The absorption of 2, 4-D by protoplasts enzymatically isolated from the cortex of corn (Zea ways L. cv Pioneer 3377) root was investigated in relation to their extracellular and intracellular pH. The 2, 4-D absorption increased as the extracellular pH (pH^o) decreased. The ratio of the 2, 4-D concentration inside to outside the protoplasts (cⁱ/c^o) reached ca. 1.2, 4.0, 10-12 and 30-40 at pH^o 7.5, 6.5, 5.5 and 4.5 respectively, within 75min incubation. At pH^o 3.5 and 2.8, cⁱ/c^o ratio reached ca. 90 and 150 respectively within 15min, but the absorbed 2, 4-D was rapidly released after 15min incubation due to the death of the protoplasts. The determination of the overall intracellular pH (pHⁱ) by the 5, 5-dimethyloxa-zolidine-2, 4-dione distribution technique indicated that the change of the pHⁱ as influenced by the pH^o variation (7.5 to 4.5) was relatively small (6.9 to 6.6). Using the measured pHⁱ, the theoretical cⁱ/c^o ratio at each pH^o was estimated based on the weak acid hypothesis. Although the pH^o dependence of the theoretical cⁱ/c^o roughly resembled the observed tendency, the measured cⁱ/c^o ratios at pH^o 6.5 and 7.5 were significantly greater than their respective theoretical maxima. These results suggest that the absorption of 2, 4-D by protoplasts cannot be accounted for simply by the weak acid hypothesis and that an active transport is involved at least at pH^o 6.5 and 7.5.

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The effects of metabolic inhibitors and antiauxins on the absorption of 2, 4-D by protoplasts enzymatically isolated from the cortex of corn (Zea mays L. cv Pioneer 3377) root were investigated in relation to their effects on the intracellular pH (pHⁱ). Five metabolic inhibitors with different types of action inhibited 2, 4-D absorption by the protoplasts. All of these chemicals reduced the pHⁱ measured by the 5, 5-dimethyloxazolidine-2, 4-dione distribution technique, and inhibition of the 2, 4-D absorption was closely correlated with the reduction in pHⁱ regardless of their type of action. This suggests that the pH gradient across the plasmalemma is a crucial factor for 2, 4-D absorption. In contrast, an antiauxin, p-chlorophenoxyisobutyric acid (PCIB), inhibited 2, 4-D absorption without affecting pHⁱ. Another antiauxin, N-1-naphthylphthalamic acid (NPA), did not affect either 2, 4-D absorption or pHⁱ. These results suggest that the mechanism of 2, 4-D absorption by the corn root protoplasts is composed of two components: 1) the passive diffusion of the undissociated 2, 4-D molecules according to the weak acid hypothesis and 2) the active transport of dissociated 2, 4-D anions mediated by a PCIB-sensitive and NPA-insensitive H⁺/auxin anion cotransport carrier.

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An inhibitor of larval growth of the azuki bean weevil was isolated from kidney beans. The growth inhibitor was a glycosylprotein with a molecular weight of ca. 48, 000 and an isoelectric point of 4.46, which had several subunits and moieties sensitive or insensitive to endo-N-acetyl-, β-D-glucosaminidase H. Its carbohydrate content was 15.2%, based on 20 mannose, 1 fucose, 2 xylose and 15 N-Ac-glucosamine residues per one mole (8.5% by the phenol-sulfuric acid method). It inhibited α-amylases of animal origins, but not those of plant and microbial origins. It had no trypsin inhibitory and lectin activities. Both carbohydrate and native protein moieties seemed to be required for growth and α-amylase inhibition. The overall results indicated that the growth inhibitor was identical with or similar to the α-amylase inhibitors previously isolated from kidney beans.

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Chlorfluazuron, a new benzoylphenyl urea insecticide developed by Ishihara Sangyo Kaisha Ltd., was very effective on Spodoptera litura larvae. At lethal dosages, larvae turned translucent and then black, or split the old cuticle but failed to exuviate. At lower dosages, most of the larvae had pale regions with dark stripes and stains. The integument of a larva enclosed in the translucent old skin was torn at regions close to the sutures between the segments, which seemed arthrodial membranes, and at the last and thoracic dorsal segments. These rips appeared at positions corresponding to stripes and stains in a successfully molted larva. Based on these observations, various malformations of the cuticles were judged to be results from the disruption in forming pre-ecdysal new cuticles. The regions close to the sutures and the last and thoracic dorsal segments were more susceptible to the compound than other regions. Larvae that neither fed nor grew after successful molt were also observed, probably because the mouthparts were disrupted by the compound.

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Degradation of isouron in soil fitted the first-order kinetics with half-lives of 42 to 203 days under various conditions in the laboratory experiments. Close correlations between the degradation rate and the soil moisture and temperature were observed. Under laboratory condition, the half-lives of isouron in autoclaved (148 to 208 days) and nonautoclaved soils (58 to 70 days) showed that soil microbes played an important role in the degradation. Under model field conditions, the half-life was about 90 days in sandy loam. One unknown metabolite (VIII), six degradation products, namely 3-(5-tert-butyl-3-isoxazolyl)-1-methylurea (II), 3-[5-(1, 1-dimethyl-2-hydroxyethyl)-3-isoxazolyl]-1, 1-dimethylurea (III), [3-(5-tert-butyl-3-isoxazolyl)] urea (IV), 3-[5-(1, 1-dimethyl-2-hydroxyethyl)]-1-methylurea (V), 3-[5-(1, 1-dimethyl-2-hydroxyethyl)-3-isoxazolyl] urea (VI), and 3-amino-5-tert-butylisoxazole (VII) were identified by autoradiography and mass spectrometry.

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Metabolism of (R)-(E)-1-(2, 4-dichlorophenyl)-4, 4-dimethyl-2-(1, 2, 4-triazol-1-yl)-1-penten-3-ol [designated as (R)-E], a major ingredient of diniconazole, was examined in rats after repeated oral administration. Effect of diniconazole on hepatic enzymes was also examined in vivo and in vitro. When (R)-E labeled with ¹⁴C at the triazole ring was orally administered once daily to male and female rats at 125mg/kg for 14 successive days (Day 0 to Day 13), ¹⁴C was slowly excreted into urine and feces for an initial few days, and faster thereafter to be completely eliminated from the body by Day 20. ¹⁴C-Tissue levels were higher on Days 1 and 6 but decreased to constant levels on Days 10 and 14. The ¹⁴C-tissue levels decreased to 1μg (R)-E equivalent/g tissue or less on Day 20, and no bioaccumulation or bioretention of ¹⁴C was observed in any tissues. Main metabolites produced by oxidation at the methyl group were found in Day 0-3 excreta as well as in Day 11-14 excreta. After single oral administration of diniconazole at 125mg/kg, the content of hepatic microsomal cytochrome P-450 had markedly decreased at 3hr, recovered at 8hr, and significantly increased at 24hr. Comparative effects were observed on microsomal drug-metabolizing activities, while no effects were found on the contents of hepatic mitochondrial cytochromes and enzyme activities of the electron transfer system. Diniconazole yielded typical Type II binding spectra with rat hepatic microsomal cytochrome P-450 and inhibited aniline hydroxylation non-competitively.

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On single oral administration of (S), (E)-1-(p-chlorophenyl)-4, 4-dimethyl-2-(1, 2, 4-triazol-1-yl)-1-penten-3-ol [designated as (S)-E], a main ingredient of uniconazole, labeled with ¹⁴C at the triazole ring at dose levels of 1 and 200mg/kg to male and female rats, radiocarbon was excreted into urine and feces nearly 100% within 7 days. The urinary and fecal excretion accounted for 43-67% and 34-55% of the dose, respectively. Males excreted more radio-activity into feces than females. ¹⁴C-Concentration reached a peak in tissues 1 to 8hr after administration. Adrenal and liver showed relatively higher peak concentrations of 4.10-4.57 and 2.34-2.59μg (S)-E equivalents/g tissue decreasing with half-lives of 8-11 and 5-6hr, respectively. ¹⁴C-Levels in tissues were generally low on the 7th day after administration. Urinary and fecal metabolites amounting to 83-91% of the dosed ¹⁴C were identified, and metabolic pathways for (S)-E were proposed. Recovery of unchanged (S)-E in feces was 7 to 13% of the dose. A main biotransformation was oxidation of the methyl group to form hydroxymethyl and carboxy metabolites, which amounted to 58-75% of the total dose. Slight sex-related differences were observed in amounts of excreted metabolites: females excreted a larger amount of the carboxy metabolite and a smaller amount of 1, 2, 4-triazole into urine than males. Although main metabolic reactions of (S)-E were similar to those of (R)-(E)-1-(2, 4-dichlorophenyl)-4, 4-dimethyl-2-(1, 2, 4-triazol-1-yl)-1-penten-3-ol, diniconazole, there was a difference in the excretion route of the main carboxy metabolites. Rats receiving Tr-¹⁴C-(S)-E excreted more ¹⁴C into urine than those receiving ¹⁴C-diniconazole. It is considered that ¹⁴C in the former was excreted into urine more easily than that in the latter because the octanol-water partition coefficient (P_o/w) of the carboxy metabolite from (S)-E is smaller than that from diniconazole.

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SD male rats 7 weeks old were fed on a diet containing (R)-(E)-1-(2, 4-dichlorophenyl)-4, 4-dimethyl-2-(1, 2, 4-triazol-1-yl)-1-penten-3-ol (diniconazole) or phenobarbital (PB) at 0ppm or 2000ppm for 2 weeks. The compounds changed various parameters relating to liver functions. They increased in liver weight, UDP-glucuronyltransferase activity, protein content, cytochrome P-450 and b₅ contents, and bile flow rate. The increased UDP-glucuronyltransferase activity and bile flow rate induced an increase in thyroxine(T₄)-glucuronide excretion into bile. As a result, T₄ levels in serum decreased significantly. Hypertrophy of the liver and thyroid in rats receiving a higher dose of diniconazole was attributable to the elevated liver function.

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The dissipation of chlorothalonil (TPN, 2, 4, 5, 6-tetrachloro-1, 3-isophthalonitrile, Daconil^®, Dacosoil^®) in soils was suppressed by the repeated applications of this fungicide to the soils. The dissipation was due to microbial actions, since chlorothalonil disappeared in a nonsterile soil but not in an autoclaved soil. The amendments of the soil with easily decomposable organic materials recovered the suppressed dissipation ability of the soil. The results suggested that easily decomposable organic materials play an important role in the microbial degradation of chlorothalonil in soil.

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Bacteria that degraded chlorothalonil (TPN, 2, 4, 5, 6-tetrachloro-1, 3-isophthalonitrile) were present in soils with no history of chlorothalonil application at population levels of 6×10⁶ to 12×10⁶ CFU/g-soil and were enriched by chlorothalonil application to higher population levels of 2×10⁷ to 8×10⁷ CFU/g-soil. The chlorothalonil-degrading bacteria were tolerant to high concentrations of chlorothalonil. Eleven strains of chlorothalonil-degrading bacteria were isolated and tentatively identified as Azomonas, Flavobacterium, Moraxella and Pseudomonas spp. The isolates required other carbon sources for degradation, except for those two from Flavobacterium strains that degraded chlorothalonil as a carbon and energy source. Substrate specificity was high in the two strains from Azomonas and Flavobacterium.

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Solid-phase extraction with bonded silica sorbents has been developed as an alternative to liquid-liquid partitioning cleanup in the pesticide residue analysis of crops. Elution patterns and recovery from an octadecyl (C18) sorbent were determined for 17 organophosphorus, nine carbamates and three other pesticides with the sample matrix from rice grain. The recovery was good for all compounds whose properties were water-soluble at below 25, 000mg/l. The sample loading test proved that a cartridge containing only 0.5g of sorbent mass was capable of trace enrichment of analytes with a sample matrix from 20g of grain. Among various types of bonded silica sorbents, octyl (C8), ethyl (C2) and cyclohexyl (CH) sorbents were superior to others in retention and selective elution. The new technique was compared with the conventional method which requires liquid-liquid partitioning. The availability of the technique was also evaluated in several fruits and vegetables.

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Various methods were compared to determine kinetic constants for the inhibition of carboxylesterase (CE) and acetylcholinesterase (AChE) with insecticides, i. e. dissociation constant (K_d), acylation constant (k₂) and deacylation constant (k₃). The results showed that the constants could be determined more accurately and easily by selectively employing appropriate equations derived from presteady state kinetics, steady state kinetics or modification of Hart & O'Brien's method, depending upon the reaction conditions and the parameter relationships. Namely, the k₃ can be estimated by combination of presteady state and steady state equations, without using the conventional methods of gel-filtration or dilution. The K_d and k₂ can be obtained from either presteady state or steady state equation. On the other hand, the K_d, k₂ and k₃ can be obtained by the modified method of Hart & O'Brien even at a single dose of inhibitor. The employment of steady state equation results in values with less errors than the other two methods, when the k₃ and k₂ are approximately in the same order. In contrast, when the k₃ is much smaller than k₂, the presteady state equation and the modified method of Hart & O'Brien give less errors than the other method. It was also shown that the K_d can be calculated using an equation derived from steady state kinetics without measuring the change in AChE activity in progress of time. Therefore, if a sufficient amount of a purified enzyme is available, this method enables the determination of the K_d even when k₂ is large (k₂>30min^-1) without using the tedious stopped-flow method.

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[in Japanese]

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[in Japanese]

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Advances in plant genetic engineering have allowed to create the transgenic plants resistant to herbicides, insects and viruses by transfer of single dominant gene. Such strategies could be used to achieve plant protection against bacterial and fungal diseases, especially caused by toxin-producing microorganisms. We have been interested in the tobacco wildfire disease caused by Pseudomonas syringae pv. tabaci, which produces a phytotoxic tabtoxin. It is known that tabtoxin has an important role in the development of chlorotic symptoms typical to the wildfire disease. An attempt was made to screen and clone the tabtoxin resistance genes from the wildfire pathogen itself, because some antibiotic-producing microorganisms are possessed of self-resistance genes to protect themselves from their toxic metabolite. We have isolated a tabtoxin resistance gene (ttr) coding for an acetyltransferase, which inactivates tabtoxin by acetylation. The chimeric ttr gene fused to the 35S promoter of cauliflower mosaic virus was inserted on the Ti-vector plasmid for introduction into tobacco cells by Agrobacterium-mediated transformation. The transgenic tobacco plants showed high-specific expression of the ttr gene, and became resistant to tabtoxin treatment. The inactivation of tabtoxin in the transgenic plants also conferred resistance to the bacterial attack of P. syringae pv. tabaci. These results demonstrate a successful approach to create the disease-resistant plants by genetic engineering for detoxification of pathogenic toxins. Also, such a strategy could be widely applied to other diseases caused by pathogenesis-related toxins.

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Triflumizole [E-4-Chloro-α, α, α-trifluoro-N-(1-imidazol-1-yl-2-propoxyethylidene)-o-toluidine] is a novel systemic fungicide discovered and developed by Nippon Soda Co., Ltd. We found a new synthetic method of imidazole derivatives and we used this method for the developing of new pesticide. The synthesized imidazole containing compounds, N-(1-imidazol-1-ylalkoxyalkylidene)anilines showed relatively strong and broad fungicidal activity accompanied with plant growth regulating effect. The highest fungicidal activity was obtained by phenyl substitution with halogen, methyl or trifluoromethyl in the 2-position and halogen in the 4-position, respectively. The introduction of hetero atom, 0, to alkyl chain reduced the effect on plant growth remarkably without reducing fungicidal efficacy. As the results, a compound was selected and is now marketed as a common name triflumizole. Triflumizole showed a wide range of antifungal spectrum in vitro including fungicidal activities to the isolates resistant to benzimidazoles or dicarboximides. In field trials, triflumizole showed a good performance for control of many diseases of crops, including scab, rust and powdery mildew of fruit trees, cereals or vegetables and also seed-born diseases (Gibberella, Cochliobolus, Ustilago, Tilletia and Pyrenophora). The fungicide has both protective and curative efficacies in controlling diseases and also systemic activities in plants. Triflumizole inhibited ergosterol biosynthesis in Ustilago maydis by interfering with demethylation at C-14 of sterol skeleton. Triflumizole was found to have a low toxicity to mammals and fishes and less hazard to the environment.

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Quizalofop-ethyl, ethyl 2-[4-(6-chloro-2-quinoxalinyloxy)phenoxy] propionate is a selective grass herbicide developed by Nissan Chemical Industries, Ltd. Quizalofop-ethyl possesses a wide herbicidal spectrum for grass weeds with a quick action at low application rates by foliage applications. It shows good safety for non-gramineous crops. A research for a grass killer herbicide, focussed on heterocyclicoxy phenoxy proionic acid, began in 1978. During investigations on the various condensed heterocyclic moieties, the quinoxalinyloxy phenoxy propanoic acid derivatives were found to have a high potential for grass herbicides. Through the optimization of derivertives, the title compound was selected as a candidate for development. In field trials conducted in major producing areas of soybeans, cotton, sugar beet and other broad leaf crops, quizaolfop-ethyl demonstrated sufficient control at 0.5-1.5g a.i./a for annual grass species and at 1.25-2.5g a.i./a for perennial ones. Quizalofop-ethyl was translocated from treated leaves to meristem tissues of grass weeds within a day and attacks these parts, followed by causing necrosis. Quizalofop, which is the metabolite of quizalofop-ethyl, was proved to be a potent inhibitor of fatty acids synthesis through acetyl-CoA carboxylase inhibition. A main metabolic pathway of quizalofop-ethyl was the hydrolysis to quizalofop in plants, soil and mammals. Following hydrolysis, cleavages of ester bonds, hydroxylations and conjugation of quizalofop occurred. Toxicity of quizalofop-ethyl in mammals, fish and birds was very low. Its potential hazard to the environment was minimal.

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This formulation study is conducted for unique granules with a pesticide delivery system to use the active ingredient efficiently in the paddy field. When the granules are scattered, they sink down on the bottom soil at first. Then the granules resurface and diffuse the active ingredient on the water surface. As the solid materials of the granules dissolve completely into the water, finally the concentrated active ingredient is left there. This resurfacing behavior of particle is caused by the binding agent transformed to a hydrosol with water and to hold air existing in the particle as well as by reduction of the particle weight due to dissolution of the solid material in water. In case of insecticide cycloprothrin granules of this type, about 75% of cycloprothrin was distributed on water surface and in water 2 hours after application, while in case of a ordinary granule formulation only about 20% of the active ingredient was distributed there. This type of granules containing insecticidal active ingredients showed for higher efficacy than the ordinary granules against several kinds of injurious paddy insects. Cycloprothrin 2% granules (Cyclosal 2UG^®) was completed by this granular formulation technique as a result of selection of raw materials, establishment of manufacturing process, product quality investigation, and others.

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Quinalphos is specified as a deleterious substance since its acute toxicity in mice and rats is high. However, irritation studies in rabbits revealed that quinalphos was a non-irritant to the eye and skin. Quinalphos was considered as a non-sensitizer and was not proved to have the potential to produce acute delayed neurotoxicity.
The depression of cholinesterase activity was observed at high doses in subchronic toxicity studies in rats and it was confirmed in chronic toxicity studies in rats and dogs. In those studies no other major abnormalities related to quinalphos administration were observed and the no-observed-effect levels were defined in the respective studies. No dose-related incidence of tumor was observed in oncogenicity studies in rats and mice, and no potential was revealed in mutagenic studies. In addition, the rat reproductive performance was not effected by quinalphos and no teratogenic potential was indicated in the studies of rats and rabbits.

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