Inhibition of Acid Phosphatase Isoforms Purified from Mature Soybean (Glyczne MAX) Seeds

The four soybean seed acid phosphatase isoforms AP1, AP2, AP3A and AP3B were competitively inhibited by phosphate, vanadate, fluoride and molybdate, using p-nitrophenylphos-phate as substrate. The four isoforms were not significantly affected by compounds that can interact with SH residues or by pyridoxal phosphate. These results indicated that cysteine and lysine residues are not present in the active site of the four soybean seed acid phosphatase isoforms. The inhibition constant values for phosphate, vanadate, fluoride and molybdate at pH 5.0 were respectively: API (250, 12.8, 1.7, 0.05 μM), AP2 (800,10, 500, 0.025 μM), AP3A (250, 24.2,250, 0.032 μM), AP3B (2400, 36.9,750, 0.05 μM).     

Release of juvenile nematodes at hibernation sites by overwintered queens of the hornet Vespa simillima

The nematode Sphaerularia vespae only parasitizes hornet queens and deprives them of fertility. To elucidate its transmission route, we observed the behavior of overwintered queens of Vespa simillima found around decayed logs of fallen trees—the principal hibernation sites for this species. We found that overwintered queens frequently visited those decayed logs in summer (late June to mid-August), hovering or walking on the surfaces of the logs, and sometimes entering holes or cracks there. These queens, unlike those visiting the hibernation sites in the fall, did not excavate wood to make their hibernacula, but often released juvenile nematodes there. In 25 % of the entries observed, we confirmed that juvenile nematodes had been released from the tips of the hornet’s gasters and thus transferred to the decayed logs. The timing of the host’s initial visit to decayed logs for nematode transmission corresponded well with the hatching of juveniles within the host’s body. These results suggest that the parasitic nematode manipulates its host to visit decayed logs in summer for its own transmission.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Volatile Flavor Components of Dried Bonito (Katsuobushi) II. From Neutral Fraction

The aqueous extract of dried bonito (Katsuobushi) was distilled under reduced pressure. The resulting distillate with diethyl ether and the extract was separated into acidic, phenolic, basic and neutral fractions. The neutral fraction was further fractionated into ten sub-fractions by silica gel column chromatography. All these sub-fractions were analyzed by gas chromatography and gas chromatography-mass spectrometry.

One hundred and sixty-five compounds were identified and 12 compounds were tentatively identified from the neutral fraction. Among them, 111 compounds were newly identified as flavor components of Katsuobushi.     

Cold Shock of Germinating Bacillus subtilis Spores

Susceptibility of germinating spores of Bacillus subtilis to a rapid chilling was examined by viable countings. Dormant spores were quite resistant to the cold shock but the spores, immediately upon germination, lose viability almost completely by the same treatment. The presence of divalent cation, magnesium, calcium or manganese, in a buffer to which the germinating spores were suspended, markedly protected the cells from the death by the cold shock effect. When the shocked cells were incubated in the buffer containing casein acid hydrolyzate, glucose and magnesium ion for short period of time, a remarkable increase in viable counts was observed. The existence of two critical temperature zones, which were determined by the initial temperature of cell suspension, was confirmed in the cold shock of germinating spores.     

Transformation of Terpenoids in Grape Must by Botrytis cinerea

Experiments were done to investigate the volatile components in botrytized grape must and transformation of terpenoids in terpene-supplemented grape must by Botrytis cinerea. Twenty-eight compounds were identified in the volatile concentrate of botrytized must with a combined gas chromatograph-mass spectrometer. No terpenoids were detected in the concentrate. Linalool or terpinen-4-ol decreased a lot when Botrytis cinerea was cultured in the must with these terpenes for 15 days. In linalool-supplemented botrytized must 9 identified and 3 unidentified terpenes were found, while only geranial was detected in terpinen-4-ol-supplemented botrytized must. Botrytis cinerea did not produce terpenoid in grape must without terpenes, but transformed linalool added to grape must into some other monoterpenes.     

Isolation of Intact Chloroplasts from Spinach Leaf by Centrifugation in Gradients of the Modified Silica “Percoll”

The isolation of the photosynthetically competent chloroplast preparations was undertaken by means of the density gradient centrifugation on the modified silica sol “Percoll.” A clear separation of the intact chloroplast sustaining the high photosynthetic activities (light dependent CO₂ fixation ca. 130μmol/mg Chl·hr) was established. The contamination of mitochondria and peroxisomes was estimated to be less than 3% by measuring the activities of their marker enzymes. The chloroplasts were proved to be free from endoplasmic reticulum and cytosol. The photosynthetic CO₂ fixation of the isolated chloroplast preparations was saturated by illumination of the light intensity of 20,000 Lux (12 mW/cm², 400~750 nm).     

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

Development of preparative and analytical methods of the hop bitter acid oxide fraction and chemical properties of its components

The bitter acids in hops (Humulus lupulus L.) and beer, such as α-, β-, and iso-α-acids, are known to affect beer quality and display various physiological effects. However, these compounds readily oxidize, and the effect of the oxides on the properties of beer or their potential health benefits are not well understood. In this study, we developed a simple preparative method for the bitter acid oxide fraction derived from hops and designated the constituents as matured hop bitter acids (MHBA). HPLC-PDA-ESI/HRMS and MS² revealed that MHBA are primarily composed of α-acid-derived oxides, which possess a common β-tricarbonyl moiety in their structures similar to α-, β-, and iso-α-acids. We also developed a quantitative analytical method of whole MHBA by HPLC, which showed high precision and reproducibility. Using our newly developed method, the concentration of whole MHBA in several commercial beers was evaluated. Our results will promote the study of bitter acid oxides.