Organophosphate inhibition of an arylesterase from Lumbricus terrestris and reversal by pralidoxime

Several enzymes of xenobiotic metabolism have been identified in soil annelids. Their roles in the physiology and ecotoxicology of the organism are diverse and may parallel those found in mammalian species. Among these are the arylesterases. An arylesterase assayed by hydrolysis of p-nitrophenylacetate was identified in Lumbricus terrestris. This arylesterase appears to be a serine esterase, since it is sensitive to inhibition by metrifonate. This mechanism of inhibition is similar to that of cholinesterase, since pralidoxime can reverse the inhibition of arylesterase by metrifonate. The presence of this esterase in soil annelids may provide insight into the processing of environmental chemicals by soil annelids as well as in vitro commercial applications of esterases based upon their mechanism of catalysis and inhibition.     

Interaction of a cationic acrylate polymer with caseins: biphasic effect of Eudragit E100 on the stability of casein micelles

When whole or skim milk was incubated with the cationic acrylate polymer Eudragit E100, a biphasic effect on the stability of casein micelles was observed. A precipitation phase was observed at low polymer/casein ratios. Strikingly, a solubilization phase of the aggregates was observed when the ratios of polymer/casein were increased. Purified alpha(s)-, beta-, and kappa-caseins or dephosphorylated caseins were equally precipitated and resolubilized by the cationic polymer, indicating no special selectivity for a particular protein or phosphate residue for these events. An increase in the size of the aggregates as the optimum precipitating amount of Eudragit E100 was reached suggests a crossbridging of the micelles by the polymer. The inhibition of the precipitation phase by high ionic strength indicates that electrostatic interactions play a critical role in complex formation. Furthermore, a dramatic reduction in size of the protein colloidal particles upon solubilization of the aggregates was observed by dynamic light scattering, indicating a dissociation of the micellar structure. Taken together, the results indicate that at low concentration Eudragit E100 may act as a precipitant of casein micelles, mainly by ionic interaction and at high concentration as an amphipathic agent, solubilizing casein micelles with a disruption of their internal structure.     

Transformation kinetics and mechanism of the sulfonylurea herbicides pyrazosulfuron ethyl and halosulfuron methyl in aqueous solutions

Pyrazosulfuron ethyl (PE) and halosulfuron methyl (HM) are two new highly active sulfonylurea herbicides that have been widely used for weed control in a variety of vegetables and other crops. These two herbicides have similar molecular structures, differing only in the substitutions on the pyrazole ring. Chemical hydrolysis is a primary process affecting the environmental fate of sulfonylurea pesticides. The hydrolytic transformation kinetics of PE and HM were investigated as a function of pH and temperature. For both herbicides, the hydrolysis rate was pH-dependent and increased with increasing temperature. The hydrolysis of both sulfonylureas was much faster in acidic or basic media than under neutral conditions. Identification of hydrolytic products by liquid chromatography-mass spectrometry (LC-MS) suggested that both PE and HM were subject to cleavage and contraction of the sulfonylurea bridge. The hydrolysis rate of HM was significantly higher than that of PE in alkaline solutions, despite their structural similarity. A chlorine substitution on HM's pyrazole ring makes HM more susceptible to bridge contraction than PE under basic conditions. The hydrolysis of HM and PE was relatively unaffected by the presence of cyclic oligosaccharides (cyclodextrins), indicating that natural OH-containing organic compounds occurring in aquatic environments may have little impact on the transformation of these sulfonylurea herbicides.     

湿筛过程中分散液的质量对土壤团聚体稳定性影响

【目的】土壤团聚体是土壤结构的基本单元,决定着土壤在生态系统中的作用和功能。保持良好的结构状态和稳定的结构性能是确保土壤水、 肥、 气、 热肥力因子协调的基本条件。现代土壤管理中大量使用的化肥无机盐以及灌溉水体富含的离子都会造成对团聚体的破坏,本文以富含有机质的黑土为研究对象,探索湿筛过程中分散溶液的质量对团聚体稳定性的影响。【方法】以氯化钠、 氯化钙和氯化铝3种不同价态的阳离子溶液为介质,采用改进的萨维若夫（Саввинов）法进行湿筛,设置0（不加盐去离子水,CK）、 0.1、 0.3、 0.5 mol/L 4个浓度水平,研究直径0.25 mm的土壤团聚体在不同阳离子盐溶液作用下的稳定性。【结果】不同盐溶液中黑土团聚体的稳定性差异很大。在氯化钠盐溶液中,黑土团聚体的盐破坏率（PAD0.25/w）均为正值,且随钠盐溶液浓度的增大而增加,在浓度为0.1、 0.3和0.5 mol/L的氯化钠溶液中分别为0.21%、 10.7%和14.4%。在氯化钙溶液和氯化铝溶液中,黑土PAD0.25/w均为负值, 氯化钙溶液中PAD0.25/w的绝对值随着盐浓度的增大而减小,氯化铝溶液中PAD0.25/w的绝对值均在51%以上。【结论】一价盐溶液加剧了0.251 mm黑土团聚体的分散,但能够保护直径1 mm以上的团聚体,而二价盐和三价盐溶液对团聚体的分散有明显的抑制和保护作用。     

Effect of carbofuran on enzymatic activities and growth of tomato plants in natural,fertilized and vermicompost-amended soils

The effects of carbofuran, a widely used carbamate pesticide, on soil enzymatic activities such as fluorescein diacetate hydrolysis (FDAH), dehydrogenase, and acid and alkaline phosphatases were studied at different time intervals in unamended soil and soil amended with inorganic fertilizers and vermicompost, cropped with tomato plants. The results showed that all enzymatic activities varied with carbofuran application rates and increased significantly up to 1.0 kg active ingredient (a.i.) ha^?1 dose of carbofuran. The most significant increase was observed at 0.20 kg a.i. ha^?1 dose both in unamended and amended soils. This showed that carbofuran was not toxic to all enzymatic activities studied upto 1.0 kg a.i. ha^?1 dose of carbofuran in both systems. A significant decrease in all enzymatic activites were observed at higher dose of carbofuran both in unamended and amended soils relative to their respective controls. Highest enzymatic activities were observed in vermicompost amended soil and minimum in fertilized soil compared to control. The results indicated that the growth of tomato plants was significantly higher at 0.20 kg a.i. ha^?1 dose of carbofuran in all the cases and followed the order: fertilized soil > vermicompost amended soil > natural soil and was positively correlated with the enzyme activities.     

Formation of soluble and micelle-bound protein aggregates in heated milk

The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.     

Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels

Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.     

Controlled release of carbofuran from an alginate-bentonite formulation: water release kinetics and soil mobility

The insecticide-nematicide carbofuran was incorporated in alginate-based granules to obtain controlled-release (CR) properties. The basic formulation [sodium alginate (1.61%)-carbofuran (0. 59%)-water] was modified by addition of sorbents. The effect on carbofuran release rate, caused by the incorporation of natural and acid-treated bentonite (0.5 and 1.0 M H(2)SO(4)) in alginate formulation, was studied by immersion of the granules in water under shaking. The time taken for 50% of the active ingredient to be released into water, t(50), was longer for those formulations containing natural bentonite (6.1 h) or acid-treated bentonite (9.0 and 11.7 h for 0.5 and 1.0 M H(2)SO(4) treatments, respectively) than for the preparation without bentonite (4.7 h). It appears from the results that the release of carbofuran from the various formulations is controlled by a diffusion mechanism according to the n values obtained, which were close to 0.5 in all cases. The mobility of carbofuran from alginate-based CR formulations was investigated by using soil columns packed with a clay soil (53% clay and 0.08% organic matter). Two alginate-based CR formulations containing natural bentonite or acid-treated bentonite (0.5 M H(2)SO(4)) were compared to technical grade carbofuran. The use of alginate-based CR formulations resulted in a reduction of the leached amount of carbofuran compared with the total amount of pesticide leached using the technical product (50 and 75% for CR granules containing natural and acid-treated bentonite, respectively). Alginate-bentonite CR formulations might be efficient systems for reducing carbofuran leaching in clay soils, which would reduce the risk of groundwater pollution.     

Sphingomonas sp. strain SB5 degrades carbofuran to a new metabolite by hydrolysis at the furanyl ring

Microorganisms capable of degrading carbofuran were isolated from soils and examined for the degradation of this pesticide at ring structure. An isolate that could degrade carbofuran and carbofuran-7-phenol was selected for further studies. The 16S rRNA analysis results showed that the isolate belongs to the genus of Sphingomonas, close to dioxin and dicamba degraders, and is named Sphingomonas sp. SB5. SB5 did not show any similarity of 16S rRNA to known carbofuran degraders. When time-course degradation of carbofuran by SB5 was examined by solvent extraction combined with liquid chromatographic analysis, almost complete disappearance of carbofuran was observed within 12 h, giving several accumulative metabolites. Bacterial cultures incubated with carbofuran-7-phenol suggested that the accumulated metabolites were derived from carbofuran-7-phenol. The control without SB5 and kanamycin-treated SB5 did not show any metabolite, suggesting a biological involvement in the degradation of carbofuran. GC/MS and LC/MS analyses identified 2-hydroxy-3-(3-methylpropan-2-ol) phenol as one of the accumulated metabolites, suggesting that the strain SB5 could degrade carbofuran-7-phenol by hydrolysis at the furanyl ring. This is the first report to identify 2-hydroxy-3-(3-methylpropan-2-ol) phenol as a new product derived biologically from carbofuran-7-phenol.     

Studies on absorption and hydrolysis of ethyl alpha-D-glucoside in rat intestine

Ethyl alpha-D-glucoside (alpha-EG) is normally contained in Sake, which has been taken by Japanese people since ancient times. In this study, the intestinal absorption of alpha-EG was investigated using rat everted intestinal sac. Furthermore, the alpha-EG hydrolytic activity in rat intestine was compared with disaccharides hydrolytic activities, and the effects of alpha-EG on disaccharides hydrolysis were examined using crude enzyme preparation from rat intestinal acetone powder. Glucose liberated from alpha-EG was detected in a serosal solution of everted rat intestinal sac, but it was only less than 4% of absorbed intact alpha-EG. alpha-EG absorption into small intestinal tissue was reduced by elimination of sodium ion from the mucosal solution or under the presence of phlorizin. The hydrolytic activity for alpha-EG was detected in crude enzyme preparation from rat intestinal acetone powder, but it showed a low value as compared to those for disaccharides. alpha-EG showed mixed type inhibition for maltose and sucrose hydrolysis, but inhibitory concentrations of alpha-EG required for 50% inhibition for the maltose and sucrose hydrolysis were higher than those of arabinose and acarbose. In conclusion, a small amount of alpha-EG was hydrolyzed and most of it was absorbed via SGLT1 as an intact form in the rat small intestine, and the inhibitory effect of alpha-EG on disaccharides hydrolysis was weak.     

Quantitative determination of diuron in ground and surface water by time-resolved fluoroimmunoassay: seasonal variations of diuron, carbofuran, and paraquat in an agricultural area

The aim of this research is to develop an ultrasensitive time-resolved fluorescence immunoassay (TR-FIA) for herbicide diuron in water samples. This method appears to be a promising approach, instead of conventional analytical techniques, in the screening procedure of organic pollutants because it is simple, rapid, and specific, and it does not require sample preconcentration or cleanup. Lanthanide chelate used as label allows to achieve sensitivity even 10 times higher than most of the other techniques. It has been applied to monitoring diuron contamination in specimens collected along a year in an agricultural area. The water specimens were collected monthly from lake, well, and irrigation ditch in the agricultural area south of Milan. Assay was performed using diuron-specific polyclonal antibody raised in sheep; as fluorescent marker, we used rabbit antisheep IgG conjugated with a chelating molecule complexed with Eu3+. The compound 4-(3-(3,4-dichloro-phenyl)-1-methyl-ureido)-butyric acid (CPD) was synthesized and conjugated with bovine serum albumin (BSA) to prepare a solid phase. Sensitivity achieved was 20 ng L-1 below the European Community limits. Paraquat (PQ) and carbofuran (CF) presence in the same samples has been also evaluated in a similar way, using immunoassays with time-resolved revelation systems. Diuron concentration shows a peak coinciding with a peak of carbofuran during summer periods. The peak of diuron was 65 pg/mL in June and 180 pg/mL in September in ditch and lake water samples, respectively; carbofuran concentration was higher than diuron in all samples: a carbofuran peak was revealed in September and October resulting in 87 ng/mL. Herbicide paraquat was not detectable in any assayed sample.     

Development of controlled release formulations of carbofuran and evaluation of their efficacy against Meloidogyne incognita

Controlled release (CR) formulations of the insecti-nematicide carbofuran have been prepared using commercially available rosin, sodium carboxymethylcellulose and sodium carboxymethylcellulose with clay (bentonite, kaolinite, and Fuller's earth). The kinetics of carbofuran release in soil from the different formulations were studied in comparison with that of the commercially available granules (3G). Release from the commercial formulation was faster than with the new CR formulations. Addition of clay in the biodegradable polymer matrix reduced the rate of release. The diffusion exponent (n value) of carbofuran in soil ranged from 0.462 to 0.740 in the tested formulations. The half-release (t1/2) values ranged between 4.79 and 25.11 days, and the period of optimum availability (POA) of carbofuran ranged from 15.10 to 43.97 days. The mean EC50 of the commercial formulation against Meloidogyne incognita was quite high as compared to those of CR formulations. The effective duration (te) of carbofuran from the CR and commercial formulations was predicted by fitting the mean EC50 values of test formulations in the model (M(infinity) - Me)/M(infinity) = Kdte. It was 0.7 day in commercial 3G in comparison with 17.8 days for CMC-bentonite. The bioassay studies revealed that with the rosin-yellow polymer, the dose of carbofuran could be reduced to half of its recommended dose for nematode control. Overall, a comparison of CR formulations with the commercial one showed an earlier degradation of carbofuran in the latter and relatively prolonged activity in the former.     

Identification of peptides in aggregates formed during hydrolysis of beta-lactoglobulin B with a Glu and Asp specific microbial protease

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity

Extensive hydrolysis of whey protein isolate by Alcalase was shown to induce gelation mainly via hydrophobic interactions. The aim of this work was to characterize the peptides released in order to better understand this phenomenon. The apparent molecular mass distribution indicated that aggregates were formed by small molecular mass peptides (<2000 Da). One hundred and thirty peptides with various lengths were identified by reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Alcalase was observed to have a high specificity for aromatic (Phe, Trp, and Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) residues. Most peptides had an average hydrophobicity of 1-1.5 kcal/residue and a net charge of 0 at the pH at which gelation occurred (6.0). Therefore, an intermolecular attractive force such as hydrophobic interaction suggests the formation of aggregates that further leads to the formation of a gel.     

Dependence of the aggregate swelling parameters in soddy-podzolic soils on their properties

The degree and rate of the aggregate swelling in loamy sandy, loamy, and clayey soddy-podzolic soils of northwestern Russia have been measured using the methods of image analysis. The results of the study have shown that the degree of swelling of the soil aggregates depended on the proportions of the fine (0.005 mm) and sandy fractions, and the rate of the swelling depended on the density of the soil solid phase. A reliable effect of the soil organic matter content on the swelling rate of the studied objects has been established. The aggregates of the arable soddy-podzolic soils with the highest content of organic matter, as well as the aggregates of the native soils, have been characterized by the lowest swelling rates. A method has been developed for the determination of the swelling rate of soil aggregates that has enabled the assessment of the soil structure. The regression dependences of the degree and rate of the aggregate swelling on the properties and composition of soddy-podzolic soils have been plotted.     

Inhibitory effects of lignans on the activity of human matrix metalloproteinase 7 (matrilysin)

Inhibitory effects of nine dibenzylbutyrolactone lignans on a human matrix metalloproteinase, matrilysin, were examined. All of the lignans examined inhibited matrilysin with the IC(50) values ranging from 50 to >280 microM. Matairesinol, which has the basic structure of the other lignans, showed the weakest inhibition. Lignans with methylenedioxy ring(s) or a hydroxyl group at the C5-position inhibited matrilysin more strongly than matairesinol. 5-Hydroxypluviatolide, which has both a methylenedioxy ring and a hydroxyl group at the C5-position, was the most potent inhibitor (IC(50) = 50 microM), suggesting that the introduction of these two elements might enhance synergistically the inhibitory activity of lignans. 5-Hydroxypluviatolide inhibited matrilysin in a competitive manner, and its inhibitory effect was greatly suppressed by the presence of another competitive inhibitor, dimethyl sulfoxide. The precursors of matairesinol, coniferyl alcohol and secoisolariciresinol, had no inhibitory activity, indicating that the dibenzylbutyrolactone structure is essential for the inhibition. It has been shown that lignans have the potential to inhibit matrilysin, and the knowledge of their structure-activity relationship might be beneficial to developing selective inhibitors for matrix metalloproteinases.     

Use of bentonite and activated carbon in controlled release formulations of carbofuran

Controlled release systems (CRS), unlike the conventional formulations, facilitate a gradual and controlled discharge of the pesticides, reducing the losses by evaporation and leaching and minimizing pesticide pollution. In this study, carbofuran-an insecticide-nematicide identified as a groundwater pollutant-was incorporated in alginate-based granules to obtain controlled release properties. The effect on carbofuran release rate caused by the incorporation of bentonite, activated carbon, and different mixtures of both sorbents in alginate basic formulation was studied by immersion of the granules in water. The water uptake, sorption capacity of the sorbent, permeability, and time taken for 50% of the active ingredient to be released into water, T(50), were calculated by the comparison of the preparations. T(50) values were higher for those formulations containing bentonite and/or activated carbon (T(50) values range from 14.76 h for the alginate formulation containing only bentonite as the sorbent to 29.5 weeks for the alginate formulation containing only activated carbon as the sorbent) than for the preparation without these sorbents (11.72 h). On the basis of a parameter of an empirical equation used to fit the insecticide-nematicide release data, it appears that the release of carbofuran from the various formulations into water is controlled by a diffusion mechanism. The sorption capacity of the sorbents for carbofuran was the most important factor modulating carbofuran release. In addition, it was observed that there is a linear correlation of the T(50) values and the content of activated carbon in dry granules.     

Release and recovery of nitrogen from winter annual cover crops in no‐till corn production

Abstract

Soil bulk density markedly influences hydrolysis of surface‐applied granular urea that is vulnerable to serious ammonia volatilization losses. In order to decrease the ammonia losses by retarding urea hydrolysis, several chemicals have been tested for their soil urease inhibition properties. Phenyl phosphorodiamidate (PPDA) is a potent soil urease inhibitor. Laboratory studies using soil column incubations were conducted to investigate the effect of soil bulk density on inhibition of hydrolysis of surface‐applied urea granules (=20 mg of urea/granule) containing 1% PPDA in unsaturated soils. The increase in soil bulk density (from 0.69 to 1.50 Mg/m³) markedly increased the rate of hydrolysis of surface‐applied urea granules and significantly decreased the apparent urease inhibition by PPDA present in the granules. These results are attributed to the probable spatial separation of urea and PPDA because of the differences in diffusive transports in unsaturated soils caused in part by differences in their solubilities in water.     

糖接枝处理改善大豆蛋白纤维聚集体泡沫稳定性

为了探究糖接枝对大豆蛋白纤维聚集行为和泡沫性质的影响,明确蛋白质结构与功能的关系,该研究以大豆蛋白(soy protein isolation,SPI)和乳糖(lactose)为原料,通过干热法制备糖接枝大豆蛋白(SPI-lactose conjugate,SPI-Lac),以及在酸性条件下加热诱导其形成纤维聚集体(p H值2.0),制备了一种糖接枝大豆蛋白纤维聚集体(SPI-lactose conjugate fibillar aggregates),并考察了糖接枝对大豆蛋白的纤维聚集行为及泡沫性质的影响。研究结果表明:大豆蛋白在酸性条件下(p H值2.0)经加热后会发生水解,同时水解产物不断聚集形成大分子的纤维聚集体。糖接枝导致大豆蛋白的水解速度下降,但荧光光强和粒径的结果表明糖接枝能增强纤维聚集能力。SPI-Lac在中性条件下的溶解度(p H值5.0—7.0)显著高于SPI(P0.05),且不同时间处理的SPI-Lac纤维聚集体均能改善SPI在酸性条件下的溶解度(p H值2.0—5.0)。此外,不同时间处理的SPI-Lac纤维聚集体在酸性条件下的起泡能力均高于SPI纤维聚集体。SPI和SPI-Lac纤维聚集体的形成会导致SPI起泡能力的下降,但是短时间酸热处理形成的纤维聚集体泡沫稳定性得到显著改善。因此,糖接枝结合短时间酸热处理制备的糖接枝大豆蛋白纤维聚集体在中性条件下的泡沫稳定性显著提高(P0.05),是合理有效的蛋白质改性方法。     

适宜含水率保持油茶籽贮藏品质

为了确定油茶籽贮藏适宜的含水率,研究了在4℃,不同含水率(7%、10%、13%、16%、20%)油茶籽贮藏期间的品质变化。结果表明,较低的含水率能较好保持油茶籽的贮藏特性及营养品质。其中,含水率为7%的油茶籽贮藏效果较好,但与10%处理效果差异不明显(P>0.05)。在整个贮藏期,含水率为7%时油茶籽可溶性蛋白下降了13.05 mg/g,油酸含量下降了2.38%,酸值、过氧化值等品质指标上升速率较慢,同时能较好保持β-谷甾醇和角鲨烯等生物活性成分;其次是10%的含水率处理。而含水率为20%的油茶籽贮藏期间可溶性蛋白下降较快,贮藏结束时为25.47 mg/g,油茶籽劣变严重,所提取的油样品质变差,营养物质含量较少,因此含水率20%的油茶籽不适宜长期贮藏。综合考虑油茶籽品质因素和处理成本,认为控制含水率在10%以下能较好保持油茶籽的贮藏品质。该研究可为科学合理地贮藏油茶籽提供参考。