Interaction of captan and folpet with mammalian DNA and histones

The reactions of captan, folpet, and thiophosgene with calf thymus histones and total rat liver histones were investigated. The binding of radioactivity from [³⁵S]captan or [¹⁴C]folpet and changes in polyacrylamide gel patterns of captan-treated calf thymus histones were pH dependent. At pH 7.5, gel patterns of captan-treated histones were not different from those of non-treated histones; whereas, at pH 9.0 the Amido Schwartz-staining bands of captan- and thiophosgene-treated histones in polyacrylamide gels were characterized by band diffusion, loss of staining definition, band migration, and aggregate formation. Nearly equal amounts of radio-activity from [¹⁴C]folpet and [³⁵S]captan were bound to lysine-rich histones at pH 9.0, while at pH 7.5 there was little binding of radioactivity to the proteins. Approximately equal amounts of radioactivity were removed from histones in the presence of the low-molecular-weight thiol, dithiothreitol. Reconstitution experiments using captan-treated lysine-rich histones and non-treated calf thymus DNA yielded less stable complexes, as well as smaller nucleohistone-nucleohistone complexes. These data suggest that the intact molecule binds to deprotonated sites on the lysine-rich histones. This binding causes changes in the configuration of the protein molecule which alters the ability of nuclear histones to stabilize DNA structure.     

The interaction of folpet with thiamine and two thiamine-dependent systems

The interaction of folpet with thiamine-thiol was studied: 1.62 μmol of folpet reacted completely with 1 μmol of thiamine-thiol in a carbonate medium. A compound releasing thiamine, a possible thiocarbonate derivative, was isolated on a Sephadex G-15 column. This substance had an ir spectrum similar to the product from the interaction of thiamine with thiophosgene. Thiamine, modified by the interaction with folpet, could not be utilized during enzyme phosphorylation catalyzed by thiaminepyrophosphokinase. The formation of the mixed disulphide of thiamine and oxidized glutathione in the presence of folpet was affected in a similar manner.     

The interaction of folpet with the glutathione-dependent demethylation enzyme system of rat liver

The interaction of folpet with the glutathione-dependent enzyme demethylation of fenitrothion was studied. In a reaction catalyzed by methyltransferase from rat liver in the presence of 0.3 μmol of glutathione, 0.21 μmol of folpet caused a 100% inhibition of the demethylation of 0.2 μmol of fenitrothion. A parallel study was made on the qualitative and quantitative course of the reaction between folpet and glutathione. Phthalimide was formed as the main degradation product of folpet, but the composition of the degradation products of glutathione was dependent on the molar ratio of the reactants. Compounds absorbing at 267 nm (probably derivatives of 2-thiazolidinethiones) were formed when the ratio of glutathione to folpet was 4.5:1. When this ratio was reversed, oxidized glutathione was the main product. From observations on the course of the enzymatic demethylation of fenitrothion and on the degradation of folpet by glutathione, it appears that in the enzyme demethylation system, the reaction between fungicide and glutathione predominates.     

Inhibition of Penicillium duponti carboxylesterase by the fungicides captan and folpet

Captan, folpet, and perchloromethylmercaptan were effective inhibitors of Penicillium duponti p-nitrophenylpropionate esterase activity (I₅₀ = 0.5 – 2 μM) whereas α-naphthyl acetate esterase activity was not affected by the presence of these compounds. Captan and folpet are both equally effective at pH 7.3 and 8.3. The ionic composition of the medium had strong effects on the degree of inhibition produced by all inhibitors but did not alter esterase activity. Neither succinamide nor phthalimide caused inhibition of the p-nitrophenylpropionate esterase activity: The trichloromethylmercaptan portion of these fungicides appears to be responsible for the observed inhibition. The rapidity of captan and folpet inhibition of esterase activity (complete in < 1 min) compared to the rates of spontaneous decomposition ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{> 1}\text{min}$) and the insensitivity of captan and folpet inhibition to hydrogen ion concentration suggest that generation of spontaneous decomposition products is not required for inhibition. The results are consistent with a mechanism in which the entire fungicide molecule binds to the protein followed by enzyme-promoted reactions of captan and folpet which result in loss of esterase activity.     

Fate of captan and folpet in rats and their effects on isolated liver nuclei

Excretion and distribution of single and multiple intraperitoneal doses of [³⁵S]captan and [¹⁴C]folpet were similar in normal and 70% hepatectomized male rats. After receiving the single dose of captan, the rats eliminate approximately 76% of the radioactivity in the urine after 72 hr. The elimination in the feces for the same time period was 13%. Normal rats administered single or multiple doses of [¹⁴C]folpet excreted nearly 100% of the total dose in the urine within the first 24 hr. Nuclei isolated from the liver of normal and 70% hepatectomized rats receiving multiple doses of [³⁵S]captan contained 0.008–0.009 μg ³⁵S/g of tissue. Appreciable amounts of the radioactivity from [³⁵S]captan were bound by isolated nuclei from the livers of normal and partially hepatectomized rats. After a 1-hr treatment with [³⁶S]captan, the nuclei were fractionated into nuclear sap protein, deoxyribonucleoprotein (including histones), acidic ribonucleoprotein, and “residual” protein fractions. These proteins in normal nuclei bound 10, 14, 39, and 16% of the total label, respectively, with essentially the same results obtained with nuclei from regenerating rat liver. When compared by polyacrylamide gel electrophoresis, acidic nuclear proteins from treated and nontreated normal nuclei were characterized by band diffusion and the presence or absence of Amido Schwartz-staining bands. None of the abovementioned effects on histones from treated nuclei were observed. Captan treatment of isolated nuclei also altered the extraction characteristics of the nuclear protein fractions, presumably because of extensive aggregation of thiol-containing nuclear proteins.     

Detection of Tobamoviruses from Soils by Non-precoated Indirect ELISA

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol. Received 4 October 1999/ Accepted in revised form 9 December 1999     

Indirect effect of 6-azauracil on Pythium debaryanum in cucumber

Protection of cucumber seedlings against damping off, caused byPythium debaryanum, was obtained by soaking the seeds in a solution of 1 ppm 6-azauracil (AzU), but not by treatment with a 10 or 100 ppm AzU solution. After the 1 ppm AzU treatment of the seeds, the rhizosphere microflora of the developing seedlings was changed; an increase of the bacterial population and a decrease of the fungal population was observed. SinceP. debaryanum is rather insensitive to AzU in vitro, it is suggested that the control of damping off is obtained in an indirect way, possibly via the microflora of the roots.Samenvatting Bescherming van komkommerzaailingen tegen aantasting doorPythium debaryanum werd verkregen na weken der zaden in een oplossing van 1 ppm 6-azauracil (AzU); 10 en 100 ppm AzU waren niet werkzaam. Na behandeling der zaden met 1 ppm AzU traden veranderingen in de rhizosfeermicroflora der kiemplanten op; de bacteriepopulatie nam toe en de schimmelpopulatie nam af. AangezienP. debaryanum in vitro vrijwel ongevoelig is voor AzU, wordt verondersteld dat de bescherming tegen aantasting door deze schimmel op indirecte wijze verkregen wordt, mogelijk via de microflora van de wortels.     

Multiresidue method for captan,folpet, captafol,vinclozolin and iprodione on italian apples and pears by capillary gas–liquid chromatography with electron-capture detection

A method is described for the simultaneous determination of residues of five fungicides used for foliar treatment of apple and pear trees, and for postharvest application. After extraction, the mixture of these fungicides is cleaned-up on a ‘SEP PAK C₁₈’ cartridge and the components determined by gas-liquid chromatography with electron-capture detection. The minimum detectable amounts in apples and pears, on a fresh weight basis, were 0.005mg kg⁻¹ for vinclozolin, 0.010mg kg⁻¹ for captan, folpet and iprodione, and 0.020 mg kg⁻¹ for captafol. The percentage recovery for each fungicide (calculated by analysing four samples of untreated apples and pears, to which varying concentrations of each active ingredient had been added) varied for vinclozolin between 70.0 and 89.2, for captan between 72.0 and 83.8, for folpet between 73.0 and 93.0, for captafol between 70.8 and 91.8, and for iprodione between 75.1 and 97.1.     

麦穗鱼脑AChE间接非竞争ELISA定量分析法的建立

用PEG2000双水相萃取、 DEAE-Sephadex A-50和Sephadex G-200方法分离纯化麦穗鱼Pseudorasbora parva脑中的乙酰胆碱酯酶(AChE), 并制备了兔抗麦穗鱼脑AChE抗血清。用兔抗麦穗鱼脑AChE抗体连接抗原, 建立了定量分析麦穗鱼脑AChE的间接非竞争酶联免疫吸附法(Indirect and non-competitive ELISA), 此法操作简便、灵敏度高, 适用于定量检测。该方法的建立有利于AChE在环境科学和农药学研究领域的进一步研究和应用。     

Indirect evidence for sexual reproduction in Cercospora beticola populations from sugar beet

Cercospora beticola is the main causal agent of cercospora leaf spot on sugar beet and has a large negative impact on the yield and quality of sugar beet production worldwide. Previous studies have shown that both mating type idiomorphs of C. beticola are present in natural populations, suggesting that C. beticola is heterothallic and may be reproducing sexually. Cercospora beticola isolates are diverse in the morphology of their conidia, onset of disease symptoms and fungicide resistance. To find the source of this diversity and to determine if sexual reproduction occurs in this fungus, C. beticola populations were collected from Western Europe, Iran and New Zealand. The mating types of these isolates were determined and AFLP analyses were used to study the genetic diversity in these populations. The mating type ratios did not deviate significantly from a 1:1 ratio in most of the populations and AFLP analyses showed high levels of genetic variation within and between the populations, with 86·4% of the isolates having unique genotypes. All populations were in significant linkage disequilibrium but levels of disequilibrium were low, and loci from only one primer pair were in significant gametic equilibrium in populations from the Netherlands and Italy. From these results there is the possibility that C. beticola reproduces sexually. High levels of gene flow among the samples from Europe demonstrated a single panmictic European population. This study confirms C. beticola to be a genetically highly diverse species, supporting the assumption that some populations are reproducing sexually.     

Indirect haemagglutination reaction with antigen of Sarcocystis gigantea (Railliet, 1886) Ashford, 1977

The water extract from cryolyzed whole muscle cysts of Sarcocystis gigantea from sheep, in spite of the high lectin content, is a suitable antigen for the detection of specific antibodies by means of indirect haemagglutination reaction (IHA). The agglutinating effect of lectin from parasitic cysts can be eliminated with a 0.5% concentration of lactose dissolved in all solutions used for IHA. In sera of slaughterhouse sheep, positive titres ranging from 1:80 to 1:1 280 were registered. Positive reactions in lower titres were observed also with antibodies against S. dispersa, S. cuniculi and Sarcocystis sp. from pigs. Sensibilized erythrocytes can be stored in refrigerator at least for 1 week.     

PM 7/97 (1): Indirect immunofluorescence test for plant pathogenic bacteria

Specific scope

This standard describes how to perform an indirect immunofluorescence test (IF) for plant pathogenic bacteria. ¹ 1 Use of brand names of chemicals or equipments in these EPPO standards implies no approval of them to the exclusion of others that may also be suitable.

Specific approval and amendment

Approved as an EPPO Standard in 2009–09.

     

The determination of trace levels of ethylenethiourea

All currently utilized herbicides have been identified by random screening and related compounds developed by subsequent imitative chemistry. Little success has as yet been achieved by rational design. Ideas for herbicide design are discussed in relationship to enzyme inhibition, enzyme regulation and the accumulation of toxic metabolites. A number of herbicides in current use function either by promoting the formation of toxic radicals or by inhibiting protective systems. There is scope for the development of protective enzyme inhibitors, as well as the modulation of other scavenging systems. Natural or synthetic photosensitizers as well as a number of allelopathic chemicals could be used as herbicides. Herbicide selectivity could be investigated by the selective inhibition of C⁴ photosynthesis enzymes, by a detailed examination of enzyme diversity, or by the modulation of mono-oxygenase enzymes or glutathione conjugation.     

The determination of the herbicide dinoseb in lentils

Residues of the herbicide dinoseb were determined gas chromatographically in lentils which had been treated at two locations in Saskatchewan with post-emergence applications of dinoseb at 1.4 and 1.7 kg ha^?1. Herbicide residues, determined at selected times after application, were not detected at the limit of detection of the analytical method (0.05 mg kg^?1) in either the seed and straw at maturity, or in the green foliage six to eight weeks after application. Recoveries of dinoseb were 76% from fortified green foliage at the 0.1 mg kg^?1 level, and 64% from fortified seed at the 0.05 mg kg^?1 level.     

Selective complexometric determination of glyphosate and related compounds

A complexometric method has been developed for the selective determination of glyphosate and related compounds. The method is based upon the different pH-dependence of tridentate and tetradentate ligand-metal complex stabilities. Glyphosate, the tridentate ligand forms a stable copper complex at pH ≥ 8 only, whereas N-carboxymethyl-N-phosphonomethylglycine and N,N-bis(phosphonomethyl)glycine, the tetradentate ligands, form sufficiently stable bismuth complexes even at low pH. The method, therefore, consists of aliquot titrations in basic and acidic media, using metal ion titrant solutions. The first aliquot containing N-(phosphonomethyl)glycine plus N-carboxymethyl-N-phosphonomethylglycine or N,N,-bis(phosphonomethyl)glycine is titrated with bismuth volumetric solution at pH 1.8–2–5 in the presence of methylthymol blue indicator. Quantities of the tetradentate ligands can be calculated from the bismuth consumption. The second aliquot is titrated with copper volumetric solution at pH 8–10, in the presence of murexide indicator. The content of glyphosate can be calculated from the difference between the copper and bismuth consumptions. Efficacy of the method is verified by analyses of standard mixtures and industrial samples.     

新生物杀线虫剂对线虫致死力室内测定

室内测定了新研制的3种生物杀线虫剂及其复配剂对根结线虫(Meloidogyen spp.)和松材线虫(Bursaphelenchusxy Lophilus)致死力,在25℃恒温、60%RH恒湿条件下4种杀线虫剂,杀线剂1、杀线剂2、杀线剂3对两种线虫的致死力都较强,药后72h根结线虫死亡率达98%,松材线虫死亡率达80%。杀线剂复配效果好于单一效果。     

水稻田样品中噻呋酰胺残留检测方法研究

建立了噻呋酰胺在稻田土壤、稻田水、水稻秸秆、水稻绿色植株、谷壳和糙米中的残留检测方法。稻田水中的噻呋酰胺用LC-18固相萃取小柱分离、净化和富集;稻田土壤、水稻秸秆、谷壳和糙米样品采用V(丙酮):V(石油醚)=1:1超声提取,硅胶柱净化;绿色植株样品用石油醚超声提取,硅胶柱净化。提取净化后的所有样品均采用气相色谱-电子捕获检测器(GC-ECD)测定。结果表明,噻呋酰胺在0.005～10 mg/L范围内线性关系良好,相关系数(r)为0.999 1。其方法检出限分别为:稻田水中0.005 mg/L,土壤、水稻秸秆、绿色稻株、谷壳及糙米中均为0.004 mg/kg。 在水样中添加水平为0.05、0.5和1 mg/L及在土壤、秸秆、绿色稻株、谷壳和糙米中添加水平为0.04、0.4和4 mg/kg时,添加回收率均在86.6%～106.2%范围内,相对标准偏差(RSD)为1.3%～9.9%。     

复方苦部微乳剂指纹图谱的建立及其活性成分含量测定

建立了高效液相色谱(HPLC)测定复方苦部微乳剂指纹图谱和微乳剂中苦参碱及氧化苦参碱含量的方法。样品经85℃水浴去除溶剂后,用0.5%的硫酸溶液溶解、静置后过滤;滤液用浓氨水调节p H至10~11,用氯仿萃取;有机相脱溶,残渣用乙醇溶解。采用Reprosil-par120-NH2色谱柱,流动相为V(乙腈)∶V(无水乙醇)∶V(3%磷酸溶液)=87∶7∶6,检测波长220 nm,柱温30℃,对微乳剂样品进行分离;运用中药色谱指纹图谱相似度评价系统(2004版)软件对10个不同批次的复方微乳剂图谱进行相似度评价和数据处理,以中位数法生成复方微乳剂对照指纹图谱;采用标准品对照法对其峰归属进行分析,结合外标法测定微乳剂中苦参碱和氧化苦参碱的含量。结果表明:10个批次微乳剂样品图谱的相似度均大于0.90,从中分离得到7个共有指纹峰,指认其中两个色谱峰分别为苦参碱和氧化苦参碱。苦参碱在3.63~58.0μg/m L(r=0.999 1)、氧化苦参碱在4.50~72.0μg/m L(r=0.999 0)范围内呈良好的线性关系,在10、20和50 mg/L 3个添加水平下,两者的添加回收率分别为98%~99%(RSD为0.77%~2.6%,n=9)和96%~99%(RSD为1.0%~2.0%,n=9),微乳剂中两者的检出限(LOD)分别为0.201和0.225 mg/L,定量限(LOQ)分别为0.671和0.750 mg/L;10个批次微乳剂样品中苦参碱、氧化苦参碱的平均含量分别为57.07和43.55 mg/L。所建立的HPLC指纹图谱特征性强、信息量大,对复方苦部微乳剂有较好的专属性;建立的HPLC检测方法操作简便、准确度高、重现性好;两者联用能全面有效地控制复方苦部微乳剂质量。