Mode of action of the plant-derived silphinenes on insect and mammalian GABAA receptor/chloride channel complex

The silphinenes are tricyclic sesquiterpenes that have antifeedant and toxic effects in insects and structural similarity to the known GABA antagonist, picrotoxinin. In murine synaptoneurosomes, silphinenes block GABA-stimulated influx of ³⁶Cl⁻ with EC₅₀s in the range of 10-30 μM. In insects, silphinenes were tested in neurophysiological recordings of central neurons from third instar Drosophila melanogaster larvae. Silphinenes reversed the blockage of neuronal firing induced by GABA, but had little effect below 100 μM. The structure-activity profile observed in the murine chloride flux assay was also observed in the larval neurophysiological assay, indicating little selectivity for the silphinenes. A reference silphinene was equally active on nerve preparations from the rdl strain of D. melanogaster, which is resistant to channel-blocking antagonists via an altered GABA receptor. This latter finding suggests that silphinenes interact with the insect GABA receptor in a manner somewhat different from PTX, and that rdl resistance in the field may have little effect on silphinene efficacy.     

Biochemical identification of putative GABA/benzodiazepine receptors in house fly thorax muscles

[³H]Flunitrazepam ([³H]Flu) was used to identify benzodiazepine binding sites in house fly thorax muscle membranes using a filter assay. [³H]Flu bound to a finite number of sites in a concentration- and time-dependent manner, reaching equilibrium in 10 min. Scatchard plots of the binding indicated a high-affinity site at 0.2 pmol/mg protein (K_d 24.3 nM) and a low-affinity site at 8.2 pmol/mg protein (K_d994nM). Binding of [³H]Flu to the high-affinity binding site was inhibited by several benzodiazepine analogs, with Flu, diazepam, and Ro 5-4864 being more potent than β-CCE, Ro 5-3027, and Ro 5-2180. Clonazepam was least potent in inhibiting [³H]Flu binding. Thus, the drug specificity of these insect muscle benzodiazepine binding sites was quite different from both the mammalian central and peripheral benzodiazepine receptor sites, though closer to the peripheral ones. GABA (γ-aminobutyric acid) and its agonists enhanced the specific binding of [³H]Flu in a dose-dependent manner, and this effect was inhibited with the GABA antagonist bicuculline. The effect was biphasic since at high GABA concentrations this stimulation was reduced. The data suggest that house fly muscles have benzodiazepine receptors, which are coupled allosterically to GABA receptors, analogous to the GABA/benzodiazepine receptors of vertebrates, but with some differences in their drug specificities.     

Alterations of offspring heart muscle electrical activity transferred by rat male genitors chronically treated with lindane (γ-hexachlorocyclohexane) trace concentrations

Using intracellular microelectrodes, we studied transmembrane resting and action potentials (AP) of left ventricle papillary muscles isolated from the heart of adult lindane-treated (TMG) and untreated (UMG) male genitor rat offspring, obtained by mating untreated female with males chronically treated and untreated with lindane (2 ppb) trace concentrations through beverage. The AP magnitude and duration (APD) were similar in both groups and their response to low temperature (22 °C) unchanged. Lowering the external Ca²⁺ concentration from 2.5 to 0.625 mM prolonged APD in the TMG group but not in the UMG group. In the TMG group, (i) cumulative addition of Sr²⁺ (1 mM) to the physiological solution prolonged APD; (ii) apamin (4 μM) and charybdotoxin (4 μM) prolonged the APD. In conclusion, our data revealed that an altered sensitivity of the Ca²⁺-induced Ca²⁺ inactivation of L-type Ca²⁺ channels and of Ca²⁺-activated K⁺ channels to Ca²⁺ has been transferred to TMG offspring.     

Quantitative structure-activity relationships of monoterpenoid binding activities to the housefly GABA receptor

BACKGROUND: Monoterpenoids are a large group of plant secondary metabolites. Many of these naturally occurring compounds have shown good insecticidal potency on pest insects. Previous studies in this laboratory have indicated that some monoterpenoids have positive modulatory effects on insect GABA receptors. In this study, the key properties of monoterpenoids involved in monoterpenoid binding activity at the housefly GABA receptor were determined by developing quantitative structure‐activity relationship (QSAR) models, and the relationship between the toxicities of these monoterpenoids and their GABA receptor binding activities was evaluated. RESULTS: Two QSAR models were determined for nine monoterpenoids showing significant effects on [³H]‐TBOB binding and for nine p‐menthane analogs with at least one oxygen atom attached to the ring. The Mulliken charges on certain carbon atoms, the log P value and the total energy showed significant relationships with binding activities to the housefly GABA receptor in these two QSAR models. CONCLUSIONS: From the QSAR models, some chemical and structural parameters, including the electronic properties, hydrophobicity and stability of monoterpenoid molecules, were suggested to be strongly involved in binding activities to the housefly GABA receptor. These findings will help to understand the mode of action of these natural insecticides, and provide guidance to predict more monoterpenoid insecticides. Copyright © 2012 Society of Chemical Industry     

Antimicrobial activity of essential oils and their components against the three major pathogens of the cultivated button mushroom, Agaricus bisporus

Essential oils of Matricaria chamommilla, Mentha piperita, M. spicata, Lavandula angusti folia, Ocimum basilicum, Thymus vulgaris, Origanum vulgare, Salvia officinalis, Citrus limon and C. aurantium and their components; linalyl acetate, linalool, limonene, α-pinene, β-pinene, 1,8-cineole, camphor, carvacrol, thymol and menthol were assayed for inhibitory activity against the three major pathogens of the button mushroom, Agaricus bisporus, i.e. the fungi Verticillium fungicola and Trichoderma harzianum and the bacterium Pseudomonas tolaasii. The highest and broadest activity was shown by the Origanum vulgare oil. Carvacrol possessed the highest antifungal activity among the components tested.     

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [³H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [³H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [³H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [³H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [³H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B_1a, were potent inhibitors of [³H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.     

Muscarinic receptor in house fly brain and its interaction with chlorobenzilate

Membranes from house fly heads were tested for the presence of mucarinic acetylcholine receptors using as a probe [³H]quinuclidinyl benzilate ([³H]QNB). Based on the presence of saturable and reversible high-affinity binding of [³H]QNB, which is inhibited by muscarinic drugs, it is suggested that these sites may be muscarinic receptors. However, these putative muscarinic receptors differ in several characteristics from the ones in mammalian brain. They have lower affinities for muscarinic drugs and lower stereoselectivity, a relatively higher affinity for the nicotinic antagonist d-tubocurarine, a lower Hill coefficient for binding of muscarinic antagonists, and a lower concentration relative to α-bungarotoxin binding sites in the same membranes. Also, unlike mammalian muscarinic receptors, they are sensitive to treatments with N-ethylmaleimide and 5,5′-dithiobis(2-nitrobenzoic acid). The effect of reduction of disulfide bonds by dithiothreitol or mercaptoethanol suggests that only the insect receptor has one or more disulfide bonds which are important to binding. On the other hand, the putative muscarinic receptors of both insect and mammalian brains have important SH group(s), whose alkylation by p-chloromercuribenzoate inhibits binding. Also, chlorobenzilate is equally effective in inhibiting [³H]QNB binding to muscarinic putative receptors of house fly and bovine brains.     

Characterization of high affinity binding of [3H]propyl bicyclic phosphate to house fly head extracts

The head of the house fly, Musca domestica L., was found to contain saturable components of specific binding of 4-n-propyl[2,3-³H]-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane 1-oxide ([³H]Pr-BP). A ratio of specific to total binding of radioreceptor assays was quite favorable, being 0.89 under the standard conditions. The apparent dissociation constant and maximal binding capacity were estimated to be about 4 nM and about 30 fmol/mg of protein, respectively, although association, dissociation, and saturation analyses suggested the presence of two or more populations of binding sites. Specific [³H]Pr-BP binding showed a marked negative temperature coefficient and was little affected by pH in incubation media. Anions which pass through chloride channels attenuated specific [³H]Pr-BP binding whereas impermeable anions enhanced it. Specific binding was selectively inhibited by insecticidal bicyclic phosphorus esters. Various neuroactive chemicals such as GABA agonists, GABA antagonists, cyclodiene insecticides, and benzodiazepines had little effect on specific binding. There was a correlation between GABA content and the density of specific Pr-BP binding sites in each part of the house fly body. In many respects, however, characteristics of the current binding site were different from those of GABA receptor-coupled sites already characterized with [³H]Pr-BP and the [³⁵S]t-butyl thiono analog in the rat brain. Bicyclic phosphorus esters may act on site(s) apart from the GABA neurotransmission system in the house fly.     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

Inhibition of Na-K-ATPase in different tissues of freshwater fish Channa punctatus (Bloch) exposed to monocrotophos

Freshwater fish, Channa punctatus, commonly known as the snakehead fish, was exposed to two sublethal concentrations (0.96 and 1.86 mg/L) (selected on the basis of 1/20 and 1/10 of 96 h LC₅₀ value) of monocrotophos for two exposure periods (15 and 60 days). Effects of monocrotophos on Na⁺, K⁺-ATPase in liver, kidney, muscle, intestine, brain, heart and gills were determined. Results indicate that Na⁺, K⁺-ATPase activity in tissues decreased as concentration of monocrotophos and exposure period increased. Monocrotophos induced significant inhibitory effects on the Na⁺, K⁺-ATPase activity of C. punctatus, ranging from gills (70%) > Kidney (63%) > brain (57%) > intestine (52%) > liver (50%) > muscle (47%) > heart (44%) inhibition at a sublethal concentration of 0.96 mg/L. Significant inhibition was detected in Na⁺, K⁺-ATPase activity, ranging from gills (90%) > heart (78%) > kidney (78%) > muscle (74%) > intestine (71%) > brain (67%) > liver (63%) at sublethal concentration of 1.86 mg/L. After subacute exposure (15 days) only gills and brain showed significant inhibition after higher concentration (1.86 mg/L). However, it is evident that exposure duration is more important than dose in the inhibition of the activity of enzyme. At lower concentration initial stimulation of the activity of Na⁺, K⁺-ATPase activity was also noticed. It is suggested that the inhibition of the ATPase by monocrotophos blocked the active transport system of the gill epithelial as well as chloride cells, glomerular and epithelial cells of the tubules and thus altered the osmoregulatory mechanism of the fish. In fact, the impairment of the activity of enzymes which carry out key physiological roles could cause alterations of the physiology of the whole organism.     

Novel mode of action of spinosad: Receptor binding studies demonstrating lack of interaction with known insecticidal target sites

The ability of spinosyn A to either enhance or displace binding to selected insecticidally-relevant receptors was investigated using a number of radioligands including, [³H]imidacloprid and [³H]ivermectin in tissues from the ventral nerve cord (VNC) membranes of the American cockroach, Periplaneta americana and head membranes from the housefly, Musca domestica. In these insect neural tissues, spinosyn A does not appear to alter the binding of a number of radioligands suggesting that spinosyn A does not interact directly with a variety of known receptors, including nicotinic or γ-aminobutyric acid (GABA)-based insecticidal target sites. However, available data are consistent with spinosyn A interacting with a site distinct from currently known insecticidal target sites, thus supporting a novel insecticidal mechanism of action for the spinosyns.     

Block of electron transport by surangin B in bovine heart mitochondria

Exposure of mitochondria isolated from bovine heart to the insecticidal coumarin surangin B results in inhibition of complex II (IC₅₀ = 0.2 μM), III (IC₅₀ = 14.8 μM), and IV (IC₅₀ = 3.1 μM), but in contrast, the NADH:ubiquinone reductase (complex I) was completely insensitive to this compound at 100 μM. Kinetic analysis of surangin B’s interaction with complex II was then investigated using sub-mitochondrial particles. With succinate as the substrate, surangin B, like carboxin, acted with non-competitive kinetics and clearly contrasted in its action with malonate, a competitive inhibitor of complex II. Likewise, surangin B acted as a non-competitive inhibitor of decylubiquinone-dependent interception of electrons at complex II. Difference spectra of reduced complex III equilibrated with surangin B were found to closely parallel those of antimycin A, but were different in nature to those of the Q_o site inhibitors myxothiazol and famoxadone. Investigation of surangin B-dependent functional perturbation of complex III used the synthetic electron acceptor 2-nitrosofluorene, which intercepts electrons specifically from the Q_i site. These experiments demonstrated that like antimycin A, surangin B acts as a selective blocker of electron diversion to 2-nitrosofluorene through Q_i within complex III. We conclude that surangin B blocks electron transport at several points in bovine heart mitochondria, however, complex I is spared. The potent inhibitory action of surangin B on complex II involves binding to a site which is distinct from both the succinate binding site and the domain responsible for interacting with ubiquinone. Surangin B apparently blocks complex III by interacting with the Q_i (antimycin A-binding) pocket.     

Influence of three diets on susceptibility of selected insecticides and activities of detoxification esterases of Helicoverpa assulta (Lepidoptera: Noctuidae)

The oriental tobacco worm, Helicoverpa assulta Guenée, is one of the most destructive pests of tobacco and peppers in China. We determined the susceptibility of H. assulta reared on an artificial diet, chili pepper and tobacco to four insecticides (fenvalerate, phoxim, methomyl, indoxacarb) under laboratory conditions associated with the activities of acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione S-transferase (GST) in its larvae. H. assulta larvae that were fed with chili pepper were more susceptible to fenvalerate, indoxacarb, and phoxim than those that were fed with tobacco and the artificial diet, but not to methomyl. The larvae that were fed with chili pepper were 3.65-, 2.49-, 1.92- and 2.44-fold more susceptible to fenvalerate, phoxim, methomyl, and indoxacarb than those fed with tobacco, respectively. The AChE activities of H. assulta larvae that were fed with chili pepper and tobacco were 2.12 and 1.07 μmol mg⁻¹ 15 min⁻¹, respectively, almost 2-fold difference. The CarE activity of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 4.12, 7.40 and 7.12 μmol mg⁻¹ 30 min⁻¹, respectively. Similarly, the GST activities of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 52.02, 79.37 and 80.02 μmol mg⁻¹ min⁻¹, respectively. H. assulta larvae that were fed with chili pepper were more resistance to the tested insecticides. The low activities of AChE and the high activities of CarE and GST lead to H. assulta become more susceptible to the tested insecticides.     

Thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants

Neonicotinoid insecticides are compounds acting agonistically on insect nicotinic acetylcholine receptors (nAChR). They are especially active on hemipteran pest species such as aphids, whiteflies, and planthoppers, but also commercialized to control many coleopteran and some lepidopteran pest species. The most prominent member of this class of insecticides is imidacloprid. All neonicotinoid insecticides bind with high affinity (I₅₀-values around 1 nM) to [³H]imidacloprid binding sites on insect nAChRs. One notable ommission is the neonicotinoid thiamethoxam, showing binding affinities up to 10,000-fold less potent than the others, using housefly head membrane preparations. Electrophysiological whole cell voltage clamp studies using neurons isolated from Heliothis virescens ventral nerve cord showed no response to thiamethoxam when applied at concentrations of 0.3 mM, although the symptomology of poisoning in orally and topically treated noctuid larvae suggested strong neurotoxicity. Other neonicotinoids, such as clothianidin, exhibited high activity as agonists on isolated neurons at concentrations as low as 30 nM. There was no obvious correlation between biological efficacy of thiamethoxam against aphids and lepidopterans and receptor affinity in electrophysiological and binding assays. Pharmacokinetic studies using an LC-MS/MS approach to analyze haemolymph samples taken from lepidopteran larvae revealed that thiamethoxam orally applied to 5th instar Spodoptera frugiperda larvae was rapidly metabolized to clothianidin, an open-chain neonicotinoid. Clothianidin shows high affinity to nAChRs in both binding assays and whole cell voltage clamp studies. When applied to cotton plants, thiamethoxam was also quickly metabolized, with clothianidin being the predominant neonicotinoid in planta briefly after application, as indicated by LC-MS/MS analyses. Interestingly, the N-desmethylated derivative of thiamethoxam, N-desmethyl thiamethoxam, was not significantly produced in either lepidopteran larvae or in cotton plants, although it was often mentioned as a possible metabolite, being nearly as active as imidacloprid. In conclusion, our investigations show that thiamethoxam is likely to be a neonicotinoid precursor for clothianidin.     

Acetylcholinesterase inhibition by nootkatone and carvacrol in arthropods

The essential oils from many botanicals have been screened for insecticidal activity. Two constituents of the Alaskan yellow cedar tree, the monoterpenoid carvacrol and the sesquiterpenoid nootkatone, both are toxic against several arthropods. The mode of action through which nootkatone and carvacrol exert their insecticidal activity remains uncertain. It has been hypothesized that they may inhibit acetylcholinesterase enzyme activity. The degree of acetylcholinesterase inhibition of carvacrol and nootkatone was compared to that of carbaryl, a known acetylcholinesterase inhibitor, in the house fly (Musca domestica), yellow fever mosquito (Aedes aegypti), American dog tick (Dermacentor variabilis) and American cockroach (Periplaneta americana). The concentration of carbaryl, at which 50% of the acetylcholinesterase activity was inhibited (IC₅₀), was less than 2 μM in all four arthropod models. Carvacrol was observed to cause slight inhibition of the acetylcholinesterase enzyme in house flies, ticks and cockroaches, but it did not inhibit the mosquito acetylcholinesterase enzyme. Nootkatone did not inhibit the acetylcholinesterase enzyme in any of the four arthropod models tested. From this study, we conclude that the acetylcholinesterase inhibition is not likely the primary mode of action for insecticidal activity by nootkatone or carvacrol.     

Protoporphyrinogen IX-oxidizing activities involved in the mode of action of a new compound N-[4-chloro-2-fluoro-5-{3-(2-fluorophenyl)-5-methyl-4,5-dihydroisoxazol-5-yl-methoxy}-phenyl]-3,4,5,6-tetrahydrophthalimide

N-[4-Chloro-2-fluoro-5-{3-(2-fluorophenyl)-5-methyl-4,5-dihydroisoxazol-5-yl-methoxy}-phenyl]-3,4,5,6-tetrahydrophthalimide (EK-5385) is an experimental substituted bicyclic herbicide. Soil-applied EK-5385 showed good rice selectivity and potent herbicidal activity on barnyardgrass (Echinochloa crus-galli var. oryzicola) at rates of 3.9-250 g a.i./ha. Barnyardgrass was exhibited normal growth under dark condition, however, the growth of shoot and root was severely inhibited under light condition (14/10 h of light/dark, 50 μmol/m²/s of photosynthetically active radiation) when treated with EK-5385, oxadiazon, and oxadiargyl. IC₅₀ of EK-5385 and oxadiargyl to chlorophyll loss in cucumber cotyledons was approximately 0.3 and 0.7 μM, respectively. IC₅₀ of EK-5385 and oxadiargyl to carotenoids loss in cucumber cotyledons was about 0.26 and 0.1 μM, respectively. IC₅₀ concentration of EK-5385 and oxadiargyl on Protox activity was approximately 5.5 and 8 nM, respectively. Cellular leakage occurred without lag period from cucumber leaf squares treated with 1 μM of EK-5385 and oxadiargyl under light exposure.     

[3H]Muscimol binding to a putative GABA receptor in honey bee brain and its interaction with avermectin B1a

A putative GABA receptor was identified in honey bee brain by virtue of its specific binding of [³H]muscimol and its drug specificity. [³H]Muscimol bound with two affinities (K_d1 of 3 nM and K_d2 of 144 nM), comparable to its affinities for binding to mammalian brain. The high-affinity binding was most sensitive to GABA agonists with the following decreasing order of potencies: muscimol>GABA>imidazole acetic acid>DL-GABOB>Zβ-guanidine propionic acid. However, it was insensitive to the antagonist bicuculline, which is potent on [³H]muscimol binding to the mammalian GABA_A receptor. It was also insensitive to baclofen, which is a potent agonist of mammalian GABA_B receptor, as well as to picrotoxinin, pentobarbital, flunitrazepam, and ethyl-β-carboxylate, which bind to allosteric sites in mammalian GABA receptor. The low-affinity [³H]muscimol binding was inhibited with GABA agonists with the following decreasing order of potencies: imidazole acetic acid = β-guanidine propionic acid>dl-GABOB. The two muscimol binding affinities may represent binding to two sites on the same GABA receptor or to two kinds of GABA receptor. The most potent inhibitor of the high-affinity [³H]muscimol binding to honey bee brain was avermectin B_1a (AVM), whose IC₅₀ was 0.01 nM. AVM also inhibited the low-affinity [³H]muscimol binding with an IC₅₀ of 2 μM.     

Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol⁻¹. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml⁻¹ for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl₂, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca²⁺ and Mg²⁺ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6-8.2, 3.2 mM and 0.013-0.018 μM, respectively. The Lineweaver-Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.     

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures,followed by modeling of ecdysteroid-EcR interactions and normal mode analysis

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC₅₀ = 3.3 μM) and Bm5 cells (EC₅₀ = 5.3 μM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC₅₀ = 0.71 μM and 0.00089 μM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC₅₀ of 0.039 μM; in Bm5 cells this effect only became visible at much higher concentrations (IC₅₀ = 18 μM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.