Development of a microtiter plate ELISA and a dipstick ELISA for the determination of the organophosphorus insecticide fenthion

In previous studies, polyclonal antibodies against the organophosphorus insecticide fenthion were obtained and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for this pesticide. In this study, using these antibodies and an enzyme tracer, direct competitive ELISAs for fenthion in microtiter plate and dipstick formats were developed. The microtiter plate ELISA showed an IC(50) value of 1.2 microg/L with a detection limit of 0.1 microg/L. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides. The use of the dipstick format using Immunodyne as a support membrane allowed the quick visual detection of fenthion in concentrations >10 microg/L. The IC(50) value of the dipstick format using reflectance detection was 15 microg/L with a detection limit of 0.5 microg/L. The recoveries of fenthion from spiked vegetable samples using the two formats without any prior enrichment or cleanup steps were 87-116%.     

New insights into the formation of aroma-active strecker aldehydes from 3-oxazolines as transient intermediates

2-Substituted-5-methyl-3-oxazolines, a novel class of aroma precursors that are able to release the respective Strecker aldehydes by hydrolysis, were identified. Hydrolysis can take place after the addition of water or with human saliva during mastication, respectively. 2-Isobutyl-, 2-sec-isobutyl-, 2-isopropyl, and 2-benzyl-5-methyl-3-oxazolines were synthesized and structurally identified by means of gas chromatography-mass spectrometry (GC-MS) in the electron impact mode and in the chemical ionization mode as well as by one- and two-dimensional NMR experiments. With these compounds at hand, a variety of stability experiments were performed using headspace-GC-MS or proton transfer reaction-MS techniques on the basis of stable isotope dilution assays, proving the ability to release the respective Strecker aldehydes was dependent on the pH value as well as on the hydrolysis time. After the addition of water at 37 °C, for example, >70 mol % of 3-methylbutanal or >40 mol % of phenylacetaldehyde was liberated from a solution of 2-isobutyl-5-methyl-3-oxazoline or 2-benzyl-5-methyl-3-oxazoline, respectively, after 5 min. Furthermore, the presence of 2-isobutyl-5-methyl-3-oxazoline in dark chocolate containing 70% cocoa was proven by GC-MS.     

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the detection of parathion-methyl (PM) was developed and optimized. Fluorescein-labeled PM derivatives (tracers) with different structures were synthesized and purified by thin-layer chromatography. The influence of immunogen and tracer structures on the assay characteristics was investigated. PM concentration determinable by the FPIA ranged from 25 to 10000 ppb. The detection limit was 15 ppb. Methanol extracts of vegetable, fruit, and soil samples were diluted 1/10 for the analysis. Recovery in spiked samples averaged between 85 and 110%. The method developed is characterized by high specificity and reproducibility (CV ranged from 1.5 to 9.1% for interassay and from 1.8 to 14.1% for intra-assay). The FPIA method can be applied to the screening of food and environmental samples for PM residues without complicated cleanup.     

Determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction cleanup

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues. The average recoveries were 86-108% for 8 fortified organochlorine pesticide residues and 61-149% for 28 fortified organophosphorus pesticide residues.     

Comparative study of methods for the extraction of eleven organophosphorus pesticide residues in rice.

Several extraction methods are compared for the simultaneous analysis of organophosphorus pesticides in unpolished rice. Four stationary phases were used for the subsequent gas-liquid chromatographic (GLC) determination of the selected pesticides. Using 3 different GLC columns, 11 pesticides were completely separated and identified. The efficiency of the cleanup and the sensitivity of the analytical method were evaluated by using powdered unpolished rice samples fortified with the pesticides and also wheat and dried bean samples. Average recoveries ranged from 74.7% for disulfoton to 97.4% for malathion in unpolished rice and from 68.1% for disulfoton to 108.3% for malathion in other crops. The method described is applicable to the analysis of selected organophosphorus pesticide residues in unpolished rice, wheat, buckwheat, and dried beans.     

New insights into controversies on the antioxidant potential of the olive oil antioxidant hydroxytyrosol

In the present study, the antioxidant profile of olive oil antioxidants was investigated. Hydroxytyrosol and oleuropein are potent scavengers of hydroxyl radicals (OH*), peroxynitrite (ONOOH), and superoxide radicals (O(2)*(-)). Homovanillic alcohol, one of the main metabolites of hydroxytyrosol, and tyrosol are less potent scavengers of these reactive species. None of the olive oil antioxidants are good hypochlorous acid (HOCl) or hydrogen peroxide (H(2)O(2)) scavengers. Hydroxytyrosol efficiently protects against LDL oxidation in vitro and in vivo. However, no protective effect of hydroxytyrosol is usually demonstrated ex vivo against the oxidation of LDL isolated from humans after hydroxytyrosol consumption. The present study shows that this controversy is due to the isolation of LDL, which greatly reduces the protective effect of hydroxytyrosol against LDL oxidation. Hydroxytyrosol is an efficient scavenger of several free radicals. The physiological relevance of the high intrinsic antioxidant activity of hydroxytyrosol is illustrated by its protection against LDL oxidation.     

New insights on the loess/paleosol Quaternary stratigraphy from key sections in the U.S. Midwest

Three key Quaternary loess/paleosol sections were examined in the Missouri Valley (Iowa), Eustis Ash Pit (Nebraska), and in the Wittsburg Quarry (Arkansas) to gain insights into the sedimentation, environment and climate change of the U.S. Midwest. Four loess units are present separated by three well-developed paleosols. Crowley's Ridge Loess (Oxygen Isotope Stage (OIS) 8) is pre-Illinoian in age, and is the oldest loess unit investigated. A well-developed paleosol, interpreted as Yarmouth Soil (OIS 7), is found in this loess in all three sections. Overlying the Yarmouth Soil is Loveland Loess (OIS 6) which has been pedogenically altered by the Sangamon Soil (OIS 5). It has luvisolic properties but a more clayey pedogenic texture than modern luvisols and has no clear eluvial horizon. The overlying Roxana Silt (OIS 4) is pedogenically altered. At Wittsburg and Missouri Valley, the Farmdale Soil (OIS 3) is developed in the Roxana Silt. Based on pedogenic features, we correlate this paleosol to that developed in the Gilman Canyon Formation at Eustis. In Missouri Valley and Eustis sections, the paleosol has chernozemic properties and therefore, the gleyic features observed at Missouri Valley are interpreted as later alterations. Overlying the Farmdale Soil is the widespread Peoria Loess (OIS 2).     

New insights into an ancient antibrowning agent: formation of sulfophenolics in sodium hydrogen sulfite-treated potato extracts

The effect of sodium hydrogen sulfite (S), used as antibrowning agent, on the phenolic profile of potato extracts was investigated. This extract was compared to one obtained in the presence of ascorbic acid (A). In the presence of A, two major compounds were obtained, 5-O-caffeoylquinic acid (5-CQA) and 4-O-caffeoyl quinic acid. With S, their 2'-sulfo-adducts were found instead, the structures of which were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Also, for minor caffeoyl derivatives and quercetin glycosides, the corresponding sulfo-adducts were observed. Feruloyl and sinapoyl derivatives were not chemically affected by the presence of S. Polyphenol oxidase (PPO) was thought to be responsible for the formation of the sulfo-adducts. This was confirmed by preparing 2'-sulfo-5-O-caffeoyl quinic acid in a model system using 5-CQA, sodium hydrogen sulfite, and PPO. This sulfo-adduct exhibited a small bathochromic shift (λmax 329 nm) as compared to 5-CQA (λmax 325 nm) and a strong hypochromic shift with an extinction coefficient of 9357±395 M(-1) cm(-1) as compared to 18494±196 M(-1) cm(-1), respectively. The results suggest that whenever S is used as an antibrowning agent, the O-quinone formed with PPO reacts with S to produce sulfo-O-diphenol, which does not participate in browning reactions.     

Incorporation of 14C into methionine as an intermediate for phytosiderophore biosynthesis in barley

In order to verify the precursory role of methionine (Met) in the biosynthesis of the mugineic acid family of phytosiderophores (MAs), feeding experiments of ¹⁴C-Iabeled compounds to barley (Hordeum vulgare L. cv. Minorimugi) roots grown hydroponically were conducted. When both ^l4C-Glucose (Glc) and unlabeled Met were fed to segmented roots, ¹⁴C was incorporated into Met and MAs in the roots. Molar-radioactivity of Met was higher than that of the amino-butanoic-acid unit in MAs in the roots. When ^l4C-Glc and unlabeled homoserine (Hse) were fed to decapitated roots, l4C was incorporated into Met but not into Hse. Therefore, it was considered that Hse might not be a major precursor of MAs. In addition, ¹⁴C was incorporated into Met and MAs in the roots when both ¹⁴C-glycerol (Gol) and unlabeled Met were fed to segmented roots. It is suggested that MAs may be synthesized from Glc via Met, bypassing Hse, and that the MAs biosynthesis may involve an unknown pathway associated with Gol and leading to Met.     

New insights into the structural characteristics of the arabinogalactan-protein (AGP) fraction of gum arabic

The structural characteristics of the gum exudate of Acacia senegal (gum arabic) have been investigated by monitoring the composition and physicochemical properties before and after treatment with proteolytic enzyme and various alkaline systems. Molecular mass ( M w) and radius of gyration ( R g) measurements were performed using gel permeation chromatography (GPC) coupled to refractive index, UV absorbance, and multiangle light scattering detectors and indicated that the macromolecules present have a compact structure. It was found that treatment with proteolytic enzyme caused the arabinogalactan-protein component (AGP) with average molecular mass approximately 2 x 10 (6) Da to degrade, yielding material of molecular mass approximately 4 x 10 (5) Da, whereas the bulk of the material corresponding to the protein-deficient arabinogalactan component (AG) with molecular mass 4 x 10 (5) remained unaffected. Barium hydroxide was found to hydrolyze the polysaccharide component (AG) itself in addition to the proteinaceous component as demonstrated in control experiments using dextran. However, sodium borohydride/sodium hydroxide treatments were unable to hydrolyze dextran and were assumed to hydrolyze only the proteinaceous component of gum arabic. The AGP component was completely degraded, yielding material of molecular mass approximately 4.5 x 10 (4) Da. It has been concluded, therefore, that the enzyme did not fully hydrolyze all of the protein present and that the AGP component of gum arabic consists of carbohydrate blocks of approximately 4.5 x 10 (4) Da linked to a polypeptide chain consistent with the wattle blossom structure. Because the AGP was degraded to differing extents using a mild and more severe sodium borohydride/sodium hydroxide treatment, it was concluded that the polysaccharide moieties were linked through both O-serine and O-hydroxyproline residues. The gum arabic sample was deglycosylated by treatment with anhydrous hydrogen fluoride and revealed the presence of two putative core proteins of approximately 3 x 10 (4) and approximately 5 x 10 (3) Da, respectively, which correspond to proteins of approximately 250 and 45 amino acids in length. A new model for the structure of the AGP component has been proposed.     

New insights into the role of iron in the promotion of lipid oxidation in bulk oils containing reverse micelles

Formation of physical structures, known as association colloids, in bulk oils can promote lipid oxidation. However, the cause of this accelerated lipid oxidation is unknown. Therefore, the aim of this study was to investigate whether transition metals were important prooxidants in bulk oils containing reverse micelles produced from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and water. The Fe(III) chelator deferoxamine (DFO) increased the oxidative stability of stripped soybean oil (SSO) containing reverse micelles from 2 to 7 days. Because phosphatidylcholine (1,2-dibutyl-sn-glycero-3-phosphocholine) that does not form reverse micelles is not prooxidative, these results suggest that the prooxidant activity of DOPC reverse micelles could be due to their ability to concentrate both endogenous iron and lipid hydroperoxides at the water-lipid interface, thereby increasing the ability of iron to decompose lipid hydroperoxides. DFO was also able to improve the activity of α-tocopherol and Trolox in SSO containing DOPC reverse micelles increasing the lag phase from 2 to 11 and 13 days, respectively. DOPC reverse micelles decreased iron-promoted α-tocopherol and Trolox decomposition and decreased the ability of α-tocopherol and Trolox to decrease Fe(III) concentrations. Overall, these results suggest that iron is an important prooxidant in bulk oils containing reverse micelles; therefore, finding ways to control iron reactivity in association colloids could provide new technologies to increase the oxidative stability of oils.     

New insights into the structure of apolipoprotein B from low-density lipoproteins and identification of a novel YGP-like protein in hen egg yolk

Apoproteins of low-density lipoproteins (LDL) and soluble proteins (livetins) contained in hen egg yolk plasma have been demonstrated as being essential to the interfacial and emulsifying properties of yolk. The knowledge of their structure is necessary to better understand these properties. Purified protein fractions were separated by SDS-PAGE or 2D-PAGE and identified through the LC-MS/MS of their trypsin peptides. Hen blood apolipoprotein B gives rise to nine different apoproteins in LDL after maturation and proteolysis. Among these apoproteins, two protein fragments appeared to be less accessible to proteases and could be enriched in beta-sheets and firmly associated with lipids. Plasma soluble proteins were constituted by approximately 45% of yolk immunoglobulins with a high heterogeneity of the variable regions of both heavy and light chains, 41% of glycoproteins constituted by YGP42 and YGP40, 14% of albumins, and one new minor protein we called YGP30, showing 75% similarity to YGP40.     

Thermal stability of organophosphorus pesticide triazophos and its relevance in the assessment of risk to the consumer of triazophos residues in food

The degradation of triazophos in aqueous solutions was monitored at 205 and 254 nm after separation using high-performance liquid chromatography. An ODS column was used with a mobile phase of 60% acetonitrile and 0.04% phosphoric acid at a flow rate of 1.4 cm(3) min(-)(1). When dissolved in distilled water, approximately 30% of the original triazophos was detected. The effect of heating time and temperature on a 0.5 mg dm(-3) standard was investigated. Over a 150 min period at 100 degrees C the peak area detected for the standard decreased by 58.67 +/- 6.19 and 65.03 +/- 4.61% when measured at 254 and 205 nm, respectively. The precision of the absorbance detected at 205 and 254 nm was 3.54 +/- 2.8 and 3.86 +/- 3.9%, respectively. There was a significant difference (P = 0.10) between the precision of the results obtained at each wavelength. The t(calcd) value was -2.236 and the t(crit) value was 1.94. The most sensitive wavelength was 205 nm. A 54% difference in the gradients of the calibration graphs obtained at each wavelength was observed. The results suggest that approximately 72% of triazophos is degraded during a 20 min cooking period at 100 degrees C, due to ambient and elevated temperature hydrolysis. Therefore, the dose to the consumer of triazophos residues in cooked food is likely to be approximately 72% lower than in the raw food, with a concomitant reduction in toxicological risk.     

Thermally generated 3-aminopropionamide as a transient intermediate in the formation of acrylamide

On the basis of the recent findings that "biogenic amines" can also be formed during thermal food processing from their parent amino acids in a Strecker-type reaction, the formation of 3-aminopropionamide, the biogenic amine of asparagine, was investigated in model systems as well as in thermally processed Gouda cheese. The results of model studies revealed that, besides acrylamide, 3-aminopropionamide was also formed in amounts of 0.1-0.4 mol % when asparagine was reacted in the presence of either glucose or 2-oxopropionic acid. Results of a second series of model experiments in which [(13)C(4)(15)N(2)]-asparagine ([(13)C(4)(15)N(2)]-Asn) and unlabeled 3-aminopropionamide were reacted together in the presence of glucose revealed a >12-fold higher efficacy of 3-aminopropionamide in acrylamide generation as compared to asparagine. Both [(13)C(3)(15)N(2)]-3-aminopropionamide and [(13)C(3)(15)N(1)]-acrylamide were formed during [(13)C(4)(15)N(2)]-Asn degradation in a ratio of about 1:4, supporting the idea that 3-aminopropionamide is a transient intermediate in acrylamide formation. In this study, 3-aminopropionamide was identified and quantified for the first time in foods, namely, in Gouda cheese. Although the fresh cheese contained low amounts of 3-aminopropionamide, its concentrations were much increased to approximately 1300 mug/kg after thermal processing. In isotope labeling studies, performed by administering to the cheese [(13)C(4)(15)N(2)]-Asn in a ratio of 1:2 as compared to the "natural" concentrations of asparagine, similar ratios of unlabeled/labeled 3-aminopropionamide and unlabeled/labeled acrylamide were determined. Thus, 3-aminopropionamide could be verified as a transient intermediate of acrylamide formation during food processing.     

Large-scale preparation of the phytoalexin elicitor glucohexatose and its application as a green pesticide

Large-scale preparation of the phytoalexin elicitor was achieved through a highly regio- and sterereoselective synthesis using 2,3,4,6-tetra-O-benzoyl-d-glucopyranosyl trichloroacetimidate (1), 1,2:5,6-di-O-isopropylidene-alpha-d-glucofuranose (2), and 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-d-glucopyranosyl trichloroacetimidate (3) as the synthons. Coupling of 1 with 2 gave the 1-->3-linked disaccharide; subsequent selective removal of 5,6-O-isopropylidene to give 5 followed by selective 6-O-glycosylation with 1 afforded the trisaccharide 6. Hydrolysis to remove the 1,2-O-isopropylidene was accompanied by ring expansion, giving 3,6-branched pyranosyl trisaccharide. Acetylation, selective 1-O-deacetylation, and activation with trichloroacetonitrile gave the trisaccharide donor 7. The trisaccharide acceptor 9 was prepared from condensation of the disaccharide 5 with 3 and subsequent 6-O-deacetylation. Coupling of the trisaccharide donor 7 with the trisaccharide acceptor 9 and subsequent deprotection afforded the glucohexatose elicitor. The cost of the produced glucohexatose should be low enough to allow its applications in agriculture as a green pesticide. At a concentration of 5-10 mg/L, the resultant elicitor was used to treat growing orange trees and harvested oranges, giving very encouraging results, comparable with those obtained using commercial pesticides at a concentration of 1400 mg/L (Topsin-M) for growing trees and 900 mg/L (Tecto) for harvested oranges, respectively. Treatment of tomato leaves against Botrytis cinerea with the synthetic elicitor at a concentration of 10 mg/L gave 82% inhibition, comparable with the inhibition of 84% by Wanmeiling at a concentration of 1000 mg/L. Treatment of tea leaves also showed promising results.     

Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody

Phosphonic acid (trans-4-phosphono-2-butenic acid; TPB) was used as a generic hapten in order to generate broad specificity antibodies against a group of organophosphorus pesticides. The polyclonal antiserum showed, in an indirect enzyme-linked immunosorbent assay (ELISA) format, preferential binding toward pesticides containing unsaturated diethyl-phosphate functionalities rather than the equivalent thiophosphate or dimethyl structures. The level of detection in the ELISA using a heterologous system was investigated and showed a 20-fold improvement when a conjugate for which the antibody had lower affinity was immobilized on the plate. Biosensor assays using parathion as a standard indicated that the antibody had a relatively high dissociation rate, and reproducible cycles of regeneration were achieved. The potential for using TPB as a generic hapten is discussed.     

New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

把小流域治理提高到一个新水平

1980年4月水利部在山西省吉县召开的13省、区水土保持小流域治理座谈会上,明确了小流域的概念和标准,提出了我国水土保持要以小流域为单元进行综合治理的要求。从此,我国的水土保持工作很快地进入了一个新的发展阶段,积累了丰富经验,促成了规模治理,显示了整体效益,加快了治理速度,在小流域治理内涵上也得到了不断地深化。1984年以来,水利部有关领导同志又先后提出:把小流域建设成为发展商品生产的基地,寓经济效益于治理措施之中;措施配置和发展方向要与农村产业结构调整相吻合,突出经济效益;小流域治理要向“高、深、细”发展;并制定了“水土保持小流域管理办法”,使小流域治理工作逐步走向规范化、制度化的轨道。小流域治理如何向“高、深、细”发展,这是当前小流域治理深化的一个重大课题。笔者根据这几年小流域治理经验和10年试点实践,提出了“高、深、细”的应有内容。供同志们深入探讨,以把小流域治理水平再推上一个新台阶。