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Arbuscular mycorrhizal (AM) fungi are obligate symbionts that depend on living host plants to complete their life cycle1,2. This feature, which leads to their unculturability in the absence of plants, strongly hinders basic research and agricultural application of AM fungi. However, at least one AM fungus can grow and develop fertile spores independently of a host plant in co-culture with the bacterium Paenibacillus validus3. The bacteria-derived substances are thought to act as stimulants or nutrients for fungal sporulation, but these molecules have not been identified. Here, we show that (S)-12-methyltetradecanoic acid4,5, a methyl branched-chain fatty acid isolated from bacterial cultures, stimulates the branching of hyphae germinated from mother spores and the formation of secondary spores in axenic culture of the AM fungus Rhizophagus irregularis. Extensive testing of fatty acids revealed that palmitoleic acid induces more secondary spores than the bacterial fatty acid in R. irregularis. These induced spores have the ability to infect host plant roots and to generate daughter spores. Our work shows that, in addition to a major source of organic carbon6–9, fatty acids act as stimulants to induce infection-competent secondary spores in the asymbiotic stage and could provide the key to developing the axenic production of AM inoculum. Palmitoleic acid as well as fatty acids isolated from bacterial cultures can be used in axenic culture of the arbuscular mycorrhizal fungus Rhizophagus irregularis to stimulate the formation of secondary spores that are able to colonize host plant roots.

Three peptaibols, trichorzins HA II (1), HA V (2), and HA VI (3), were isolated from okara fermented with Trichoderma harzianum HK-61 as anti-plant viral agents. Their structures were confirmed by spectroscopic and chemical methods. At micro molar concentrations, the trichorzins inhibited infections by Cucumber mosaic virus in the cowpea plant Vigna sesquipedalis.

The genus Aglaia is a a rich source of different compounds with interesting biological activities. A part of our continuing search for novel biologically active compounds from Indonesia Aglaia plants, the ethyl acetate extract of bark of Aglaia eximia showed significant antioxidant activity. Four antioxidant compounds, kaempferol ( 1 ), kaempferol-3- O- α-L-rhamnoside (2), kaempferol-3- O- β-D-glucoside (3) and kaempferol-3- O -β-D-glucosyl-(1→4)-α-L-rhamnoside (4) were isolated from the bark of Aglaia eximia (Meliaceae). The chemical structures of compounds 1 - 4 were identified on the basis of spectroscopic datas including UV, IR, NMR and MS along with by comparison with those spectra datas previously reported. All compounds showed DPPH radical-scavenging activity with IC 50 values of 1.18, 6.34, 8.17, 10.63 m g/mL, respectively.

Didodecyldimethylammonium bromide (DDAB)-modified montmorillonite (MT) organoclay was used for the sorption and degradation of an organophosphate pesticide, fenitrothion, in water. The degree of sorption increased by the modification of MT with DDAB, because of the formation of hydrophobic DDAB assemblies for the sorption of hydrophobic fenitrothion. Fenitrothion ([M + 1] = 278) sorbed on the organoclay rapidly converted to the degraded product anion ([M] = 262) followed by the further degradation into 3-methyl-4-nitrophenol ([M-1] = 152). The activation energy for the first-order reaction of the primary degradation of fenitrothion (79.9 kJ mol-1) in the organoclay was lower than the value (94.1 kJ mol-1) in water. Organoclay-mediated catalytic activity expressed over a wide pH region (pH 5-9), being advantageous for the application to different wastewaters and environmental waters. Continuous sorption and degradation of fenitrothion in contaminated water was examined by using a laboratory-scale organoclay-packed column. Fenitrothion-free effluent containing its degraded product, 3-methyl-4-nitrophenol, having lower toxicity to aquatic organisms outflowed from the bottom of the column. These results strongly suggests the usefulness of DDAB-MT organoclay as a barrier material for diffusion control of organophosphate pesticides.

Flavonoids compounds such as kaempferol (1), kaempferol-3-O-β-D-glucopyranoside or astragalin (2) and kaempferol-3-O-α-L-rhamnoside or afzelin (3) have been isolated from the fresh leaves of Kalanchoe tomentosa (Crassulaceae). The chemical structure of isolated compounds 1-3 were identified by spectroscopic evidences and comparison with those compound previously reported. Compounds 1-3 showed cytotoxic activity against P-388 murine leukimia cells with IC50 values of 51.8, > 100 and 3.32 µg/mL, respectively.

Two new bisamide compounds, eximiamide A ( 1 ) and eximiamide B ( 2 ) were isolated from the bark of Aglaia eximia (Meliaceae). The chemical structures of the new compound were elucidated on the basis of spectroscopic data. All of the compounds were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1 and 2 exhibited cytotoxic activity against P-388 murine leukemia cells with IC 50 values of 7.6 and 8.5 μg/mL, respectively.

Prenylated indole alkaloid okaramines selectively target insect glutamate-gated chloride channels (GluCls). Because of their highly complex structures, including azocine and azetidine rings, total synthesis of okaramine A or B has not been achieved, preventing evaluation of the biological activities of okaramines. Biosynthetic approaches provide alternatives to accessing structurally diverse derivatives and enabling the elucidation of structure-activity relationships. To explore the biosynthetic potential of okaramines, gene knockout experiments of an okaramine-producer fungus were performed. The deletion mutants of the oxygenase genes okaB, okaD, okaE, and okaG provided analogues that were unlikely to be accumulated in the normal biosynthetic process of the wild-type strain. Analysis of the structure-activity relationships of okaramines collected from the fungal cultures revealed that 1,4-dihydroazocine and N-aliphatic group attached to the indole were crucial for GluCl-activating activity. This provided insights into further derivatization of the complex structure of okaramines in order to facilitate the development of new insecticides.

Pseudomonas cichorii s train SPC9018 was found to produce the siderophore pyoverdine. Application of the phytosiderophore mugineic acid (MA) led to a decrease in the in vitro growth of SPC9018. MA application resulted in increased expression of fecA, which is involved in citrate-mediated iron uptake and in pyoverdine production-related pvdL and pvdR, suggesting that the MA addition may lead to iron-limited conditions in SPC9018. MA application also reduced SPC9018 growth in eggplant leaves, leading to loss of its virulence on eggplant. MA also reduced adhesion of SPC9018. Considering these results together, iron acquisition may play a specific role in SPC9018 virulence on eggplant.

The new stigmastane steroid, 3,4-epoxy-(22R,25)-tetrahydrofuran-stigmast-5-en (1), along with two known triterpenoids (2–3) and two known steroids (4–5) were isolated from the stem bark of Aglaia eximia. The chemical structures of the new compound were elucidated on the basis of spectroscopic data. All of the compounds were tested for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1–5 showed cytotoxicity against P-388 murine leukemia cells with IC50 values of 11.42, 4.26, 29.10, 45.96, and 52.09 μg/mL, respectively.

(-)-Indolactam-V (1) has the fundamental structure of potent tumor-promoting teleocidins. A new teleocidin-related metabolite, (-)-14-O-malonylindolactam-V (2), was isolated from the culture broth of Streptomyces blastmyceticum NA34-17 which produced a large quantity of 1 along with a small amount of (-)-14-O-acetylindolactam-V (3). Heat treatment of 2 in methanol readily produced 1 and 3, suggesting that S. blastmyceticum NA34-17 excreted 1 as a malonic acid conjugate to accumulate 1 and 3 in the culture broth during cultivation.

A new lanostane-type triterpenoid, 3β-hydroxy-25-ethyl-lanost-9(11),24(24′)-diene (1), along with 3β-hydroxy-lanost-7-ene (2) and β-sitosterol-3-O-acetate (3) was isolated from the stem bark of C. cumingianus. The chemical structure of the new compound was elucidated on the basis of spectroscopic data. All of the compounds were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1-3 showed cytotoxicity against P-388 murine leukemia cells with IC50 values of 28.8 ± 0.10, 4.29 ± 0.03, and 100.18 ± 0.16 μg/ml, respectively.

N -Acylhomoserine lactones (AHLs) are the conserved quorum-sensing signal molecules in Gram-negative bacteria. ( Z )- N -(4-Decenoyl)homoserine lactone ( 1 ), a new AHL, was isolated from the culture broth of the fungus Mortierella alpina A-178 harboring bacterial endosymbionts, called endobacteria. The structure and absolute configuration of 1 were elucidated by EI-MS, chemical synthesis, and chiral GC analysis. The compound induced the expression of a QS-regulated reporter gene in Agrobacterium tumefaciens NTL4, although its activity was lower than that of N -decanoylhomoserine lactone ( 6 ).

Two new dammarane triterpenoids, aglinone (1) and aglinin E (20S,24S-epoxy-25-hydroxy-1-en-dammarene) (2) along with three known compounds, 3-epiocotillol (3), aglinin A (4), and eichlerianic acid (5), were isolated from the bark of Aglaia smithii. The chemical structures of the new compound were elucidated on the basis of spectroscopic data interpretation. All the compounds isolated were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1, 2, 4 and 5 showed cytotoxicity against P-388 murine leukemia cells with IC50 values of 21, 42, 34, and 11 μg/mL, respectively.

Erythrina plants, locally known as “dadap ayam”, are higher plant species and have been used as a folk medicine for treatment of cancer. To prove the effectiveness of the leaves and stem bark of E. variegata as an anti-cancer agent, the assay in this research was focused on in vitro test towards breast cancer cell T47D . In the course of our continuing search for novel anti-cancer agent from Erythrina plants, the methanol extract of the leaves and stem bark of E. variegata showed significant anti-cancer activity against breast cancer cell T47D in vitro using the Sulphorhodamine B (SRB) assay. By using the anti-cancer activity to follow the separations, the methanol extract was separated by combination of column chromatography. The chemical structure of an anti-cancer compounds were determined on the basis of spectroscopic evidence and comparison with the previously reported and identified as an erythrina alkaloid ( 1 ) and isoflavonoid ( 2 ). Compounds ( 1 - 2 ) showed anti-cancer activity against breast cancer cell T47D used with IC 50 of 1.0 and 3.3 µg/mL, respectively. This results strongly suggested that E. variegata is promising sources for anti-cancer agents. Keywords : Anti-cancer, Erythrina variegata, Leguminoseae

Changes in filling characteristics when adding oxygen to sputtering gas (1 at.%N2–Ar) were investigated using high-vacuum planar magnetron sputtering equipment having little residual gas effects. It was found that copper filling accelerates for oxygen partial pressure in sputtering gas of P O 2 = 5 × 10 − 5 to 1 × 10 − 4 Pa and a substrate temperature of 300–320 °C. Under these conditions, 70% copper filling in fine holes of diameter ϕ = 100 nm (AR = 4.5) was obtained.

4-｛（4-ジエチルアミノ-2-ヒドロキシフェニル）アゾ｝-5-ヒドロキシナフタレン-2,7-ジスルホン酸（ベリロンIII）とホウ素（III）との錯形成を利用する吸着ストリッピングボルタンメトリーを環境水中微量ホウ素の高感度定量に適用した．0.06 M硝酸－0.04 M硫酸溶液（pH 4.5）をかき混ぜながら，－0.35 V vs. 飽和カロメル電極（SCE）で60秒間前電解してつり下げ水銀滴電極上にホウ素（III）－ベリロンIII錯体を濃縮後，10 mV s－1の速度で示差パルス法により電位を－0.8 V vs. SCEまで走査した．錯体の還元溶出ピークは－0.4 V vs. SCE付近に得られた．共存元素の影響について検討し，アルミニウム（III），鉄（III）及びケイ素（IV）の妨害はメタリン酸の添加により抑制できた．ホウ素（III）濃度0.2～100 ng mL－1の範囲で直線（相関係数＞0.999）の検量線（ピーク高さ対濃度）が得られ，相対標準偏差（n＝4）は2～7％ であった．煩雑な前濃縮操作を必要としない本法を環境試料水中の微量ホウ素定量に応用し，十分な精度と正確さが得られた．

Two sucrose phosphorylases were employed for glycosylation of carboxylic acid compounds. Streptococcus mutans sucrose phosphorylase showed remarkable transglycosylating activity, especially under acidic conditions. Leuconostoc mesenteroides sucrose phosphorylase exhibited very weak transglycosylating activity. Three main products were detected from the reaction mixture using benzoic acid and sucrose as an acceptor and a donor molecule, respectively. These compounds were identified as 1-O-benzoyl α-d-glucopyranoside, 2-O-benzoyl α-d-glucopyranose, and 2-O-benzoyl β-d-glucopyranose by 1D-and 2D-NMR analyses of the isolated products and their acetylated products. Time-course analyses proved that 1-O-benzoyl α-d-glucopyranoside was initially produced by the transglycosylation reaction of the enzyme. 2-O-Benzoyl α-d-glucopyranose and 2-O-benzoyl β-d-glucopyranose were produced from 1-O-benzoyl α-d-glucopyranoside by intramolecular acyl migration reaction. S. mutans sucrose phosphorylase showed broad acceptor-specificity. This sucrose phosphorylase catalyzed transglycosylation to various carboxylic compounds such as short-chain fatty acids, hydroxy acids, dicarboxylic acids, and phenolic carboxylic acids. 1-O-Acetyl α-d-glucopyranoside was also enzymatically synthesized by transglucosylation reaction of the enzyme. The sensory test of acetic acid and the glucosides revealed that the sour taste of acetic acid glucosides was significantly lower than that of acetic acid.

Transglucosylation from sucrose to acetic acid by sucrose phosphorylase (EC 2.4.1.7) was studied. 1-O-Acetyl-alpha-D-glucopyranose was isolated as the main product of the enzyme reaction. We also compared the pH-dependence of transglycosylation catalyzed by sucrose phosphorylase toward carboxyl and hydroxyl groups. With hydroquinone as an acceptor molecule, the transfer ratio of glucose residue was higher at neutral pH. This pH-activity profile was similar to that of the phosphorolysis of sucrose by sucrose phosphorylase, but with acetic acid as an acceptor molecule, the transfer ratio of glucose residue was higher at low pH. These findings suggest that the undissociated carboxyl group is essential to the acceptor molecule for the transglycosylation reaction of sucrose phosphorylase. In a sensory test, the sour taste of acetic acid was markedly reduced by glucosylation. The threshold value of the sour taste of acetic acid glucosides was approximately 100 times greater than that of acetic acid.

Background/Aim: Indoxyl sulfate (IS) is a uremic toxin that accelerates the progression of chronic renal failure (CRF). This study aimed at determining whether IS impairs antioxidative systems (redox status) in the kidney. Methods: IS was orally administered to normal and subtotally nephrectomized (three fourths and five sixths) rats (CRF rats) for 2 weeks. By use of in vivo and ex vivo electron spin resonance spectroscopy, the kidney redox status was evaluated using carbamoyl-PROXYL as a radical spin probe in living rats, and the kidney superoxide scavenging activity was measured. Immunohistochemistry of superoxide dismutase (SOD) in the kidney was performed. Results: Administration of IS increased serum and kidney levels of IS and serum creatinine and decreased creatinine clearance. CRF rats showed reduced spin reduction rate, prolonged half-life of the spin probe, and reduced superoxide scavenging activity and SOD-positive areas in the kidney as compared with normal rats. Administration of IS further reduced radical spin reduction rate, prolonged half-life of the spin probe, and reduced superoxide scavenging activity and SOD-positive areas in the kidneys. Conclusions: Administration of IS reduced superoxide scavenging activity in the kidneys of normal and CRF rats. Thus, the nephrotoxicity of IS may be induced by impairing the antioxidative systems in the kidney.

Three flavonoid compounds, kaempferol (1) , kaempferol-3- O- α-L-rhamnoside (2) , and kaempferol-3- O -β- D -glucosyl-α- L -rhamnoside (3), were isolated from the bark of Aglaia eximia (Meliaceae). The chemical structures of compounds 1–3 were identified with spectroscopic data, including UV, IR, NMR ( 1 H, 13 C, DEPT 135°, HMQC, HMBC, 1 H- 1 H-COSY NMR), and MS, as well as a compared with previously reported spectra data. All compounds were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1–3 showed cytotoxicity against P-388 murine leukemia cells with IC 50 values of 1.22, 42.92, and >100 mg/mL, respectively

Two new glucosides, capsaicin-β-d-glucopyranoside (1) and dihydrocapsaicin-β-d-glucopyranoside (2), were discovered in the fruit of the Capsicum annuum cultivar ‘High Heat'. They were sequentially purified by acetone extraction, n-hexane extraction, and acetonitrile extraction, followed by medium-pressure liquid chromatography and high-performance liquid chromatography performed on an octadecylsilane column. Their chemical structures were elucidated by proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and hydrolysis with α- and β-glucosidases. The glucosides were also detected in various pungent cultivars of C. annuum, Capsicum frutescens, and Capsicum chinense by liquid chromatography−mass spectrometry. However, they were not detected in nonpungent cultivars of C. annuum. Furthermore, a positive correlation was observed between the quantity of the capsaicinoids, capsaicin, and dihydrocapsaicin and their glucosides. Keywords: Capsicum; pepper; capsaicin; dihydrocapsaicin; glucoside

Adsorptive stripping voltammetry is proposed for determination of boron at low μg g−1 level in iron and steel. Beryllon III was used as a complexing agent for boron(III). Optimum conditions for the formation of the boron(III)–Beryllon III complex were as follows: 0.06 M nitrate–0.04 M sulfate supporting electrolyte at pH 4.5; 1.0×10−5 M Beryllon III; 24 h complexation time. The complex was accumulated on a hanging mercury drop electrode at −0.35 V vs. SCE for 60 s under stirring, and was subsequently cathodically stripped to −0.8 V vs. SCE at a scan rate of 10 mV s−1 using a differential pulse mode. Interference by iron(III) was eliminated by the complexation with metaphosphoric acid. After cathodic stripping, a single well-defined peak was observed at around −0.5 V vs. SCE. The influence of foreign elements on the determination was also evaluated. The calibration graph (peak height vs. boron(III) concentration) in the presence of 1 mg mL−1 iron(III) was linear over a concentration range of 1–25 ng mL−1 (correlation coefficient >0.999), with a relative standard deviation (n=4) of 2.6% at 10 ng mL−1. The minimum limit of determination was 1 μg g−1 of boron in iron and steel. The proposed method has been proved to be applicable to the determination of trace boron in iron and steel with good precision and accuracy.

The ethyl acetate extract of the whole aerial parts of Varthemia iphionoides (Compositae) showed a pronounced antibacterial activity. The bioassay-guided fractionation of the ethyl acetate extract and further purification of the most antibacterially active fraction led to the isolation and identification of an antibacterial sesquiterpene, selina-4,11(13)-dien-3-on-12-oic acid. This compound exhibited potent antimicrobial activity against six bacterial species (Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Bacillus cereus and Salmonella enteritides). The minimum inhibitory concentrations (MICs) of this compound which was determined by the agar dilution method ranged between 250 and 500 microg/ml.

The purpose of this study was to elucidate the influence of otitis media on blood flow in the lateral wall of the cochlea by means of a model of endotoxin-induced otitis media. The cochlear blood flow (CBF) following lipopolysaccharide inoculation into the middle ear cavities of rats was measured by laser-Doppler flowmetry and compared with that of untreated ears. After this evaluation, the influence on CBF of concomitant use of a nitric oxide synthase inhibitor was also investigated. The first day after inoculation, the CBF of treated ears decreased significantly. This decrease recovered gradually between the 7th and 14th days. With concomitant use of a nitric oxide synthase inhibitor, the decrease in CBF was prevented to some extent. The results showed a functional influence upon CBF by endotoxin-induced otitis media. The significance of prophylactic use of the drug is also discussed in regard to the effect on CBF following otitis media.

通常の大気圧アルゴン誘導結合プラズマ質量分析法（ICP-MS）では，アルゴンや大気成分に起因したバックグラウンドスペクトルによる定量妨害，及びイオン化電圧の高い元素の感度不足がしばしば問題となる．本研究では，これらの点を解決するため，減圧ヘリウムICP-MSを基礎及び応用の両方面から研究した．すなわち，通常のプラズマトーチを水冷式に変え，インターフェース部を銅からアルミニウム製に変え，整合回路を調節し，装置を最適化した．また，キャピラリーインターフェースを備えた超音波ネブライザーシステムを開発し，溶液試料を減圧下のプラズマに安定に導入できるようにした．更に，電熱気化法，水素化物発生法及びレーザーアブレーションによる試料導入法も検討した．本法を水，鉄鋼，頭髪試料などの分析に応用し，その有用性を実証した．

Three new brasiliamide congeners, brasiliamides C, D and E, were isolated from okara fermented with Penicillium brasilianum Batista JV-379. Their structures were elucidated on the basis of spectral data and chemical evidence. NMR spectra of these brasiliamides exhibited a mixture of four or two conformers due to the restricted rotation of an amide bond in a solution. The (1)H- and (13)C-NMR spectral data were analyzed for a major rotamer at an appropriate temperature, since the signals were broadened at room temperature. Both brasiliamides C and D showed convulsive activity against silkworms with an ED(50) value of 400 microg/g of diet, whereas brasiliamide E showed less activity than the others.

We studied the damage to the cochlear lateral wall induced by otitis media and the therapeutic effects of intratympanic administration of steroid and nitric oxide (NO) synthase inhibitor. In Sprague-Dawley rats, right middle ear cavities were inoculated with lipopolysaccharide, followed after 30 min by intratympanic administration of dexamethasone, NOS-inhibitor or PBS. Twenty-four hours after lipopolysaccharide inoculation, cochlear blood flow was measured by laser-Doppler flowmetry. Prostaglandin E(1) was topically applied to the round window membrane of the right ear and changes in cochlear blood flow were calculated. Changes of cochlear blood flow were significantly different among the three groups. Increases in cochlear blood flow following PGE(1) application were higher in the group that received NOS-inhibitor. Electron microscopic examination revealed that changes in the stria vascularis were less severe in rats treated with dexamethasone or NOS-inhibitor. Our results show the effectiveness of intratympanic dexamethasone or NOS-inhibitor in treating cochlear lateral wall damage caused by otitis media.

A low-pressure ICP formed with helium has been utilized as an ion source for MS, which is capable of providing information about traces of iron, arsenic and selenium without any interference from ArO+, ArCl+ and Ar2+, respectively. The present paper describes the application of a novel ICP for the determination of fluorine, chlorine, bromine and iodine, because the ionization of these halogens is highly suppressed in the case of conventional argon ICP (e.g., 9×10−4% for fluorine). A 5 μl aliquot of an aqueous sample was placed on a looped tungsten filament and heated electrothermally at ca. 2500°C . The resulting sample vapor was transported to the ICP with a stream of helium. The analytes (19F+, 35Cl+, 79Br+ and 127I+) were then determined by a quadrupole mass analyzer. The detection limits (ng ml−1, based on 3σ) were 23 (F), 2.4 (Cl), 0.13 (Br) and 0.05 (I), which were much better than those of argon ICP. The absolute detection limits using 5 μl of a sample reached to the sub-picogram range. Especially, the detectability for fluorine was significantly improved, which allowed a direct determination of fluorine at the ng ml−1 level in water.

Two cyclic peptides, PF1171F ( 1 ) and PF1171G ( 2 ), were isolated from okara fermented with the unidentified ascomycete OK-128, and their structures were determined by NMR, MS, and Marfey amino acid analysis. Both peptides showed paralytic activity against silkworms.

Three new dimeric naphthopyrones, asperpyrones A (1), B (2), and C (3), together with two known compounds, fonsecinone A (4) and aurasperone A (5), have been isolated from okara that was fermented with Aspergillus niger JV-33-48. Compounds 1, 4, and 5 showed inhibitory activity on Taq DNA polymerase.

A high-speed digital signal processing system has been developed as an individual particle analyzer combined with ICP-MS to achieve signal profiling with a time resolution of 20 µs and a lower detection limit. When air samples are directly introduced into the plasma, the ICP-MS generates a train of pulses corresponding to the ion population of a given element in the plasma. The present system can digitally count pulses from a channel electron multiplier used as a detector in ICP-MS in a duration time of 20 µs each. A time-resolved signal profile stored in the computer memory shows that each flash signal derived from individual airborne particles has a sharp peak with a time-width of 0.1–0.5 ms. The detection limit for zinc in a particle is lowered to sub-fg levels by precisely measuring the signal strength for each particle under a condition of low background noise. The profile of the flash signal for individual particles reflects the evaporation and ionization processes of each particle in the plasma and the extraction process of ions through the sampling orifice of the mass spectrometer.

The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens .

Summary A Gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI-2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell-free culture supernatant from wild-type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI-2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha-, kappa- and theta-toxins. In the luxS mutant, the transcription of the theta-toxin gene (pfoA) was lower at mid-exponential growth phase, whereas alpha- and kappa-toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild-type cells, possibly because of the presence of AI-2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild-type C. perfringens, Escherichia coli DH5α carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell–cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell–cell signalling by AI-2 plays an important role in the regulation of toxin production in C. perfringens.

Melon (Cucumis melo) roots were inoculated with or without the arbuscular mycorrhizal (AM) fungus Glomus caledonium under low phosphate conditions. High-performance liquid chromatography analysis of the secondary metabolites in butanol extracts from roots revealed that the level of one compound in noninoculated roots showed a significant increase from 30 days postinoculation. No accumulation was observed in mycorrhizal roots and high-phosphate-supplemented roots, indicating that the accumulation of the compound was caused by a phosphate deficiency. The compound was isolated by column chromatography and identified by spectroscopic methods to be a C-glycosylflavone, isovitexin 2″-O-β-glucoside. The effect of the compound on mycorrhizal colonization in melon roots was examined under low (0.05 mM) and high (2 mM) phosphate conditions. The degree of mycorrhizal colonization in control roots grown under high phosphate conditions (8.8%) was much lower than when grown under low phosphate conditions (22%). The treatment of roots with the compound at concentrations of 20 and 50 μM increased root colonization under both low and high phosphate conditions. In particular, the degrees of mycorrhizal colonization in treated roots grown under high phosphate conditions (25 and 22% at 20 and 50 μM, respectively) were comparable to that in untreated control roots grown under low phosphate conditions (22%). These findings suggest that the phosphate deficiency-induced C-glycosylflavonoid is involved in the regulation of AM fungal colonization in melon roots.

A novel gene that regulates the alpha-toxin (plc), kappa-toxin (colA), and theta;-toxin (pfoA) genes was identified using toxin-negative mutant strains of Clostridium perfringens. The cloned 3.2-kb fragment contained the virX gene encoding a 51-amino acid polypeptide of unknown function that seemed to be responsible for the activation of toxin genes. The virX knock out mutant of wild-type strain 13 showed a reduced expression of the plc, colA, and pfoA genes, which was complemented by the transformation of the intact virX gene. Deletion and site-directed mutagenesis studies suggested that the virX gene acts as a regulatory RNA rather than as a peptide regulator. The virX locus found in this study might play a part in the signal transduction to regulate toxin production in C. perfringens.

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that causes life-threatening gas gangrene and mild enterotoxaemia in humans, although it colonizes as normal intestinal flora of humans and animals. The organism is known to produce a variety of toxins and enzymes that are responsible for the severe myonecrotic lesions. Here we report the complete 3,031,430-bp sequence of C. perfringens strain 13 that comprises 2,660 protein coding regions and 10 rRNA genes, showing pronounced low overall G + C content (28.6%). The genome contains typical anaerobic fermentation enzymes leading to gas production but no enzymes for the tricarboxylic acid cycle or respiratory chain. Various saccharolytic enzymes were found, but many enzymes for amino acid biosynthesis were lacking in the genome. Twenty genes were newly identified as putative virulence factors of C. perfringens , and we found a total of five hyaluronidase genes that will also contribute to virulence. The genome analysis also proved an efficient method for finding four members of the two-component VirR/VirS regulon that coordinately regulates the pathogenicity of C. perfringens . Clearly, C. perfringens obtains various essential materials from the host by producing several degradative enzymes and toxins, resulting in massive destruction of the host tissues.

減圧ヘリウムICP-MSは, アルゴンに起因したスペクトル干渉を除くことができる. しかし, m/z=79～82に小さなバックグラウンドピークがなお存在し, 微量な臭素及びセレンの定量を妨害した. 本研究では, これらのピークがインターフェース部材質の銅に起因した分子イオンであることを突き止めた. これらのイオンを抑制するため, ニッケル及びアルミニウム製インターフェース部を試作した. その結果, ニッケル製では, 広い質量範囲にわたりバックグラウンドピークが生じたが, アルミニウム製ではほとんど観測されなかった. そのため, 減圧ヘリウムICP-MSのインターフェース部材質としてアルミニウムが最適であるとの結論を得た. 電熱気化法により臭素及びセレンの溶液試料 (10ng ml-1, 5μl) を導入して得られた相対標準偏差 (n=10) は, いずれも約10%であった. また, 臭素及びセレンの検出下限 (3σ) はそれぞれ0.2及び0.09ng ml-1であり, 通常のICP-MS (Br 20ng ml-1, Se 0.25ng ml-1) に比べ高感度な定量が可能になった.

電熱気化 (ETV)/ICP-MSにより, 頭髪試料1本相当 (0.5mg) を用いてAs及びSeの定量を試みた. 頭髪試料に14mol l-1硝酸10μl及び30%過酸化水素水2μlを添加して分解後, 1μmol l-1過マンガン酸カリウム溶液25μlを添加して蒸発乾固し, 残留物を1mol l-1硝酸500μlに溶解した. この溶液の5μlを分取し, ETV用のタングステンフィラメントに載せ, プラズマトーチ内にセットした. タングステンフィラメントに電流を流して試料溶液を乾燥させた後, コンデンサーの放電を利用してフィラメントを約2500℃に加熱し, 残留物を気化させた. 試料蒸気をキャリヤーガスによりICPに導入し, 75As+及び82Se+の信号強度を測定した. 試料の前処理においては, 過マンガン酸カリウムを添加することにより, 蒸発乾固の際の目的元素の損失 (30～50%) を防ぐことができた. これは, 過マンガン酸カリウムの酸化作用とともに蒸発乾固後の残留物の器壁への強い吸着が抑制されたためと考えられる. 本法により頭髪標準試料中のAs (0.31±0.02μg g-1) 及びSe (2.0±0.08μg g-1) を定量したところ, 分析結果 (n=4) はAs0.30±0.01μg g-1及びSe2.0±0.2μg g-1であり, 認証値とよく一致した.

Cucumber (Cucumis sativus L.) roots were analyzed by HPLC and TLC for their levels of secondary metabolites upon inoculation with the arbuscular mycorrhizal fungus, Glomus caledonium. Three compounds in EtOAc extracts from the mycorrhizal roots showed significant increases six weeks after inoculation. These compounds were isolated by column chromatography and determined to be two novel triterpenes, 2beta-hydroxybryonolic acid (2beta,3beta-dihydroxy-D:C-friedoolean-8-en-29-oic acid) and 3beta-bryoferulic acid [3beta-O-trans-ferulyl-D:C-friedooleana-7,9(11)-diene-29-oic acid], and the known triterpene, bryonolic acid, by spectroscopic methods. Time-course experiments showed that the levels of the three terpenoids in cucumber roots were significantly increased by the application of a 53-microm sieving from a soil inoculum of the arbuscular mycorrhizal fungus containing soil microbes but no mycorrhizal fungi, and that mycorrhizal colonization further promoted the terpenoid accumulation. Inoculation with Glomus mosseae also enhanced the accumulation of the triterpenes, whereas no accumulation was observed by inoculating with the fungal pathogen, Fusarium oxysporum f. sp. cucumerinum. 2Beta-hydroxybryonolic acid was also isolated from the roots of melon and watermelon.

New convulsive compounds, brasiliamides A (1) and B (2), were isolated by activity-guided fractionation from okara fermented with a soil isolate of Penicillium brasilianum Batista JV-379. Their structures were elucidated on the basis of spectral and chemical evidence and by X-ray crystallography of the hydrogenated product of 2. In the 1H- and 13C-NMR spectra of 2, the signals were complicated, all being doubled or broadened in several deuterated solvents at room temperature. The conformational change of 2 was clarified as the rotational isomerization of amide bonds in solution by NMR measurements at various temperatures. Four rotamers of 2 at two amide bonds were presented at -60 degrees C in CDCl3, whereas only two isomers were apparent at room temperature, owing to rapid rotation of one of the amide bonds. Brasiliamides A and B respectively showed convulsive activity against silkworms with ED50 values of 300 and 50 microg/g of diet.

A new synthetic analog of bufadienolide, methyl isobryophyllinate A (1), and a known synthetic analog, methyl isobersaldegenate-1,3,5-orthoacetate (2), were obtained by methanolysis of bryophyllin A (3) and bersaldegenin-1,3,5-orthoacetate (5) in basic solution. Structure-insecticidal activity relationship studies revealed both orthoacetate and α-pyrone moieties seemed to be essential structural elements for exhibiting insecticidal activity, whereas oxygenated substituents in the C ring enhanced the insecticidal activity against the third instar larvae of silkworm (Bombyx mori).

In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) γ-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 μM on human α1β2γ2 GABACl and α1β glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.

Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.