Enzymatic dehydrogenative polymerization of urushiols in fresh exudates from the lacquer tree, Rhus vernicifera DC

Fresh exudates from the lacquer tree, Rhus vernicifera DC, were extracted with acetone and the solution was chromatographed to isolate monomer, dimer, trimer, and oligomer fractions of urushiols. Constituents of the monomeric and dimeric fractions were then identified by two-dimensional (2D) 1H-13C heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC) NMR spectroscopic techniques. The results showed that the monomeric fraction contained 3-[8'Z,11'E,13'Z-pentadecatrienyl]catechol (1), 3-[8'Z,11'Z,14'-pentadecatrienyl]catechol (2), and 3-pentadecanyl]catechol (3), which was verified by HPLC analysis. The dimeric fraction contained 8'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[9'E,11'E,13'Z-pentadecatrienyl]catechol (4), 14'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecatrienyl]catechol (5), 2-hydroxyl-3- or -6-alkenylphenyl ethyl ether (6), 14'-(3' ',4' '-dihydroxy-2' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadeca-trienyl]catechol (7), 15'-(2' '-hydroxy-3' '- or -6' '-alkenyl)phenyloxy-3-[8'Z,11'Z,13'E)-pentadecatrienyl]catechol (8), 14'-(2' ',3' '-dihydroxy-4' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecantrienyl]catechol (9), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,3'-biphenyl (10), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,4'-biphenyl (11), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-5,4'-biphenyl (12), and 1,2,1'-trihydroxy-6,6'-dialkenyldibenzofuran (13) as constituents. In addition, dimeric ethers and peroxides, such as compounds 14 and 15, were produced by autoxidation of monomeric urushiols in atmospheric air. The possible reaction mechanisms for the dehydrogenative polymerization of urushiols by Rhus laccase present in the fresh raw exudates under the atmospheric oxygen are discussed on the basis of structures identified. This is of primary importance because the use of the urushi exudates as coating materials does not involve organic solvents and is an environmentally friendly process.     

Analysis of methoxyfenozide residues in fruits, vegetables, and mint by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Methoxyfenozide [3-methoxy-2-methylbenzoic acid 2-(3,5-dimethylbenzoyl)-2-(1,1-dimethylethyl) hydrazide; RH-2485], in the formulation of INTREPID, was applied to various crops. Analysis of methoxyfenozide was accomplished by utilizing liquid-liquid extraction and partitioning, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method validations for fruits, vegetables, and mint are reported. Methoxyfenozide mean recoveries ranged from 72 to 129% over three levels of fortification. The overall average of mean recoveries is 97 +/- 10%. The limit of quantitation for fruits, artichoke, cucumber, squash, and refined sugar was 0.010 ppm, with a detection limit of 0.005 ppm. For all other crops, the limit of quantitation was 0.050 ppm, with a detection limit of 0.025 ppm. No residues were found greater than the limit of quantitation in control samples. Residues above the limit of quantitation were found in all matrices except refined sugar. Foliage (bean, beet, pea, and radish) had greater residue levels of methoxyfenozide residue than their corresponding roots or pods. Other crop matrices contained <1.0 ppm of methoxyfenozide except artichoke, which had a mean of 1.10 ppm.     

Determination of antioxidant potential of volatile extracts isolated from various herbs and spices

Antioxidant activities of volatile extracts isolated from thyme, basil, rosemary, chamomile, lavender, and cinnamon were evaluated by two independent assays: the aldehyde/carboxylic acid assay and the conjugated diene assay. The volatile extracts were prepared from dried herbs and spices using liquid-liquid continuous extraction following steam distillation under reduced pressure (55 degrees C and 95 mmHg). The antioxidant activities of the extracts decreased in the following order in both of the lipophilic assay systems: thyme > basil > rosemary > chamomile > lavender and cinnamon. Thyme and basil extracts inhibited the oxidation of hexanal for 40 days at the levels of 10 microg/mL and 50 microg/mL, respectively. The extracts of thyme and basil were effective in retarding methyl linoleate deterioration at 40 degrees C, with activity increasing with concentration in the range 10-200 microg/mL. At a concentration of 50 microg/mL, thyme extract was similar in antioxidant activity to BHT and alpha-tocopherol in the conjugated diene assay. The antioxidant potentials of the volatile extracts used in this study were accurately measured by the lipophilic systems, such as the aldehyde/carboxylic acid assay and the conjugated diene assay.     

Application of ELISA-based kit for detecting AOZ and determining its clearance in eel tissues

Furazolidone, an antibacterial drug that was once widely used in the livestock industry and aquaculture, is now prohibited in numerous countries. It is difficult to detect residual furazolidone because it is readily metabolized in animal tissues but, by using and liquid chromatography coupled with tandem mass spectrometry, its metabolite, 3-amino-2-oxazolidinone (AOZ) can be detected. Here we describe the validity of an enzyme-linked immunosorbent assay (ELISA) kit to detect AOZ in Japanese eel Anguilla japonica tissue. ELISA is capable of detecting AOZ at 1.0 μg/kg in an eel sample with excellent accuracy and precision. Our results show that ELISA is suitable for regulatory purposes and for studying the fate of AOZ residues in eel treated with furazolidone. To measure the persistence of AOZ in eel tissues, eels (1.4–6.5g) were immersed in tanks containing 2 and 10 mg furazolidone/L for 3 h, and then maintained in a tank supplying well water for the next 160 days. The half-lives of AOZ, calculated from the linear terminal part of the excretion curve, were 25.0 days in muscle and 21.6 days in liver from fish exposed to 2 mg/L furazolidone. In the eels treated with 10 mg/L furazolidone, by contrast, high levels of AOZ were detected in liver and muscle, but the half-lives of AOZ were similar to those in fish treated with 2 mg/L furazolidone. The half-lives of AOZ in eel tissues were prolonged by the condition of low water temperature.     

Characterization of the perylenequinone pigments in Japanese Andosols and Cambisol

The green fraction of humic acid (Pg) and the chloroform-extractable green fraction (CEGF) are characteristic soil organic matter (SOM) components. These alkaline solutions are green-colored due to the presence of 4,9-dihydroxyperylene-3,10-quinone (DHPQ) chromophore. While both of which are potential indicators for the effect of land use and paleoclimatic environment in the fields of soil science as well as geochemistry, CEGF as well as its relationship with Pg in soils are not yet fully documented. In this study, we firstly investigated the chemical properties of soil CEGF fractions by ultraviolet–visible (UV–Vis) and infrared (IR) method. Two CEGF components were separated by sequential liquid-liquid extraction using aqueous ammonium hydroxide (NH₄OH) followed by aqueous sodium hydroxide (NaOH). Results showed that the UV–Vis spectral shape of NH₄OH-extractable component is very similar to that of DHPQ, except that it is red-shifted. The solubility and UV–Vis spectrum of the NaOH-extractable fraction were completely identical with those of synthesized DHPQ. Their IR spectral shapes were also almost the same. Subsequently, the distribution of CEGF in humic acid (HA), fulvic acid (FA) and humin (HN) from Japanese Andosols and Cambisol was quantitatively evaluated by sequential extraction. Most of CEGF was detected in the HA (60–78%) and HN (22–40%), but not in the FA. While the UV–Vis spectral shape of CEGF extracted from Andosols HAs showed a relatively higher proportion of DHPQ than its derivative, the opposite was observed in Cambisol HA, whose CEGF is similar to that of sclerotium grain (one of the possible origin of CEGF). These results suggest the diversity of CEGF-producing soil fungi. Quantitative data also indicated that 35–49% of Pg consisted of a chloroform-soluble fraction (i.e., CEGF) and the remaining 51–65% of Pg was chloroform-insoluble. Based on these results, we propose that CEGF is composed of DHPQ and DHPQ-derivatives and that CEGF is one of the major fractions of Pg.     

Model-based forecasting of twig canker incidence of bacterial spot of peach in Fukushima Prefecture

Bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is the most important disease that affects peach production. A disease-forecasting model was developed to help growers decide when to apply bactericides and remove diseased, last-year twigs. To predict the incidence of “spring cankers”, peach twigs damaged by Xap, we used 12 years (2009–2020) of data from Fukushima Prefecture to develop a forecasting system using a hierarchical Bayesian model (HBM). The model included the number of fields with a bacterial spot incidence (BSI) on leaves?≥?10% in late September of the previous season and the number of days with rain (≥?10 mm/day) and maximum wind speed (≥?5 m/s) during the previous October as predictors. Using a best-fit cutoff value based on a receiver operating characteristic (ROC) curve, the model achieved a 0.836 accuracy, 0.804 sensitivity, 0.847 specificity, 0.847 precision, and 0.712 F-measure. The model was validated using a fourfold cross-validation (CV) procedure and achieved an average accuracy of 0.847. Thus, the model explained 65.7% of the variability compared to observed frequencies with predicted probabilities of twig canker incidence (TCI)?≥?2% from April to May 2009 to 2020 in Fukushima Prefecture. These results suggest that this disease-forecasting model using HBM based on 12 years of historical data can be used to predict the risk of twig cankers of bacterial spot of peach.

     

Production of a desirable Brassica oleracea CMS line using an alloplasmic B. rapa CMS line carrying Diplotaxis erucoides cytoplasm as a bridge plant

In Brassica oleracea, production of F₁ hybrid seeds mainly makes use of the improved Ogura cytoplasmic male sterile (CMS) line. However, reliance on one particular line is a risk, and it would be advantageous to develop other CMS lines. In this study, we transferred Diplotaxis erucoides cytoplasm to B. oleracea cultivars using an alloplasmic B. rapaCMS line as a bridge plant to avoid incompatibility between donor and recipient plants. The new B. oleraceaCMS lines, which were derived by four generations of backcrossing, had small rudimentary anthers with no pollen grain and showed complete male sterility. There was no functional defect in other floral organs, and the ability to receive normal pollen did not appear to be impaired. Moreover, the B. oleraceaCMS lines carrying D. erucoides cytoplasm had larger leaf areas and a normal plastochron. As a consequence, the B. oleraceaCMS lines carrying D. erucoides cytoplasm have the potential to be valuable alternatives for use in commercial B. oleracea hybrid seed production.     

Yield estimation of 1-year-old asparagus grown using rootstock-planting forcing culture

Rootstock-planting forcing culture was developed in asparagus to harvest spears even during the seasons when the plants become dormant, but the demand for them high. In this study, cumulative hours during which the air temperature remained lower than 5°C, i.e. chilling hours (CHs), were calculated to determine dormancy breakage for asparagus cultures. We also measured CIELab colour values for cut stems immediately before rootstock digging, and determined whether they could be substituted and/or compensated for CHs while evaluating asparagus plant productivity in different low-temperature backgrounds, and obtained regression equations for yield estimation. Asparagus seedlings were cultivated in seven different regions across Japan and brought to the study site for harvesting. Our regression equation based on CHs and rootstock weight for yield estimation had relatively high fitness (adjusted R² = 0.5795). The colour values of cut stalks at rootstock digging can also be used to evaluate their productivity. These values can be useful in regions where CHs cannot be determined, although their effectiveness was slightly lower than that of CHs of areas adjacent to the study sites.     

Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by Maillard reaction

Typical heterocyclic compounds substituted with various functional groups found in Maillard reaction products were examined for antioxidant activity. Pyrroles exhibited the greatest antioxidant activity among all heterocyclic compounds tested. All pyrroles inhibited hexanal oxidation by almost 100% at a concentration of 50 microg/mL over 40 days. Addition of formyl and acetyl groups to a pyrrole ring enhanced antioxidative activity remarkably. Pyrrole-2-carboxaldehyde, 2-acetylpyrrole, 1-methyl-2-pyrrolecarboxaldehyde, and 2-acetyl-1-methylpyrrole inhibited hexanal oxidation by >80% at 10 microg/mL. Unsubstituted furan exhibited the greatest antioxidant activity among furans tested. Addition of all functional groups used in this study to furan decreased antioxidative activity. The antioxidant activity of thiophene increased with the addition of methyl and ethyl groups, but the addition of formyl or acetyl groups to thiophene decreased antioxidant activity. Thiazoles and pyrazines were ineffective antioxidants at all concentrations tested. Reaction of all heterocyclic compounds with hydrogen peroxide resulted in the formation of various oxidized products.     

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.