Hiroto Tamura

Effect of the methoxyiminoacetamide fungicide, SSF129, on respiratory activity in Botrytis cinerea

(E)-2-Methoxyimino-N-methyl-2-[2-(2,5-dimethylphenoxymethyl)phenyl]acetamide (SSF129) has been developed as a broad-spectrum systemic fungicide for control of cereal and fruit diseases. This compound inhibited NADH-oxidation by submitochondrial particles from mycelial cells of Botrytis cinerea, with an EC 50 value of 14.5 nM, due to blockage of electron transport through the cytochrome bc 1 complex in the mitochondrial respiratory chain. However, SSF129 did not suppress, but rather increased, oxygen consumption by mycelial cells of the fungus. This was because mycelial cells contain an alternative oxidase protein and the cells have the ability to rapidly switch electron flux from the main cytochrome pathway to the alternative pathway on blockage of the former by SSF129. The alternative pathway of the mycelia seems not to be operative when the cytochrome pathway is functional. Naturally occurring flavonoids inhibited the alternative oxidase of the mycelial cells in a dose-dependent manner, with EC 50 values of 68.4 μM for flavone and 63.7 μM for flavanone. These observations suggested that plant components play an important role in control of gray mould by SSF129.

Characterization of biodegradation intermediates of non-ionic surfactants by matrix-assisted laser desorption/ionization–mass spectrometry 1. Bacterial biodegradation of octylphenol polyethoxylate under aerobic conditions

Bacterial biodegradation intermediates of octylphenol polyethoxylate (OPEO) by a pure culture of Pseudomonas sp. under aerobic conditions were characterized by means of matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). The MALDI mass spectrum of the original OPEO sample showed that almost all of the OPEO constituents have regular structure with a hydroxyl end-group, which were distributed mostly from n=4 to 17 with the maximum at n=9 (n=numbers of repeating ethylene oxide (EO) units). Through biodegradation tests, the gradual shortening process of the EO chain from their hydroxyl terminal side could be successfully monitored by MALDI–MS, where the maximum of the ion peak distributions of OPEO decreased from n=9 finally down to 3. Furthermore, among the biodegradation intermediates, the formation of OPEO oligomers with a carboxyl terminal of EO chain was also clearly observed mainly under m/z 600. Based on these observations, the biodegradation of OPEO in this study would be proceeded mainly according to the exo-scission of EO chain accompanied with oxidation of the hydroxyl terminal side.

Classification of the Genus Bacillus Based on MALDI-TOF MS Analysis of Ribosomal Proteins Coded in S10 and spc Operons

A rapid bacterial identification method by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal proteins coded in S10 and spc operons as biomarkers, named the S10-GERMS (the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum) method, was applied for the genus Bacillus a Gram-positive bacterium. The S10-GERMS method could successfully distinguish the difference between B. subtilis subsp. subtilis NBRC 13719(T) and B. subtilis subsp. spizizenii NBRC 101239(T) because of the mass difference of 2 ribosomal subunit proteins, despite the difference of only 2 bases in the 16S rRNA gene between them. The 8 selected reliable and reproducible ribosomal subunit proteins without disturbance of S/N level on MALDI-TOF MS analysis, S10, S14, S19, L18, L22, L24, L29, and L30, coded in S10 and spc operons were significantly useful biomarkers for rapid bacterial classification at species and strain levels by the S10-GERMS method of genus Bacillus strains without purification of ribosomal proteins.

Classification of genus Pseudomonas by MALDI-TOF MS based on ribosomal protein coding in S10-spc-alpha operon at strain level.

We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.

Isolation of Bacterial Strains that Produce the Endocrine Disruptor, Octylphenol Diethoxylates, in Paddy Fields

Topsoil samples were collected from 36 different paddy fields in West Japan. Each soil sample was incubated with a basal salt-medium containing 0.2% OPPEO. Twelve samples possessed OPPEO-degrading activity, from which twelve cultures of OPPEO-degrading bacteria were isolated. The isolated bacteria grew on a medium containing 0.2% OPPEO as the sole carbon source, and OP2EO and OP3EO were accumulated in the medium under aerobic conditions. OP1EO and octylphenol, which have often been identified in surface water together with OP2EO, were not observed in this experiment. The bacterial isolates were gram negative and tentatively identified as Pseudomonas putida (10 isolates) and Burkholderia cepacia (one isolate) by BIOLOG and 16S rDNA RFLP analyses.

Comparison of antiandrogenic activities of vinclozolin and D, L-camphorquinone in androgen receptor gene transcription assay in vitro and mouse in utero exposure assay in vivo

A chemical substance used as a photoinitiator for light-cure resin compositions, D,L-camphorquinone (CQN) was found to be weakly antiandrogenic in vitro. It competitively antagonized dihydrotestosterone (DHT)-induced transcriptional activity on the yeast-based androgen receptor gene transcription assay (YAA). Antiandrogenic activity of CQN was shown at higher concentration than 10(-4) M while the well-known antiandrogen, vinclozolin (VCZ) showed the activity at concentrations of 10(-6) M and above in YAA. The antiandrogenic activity of CQN was reconfirmed in the human cell line-based androgen receptor gene transcription assay (HCAA). To determine whether CQN affect male reproductive development, CQN or VCZ was administered to pregnant mice daily from gestational days 10 to 18 by gavage. In utero exposure to VCZ at 100 mg/kg/day caused a significant decrease in anogenital distance (AGD) of F1 neonates and reduced spermatogenesis in F1 males at 42 days of age. In contrast, maternal doses (100 and 300 mg/kg/day) of CQN had no affect on these endpoints in F1 offspring. Further, VCZ or CQN had no adverse affect on F1 male fertility. From these observations, CQN is potentially antagonistic to androgen receptor (AR) in vitro, but is estimated to be less antiandrogenic in vivo when it is administered to pregnant mice by gavage. Furthermore, these findings are the first to demonstrate that VCZ exerts significant antiandrogenic effects on reproductive tract development during gestation in mice.

The Biological and Structural Similarity between Lunularic Acid and Abscisic Acid

Lunularic acid (LA) inhibited not only the germination and the growth of cress and lettuce at 1 mM but also the gibberellic acid (GA3)-induced α-amylase induction in embryoless barley seeds at 120 μM, which was recognized as a specific activity of abscisic acid (ABA). Moreover LA and ABA equally inhibited the growth of Lunularia cruciata A18 strain callus at 40 and 120 μM. A computational analysis revealed that the stable conformers of LA could be superimposed on the stable ABA conformers. In addition, the antibody raised against the conjugate of C1-ABA-bovine serum albumin (ABA-BSA) reacted with LA-horse-radish peroxidase (LA-HRP) conjugate as well as ABA-HRP conjugate, apparently. These results can explain why LA has ABA-like activity in higher plants. Moreover the results suggest that LA and ABA bind to the same receptor in higher plants.

Effect of flavonoids on androgen and glucocorticoid receptors based on in vitro reporter gene assay.

The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC(50) value of 0.3 microM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC(150) value of 0.7 microM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC(150) values of 4.4 and 10.1 microM, respectively. These findings provide evidence of a fundamental property of their structure-activity relationship with AR and/or GR.

Isolation and identification of Sphingomonas sp. That yields tert-octylphenol monoethoxylate under aerobic conditions

Topsoil samples were collected from eight golf courses in Yamaguchi Prefecture, Japan, and enrichment cultures were carried out with a basal-salt medium containing 0.2% 4-tert-octylphenol polyethoxylate (OPPEO) as sole carbon source. OPPEO-degrading activity was detected in one of the samples, from which a strain of OPPEO-degrading bacterium was isolated. The isolated bacterium grew on a nutritionally enriched medium (NE medium) containing 0.2% OPPEO as sole carbon source, and accumulated 4-tert-octylphenol diethoxylate (OP2EO) (63%), 4-tert-octylphenol triethoxylate (OP3EO) (14%), and 4-tert-octylphenol monoethoxylate (OP1EO) (2%) after 7 d cultivation under aerobic conditions. The addition of clay mineral (vermiculite) to the medium accelerated the degradation of OP2EO (40%) and OP3EO (4%) to OP1EO (23%). This is the first report about bacteria that can degrade OPPEO to OP1EO under aerobic conditions. The strain was identified as Sphingomonas macrogoltabidus, based on the homology of a 16S rDNA sequence.

Effect of essential oils, such as raspberry ketone and its derivatives, on antiandrogenic activity based on in vitro reporter gene assay.

The effect of essential oils, such as raspberry ketone, on androgen (AR) receptor was investigated using a MDA-kb2 human breast cancer cell line for predicting potential AR activity. Among them, eugenol had the highest AR antagonistic activity with its IC(50) value of 19 microM. Raspberry ketone, which has threefold higher anti-obese activity than that of capsaicin, also had AR antagonist activity with its IC(50) value of 252 microM. Based on these findings, a more precise CoMFA model was proposed as follows: pIC(50) [log (1/IC(50))]=3.77+[CoMFA field terms] (n=39, s=0.249, r(2)=0.834, s(cv)=0.507, q(2)=0.311 (three components).