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Plant R2R3-MYB transcription factors are encoded by more than 100 copies of genes. In this study, we attempted to isolate some members of the R2R3-MYB superfamily involved in regulation of nitrogen fixation in legumes. A library of 300 recombinant plasmid clones containing the R2R3-MYB fragments of the superfamily was screened by differential hybridization to isolate R2R3-MYB genes whose expression was up-regulated under nitrogen nutrient-limited conditions. Two groups of clones were identified, each of which seemed to represent a gene responsive to nitrogen starvation. The entire coding regions for the genes were further isolated by PCR and were designated LjMYB101 and LjMYB102. By screening a genomic library of Lotus japonicus with a probe derived from LjMYB101, the third gene, LjMYB103, was isolated. In addition, a candidate for the soybean orthologue of LjMYB101 was isolated and designated GmMYB101. Sequence alignment of the genes with members of the plant R2R3-MYB superfamily showed that they all belonged to the subgroup 10 of the superfamily. The expression analysis of the genes showed that the organ-specific and nitrate-regulated expression profile of MYB101 was very similar to that of CHS in Lotus as well as in soybean, suggesting a possible role for MYB101 in regulation of flavonoid biosynthesis in response to nitrate starvation. On the other hand, an interesting relationship, in structure and function, was found between LjMYB101 and LjGln1, suggesting an alternative role for MYB101 in regulation of nitrogen metabolism.

Seed coat color in soybeans is determined by the I (Inhibitor) locus. The dominant I allele inhibits seed coat pigmentation, and it has been suggested that there is a correlation between the inhibition of pigmentation by the I allele and chalcone synthase (CHS) gene silencing in the seed coat. Analysis of spontaneous mutations from I to i has shown that these mutations are closely related to the deletion of one of the CHS genes (designated ICHS1). In soybeans with the I/I genotype (cv. Miyagi shirome), a truncated form of the CHS gene (CHS3) is located in an inverse orientation 680 bp upstream of ICHS1, and it was previously suggested that the truncated CHS3- ICHS1 cluster might be involved in CHS gene silencing in the seed coat. In the current study, the truncated CHS3- ICHS1 cluster was compared with the corresponding region of pigmented seed coat mutants in which I had changed to i in Miyagi shirome and in the strain Karikei 584. In the Karikei 584 mutant, the truncated CHS3-ICHS1 cluster was retained and the sequence diverged at a point immediately upstream (32 bp) of this cluster. The sequences upstream of the points of divergence in both mutants almost perfectly matched a part of the registered sequence in a soybean BAC clone containing the soybean cyst nematode resistance-associated gene, and inspection of the sequences suggested that the sequence divergence of the CHS gene in the Karikei 584 and Miyagi shirome mutants was due to an unequal crossing-over via 4-bp or 5-bp short repeats, respectively.

Unlabelled: Tominaga K, Kataoka Y, Sendo T, et al. Contrast medium-induced pulmonary vascular hyperpermeability is aggravated in a rat climacterium model. Invest Radiol 2001;36:131-135., Rationale and Objectives: To test whether climacterium influences adverse pulmonary reactions to contrast media, the authors investigated the effect of ioxaglate on pulmonary vascular permeability in ovariectomized rats as a climacterium model., Methods: From 7 days after surgery, ovariectomized rats were treated with estradiol valerate or vehicle once per week for 3 weeks. At 28 days after surgery, ioxaglate, an ionic contrast medium, was intravenously injected at 1.5 mL/min in rats. Pulmonary vascular permeability was evaluated by measuring the amount of Evans blue dye in the lung tissue., Results: Ioxaglate dose-dependently increased pulmonary vascular permeability in sham-operated and ovariectomized rats. Ovariectomized rats showed a 2.6-fold increased aggravation of vascular permeability by ioxaglate 4 g I/kg compared with sham-operated rats. Estradiol valerate (0.2-5.0 mg/kg) dose-dependently blocked ioxaglate-increased vascular permeability in ovariectomized rats., Conclusions: These findings suggest that climacterium is included, at least in part, in the risk factors for contrast-induced adverse pulmonary reactions, and this risk is lowered by estrogen replacement therapy.

A simple and sensitive semi-micro high-performance liquid chromatography (HPLC) was established for determining the serum levels of glycyrrhizin (GL) in humans. Butyl p-hydroxybenzoate was used as the internal standard and serum was deproteinized by methanol. The samples were separated on a Capcell Pak C18 UG120 column (150 x 1.5 mm i.d.; particle size, 5 microm). The detection limit of GL in serum was 100 ng/ml, which enables determination of serum levels of GL after administration of a therapeutic dose. The time-course study suggested that the elimination rate of GL differed between subjects for the same administered dose, although the sample was too small to allow a meaningful comment. In clinical practice, GL is used for its antiviral and anti-inflammatory effects. Excessive administration of GL can induce pseudoaldosteronism; however the optimal GL concentration in serum remains to be determined. The determination method reported here is expected to aid in the safe and efficient use of the drug in clinical practice.

We demonstrate that glycyrrhizin (GL) enhanced Fas-mediated apoptotic body formation and DNA fragmentation in T cell lines although GL alone did not induce apoptosis. The enhancement effect of Fas-mediated apoptosis by GL was dose-dependent above 0.3 microM. Time course study revealed that simultaneous co-treatment of GL and anti-Fas antibody was crucial for the enhancement of apoptosis and pretreatment with GL was not effective. Anti-Fas antibody elicited caspase-3-like activity. However caspase-3-like activity with co-treatment of GL and anti-Fas antibody was the same level as the antibody alone. Glycyrrhetic acid, the aglycon of GL, did not enhance Fas-mediated apoptosis. The amphipathic property of GL might enable it to interact with the plasma membrane and lead to the enhancement of apoptosis.

By developing gene-specific RT-PCR and using filters to allow transmission down to 290 nm (UV-B+) or blocking all radiation below 320 nm (UV-B(-)), the effect of UV-B+ and UV-B- light on expression of each of the presently known seven members of soybean chalcone synthase (CHS) gene family in dark-grown seedlings was analyzed. Dark expression was detectable already in 18 h dark-germinating embryos, with progressive increases on successive days, suggesting that chs belongs to a class of genes expressed very early during germination, and that the expression at this stage is either constitutive or induced by non-light-dependent factors present in the seed or made available following imbibition. Exposure of 18 h dark-germinating embryos to UV-B- or to UV-B+ light did not lead to an increase in chs signal. However, the 24 h dark-germinating embryos showed a distinct effect of UV-B+, interestingly coinciding with the stage when the head of seedlings was in the process of being pushed up above ground by stem elongation, suggesting the possibility of a developmental switch modulating the appearance of UV-B response. The response to UV-B- was most prominent in chs1 and almost silent in chs2, while the up-regulation by UV-B+ was most prominent in chs5 and chs6 and much less so in chs2. Interestingly, chs2 was noted to be the only member of the Gmchs gene family devoid of H-box, raising the possibility that the H-box may be a good indicator of the photo-inducibility of a chs gene.

Molecularly imprinted polymer (MIP) was prepared against phenobarbital using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross linking monomer. We analyzed the recognition properties of the phenobarbital MIP. In some organic solvents, imprinted polymer showed selective binding to phenobarbital. Two dissociation constants of binding were calculated by Scatchard plot analyses; Kd values were 1.8, 121.7 microM, and the number of binding sites was 8.3, 92.3 mumol/g MIP in toluene-heptane-acetic acid (25 : 75 : 1, v/v), respectively. The relationship between the binding affinity to phenobarbital MIP and the polarity of the solvent system, as well as the structure of the template molecule is also discussed.

We found that ibuprofen (IBU) had a potential for releasing serum albumin-bound glycyrrhetic acid (GA). Based on this observation, IBU was used to pretreat samples for the determination of serum GA levels by an inhibition ELISA. This method, termed IBU method was evaluated by the recovery of GA from human serum albumin (HSA) or normal human serum (NHS) that contained the exogeneously added GA (37-1000 ng/ml). The mean recovery of GA from HSA and NHS samples treated with IBU were 104.7 and 105.2%, respectively, whereas those without IBU pretreatment were 2.8 and 10.7%, respectively. Comparison of IBU method and chloroform extraction method revealed that the GA content of serum samples pretreated by each method were almost the same. These results suggest that IBU method is useful as a serum processing procedure for the determination of serum GA levels by an inhibition ELISA.

Albino rice plants derived from pollen contain plastid genomes that have suffered large-scale deletions. From the roots of albino plants, we obtained several calli containing homogeneous plastid DNA differing in the size and position of the deletion. DNA differing in the size and position of the deletion. Southern blotting and pulsed field gel electrophoresis experiments revealed that the DNAs were linear molecules having a hairpin structure at both termini, existing as monomers (19 kb) or dimers, trimers and tetramers linked to form head-to-head and tail-to-tail multimers. This characteristic form is similar to that of the vaccinia virus, in which the replication origin is thought to lie at or near the hairpin termini. Furthermore, polymerase chain reaction experiments revealed complete loss of the ribosomal RNA genes of the plastid DNA. The results suggest that plant cells can grow without translation occurring in plastids. All of the deleted plastid DNAs commonly retained the region containing the tRNA(Glu) gene (trnE), which is essential for biosynthesis of porphyrin. As porphyrin is the precursor of heme for mitochondria and other organelles, it is considered that trnE on the remnant plastid genome may be transcribed by an RNA polymerase encoded on nuclear DNA.

Amounts of particulate matter present in 17 small volume antibiotic injections marketed in Japan were determined using light obscuration particle analyzer (HIAC). The vial volume range of each batch of product was 7-20 ml, and each vial contained 1 g as antibiotic potency. In 4 products, contents of particles between 2.5 and 10 microns in diameter were counted 2,000-7,000 per vial, and particles in other products were counted less than 2,000 per vial. Numbers of particles greater than or equal to 10 microns in diameter were much less than the USP XXI criteria for particulate matter in small volume injections. The product of the highest counts for particles between 10 and 25 microns in diameter showed counts amounted to 0.13% of the USP XXI criteria. In the 25-50 microns particulate diameter range, particulate matters were detected only in 2 products, and they were less than 0.2% of the USP XXI criteria. Particles over 50 microns in diameter were not detected in any products. These results showed that 17 small volume antibiotic injections marketed in Japan had good qualities regarding contents of particulate matter.

The antibacterial activity of MT-141 against Escherichia coli and Proteus morganii in compromised mice was investigated and compared with that of latamoxef, cefmetazole and cefoxitin. The bactericidal activity of MT-141 in short-term contact with E. coli and P. morganii was markedly enhanced when combined with mouse serum, and the activity of MT-141 was greater than the activities of the three reference drugs. The antibacterial activities of MT-141 in the liver, spleen and kidney of neutropenic and Sarcoma 180 tumor-bearing mice infected with E. coli and P. morganii were superior to the activities of the reference drugs. MT-141 was more potent than cefmetazole and cefoxitin, and similar to latamoxef in potency against systemic P. morganii infection in Sarcoma 180 tumor-bearing mice.