Distribution and Inhibition of Esterases in Various Body Tissues of Susceptible and Resistant German Cockroaches (Dictyoptera: Blattellidae)

The distribution and inhibition of esterase activity among body of the resistant Crawford and susceptible CSMA strains of male German cockroach, Blattella germanica (L.), were evaluated with α- and β-naphthyl esters using a spectrophotometric assay and native polyacrylamide gel electrophoresis. The esterase activities in the Crawford strain were significantly higher than in the CSMA strain in most tissues except midgut and male genitalia, where the activities were similar. In both strains, isozyme a was dominant in the brain and fat body and isozymes c and d were more abundant in the midgut; but in addition to isozyme a, isozyme b was observed only in the Crawford strain. The α-naphthyl butyrate (α-NB) and β-naphthyl acetate (β-NA) esterase activities in the brain of both strains were completely inhibited by most inhibitors (DEF, 3,4-dichloroisocoumarine, paraoxon, pepstatin A, and propoxur). However, in the midgut tissue and contents, the activities and inhibition patterns of the α-NB and β-NA esterase were apparently different from the brain and between strains.The complete inhibition by paraoxon was observed regardless of the strain, substrate type, and body part for enzyme source. In esterase inhibition on native PAGE gels, paraoxon completely inhibited the activity of most esterases similar to the results from spectrophotometric assay. In addition, the esterase b was observed from the whole body homogenates of several German cockroach resistant strains on native PAGE analysis

Decapitation Impacting Effect of Topically Applied Chlorpyrifos on Acetylcholinesterase and General Esterases in Susceptible and Resistant German Cockroaches (Dictyoptera: Blattellidae)

The effect of topically applied chlorpyrifos on acetylcholinesterase and other esterases in heads and decapitated bodies of CSMA and Crawford German cockroaches was examined with spectrophotometric enzyme assay and native polyacrylamide gel electrophoresis. The toxicity of chlorpyrifos was greatly reduced in decapitated CSMA male cockroaches with LD50 value 17.1-fold higher than that of normal CSMA cockroaches. Acetylcholinesterase activity from heads was significantly higher in the Crawford compared with the CSMA strain and did not change until 24 h after chlorpyrifos in vivo treatment in both strains. The p-nitrophenyl butyrate (NPB) esterase activities from both heads and decapitated bodies of the resistant Crawford strain were significantly greater than the susceptible CSMA strain. The p-NPB esterase activity was significantly inhibited by chlorpyrifos in vivo treatment, and total p-NPB esterase activity was significantly reduced in decapitated bodies compared with heads of both strains. Native polyacrylamide gel electrophoresis (PAGE) analysis of extracts solubilized with Triton X-100 from heads and decapitated bodies revealed five major esterase bands and an acetylcholinesterase (AChE) band with a high capability of hydrolyzing α-naphthyl butyrate and acetylthiocholine, respectively. In the heads of susceptible CSMA male cockroaches, the activity of mobile isozymes d1 and d2 was completely inhibited at 24 h after chlorpyrifos application, and isozyme e was partially inhibited. In contrast, isozymes c1 and c2 from the decapitated bodies of CSMA cockroaches were mostly affected at 24 h after the topical application of chlorpyrifos. The activities of acetylcholinesterase and esterase isozymes a and b from the decapitated body remained uninhibited in both strains. Inhibition of isozymes d1 and d2 seems to be more important in chlorpyrifos intoxication than acetylcholinesterase

Comparison of Esterases Between Life Stages and Sexes of Resistant and Susceptible Strains of German Cockroach (Dictyoptera: Blattellidae)

Esterase activity between the resistant Crawford and susceptible CSMA strains of German cockroach, Blattella germanica (L.), was compared with the substrates α- and β-naphthyl acetate across sex and nymphal age classes. Esterase isozyme analysis with native polyacrylamide gel electrophoresis also was conducted to identify quantitative and qualitative differences between strains, sexes, and age classes. The Crawford strain was highly resistant to cypermethrin, propoxur, and permethrin with a resistant ratio (RR) of 17.26,15.75,and 13.53, respectively, and mildly resistant to chlorpyrifos (RR 5.62). The α-NA and β-NA esterase activities in the Crawford strain were significantly higher than those activities in the CSMA strain in both nymphal and adult stages. In the Crawford strain, the enzyme activity in nymphs was significantly higher than that in adults, but such differences were not observed in the CSMA strain. The mobile isozymes a and c stained more intensely than others in every developmental stage and sex of both strains but showed greater intensity in the Crawford strain. Another intensely stained isozyme b was observed only in the homogenates from the Crawford strain. The combination of isozyme b and the overproduced isozyme II and c in the Crawford strain seems to be responsible for the difference in total esterase activity between the CSMA and Crawford strains

Distribution and Inhibition of Esterases in Various Body Tissues of Susceptible and Resistant German Cockroaches (Dictyoptera: Blattellidae)

The distribution and inhibition of esterase activity among body of the resistant Crawford and susceptible CSMA strains of male German cockroach, Blattella germanica (L.), were evaluated with α- and β-naphthyl esters using a spectrophotometric assay and native polyacrylamide gel electrophoresis. The esterase activities in the Crawford strain were significantly higher than in the CSMA strain in most tissues except midgut and male genitalia, where the activities were similar. In both strains, isozyme a was dominant in the brain and fat body and isozymes c and d were more abundant in the midgut; but in addition to isozyme a, isozyme b was observed only in the Crawford strain. The α-naphthyl butyrate (α-NB) and β-naphthyl acetate (β-NA) esterase activities in the brain of both strains were completely inhibited by most inhibitors (DEF, 3,4-dichloroisocoumarine, paraoxon, pepstatin A, and propoxur). However, in the midgut tissue and contents, the activities and inhibition patterns of the α-NB and β-NA esterase were apparently different from the brain and between strains.The complete inhibition by paraoxon was observed regardless of the strain, substrate type, and body part for enzyme source. In esterase inhibition on native PAGE gels, paraoxon completely inhibited the activity of most esterases similar to the results from spectrophotometric assay. In addition, the esterase b was observed from the whole body homogenates of several German cockroach resistant strains on native PAGE analysis

Comparison of esterase between insecticide resistant and susceptible strains of German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae)

The Crawford strain of German cockroach, Blattella germanica (L.) was determined to be highly resistant to chlorpyrifos, propoxur, cypermethrin, and permethrin and esterase activities were higher than susceptible CSMA strains at both nymphal and adult stages. The most mobile isozyme a and c that were stained more intensely in every developmental stage and sex of both strains showed much greater intensity in the Crawford strain. Another intensely stained isozyme b was observed only in homogenates from the Crawford strain. The distribution and inhibition of esterase activity among body of the resistant Crawford and susceptible CSMA strains of male German cockroach, Blattella germanica (L.), were evaluated with α- and β-naphthyl esters using a spectrophotometric assay and native polyacrylamide gel electrophoresis. The esterase activities in the Crawford strain were significantly higher than in the CSMA strain in most tissues except midgut and male genitalia, where the activities were similar. In both strains, isozyme a was dominant in the brain and fat body and isozymes c and d were more abundant in the midgut; but isozyme b was observed only in the Crawford strain. In addition, the esterase b was observed from the whole body homogenates of several German cockroach resistant strains on native PAGE analysis. The complete inhibition by paraoxon was observed regardless of the strain, substrate type, and body part for enzyme source in spectrophotometric assay and native PAGE gels. The brain esterase preferred ester substrates with chain length of carbons, less than 5 in carbon number. Maximum activity of the p-NPB esterase appeared at 40°C and pH 7.4 at 38°C in both strains. The addition of Triton X-100 into extraction buffer enhanced the p-NPB esterase activity. The addition of sucrose and dithiothreitol was also favorable in minimizing the spontaneous inactivation of enzyme activity during homogenization and storage. The p-NPB esterase in brain was extremely sensitive to paraoxon and strongly inhibited by eserine and propoxur. The toxicity of chlorpyrifos was greatly reduced in decapitated CSMA male cockroaches with LD50 values 17.1-fold higher than that from normal CSMA roaches. The acetylcholinesterase activity from heads were significantly higher in Crawford stain than CSMA strain and were not changed till 24 h after in vivo treatment chlorpyrifos in both strains. The p-NPB esterase activities from both heads and decapitated bodies of resistant Crawford strain were significantly greater than those from susceptible CSMA strain. The p-NPB esterase activity was significantly inhibited after chlorpyrifos treatment, and the total p-NPB esterase activity was more significantly declined in the decapitated bodies than that in the heads of both strains. In native PAGE analysis, the isozyme d1 and d2 appear to be more sensitive to the chlorpyrifos intoxication than acetylcholinesterase and the isozyme c1 and c2 might be responsible for detoxification of chlorpyrifos

Pungent chemicals increase cytoplasmic free calcium concentration in mammalian taste receptor cells

Pungent compounds are widely used for seasoning. Hot sensation evoked by pungent chemicals including capsaicin, has been known that it is the result of stimulation of free nerve endings in sensory neurons. However, there has not been rigorous study of its direct effects on the taste receptor cells (TRCs). In the present study, we investigated direct effect of two kinds of pungent chemicals, capsaicin and piperine, on the cytoplasmic free calcium concentration ([Ca^2+]_i) in the isolated rat taste receptor cells using micrspectrofluorimetry technique. The effect of bitter taste and sweets on [Ca^2+]_i was also examined for comparision with those of pungent chemicals. Epithelial sheets rich in taste buds and free of muscle tissue were isolated from the circumvallate papilla of the rat tongue by mixed enzyme treatment following microdissection. After exposure to Ca^2+ -free Tyrode solution, we could get taste buds or single TRCs from the epithelial sheets. In the high K^+ bath solution, [Ca^2+]_i transient was observed, suggestion existence of voltage dependent Ca^2+ channels in TRCs. Capsaicin increased [Ca^2+]_i in a dose dependent manner in these cells. 10㎍M capsaicin little affected on [Ca^2+]_i, but 50㎍M capsaicin increased a small amount of [Ca^2+]_i, 100㎍M capsaicin increased a large amount of [Ca^2+]_i, from 1.04±0.05(Mean±S.E.) to 1.40±0.059(n=28). The effect of piperine on [Ca^2+]_i responses was similar to those of capsacin, but poperine was more potent than capsaicin. 1μM piperine little affected on [Ca^2+]_i responses, but 5μM piperine increased a small amount of [Ca^2+]_i. 10μM piperine increased a large amount of [Ca^2+]_i, from 0.99±0.03 (mean ± S.E.) to 1.43±0.05(n=20). The increase of [Ca^2+]_i evoked by both capsaicin and piperine appears to be due to Ca^2+ influx from the extracelllular medium, since the capsaicin induced [Ca^2+]_i increase was not observed in Ca^2+ free bath solution. Furthermore, 10mM ruthenium red also inhibited this capsaicin induced [Ca^2+]_i increase, suggestion it's mediated by vanilloid receptors. We also confirmed that denatonium, one of bitter taste and sucrose, sweet compound, both increase [Ca^2+]_i in these cells. But the Ca^2+ responses to these two substances were quite different to those evoked by pungent chemicals; Ca^2+ response was even observed in the Ca^2+ free bath solution. All our results suggest that pungent chemicals including capsaicin and poperine could increase [Ca^2+]_i responses of TRCs, presumably via vanilloid receptors

7,7,8,8-Tetracyanoquinodimethane-assisted onestep electrochemical exfoliation of graphite and its performance as an electrode material

A green approach for the preparation of water-dispersible functionalized graphene via one-step electrochemical exfoliation of graphite using 7,7,8,8-tetracyanoquinodimethane (TCNQ) anions as surface modifiers and electrolytes was described. TCNQ is an organic charge-transfer complex with electron accepting and noteworthy electrical properties. The exfoliation of graphite to a few-layer graphene sheets was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM) image analysis. The chemical state, surface functional groups and chemical compositions of bulk graphite as well as TCNQ-functionalized graphene sheets were investigated by Fourier-transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) analysis. Adsorption of TCNQ onto the surface of graphene sheets was confirmed by the appearance of the N1s peak at _399.4 eV in the XPS of TCNQ-functionalized graphene. Exfoliation of bulk graphite to functionalized graphene sheets was further confirmed by the appearance of a sharp single peak at _2695 cm_1 along with increased intensity ratios of the D-band to the G-band. Electrochemical performance of a TCNQfunctionalized graphene sheet was investigated using 1 M Na2SO4 and 1 M KOH aqueous solutions. Cyclic voltammetry (CV) and galvanometric charge–discharge experiments revealed that TCNQfunctionalized graphene could be used as a supercapacitor electrode material. The specific capacitance values of TCNQ-modified graphene measured with electrolytes (1 M KOH and 1 M Na2SO4) were 324 and 140 F g_1, respectively, at a current density of 1 A g_1. Impedance spectroscopic analysis revealed that the charge transfer process was dependent on surface functionalization and interaction between the electrode and the electrolyte

Highly regioselective and sustainable solar click reaction: a new post- synthetic modified triazole organic polymer as a recyclable photocatalyst for regioselective azide- alkyne cycloaddition reaction

The synthesis of pharmaceutically active 1,2,3-triazoles has been continuously scrutinized in the search for unique and effective catalysts to make the process efficient, green, and sustainable. Here, we are presenting a new visible light active Ni(ii) cyclam-integrated triazole-linked organic polymer (Ni-TLOP) photocatalyst for the synthesis of 1,2,3-triazole compounds with excellent efficiency and regioselectivity. The reaction was studied for a series of substrates and the absolute regioselectivity of a representative triazole product has also been confirmed by X-ray crystallography. The proficiency and chemical orthogonality of the Ni-TLOP are remarkable and it shows enhanced efficiency and regioselectivity. The use of a recyclable photocatalyst and non-hazardous reagents makes the catalytic system sustainable and environmentally friendly. This photocatalyzed click reaction technique has been successfully applied to the expedient synthesis of one of the most sold anti-epileptic drugs rufinamide. © The Royal Society of Chemistry 201