P-nitrophenyl Acetate Esterase Research Articles

Resistance to commonly used insecticides and underlying mechanisms of resistance in Aedes aegypti (L.) from Sri Lanka

BackgroundDrastic increases of dengue fever (DF) over the past few years have prompted studies on the development of resistance to insecticides in the mosquito vector, Aedes aegypti (Linnaeus). In Sri Lanka control of the vector population is essentially achieved using larvicides (temephos) and adulticides (principally pyrethroids). The present study investigates resistance to commonly used insecticides and underlying mechanisms of Ae. aegypti in selected sites in Sri Lanka.MethodsIn this study, susceptibility to three commonly used adulticides (malathion, permethrin and deltamethrin) and the larvicide temephos were tested for Ae. aegypti sampled from five localities in Sri Lanka using WHO dose diagnostics tests. In addition, we performed dose-response tests for permethrin to determine lethal concentrations (LCs) with CDC bottle bioassays. An assessment of the activity of metabolic detoxifying enzymes (multifunction oxidases (MFOs), glutathione S-transferases (GSTs) and esterases) and determination of frequency of the kdr mutations (F1534C, V1016G and S989P) were also carried out to ascertain the associated resistance mechanisms. Kdr genotype frequencies were compared with samples collected from the same sites in 2015 to determine the change of allele frequencies over the years.ResultsThe present study revealed resistance in all Ae. aegypti populations studied, with low mortality percentages for both permethrin (10–89%) and deltamethrin (40–92%). Dose response tests revealed highest resistance ratios (RR) for permethrin and temephos from Colombo district whereas Puttalum district exhibited the lowest. High frequencies of the 1534C allele (0.052–0.802) were found in the study sites in 2017. Comparison with samples collected in 2015 revealed a substantial increase in this allele. The activity of MFOs and p-nitro phenyl-acetate esterase was significantly greater in most Sri Lankan populations in comparison to that of the New Orleans (NO) susceptible strain. In contrast, the activity of α-esterase and β-esterase was similar or lower than that in the NO strain.ConclusionsAedes aegypti from Sri Lanka is resistant to pyrethroid insecticides showing rapid selection for kdr mutations and varying metabolic mechanisms. Continued monitoring of vector populations is crucial to mitigate the development of resistance to commonly used insecticides and in turn, controlling the vector population.

Relationship between plasma biochemistry values and metal concentrations in nesting olive ridley sea turtles.

A hundred nesting olive ridley turtles were sampled to determine biochemical parameters (ALP, AST, ALT, creatinine, albumin, cholesterol, glucose, proteins, triglycerides, urea, and P-nitrophenyl acetate esterase activity). Esterase activity (EA) is a new biomarker very sensitive to metals. Most of the samples showed detectable levels. We also analyzed the concentration of 11 inorganic elements (As, Cd, Cr, Cu, Mn, Ni, Pb, Sr, Ti, Se, and Zn), some of them previously reported with very high concentrations in this population (especially cadmium with 82 and 150μgg-1 ww in liver and kidney, respectively). Cadmium presented two negative relationships with creatinine and glucose. Some other understudied elements, Sr and Ti, for instance, presented five and four significant relationships with some biochemical parameters, respectively (most of them positive). EA was the parameter with most negative relationships (with Pb, Ti, As, Cr, and Se), reinforcing the results of other researchers in humans regarding the possible inhibition of EA by metals.

P-Nitrophenyl Acetate Esterase Activity and Cortisol as Biomarkers of Metal Pollution in Blood of Olive Ridley Turtles (Lepidochelys olivacea).

This study was designed to determine the concentrations of p-nitrophenyl acetate esterase activity (EA) and cortisol in serum of marine Olive Ridley turtles (Lepidochelys olivacea) from a Mexican Pacific population ("La Escobilla" beach) and to evaluate the possible relationship of inorganic elements with these biomarkers. EA, cortisol, and selected chemical elements (Cd, Pb, Ti, Sr, Se, Al, As, and Zn) were measured in the blood of 44 sea turtles from the Eastern Pacific (Southeast Mexico). Serum EA ranged from 0.4 to 3.9 UI mL-1, and cortisol concentrations ranged from 0.07 to 2.5 μL dL-1. A strong negative correlation between EA and cortisol was observed (r=-0.59, p<0.01), and significant correlations also were found between EA and important metals, such as Cd (r=-0.31, p<0.05) and Pb (r=-0.27, p<0.05), and elements of growing concern like Ti (r=-0.37, p<0.01) or Al (r=-0.34, p<0.05) and between cortisol and Sr (r=0.29, p<0.05), Se (r=-0.38, p<0.01), and As (r=-0.26, p<0.05). These results suggest that turtles chronically exposed to different inorganic elements (such as Pb and Cd), driving to a highly consume of esterase and to a prolonged cortisol elevation. The obtained results indicate the usefulness of these biomarkers in the assessment of inorganic elements pollution in this species.

Measurement of p-nitrophenyl acetate esterase activity (EA), total antioxidant capacity (TAC), total oxidant status (TOS) and acetylcholinesterase (AChE) in gills and digestive gland of Mytilus galloprovincialis exposed to binary mixtures of Pb, Cd and Cu.

The aims of the present work were (1) to evaluate oxidative stress biomarkers and AChE in two tissues of wild mussel (Mytilus galloprovincialis) of high biochemical activity and accumulation capacity (gills and digestive gland) and (2) to study the behaviour of these biomarkers in presence of heavy metals. For this, EA, TOS, TAC and AChE were measured in tissues of mussels exposed to binary combination of Pb, Cd and Cu. Mussels (n=36) were exposed to one of the binary mixtures of Pb (1000μgL-1), Cd (100μgL-1) and Cu (100μgL-1) for 7days, under controlled conditions. Gills and digestive gland were extracted and frozen at -80°C until analysis. The automatic methods employed for the measurement of EA, TAC, TOS and AChE in M. galloprovincialis revealed higher levels of these biomarkers in digestive gland than gills. Study results suggest that gills would be the tissue of election for study oxidative stress markers, whereas digestive tissue should be selected for AChE measurements in case of evaluation of combined metal toxicity in mussels.

Increased alanine concentration is associated with exposure to fenitrothion but not carbamates in Chironomus riparius larvae

Chironomus riparius Meigen were exposed to three different insecticides, the organophosphorous fenitrothion and the carbamates carbaryl and carbofuran (0, 1, 10, and 100 μg/L) for 24 h as fourth-instar larvae. Acetylcholinesterase (AChE), naphtylacetate esterase (NAE), p-nitrophenylacetate esterase (PNPAE), glutathione-S-transferase (GST), and a number of metabolites (alanine, pyruvate, lactate, trehalose, aspartate, oxalacetate) were measured to determine which was the most valuable biochemical biomarker of exposure. AChE activity was significantly reduced by all three insecticides, PNPAE by fenitrothion, carbofuran and carbaryl, whereas NAE activity was stimulated by carbaryl and unaffected by fenitrothion and carbofuran. Metabolites analysis revealed a strong accumulation of alanine in larvae exposed to fenitrothion, but not in larvae exposed to carbamates. This accumulation was accompanied by a significant increase of lactate and a significant decrease of pyruvate and trehalose. No variations were observed with carbofuran and carbaryl. No change of aspartate concentration was detected. We conclude that the association of alanine accumulation with a significant inhibition of AChE activity can be used as a valuable biochemical biomarker of exposure.

Usefulness of Soil p-Nitrophenyl Acetate Esterase Activity as a Tool to Monitor Biodegradation of Polybutylene Succinate (PBS) in Cultivated Soil

Degradation behaviors of polybutylene succinate (PBS) and p-nitrophenyl acetate (pNPA) esterase activity in soil were monitored for six months in the experimental field located at Fujieda in Japan. The PBS used in this experiment was an aliphatic polyester which was under trial product as a new thermoplastic material (Mn ≒ 7.0×10.4 This PBS was prepared through direct dehydration polycondensation without diisocyanate as coupling agent for increasing molecular weight. Cellulose filter paper was used as a control for the degradation test. This PBS strips (50 mm × 30 mm, thickness of 0.3 mm) mixed with field moist soil sample which had been sieved (<2 mm mesh) were packed in bags made of glass-fiber filter papers. The bags were buried in the surface soil layer (0–10 cm) of experimental field. Weight of PBS strip was decreased by approximately 20% in six months. Scanning electron microscopy (SEM) photographs showed that the surface of the PBS strip was gradually corroded in soil. During PBS degradation, pNPA esterase activity in the peripheral soil was higher than that of the soil without PBS addition. Actinomycetous PBS degrader was isolated from peripheral soil of degrading PBS strip using PBS agar plate. The isolate exhibited pNPA esterase activity which was competitively inhibited by PBS particles. These results suggested that pNPA esterase activity of soil is an useful indicator of PBS biodegradation in soil.

An Improved Method for Determination of p-Nitrophenyl Acetate Esterase Activity in Soil

An Improved Method for Determination of p-Nitrophenyl Acetate Esterase Activity in Soil

The [formula omitted] phosphatase activity of ubiquitin from bovine erythrocytes

1.1. Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity.2.2. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 × 10−3M.3.3. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 × 10−3M), molybdate (Ki = 1.2 × 10−6M), and phosphate (Ki = 1.4 × 10−3M).4.4. These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin.5.5. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.

Specific arginine modification at the phosphatase site of muscle carbonic anhydrase.

Mammalian carbonic anhydrase III has previously been shown to catalyze the hydrolysis of p-nitrophenyl phosphate in addition to possessing the conventional CO2 hydratase and p-nitrophenylacetate esterase activities. Modification of pig muscle carbonic anhydrase III with the arginine reagent phenylglyoxal yielded two clearly distinctive results. Reaction of the enzyme with phenylglyoxal at concentrations equivalent to those of the enzyme yielded stoichiometric inactivation titration of the enzyme's phosphatase activity, approaching 100% loss of activity with the simultaneous modification of one arginine residue, the latter based on a 1:1 reaction of phenylglyoxal with arginine. At this low ratio of phenylglyoxal to enzyme, neither the CO2 hydratase activity nor the acetate esterase activity was affected. When the modification was performed with a significant excess of phenylglyoxal, CO2 hydratase and acetate esterase activities were diminished as well. That loss of activity was accompanied by the incorporation of an additional half dozen phenylglyoxals and, presumably, the modification of an equal number of arginine residues. The data in their entirety are interpreted to show that the p-nitrophenylphosphatase activity is a unique property of carbonic anhydrase III and that excessive amounts of the arginine-modifying reagent lead to unspecific structural changes of the enzyme as a result of which all of its enzymatic activities are inactivated.

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO 2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K m of 2.8 m m. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate ( K i = 1.22 × 10 −3M), arsenate ( K i = 1.17 × 10 −3M), and molybdate ( K i = 1.34 × 10 −7M), with these inhibitors having no effect on the CO 2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO 2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO 2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.

The effect of zinc on the activity and fluorescence of carbonic anhydrase holoenzymes.

The addition of Zn2+ to human carbonic anhydrase B holoenzyme was shown to enhance the protein fluorescence, and this enhancement was correlated with the inhibition of the p-nitrophenyl acetate esterase activity. The affinity for the inhibitory Zn2+ was increased when the ionic inhibitors, acetate or chloride, were added, suggesting that the inhibitory Zn2+-binding site is within the region of the protein that undergoes an anion-induced conformational change. A similar fluorescence enhancement was observed when Zn2+ was added to human carbonic anhydrase C and to bovine carbonic anhydrase, demonstrating that the binding site is not a thiol group. Circular-dichroism studies showed that the C isoenzyme but not the B isoenzyme underwent a major conformational change in the presence of Zn2+. A mechanism for the Zn2+-induced fluorescence enhancement was suggested on the basis of studies with simple compounds.

Distribution, specificity and function of some proteases, general esterases and cholinesterases from several species of starfish

1. 1. The distribution and pattern of proteases, lipases and esterases in extracts from the pyloric caeca and skin of several species of starfish have been determined by starch gel electrophoresis coupled with specific staining. In Asterias rubens the cardiac stomach, intestine and rectal sacs were also investigated. 2. 2. Most protease activity originates in the caecae. The skin of A. rubens contained insignificant activity towards artificial protease substrates, but while that of Solaster papposus was about 10 per cent of the caecal level, it would not digest gelatin. The significance of these findings for external digestion is discussed. 3. 3. The proteases are first formed as inactive zymogens and become spontaneously active after extraction. The number of bands resolved by electrophoresis increases in the first day of extraction and then remains constant. 4. 4. Using artificial substrates and specific inhibitors, distinct trypsin and chymotrypsin-like activities could be demonstrated in tissue extracts. Their distribution in various species is reported. Two bands of trypsin, two bands of chymotrypsin and one band of p-nitrophenylacetate esterase could be resolved in extracts of A. rubens by electrophoresis on Sephadex blocks. Michaelis constants and other properties of the partially purified enzymes and those in crude extracts are reported. 5. 5. In caecal extracts tributyrin esterase showed two bands of activity on starch gel and these corresponded to the major bands of p-nitrophenyl acetate esterase activity. Triolein esterase activity was weaker and corresponded with the most electropositive of the other esterase bands. These esterase activities were distinct from the protease activity. A. rubens skin esterase was inactive with p-nitrophenyl acetate. 6. 6. Extracts were also investigated for the presence of enzymes similar to human acetylcholine esterase and serum cholinesterase. Evidence was obtained for an acetylcholinesterase which is tentatively suggested to be involved in nervous function.