Evaluation and validation of a commercially available enzyme-linked immunosorbent assay for the neonicotinoid insecticide imidacloprid in agricultural samples

The performance of a commercially available microtiter plate ELISA kit for the determination of the neonicotinoid insecticide imidacloprid was evaluated for sensitivity, selectivity, influence of organic solvent used for extraction procedure, matrix interference originated from agricultural sample, accuracy, and method comparison with conventional HPLC analysis. The limit of detection for the kit (0.1 or 0.5 ng/mL) was determined. The working range (1-39 ng/mL) experimentally calculated on the basis of a criterion, which is determined as the range from I(20) to I(80), was comparable to that established by the manufacturer (1-50 ng/mL). The linearity of the standard curve based on the kit-assembled standard solutions agreed with the one based on the self-made standard solutions. Specificity studies indicate that the imidacloprid monoclonal antibody can readily distinguish the target compound from other structurally related neonicotinoid analogues and some metabolites, with the exception of clothianidin, the cross-reactivity of which was approximately 12%. To extract imidacloprid from an agricultural sample (apple) as simply and rapidly as possible, some extraction methods were examined. Consequently, the extraction method with hand-shaking for 5 min was the best among the examined methods. For the analysis of imidacloprid in apple samples, it was extracted directly with methanol and the extracts were diluted 10-fold (100-fold in the well) with water prior to ELISA analysis. No significant matrix interference was observed with the dilution factor. Recoveries of imidacloprid from fortified apple samples ranged from 87.7 to 112.0%. The results obtained with the ELISA kit correlated well with those by the reference method (conventional HPLC analysis) for apple samples (r > 0.998). These findings strongly indicate that the ELISA kit may be employed routinely for an on-site imidacloprid residue analysis of apple samples.     

Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice

Apoptotic cells expose phosphatidylserine and are swiftly engulfed by macrophages. Milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and that enhances the engulfment of apoptotic cells by macrophages. We report that tingible body macrophages in the germinal centers of the spleen and lymph nodes strongly express MFG-E8. Many apoptotic lymphocytes were found on the MFG-E8-/- tingible body macrophages, but they were not efficiently engulfed. The MFG-E8-/- mice developed splenomegaly, with the formation of numerous germinal centers, and suffered from glomerulonephritis as a result of autoantibody production. These data demonstrate that MFG-E8 has a critical role in removing apoptotic B cells in the germinal centers and that its failure can lead to autoimmune diseases.     

Effect of 3-O-octanoyl-(+)-catechin on the responses of GABA(A) receptors and Na+/glucose cotransporters expressed in xenopus oocytes and on the oocyte membrane potential

Recently, 3-O-octanoyl-(+)-catechin (OC) was synthesized from (+)-catechin (C) by incorporation of an octanoyl chain into C in the light of (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), which are the major polyphenols found in green tea and have strong physiological activities. OC was found to inhibit the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA(A) receptors) and Na+/glucose cotransporters expressed in Xenopus oocytes in a noncompetitive manner more strongly than does C. OC also induced a nonspecific membrane current and decreased the membrane potential of the oocyte, and thus death of the oocyte occurred even at lower concentrations than that induced by C or EGCg. Although EGCg produced H2O2 in aqueous solution, OC did not. This newly synthesized catechin derivative OC possibly binds to the lipid membrane more strongly than does C, Ecg, or EGCg and as a result perturbs the membrane structure.     

Extended glucuronidation is another major path of cyanidin 3-O-beta-D-glucopyranoside metabolism in rats

We previously determined that five rather hydrophobic metabolites appeared in blood plasma after oral administration of cyanidin 3-O-beta-D-glucopyranoside, but a group of hydrophilic metabolites still remained unidentified. In the present study, 12 hydrophilic metabolites found were collected from urine and plasma samples by high-performance liquid chromatography (HPLC) and then analyzed by tandem MS spectrometry. From the MS spectra, four metabolites out of 12 were assigned as glucuronides of cyanidin 3-O-beta-D-glucopyranoside and six out of 12 were glucuronides of the primary metabolites of cyanidin 3-O-beta-D-glucopyranoside (O-methyl cyanidin 3-O-beta-D-glucopyranoside). Extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and O-methyl cyanidin 3-O-beta-D-glucopyranoside showed their maximum plasma concentrations at 15 and 60 min (or 30 min) after oral administration, respectively. Their maximum plasma concentrations ranged from 15 to 70 nM. From the profile of urinary-excreted anthocyanins after intravenous administration, it was deduced that extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and O-methyl cyanidin 3-O-beta-D-glucopyranoside were mainly produced in the liver rather than by intestinal flora. The area under the plasma concentration curve was 0.25 micromol min/L for extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and 0.14 micromol min/L for O-methyl cyanidin 3-O-beta-D-glucopyranoside, respectively, when evaluated as cyanidin 3-O-beta-D-glucopyranoside equivalent, indicating that extended glucuronidation is a critical pathway in cyanidin 3-O-beta-D-glucopyranoside metabolism in rats.     

Differentiation and elimination of uterine natural killer cells in delayed implantation and parturition mice

To clarify the roles of uterine natural killer (uNK) cells in implantation and parturition, differentiation and elimination of uNK cells in the pregnant uterus was examined using artificial delayed implantation (DI) and delayed parturition (DP) mice. To prepare DI mice, pregnant mice were ovariectomized on the third day of pregnancy (D3) and treated with 2 mg progesterone daily. The same amount of progesterone was administered on D15 or D17 of normal pregnant mice at 24 h intervals until sampling to prepare DP mice. The uNK cells contained PAS-positive granules on D8 in DI mice. The uNK cells in DI mice were smaller in size, and differentiation of these cells was delayed compared to those of the control mice. From D19 to D21 in DP mice, the metrial gland was well developed and uNK cells were present. The number of uNK cell granules decreased on D21, and there were no uNK cells in the normal pregnant mice. This result suggests that differentiation of uNK cells is not directly related to implantation, but elimination of these cells is closely involved in parturition.     

Histological study of granulated metrial gland (GMG) cells in beige (DA-bg/bg) rats

To clarify the roles of granulated metrial gland (GMG) cells for successful pregnancy in rats, GMG cells in beige rats (genotype: DA-bg/bg), whose NK cells show lysosomal dysfunction because of abnormalities in cytoplasmic granules, were examined in mid- and late-pregnancy by light and electron microscopies. The GMG cells of beige rats were significantly less in number than those of the two controls (genotypes: DA-bg/+ and DA-+/+) in mid- and late-pregnancy, and this accompanied a low reproductive performance in the beige rats. The size of intracellular granules in the GMG cells of the beige rats was larger than for the two controls on each corresponding day of pregnancy. These results suggest that the activity of rat GMG cells and peripheral NK cells might be influenced by the beige gene, which is involved in reproductive performance.     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.