Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Nitrous oxide evolved from soil covered with plastic mulch film in horticultural field

Soil solarization practice, in which soil is covered with plastic mulch film and exposed to high temperature prior to crop cultivation, is expected to be an effective method for reducing weeds and pathogenic microorganisms without using agricultural chemicals. Although the production of nitrous oxide (N₂O), a major greenhouse gas, is enhanced in fertilized soil covered with plastic mulch films, its transport route to the atmosphere has not been sufficiently elucidated to date. In this study, we investigated the N₂O evolution from plastic-mulch-film-covered agricultural soil. In a horticultural field where ridge soil was covered with a plastic mulch film after fertilization, we observed significant N₂O flux from the soil surface of the unfertilized furrow between the ridges, indicating the horizontal diffusion of N₂O from the ridge soil covered with the mulch film to the adjacent furrow soil surface. On the other hand, the measurement of the permeance (permeation coefficient) of the plastic mulch film for gaseous N₂O by laboratory experiment revealed that N₂O gradually permeated the mulch film; the permeance increased exponentially with an increase in ambient temperature, indicating possible N₂O emission by permeation through the mulch film under field conditions. In winter, the amount of N₂O emission by permeation through the mulch film was estimated to be lower than that emitted from the furrow soil surface, and it was lower than that in summer. On the other hand, it was estimated to be much higher in summer owing to the higher permeance of the film at high temperatures.     

Insecticidal and neuroblocking potencies of variants of the imidazolidine moiety of imidacloprid-related neonicotinoids and the relationship to partition coefficient and charge density on the pharmacophore

The pharmacophore of the neonicotinoid insecticide imidacloprid, nitroiminoimidazolidine, was modified to heterocycles such as thiazolidine, pyrrolidine, dihydroimidazole, dihydrothiazole, and pyridone conjugated to nitroimine (=NNO2) or nitromethylene (=CHNO2). Their 6-chloro-3-pyridylmethyl or 5-chloro-3-thiazolylmethyl derivatives were examined for insecticidal activity against the American cockroach by injection and for neuroblocking activity using the cockroach ganglion. Most of the compounds having the neonicotinoidal pharmacophore exhibited insecticidal activity at the nanomolar level, which was enhanced in the presence of synergists, and high neuroblocking activity at the micromolar level. Quantitative analysis for the compounds showed that the neuroblocking potency is proportional both to the Mulliken charge on the nitro oxygen atom and to the partition coefficient log P value. The equation for the insecticidal versus neuroblocking potencies indicated that both potencies are related proportionally with each other when the other factors are the same.     

Identification of a Germicidal Compound against Picornavirus in Bamboo Pyroligneous Acid

The germicidal activity of pyroligneous acid (PA) against a picornavirus, encephalomyocarditis virus (EMCV), was analyzed, and the component responsible for its disinfectant activity was identified. Bamboo PA (BPA) inactivated EMCV, but neutralization of BPA abolished this activity. Using liquid-liquid phase extraction and silica gel column chromatography, the hydrophobic active fraction of BPA was separated and its 12 major components were identified. The active fraction was reconstructed by mixing synthetic chemicals at the determined concentrations, and a subtraction series of one chemical from the complete mixture was prepared. An in vitro virus assay demonstrated that phenol was the sole germicidal component, and acetic acid augmented the phenol's inactivating activity resulting in >5-log decrease in EMCV infectivity. Considering the low environmental risk of PA, these findings suggest that BPA is a potentially useful agent for preventing viral epidemics in agricultural and human environments.     

Interdigital involvement in a case of primary cutaneous canine histoplasmosis in Japan

A 5-year-old male Siberian husky bred outdoor in Tokyo had a swollen paw with interdigital granulomatous lesions in the left hindlimb. The dog had no apparent pulmonary or gastrointestinal involvement. Histopathological analysis of the skin lesions demonstrated yeast-like organisms predominantly within macrophages. Sequence analysis of fungal ribosome RNA gene isolated from a paraffin sample revealed a 100% homology with the teleomorph of Histoplasma capsulatum. The present case may support the concept of primary cutaneous canine histoplasmosis as an endemic phenotype recognized in Japan.     

Angiotensin I-converting enzyme-inhibitory peptides obtained from chicken collagen hydrolysate

In this study, collagen extracted from chicken legs (which are the yellow keratin parts containing a nail) was hydrolyzed with various enzymes, and the angiotensin I-converting enzyme (ACE)-inhibitory activity of each hydrolysate was determined. The hydrolysate by treatment with an Aspergillus species-derived enzyme had the highest activity (IC 50 = 260 microg/mL). The fraction of this hydrolysate obtained by ultrafiltration with a molecular-weight cutoff of 3000 Da (low fraction) had a stronger activity (IC 50 = 130 microg/mL) than the fractionated one. This fraction was further fractionated by HPLC, and the peptides in the fraction with high ACE-inhibitory activity were identified. The amino acid sequences of the four peptides were identified using a protein sequencer. These peptides were synthesized to confirm their ACE-inhibitory activities; this showed that peptides with a Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro sequence had the highest activity (IC 50 = 29 microM). When the low fraction was administered to spontaneous hypertensive rats, a decrease in their blood pressure was observed after 2 h of administration, and a significant decrease in blood pressure (-50 mmHg) was observed after 6 h. Moreover, long-term administration studies indicated that the low fraction showed a significant suppression of increased blood pressure.     

Effect of pore conformation on dielectric anisotropy of oven-dry wood evaluated using terahertz time-domain spectroscopy and eigenvalue problems for two-dimensional photonic crystals

To clarify the effect of pore conformation on the dielectric anisotropy of wood, the relative permittivity along the longitudinal and tangential axes of flat-sawn oven-dry specimens of 12 different wood species was measured using terahertz time-domain transmission spectroscopy and compared with the values calculated using the eigenvalue problem for two-dimensional photonic crystals. The measured dielectric anisotropy, which is the ratio of the relative permittivity along longitudinal axis to that along the tangential axis, was well explained by the calculated value. It was concluded that the ratio of tangential to radial widths of wood pores affects the relative permittivity along the tangential axis, and that the dielectric anisotropy decreased with an increase in the ratio. This discussion can also be applied to the relative permittivity in frequencies below 0.15 THz. These findings show promise as a new method for evaluating the porous structure of wood.     

Structural Effects of N-Arylcarbamoylpyrazolines on Calcium Uptake in Rat Brain Synaptosomes

N-Arylcarbamoylpyrazolines with various substituents at the para position of the carbamoyl benzene ring inhibited ATP-dependent Ca²⁺-uptake in synaptosomes prepared from the rat brain. The activity of these compounds was evaluated as log(1/I₅₀), the reciprocal logarithm of half inhibitory concentration, I₅₀ (m ), from the concentration–response curve for the inhibition of Ca²⁺-uptake. Among the compounds tested, methyl 3-(4-chlorophenyl)-4-methyl-1-[N-(4-trifluoromethylphenyl)carbamoyl]-2-pyrazoline-4-carboxylate was the most potent, the I₅₀ value of which as 9·12×10⁻⁷ m . Variations in the activity in terms of log(1/I₅₀) were quantitatively analysed using a substituent parameter, showing that the higher the electron-withdrawing effect of the substituent, the higher was the activity. The substituent effects were similar to those on insecticidal activity against the Americal cockroach. The higher the inhibitory activity against Ca²⁺ uptake, the higher seemed to be the insecticidal activity. Methyl(4S) - 3 - (4 - chlorophenyl) - 4 - methyl - 1 - [N - (4 - chlorophenyl)carbamoyl] - 2 - pyrazoline -4-carboxylate had higher inhibitory activity against Ca²⁺-uptake and higher in-secticidal activity than the R-isomer, but the difference was greater in theCa²⁺-uptake system.     

Differentiation-inducing effects of small fruit juices on HL-60 leukemic cells

Epidemiological studies indicate that high intakes of fruits and vegetables are associated with a reduced risk of cancer, and several plant-derived drugs have been developed in medical oncology. Since only a small part of the flora has been tested for any kind of bioactivity, we chose small fruits as sources of differentiation-inducing activity against HL-60 leukemic cells. We have prepared juices from various small fruits that grow mainly in the northern part of Japan. Screening of 43 samples indicated that juices of Actinidia polygama Maxim., Rosa rugosa Thunb., Vaccinium smallii A. Gray, and Sorbus sambucifolia Roem. strongly induced differentiation of HL-60 cells to monocyte/macrophage characteristics in a concentration-dependent manner as indicated by histochemical and biochemical examinations.     

Determination of theaflavins including methylated theaflavins in black tea leaves by solid-phase extraction and HPLC analysis

A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.