Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of first generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats (Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory effect of different anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats (Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin (K_i = 0.72 ± 0.1 μM; 29 ± 4.1 μM), chlorophacinone (K_i = 0.08 ± 0.01 μM; 1.6 ± 0.1 μM), diphacinone (K_i = 0.07 ± 0.01 μM; 5.0 ± 0.8 μM), coumachlor (K_i = 0.12 ± 0.02 μM; 1.9 ± 0.2 μM), coumatetralyl (K_i = 0.13 ± 0.02 μM; 3.1 ± 0.4 μM), difenacoum (K_i = 0.07 ± 0.01 μM; 0.26 ± 0.02 μM), bromadiolone (K_i = 0.13 ± 0.02 μM; 0.91 ± 0.07 μM), and brodifacoum (K_i = 0.04 ± 0.01 μM; 0.09 ± 0.01 μM) on VKOR activity. Analysis of the results leads us to highlight different anticoagulant structural elements, which influence inhibition parameters in both susceptible and Y139F resistant rats.     

Expression of a wheat cytochrome P450 monooxygenase cDNA in yeast catalyzes the metabolism of sulfonylurea herbicides

A wheat cytochrome P450 cDNA (CYP71C6v1) was cloned by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain could catalyze the metabolism of some sulfonylurea herbicides such as chlorsulfuron, triasulfuron, metsulfuron-metyl, bensulfuron-metyl, and tribenuron-metyl, but not sulfonylurea herbicides such as thifensulfuron and pyrazosulfuron. Kinetic parameters K_m for chlorsulfuron and triasulfuron were 57 (±15) μM and 38 (±16) μM in vitro, respectively. Analysis of the metabolites demonstrated that the CYP71C6v1 functioned as a 5-phenyl ring hydroxylase when chlorsulfuron and triasulfuron were the substrates.     

Acetylcholinesterase in selected plant-parasitic nematodes: Inhibition, kinetic and comparative studies

The in vitro inhibition potency of some organophosphates (OPs) and carbamates (CAs) which are widely used to control plant-parasitic nematodes on acetylcholinesterase (AChE) of Meloidogyne javanica, Heterodera avenae and Tylenchulus semipenetrans, the major pathogens responsible for the damage of a wide range of crops in Al-Qassim region, Saudi Arabia was examined. AChE of H. avenae activity was 1.58- and 1.51-fold greater than that of T. semipenetrans or M. javanica, respectively. The order of inhibition potency of the tested compounds against T. semipenetrans AChE was: carbofuran > paraoxon > oxamyl > fenamiphos > phorate-sulfoxide > aldicarb, where the corresponding concentrations that inhibited 50% of the nematode AChE activity (I₅₀) were 5 × 10⁻⁸, 7 × 10⁻⁷, 7.5 × 10⁻⁷, 2 × 10⁻⁶, 2 × 10⁻⁴ and 2 × 10⁻³ M, respectively. Paraoxon, fenamiphos and carbofuran exhibited high inhibition potency against M. javanica AChE where the I₅₀ values were below 1 nM. Phorate-sulfoxide and aldicarb were potent inhibitors of M. javanica AChE with I₅₀ values of 3.8 and 8 nM, respectively, while oxamyl exhibited low inhibition potency with I₅₀ of 15 nM. Fenamiphos and paraoxon showed the highest I₅₀ values of <100 μM against H. avenae followed by oxamyl (I₅₀ < 1 mM), whereas paraoxon, carbofuran and aldicarb showed low potency with I₅₀ values >1 mM. All the tested compounds exhibited high inhibition potency to AChE of M. javanica than T. semipenetrans or H. avenae. Except phorate-sulfoxide in M. javanica the inhibition pattern and implied mechanism for all the tested compounds for the three nematodes is suggested to be a linear mixed type (a combination of competitive and non-completive type).     

Pesticide transformation by a variant of CYPBM3 with improved peroxygenase activity

The activity of the mutant CYPBM3 “21B3”, which is able to use hydrogen peroxide as the final electron acceptor, was evaluated against two major environmental pollutants; organochlorine and organophosphorus pesticides. This evolved CYP from Bacillus megaterium is able to transform a variety of structurally different pesticides. The catalytic parameters for two organochlorine; dichlorophen (k_cat = 9.2 min⁻¹, K_M = 64.1 μM) and linuron (k_cat = 226.5 min⁻¹, K_M = 468.2 μM), and two organophosphorus compounds; parathion (k_cat = 10.9 min⁻¹, K_M = 59.3 μM) and chlorpyrifos (k_cat = 9.2 min⁻¹, K_M = 226.5 μM) were determined giving catalytic efficiencies between 0.143 and 1.107 min⁻¹ μM⁻¹. CYPBM3 “21B3” has the ability to both activate and detoxify organophosphorus pesticides, as demonstrated by the chemical nature of the reaction products. The capacity to transform structurally diverse compounds together with the great stability, easy production and relatively inexpensive cofactors needed, makes CYPBM3 “21B3” an enzyme with a potential use on the environmental field.     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Leaf tissue pigments and chlorophyll fluorescence parameters vary among sweet corn genotypes of differential herbicide sensitivity

Herbicide applications are meant to eliminate weed competition; however, herbicides may also impose abiotic stress on registered crops. Leaf tissue carotenoid pigments play vital roles in the photoprotection of photosynthetic membranes and contribute to non-photochemical quenching (NPQ) of excitation energy, both important to plant environmental stress tolerance. Our research objectives were to characterize leaf tissue pigments and chlorophyll fluorescence parameters following post-emergence herbicide applications (simulating an abiotic stress) to sweet corn (Zea mays var. rugosa) genotypes of differential herbicide sensitivities. Post-emergence herbicide applications of combinations of mesotrione (105 g ai/ha) and atrazine (560 g ai/ha) were applied to ‘Merit’ (sensitive), ‘Temptation’ (tolerant), and ‘Incredible’ (moderately sensitive) sweet corn genotypes. Leaf tissues were sampled after herbicide applications and measured for chlorophyll fluorescence parameters, and the same tissues were analyzed for carotenoid and chlorophyll pigments. Leaf pigments and chlorophyll fluorescence were not affected by any herbicide treatment; however, data revealed significant differences between genotypes for leaf tissue antheraxanthin, β-carotene, zeaxanthin, chlorophyll a/b ratios, and for values of F_o, F_m, F_v, and NPQ, with ‘Merit’ leaf tissue having higher values than the other two genotypes evaluated. Results demonstrate that genotypic sensitivities to certain post-emergence herbicides may be related to concentrations of photo-protective carotenoids in sweet corn leaf tissues.     

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.     

Multiple effects of a naturally occurring proline to threonine substitution within acetolactate synthase in two herbicide-resistant populations of Lactuca serriola

Two populations of Lactuca serriola L. with resistance to acetolactate synthase (ALS)-inhibiting herbicides were discovered in wheat fields at two locations more than 25 km apart in South Australia. Both resistant populations carried a single base change within a highly conserved coding region of the ALS gene that coded for a single amino acid modification within ALS. The modification of proline 197 to threonine resulted in an enzyme that was highly resistant (>200-fold) to inhibition by sulfonylurea herbicides and moderately resistant to triazolopyrimidine and imidazolinone herbicides. The herbicide-resistant ALS was also less sensitive to inhibition by the branched-chain amino acids valine and leucine. In addition, the resistant enzyme had a lower K_m for pyruvate. However, extractable ALS activity was similar between resistant and susceptible plants. The substitution of threonine for proline 197 within ALS has multiple impacts on ALS enzyme activity in L. serriola that may influence the frequency of this resistant allele in the environment.     

Inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase

Valienamine, an aminocyclitol with similar configuration to α-glucose, has a strong inhibitory effect on α-glucosidase. α-Glucosidase plays an important role in insect carbohydrate metabolism. The inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase was investigated. Our results show that valienamine inhibition of honeybee α-glucosidase was pH- and dose-dependent, but temperature-independent. Valienamine is shown to be a potent and competitive reversible inhibitor of honeybee α-glucosidase in vitro with an IC₅₀ value of 5.22 × 10⁻⁵ M and K_i value of 3.54 × 10⁻⁴ M at pH 6.5, 45 °C. Valienamine has the potential to be developed into novel insecticides.     

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.     

In vitro metabolism and interaction of profenofos by human, mouse and rat liver preparations

Biotransformations of profenofos were studied in vitro. Two metabolites, desthiopropylprofenofos and hydroxyprofenofos, were detected by LC-MS after incubation of profenofos with human liver homogenates and different mammalian liver microsomes. The rank order of desthiopropylprofenofos formation in liver microsomes based on intrinsic clearance (V_max/K_m) was mouse > human > rat, while for profenofos hydroxylation it was mouse > rat > human. In view of the ratio between desthiopropylation and hydroxylation intrinsic clearance rates, human liver microsomes were most active in profenofos bioactivation. The interspecies differences and interindividual variation were within range of the default uncertainty/safety factors for chemical risk assessment. CYP3A4, CYP2B6 and CYP2C19 were identified as profenofos-oxidizing enzymes in human liver on the basis of recombinant expressed enzymes and correlation with CYP model activities. The rank order of CYPs in profenofos activation was CYP3A4 > CYP2B6 > CYP2C19, whereas it was the contrary for profenofos hydroxylation. Profenofos inhibited relatively potently several human liver microsomal activities: the lowest IC₅₀ values were about 3 μM for CYP1A1/2 and CYP2B-associated activities. Profenofos is extensively metabolized by liver microsomal CYP enzymes and its interaction potential with several CYP activities is considerable.     

Synergism of resistance to phosalone and comparison of kinetic properties of acetylcholinesterase from four field populations and a susceptible strain of Colorado potato beetle

The susceptibility to phosalone and biochemical characteristics of acetylcholinesterase (AChE) were compared between susceptible (SS) strain and four field populations of Colorado potato beetle (CPB) collected from commercial potato fields of Hamedan Province in west of Iran. Bioassays involving topical application of phosalone to fourth instars revealed up to 252 fold resistance in field populations compared with the SS strain. Synergism studies showed that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not have a major role in resistance. It is likely that piperonyl butoxide (PBO) reduced phosalone toxicity by inhibiting bio-activation of phosalone. The affinity (K_m) and hydrolyzing efficiency (V_max) of AChE to selected substrates, namely, acetylthiocholine iodide (ATC), propionylthiocholine iodide (PTC), and butyrylthiocholine iodide (BTC) were examined. AChE inhibition by higher substrate concentration was evident only in the SS strain. In resistant field populations, Aliabad (Aa), Bahar (B) and Dehpiaz (Dp), substrate inhibition at higher concentrations was not seen. There was no definite optimal concentration found for any of the substrates examined. When ATC, PTC, and BTC were used as substrate, the reaction rates of AChE from Yengijeh (Yg) population increased as the concentration of all three substrates were increased, but were almost constant at concentration of ATC ? 3.98, PTC ? 2.8, and BTC ? 5 mM. The susceptible form of AChE had the most efficient ATC hydrolysis but very low BTC hydrolysis activity. In contrast, AChEs from field populations elicited relatively reduced ATC hydrolysis, but relatively increased BTC hydrolysis. The in vitro inhibition potency of some organophosphates (OPs), on AChEs of the field populations and SS strain was determined. The rank order from the most potent inhibitor to the least as determined by their bimolecular reaction constants (K_i) was ethyl paraoxon > diazoxon > methyl paraoxon for AChE from Aa, B, Dp, and Yg populations, respectively, whereas the rank order for the susceptible strain was methyl paraoxon > ethyl paraoxon > diazoxon.     

Selectivity, acetylcholinesterase inhibition kinetics, and quantitative structure-activity relationships of a series of N-(2-oxido-1,3,2-benzodioxa-phosphol-2-yl) amino acid ethyl or diethyl esters

The inhibitory effects of a recently introduced series of the titled compounds on insect and mammalian acetylcholinesterase (AChE) activity were examined, where the median inhibition concentration (I₅₀) and the inhibition kinetic parameters, bimolecular inhibition rate constant (k_i), affinity constant (K_a), and phosphorylation rate constant (k_p), were determined for each compound. Results indicated that all examined dioxaphospholenes had less inhibitory effects on mammalian AChE than fenitrothion, a commercial pesticide with moderate mammalian toxicity. The highest selectivity was obtained with compounds containing glutamic and leucine moieties (2.70 and 2.18, respectively) while selectivity of fenitrothion was 0.93. The low inhibitory effects of the examined dioxaphospholenes on mammalian AChE were attributed to their low phosphorylation rates (k_p < 2.2 min⁻¹) compared to that of fenitrothion (k_p = 4.84 min⁻¹). QSAR equations indicated that the inhibition process is controlled mainly by both the phosphorylation rate (direct effect) and the affinity of compounds toward the enzyme (inverse effect). Although the compounds’ hydrophobicity had no effects on the inhibition process, it affects the compounds’ toxicity since it affects the ability of compounds to penetrate insects to reach the enzyme active site.     

Comparison of kinetic properties of a hydrophilic form of acetylcholinesterase purified from strains susceptible and resistant to carbamate and organophosphorus insecticides of green rice leafhopper (Nephotettix cincticeps Uhler)

A hydrophilic form of acetylcholinesterase (AChE) was purified from N-methyl carbamate susceptible (SA) and highly N-methyl carbamate-resistant (N3D) strains of the green rice leafhopper (GRLH), Nephotettix cincticeps Uhler. Both of purified AChE from SA and N3D strains displayed the highest activities toward acetylthiocholine (ATCh) at pH 8.5. In the SA strain, the optimum concentrations for ATCh, propionylthiocholine (PTCh), and butyrylthiocholine (BTCh) were about 1 × 10⁻³, 2.5 × 10⁻³, and 1 × 10⁻³ M, respectively. However, in the N3D strain, substrate inhibition was not identified for ATCh, PTCh, and BTCh to 1 × 10⁻² M. The K_m value in the SA strain was 51.1, 39.1, and 41.6 μM and that in the N3D strain was 91.8, 88.1, and 85.2 μM for ATCh, PTCh, and BTCh, respectively. The K_m value in the N3D strain indicated about 1.80-, 2.25-, and 2.05-fold lower affinity than that of the SA strain for ATCh, PTCh, and BTCh, respectively. The V_max value in the SA strain was 70.2, 30.5, and 4.6 U/mg protein and that in the N3D strain was 123.0, 27.0, and 14.5 U/mg protein for ATCh, PTCh, and BTCh, respectively. The V_max value in the N3D strain was 1.75- and 3.15-fold higher for ATCh and BTCh than that in the N3D strain. However, it was 1.13-fold lower for PTCh. The increased activity of AChE in the N3D strain is due to the qualitatively modified enzyme with a higher catalytic efficiency. The bimolecular rate constant (k_i) for propoxur was 27.1 × 10⁴ and 0.51 × 10⁴ M⁻¹ min⁻¹ in the SA and N3D strain and that for monocrotophos was 0.031 × 10⁴ and 2.0 × 10⁴ M⁻¹ min⁻¹ in the SA and N3D strain. AChE from the N3D strain was 53-fold less sensitive than SA strain to inhibition by propoxur. In contrast, AChE from the N3D strain was 65-fold more sensitive to inhibition by monocrotophos than AChE from the SA strain. This indicated negatively correlated cross-insensitivity of AChE to propoxur and monocrotophos.     

Insensitivity of acetylcholinesterase in a field strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith)

Acetylcholinesterase (AChE) was purified from adult heads of the fall armyworm (Spodoptera frugiperda) by using a two-step procedure involving gel filtration on a Sephadex G-200 column and affinity chromatography on a procainamide-ECH Sephadex 4B column. Both susceptible and field strains possessed two AChE isozymes, namely, AChE-1 and AChE-2, with subunit molecular weights of 63.7 and 66.1 kDa. The purified AChE had an apparent K_m value of 33.5 μM and a V_max of 7.07 μmol/min/mg protein in the susceptible strain. The apparent K_m and the V_max were 2.2- and 2.0-fold higher, respectively, in the field strain than in the susceptible strain. The purified AChE from the field strain was 17- to 345-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, eserine, methomyl, and bendiocarb) and organophosphates (methyl paraoxon and paraoxon), insensitivity being highest toward carbaryl. The results further support the notion that insensitive AChE played an important role in the insecticide resistance observed in the field strain.     

Bio-potency of serine proteinase inhibitors from Acacia senegal seeds on digestive proteinases, larval growth and development of Helicoverpa armigera (Hübner)

Proteinase inhibitors (AsPIs) with high activity against serine proteinases were purified from seeds of the tree legume, Acacia senegal by ammonium sulfate precipitation followed by DEAE-Sephadex A-25 column and evaluated against Helicoverpa armigera larvae by in vitro and in vivo methods. The molecular weight of AsPIs was found to be approximately 19.58 ± 1.00 and 21.23 ± 1.00 kDa for PI and 18.16 ± 1.00 kDa for PII on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AsPIs (5 μg/ml) inhibited approximately 70% of midgut trypsin and 61% of elastase-like chymotrypsin. In vitro studies showed that AsPIs have remarkable inhibitory activity towards total gut proteolytic enzymes followed by trypsin and chymotrypsin. The IC₅₀ of AsPIs for midgut trypsin was 0.1 μg/ml and for chymotrypsin was 2.0 μg/ml. The inhibition of gut proteinase enzymes was of the non-competitive type. In larval feeding studies, AsPIs were found to retard growth and development of H. armigera and also affects the fecundity of the pest. The results advocate the use of AsPIs in transgenic technology to develop plant resistance to H. armigera.     

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.     

Use of Myxococcus xanthus protoporphyrinogen oxidase as a selectable marker for transformation of rice

Protoporphyrinogen oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO (Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T₀ generation by Northern blot and Western blot analysis. The T₀ transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.     

Studies on glutathione transferase from grasshopper (Zonocerus variegatus)

Glutathione transferase (GST) was purified from the hindgut of grasshopper (Zonocerus variegatus) a polyphagous insect. The purified enzyme had a native molecular weight of 40 kDa and a subunit molecular weight of 19 kDa. The purified enzyme could conjugate glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB), paranitrobenzylchloride, paranitrophenylacetate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl), and 1,2-dichloro-4-nitrobenzene (DCNB) with specific activities of 3.3 ± 0.3, 0.49 ± 0.10, 0.10 ± 0.002, 1.2 ± 0.2, and 1.7 ± 0.4 μmol/min/mg protein, respectively. CDNB appears to be the best substrate with a specificity constant, k_cat/K_m, of 1.8 ± 0.1 × 10⁻⁴ M⁻¹ S⁻¹. The kinetic mechanism of Z. variegatus GST (zvGST) in the conjugation of GSH with some electrophilic substrates appears complex. Conjugation of GSH with DCNB was inhibited by high DCNB concentration, while with NBDCl, as the electrophilic substrates, different values of K_m were obtained at high and low concentrations of the substrates. Cibacron blue, hematin, S-hexylglutathione, and oxidized glutathione inhibited the enzyme with I₅₀ values of 0.057 ± 0.004, 0.80 ± 0.2, 33 ± 2 μM, and 5.2 ± 0.3 mM, respectively. The nature of inhibition by each of these inhibitors is either competitive or non-competitive at varying GSH or CDNB as substrates. NADH and NAD⁺ inhibited the enzyme with an I₅₀ value of 0.4 ± 0.01 and 11 ± 1 mM, respectively. NADH at a concentration of 0.54 mM completely abolished the activity. As part of its adaptation, the flexible kinetic pathway of detoxication by zvGST may assist the organism in coping with various xenobiotics encountered in its preferred food plants.