium salts, with sodio diethyl malonate was described

Unexpected O → S and N → S alkyl transfer reactions were observed in the reaction of 3-alkyl-2-(N-cyanoimino)thiazolidines with sodium alkoxides, and a new synthetic method for cimetidine was developed using the N → S alkyl transfer reaction as a key step.