Comparative inhibition of the juvenile hormone esterases from Trichoplusia ni,Tenebrio molitor, and Musca domestica

Twenty-seven compounds were screened as potential inhibitors of juvenile hormone esterases. Of these compounds O-ethyl-S-phenyl phosphoramidothiolate provided the best inhibition for the cabbage looper, Trichoplusia ni (Hubner), and the yellow mealworm, Tenebrio molitor L., while the juvenile hormone esterases of the house fly, Musca domestica L., were best inhibited by a juvenoid carbamate (1-(m-phenoxy-N-ethyl carbamate)-3,7-dimethyl-7-methoxy-2E-octene). The inhibition patterns of T. ni and T. molitor are similar, while those of M. domestica are relatively different. Further studies on the juvenile hormone and α-napthyl acetate esterases of T. ni showed that they could be differentially inhibited. Diisopropyl phosphorofluoridate and an alkyl trifluoromethyl ketone selectively inhibit the hydrolysis of α-naphthyl acetate and juvenile hormone, respectively, while O-ethyl-S-phenyl phosporamidothiolate inhibits both enzymes. The juvenile hormone esterases of T. ni also appear to be unique enzymes that are selective for juvenile-hormone-like molecules. The in vivo inhibition of T. ni juvenile hormone esterases by O-ethyl-S-phenyl phosphoramidothiolate slows the in vivo hydrolysis of juvenile hormone and results in delayed pupation and malformed larvae that resemble larval-pupal intermediates. Thus, the esterases involved in juvenile hormone metabolism appear to be important in juvenile hormone regulation.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid cypermethrin

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of cypermethrin was developed. Two haptens, the trans- and cis-isomers of 3-[(+/-)-cyano-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarbonyloxy]methyl]phenoxyacetic acid, were conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for cypermethrin was optimized and characterized. The IC(50) for cypermethrin was 13.5 +/- 4.3 microg/L, and the lower detection limit (LDL) was 1.3 +/- 0.5 microg/L. This ELISA had relatively low cross-reactivities with other major pyrethroids, such as deltamethrin, phenothrin, resmethrin, fluvalinate, and permethrin. Methanol was found to be the best organic cosolvent for this ELISA, with an optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was applied to various domestic and environmental water samples. The water samples, fortified with cypermethrin, were analyzed according to this method. Good recoveries and correlation with spike levels were observed.     

Development of an enzyme-linked immunosorbent assay for the detection of the organophosphorus insecticide acephate

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the organophosphorus insecticide acephate, O,S-dimethyl acetylphosphoramidothioate, was developed using a polyclonal antibody. Five different haptens mimicking the analyte were synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) by the N-hydroxysuccinimide active ester and diazotization methods. Polyclonal antibodies raised against hapten-KLH conjugates in rabbits and hapten-BSA conjugates as coating antigens were screened and selected for the assay in the homologous and heterologous ELISA systems. The effects of various assay conditions such as detergent, organic solvents, pH, and preincubation of the mixture of the polyclonal antibody and the analyte on the sensitivity were evaluated. The IC(50) value for acephate was 25 ng/mL in an optimized heterologous system using hapten-4-BSA as a coating antigen and a polyclonal antibody no. 8377 against hapten-1-KLH, showing the detection range of 5-140 ng/mL and the lowest detection limit of 2 ng/mL. The cross-reactivities of the structurally related organophosphorus insecticides, including the major metabolite of the analyte, methamidophos, were less than 1%. Recoveries from the analyte-fortified tap water, mulberry leaves, and lettuce samples in the assay were in the range of 72-121% by simple extraction, concentration, and dilution. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring acephate residues in environmental and agricultural samples.     

Cloning and characterization of two glutathione S-transferases from pyrethroid-resistant Culex pipiens

BACKGROUND: The Marin strain of Culex pipiens Say is a pyrethroid‐resistant population that was collected in Marin County, California, in 2001 and subsequently maintained in the laboratory under regular permethrin exposure. RESULTS: In this study, two cDNAs, CpGSTd1 and CpGSTd2, encoding glutathione S‐transferase (GST) were cloned from Cx. pipiens Marin. Phylogenetic analysis of the deduced amino acid sequences, CpGSTD1 and CpGSTD2, of these genes indicated that they belong to the Delta class of insect GSTs. The nucleotide and deduced amino acid sequences of CpGSTd1 and CpGSTd2 were 59 and 48% identical respectively. CpGSTD1 and CpGSTD2 were expressed in Escherichia coli and purified by affinity chromatography. The recombinant GSTs exhibited unique selectivity towards the general GST substrates 1‐chloro‐2,4‐dinitrobenzene (CDNB) and 1,2‐dichloro‐4‐nitrobenzene (DCNB), and also differed in their sensitivity to known inhibitors of GSTs. CpGSTD1 exhibited peroxidase activity with cumene hydroperoxide, while CpGSTD2 appeared to lack this activity. CpGSTD1 was able to metabolize 1,1,1‐trichloro‐2,2‐bis(4‐chlorophenyl)ethane (DDT), while DDT metabolism by CpGSTD2 was not detectable. CpGSTD1 and CpGSTD2 showed no detectable metabolism of permethrin. Gene expression of CpGSTd1 and CpGSTd2 in Marin mosquitoes was elevated about twofold in comparison with that found in a pyrethroid‐sensitive mosquito strain. CONCLUSION: The results indicate that CpGSTD1 and CpGSTD2 have unique biochemical characteristics, but they do not appear to play major roles in permethrin resistance in Marin mosquitoes. Copyright © 2012 Society of Chemical Industry     

The apparent in vivo neuromuscular effects of the δ-endotoxin of Bacillus thuringiensis var. israelensis in mice and insects of four orders

Alkaline-dissolved crystal δ-endotoxin from Bacillus thuringiensis var. israelensis (serovar H 14) was injected into mice and seven species of insects representing the orders Lepidoptera, Orthoptera, Coleoptera, Hemiptera, and Diptera. High in vivo toxicity, at 1 to 5 ppm (μg toxin/g body wet wt), was observed with mice and some insects, including some that are not sensitive to the toxin when administered orally. Neuromuscular effects were observed when the toxin was injected directly into the body cavity of the test animals. Biochemical studies suggested that different protein fragments within the crystal δ-endotoxin may be responsible for the majority of the mosquito larvacidal activity and the neurotoxic symptoms observed in larvae of Trichoplusia ni.     

Clinical signs and treatment of chronic uterine torsion in two mares

Two mares were evaluated because of chronic uterine torsions of 2 and 4 weeks' duration; both were pyrectic, anemic, tachycardic, and anorectic, had signs of depression, and had an abnormal uterus and contents on transrectal examination. Both mares underwent cesarean section for lysis of adhesions from the uterus to the peritoneum, correction of the torsion, and ovariohysterectomy Both mares recovered with only minor complications and returned to be used as riding horses. Chronic uterine torsion should be considered in mares in late stages of gestation that have vague clinical signs and transrectal palpation findings that are unlike those described in typical cases of uterine torsion. Prognosis for life can be good after treatment by ventral midline celiotomy, cesarean section, correction of the torsion, and ovariohysterectomy.     

Use of immunochemical techniques for the analysis of pesticides

Immunochemical assays for small molecules such as pesticides are rapidly gaining acceptance among analytical chemists. These techniques are rapid, sensitive, cost effective and can easily cope with large sample loads. This review lists the advantages and disadvantages of the technique and describes the steps in assay development using examples from this laboratory, particularly the thiocarbamate herbicide molinate and the triazine herbicides. The focus is primarily on hapten synthesis strategies, assay format considerations, sample preparation and assay validation.     

Inhibition of epoxidation of methyl farnesoate to juvenile hormone III by cockroach corpus allatum homogenates

Radiolabeled methyl farnesoate is epoxidized to juvenile hormone III by an NADPH-dependent reaction occurring in corpus allatum homogenates from the cockroach Blaberus giganteus L. Most of the enzymatically produced juvenile hormone has the 10R configuration described for previously isolated natural juvenile hormones. The unnatural 2Z geometrical isomer of methyl farnesoate is epoxidized by the above system faster than the natural 2E isomer. Several series of chemicals known to be inhibitors of mixed-function oxidases were surveyed as inhibitors of methyl farnesoate epoxidation. The anti-juvenile hormone precocene II caused negligible inhibition at 1 · 10^?4M, whereas the best inhibitor was o-bromophenoxymethyl-imidazole with an apparent I₅₀ of 4 · 10^?7M. None of the inhibitors tested were potent morphogenetic agents on Tenebrio molitor pupae, and they failed to cause precocious development of Oncopeltus fasciatus nymphs. The inhibition of in vitro juvenile hormone biosynthesis suggests the possibility of finding an anti-hormone which acts by blocking juvenile hormone biosynthesis.     

Trifluoromethylketones as possible transition state analog inhibitors of juvenile hormone esterase

A series of compounds containing the trifluoromethylketone group have been synthesized utilizing either a modified Grignard procedure or by reacting selected aliphatic bromides or tosylates with the Collman reagent [Na₂Fe(CO)₄]. When tested in vitro as inhibitors of crude juvenile hormone esterase from the hemolymph of the cabbage looper, Trichoplusia ni (Noctuidae), the most active compounds were trifluoromethylketones possessing either a juvenoid-like structure or a straight aliphatic chain. The logarithm of the inhibitory potency of the aliphatic compounds was proportional to their chain length, up to 1,1,1-trifluorotetradecan-2-one (I₅₀ = 1 × 10^?7M). This powerful inhibition was found to be highly selective for JHE, reversible, competitive by Lineweaver-Burk analysis, and was characterized by high affinity of the inhibitor for the esterase (K_i = 3.2 × 10^?9M, K_m JH III = 2 × 10^?7M). Other trifluoromethylketones were shown to be inhibitors of T. ni α-naphthylacetate esterase and bovine trypsin. By analogy with the mechanism of trypsin action, trifluoromethylketones are probably potent inhibitors due to their resemblance to a tetrahedral transition state on the reaction coordinate to the acylated enzyme.