Increase In Esterase Activity Research Articles

Resistance to common organophosphate and carbamate insecticides inAphis pomi(Hemiptera: Aphididae)

Introduction. Aphis pomi (De Geer) has developed resistance to organophosphate and carbamate insecticides, as a result of long-term application of these insecticides in conventional apple orchards. For many years, the only mechanism of resistance identified in aphids was overproduction of insecticide-detoxifying esterases. Materials and methods. Insecticide resistance of A. pomi , collected from two conventional apple orchards (localities of Radmilovac – RA and Bela Crkva – BC) and one organic apple orchard (locality of Surcin – SU), was tested by bioassays and biochemical assays. Results and discussion. Compared with LC 50 values for the susceptible population (organic orchard), both populations from the conventional orchards were highly resistant to pirimicarb (234.5 and 52.9 times) and moderately resistant to dimethoate (10.7 and 9.0 times). Increased esterase activity was determined in these two resistant aphid populations. Each of them also produced one esterase isoform more than the susceptible population, when 1-naphthyl acetate was used as a substrate for zymographic detection; when 2-naphthyl acetate was used as a substrate, only one resistant population produced two new esterase isoforms. In one of the resistant populations acetylcholinesterase (AChE) was significantly less inhibited by pirimicarb than in the other resistant population and the susceptible population, which indicates that this population developed another resistance mechanism – Modification of AChE (MACE). Conclusion. Detoxification of insecticides by the metabolic resistance mechanism of esterase enzymes and mechanism of modification of AChE was proven in one aphid population (RA). The other population (BC) has developed only metabolic resistance (enhanced metabolism by esterases), without modification of the insecticide target site (AChE). Development of insecticide resistance was caused by long-term application of acetylcholinesterase inhibitors (organophosphates and carbamates) in these conventional orchards.

Characterisation of a cold-active and salt-tolerant esterase from Lactobacillus plantarum with potential application during cheese ripening

During cheese ripening, the metabolic activity of microorganisms influences cheese flavour. Lactobacillus plantarum is a non-starter lactic acid bacterium that can be found during cheese ripening. An esterase-encoding gene from L. plantarum, lp_3505, was cloned and expressed in Escherichia coli BL21 (DE3). The biotechnologically produced Lp_3505 protein was purified as an active soluble form using His-tag affinity chromatography. The enzyme has an optimal pH and temperature of 6 and 5 °C, respectively. The enzyme showed remarkable stability at 20 °C, retaining more than 60% of its maximal activity after 20 h incubation at this temperature. In addition, NaCl concentrations lower than 20% increased esterase activity. The cold-activity and salt-tolerance exhibited by Lp_3505 indicated that this esterase could be a useful exogenous enzyme to be added for cheese ripening.

Link between domoic acid production and cell physiology after exchange of bacterial communities between toxic Pseudo-nitzschia multiseries and non-toxic Pseudo-nitzschia delicatissima.

Bacteria are known to influence domoic acid (DA) production by Pseudo-nitzschia spp., but the link between DA production and physiology of diatoms requires more investigation. We compared a toxic P. multiseries to a non-toxic P. delicatissima, investigating links between DA production, physiological parameters, and co-occurring bacteria. Bacterial communities in cultures of both species were reduced by antibiotic treatment, and each of the diatoms was inoculated with the bacterial community of the other species. The physiology of P. delicatissima was minimally affected by the absence of bacteria or the presence of alien bacteria, and no DA was detected. P. multiseries grew faster without bacteria, did not produce a significant amount of DA, and exhibited physiological characteristics of healthy cells. When grown with alien bacteria, P. multiseries did not grow and produced more DA; the physiology of these cells was affected, with decreases in chlorophyll content and photosynthetic efficiency, an increase in esterase activity, and almost 50% mortality of the cells. The alien bacterial community had morphological and cellular characteristics very different from the original bacteria, and the number of free-living bacteria per algal cell was much higher, suggesting the involvement of bacteria in DA production.

Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase.

Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min-1 for hydrolysis of p-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat ~ 1.6 min-1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the corresponding location of Candida lipases is uncharged due to other active site differences and does not cause the same distortion. This example of the fine tuning of the same catalytic triad for different types of catalysis by subtle interactions with other active site residues shows how difficult it is to design new catalytic reactions of enzymes.

Apoptosis of tiger shrimp (Penaeus monodon) haemocytes induced by Escherichia coli lipopolysaccharide

This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca2+ (CF-Ca2+) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10μgmL−1) were used. Results showed that ROS production, NO production and CF-Ca2+ concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca2+ release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca2+-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes.

Synthesis and characterization of phenolic Mannich bases and effects of these compounds on human carbonic anhydrase isozymes I and II

Mannich bases 2a-f derived from 3,4-dimethylphenol (1), formaldehyde and different amines are prepared and subjected to spectral (IR, 1H and 13C NMR) and elemental analyses. The inhibition of two human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with 1 and synthesized Mannich bases 2a-f and acetazolamide (AAZ) as a control compound was investigated in vitro by using the hydratase and esterase assays. In relation to hydratase and esterase activities of the half maximal inhibitory concentration (IC50) and the inhibition equilibrium constants (Ki)values were determined. Only two compounds (2a and 2e)exhibit weak hCA II inhibitory effects on esterase activity. IC50 and Ki values for 2a and 2e with respect to esterase activity of hCA II are0.88 × 103 and 6.3–7.6 µM and 0.44 × 103 and 19.0–96.4 µM,respectively. On the contrary, compounds 2b and 2d might be used as CA activators due to increasing esterase activity of hCA I and hCA II isozymes.

Esterase profile of Rhipicephalus (Boophilus) microplus populations collected from Northern India exhibiting varied susceptibility to deltamethrin

Rhipicephalus (Boophilus) microplus is an economically important ectoparasite of cattle. Chemical acaricides remain the most practical method for control of these pests. During past two decades there have been increasing reports of resistance development against synthetic pyrethroids in tick populations of this species throughout the world. A study was conducted to determine the level of susceptibility of R. (B.) microplus to deltamethrin collected from different geographical locations of northern India. LPT bioassay results revealed LC(50) values of deltamethrin ranging from 0.035 to 0.00037% A.I. Esterase profile of the tick larval extracts using native PAGE, revealed 5 bands of esterase activity designated EST-5 to EST-1A. Inhibitory tests recognized EST-1, EST-2 and EST-3 as Acetylcholinesterases (AchEs), EST-4 and EST-5 as Carboxylesterases (CaEs). The band intensity varied between tick populations of various locations, being more intense in case of the resistant populations. An extra band of esterase activity (EST-1A) was obtained in larval extracts of ticks from 3 locations. This increased esterase activity may be involved in the resistance development in these tick populations. Acaricide resistance is a multi-factorial phenomenon, thus other causes of increased resistance like sodium channel mutation and reduced drug penetration (e.g. cuticle thickening) and behavioural changes (e.g. avoiding the pesticides) are to be tested in future in order to confirm the basic cause of the resistance development in these acaricide resistant tick populations.

Association of Esterases With Insecticide Resistance in <I>Culex quinquefasciatus</I> (Diptera: Culicidae)

The southern house mosquito, Culex quinquefasciatus Say, is a competent vector of human disease and an important target of mosquito abatement programs. However, these management programs have been compromised by development of insecticide resistance. In the current study, susceptibilities to naled and resmethrin, two adulticides used in mosquito abatement, were monitored using a topical and contact bioassay, respectively, in five field- collected populations of C. quinquefasciatus (MARC, HOOD1, HOOD2, MINLOVE, and THIB). Frequencies of resistance, measured as survival after treatment with discriminating concentrations (i.e., sufficient to kill > 90% of a reference susceptible strain) were high (88.0-96.8%) in all field collections treated with naled, but were variable (3.3-94.2%) with resmethrin. In addition, esterase activities in mosquitoes from these collections were quantified using alpha-naphthyl acetate and ranged from 1.08 to 3.39 micromol alpha-naphthol produced min(-1) mg prot(-1). Heightened activities were associated with decreased insecticide susceptibility in HOOD1, THIB, and MINLOVE but not HOOD2. Esterases were visualized using native polyacrylamide gel electrophoresis, and intra- and interstrain differences in banding patterns were detected. In addition, esterases from MINLOVE mosquitoes were more numerous and intensely staining when compared with those from a laboratory-susceptible strain. Finally, naled synergized the toxicity of resmethrin in populations with decreased insecticide susceptibility and increased esterase activity by 2.5-(MINLOVE) to three-fold (THIB). Results from this study will allow management strategies for populations of C. quinquefasciatus to be optimized, and provide a foundation for further studies exploring use of esterase inhibitors as synergists of pyrethroid toxicity.

Esterases and glutathione S‐transferases contribute to pyrethroid resistance in western flower thrips, Frankliniella occidentalis

AbstractWestern flower thrips, Frankliniella occidentali s (Pergande), has developed a high level of resistance to pyrethroid insecticides in Australia. Biochemical examination of those pyrethroid‐resistant populations shows esterase activity 2.0‐ to 6.5‐fold higher in six of the seven resistant field populations compared with the susceptible population. This increase in esterase activity in the resistant populations was further supported by native polyacrylamide gel electrophoresis and inhibition studies with the organophosphate insecticide profenofos. The polyacrylamide gel electrophoresis analysis showed differences in the banding pattern and intensity of the esterase isozymes between the pyrethroid‐resistant and susceptible F. occidentalis populations. Glutathione S‐transferase activity was significantly higher (up to 1.7‐fold) in five of the seven resistant field populations when compared with the susceptible populations. Esterases appear to be an important mechanism that contributes to pyrethroid resistance. Glutathione S‐transferases may also play a role in pyrethroid resistance in Australian F. occidentalis populations.

Resistance Mechanisms to Chlorpyrifos and F392W Mutation Frequencies in the Acetylcholine Esterase Ace1 Allele of Field Populations of the Tobacco Whitefly,Bemisia tabaciin China

The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides.

Cross-Resistance to Insecticides in a Malathion-Resistant Strain of Ceratitis capitata (Diptera: Tephritidae)

Resistance to malathion has been reported in field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in areas of Spain where an intensive use of this insecticide was maintained for several years. The main goal of this study was to determine whether resistance to malathion confers cross-resistance to different types of insecticides. Susceptibility bioassays showed that the malathion-resistant W-4Km strain (176-fold more resistant to malathion than the susceptible C strain) has moderate levels of cross-resistance (three- to 16-fold) to other organophosphates (trichlorphon, diazinon, phosmet and methyl-chlorpyrifos), the carbamate carbaryl, the pyrethroid lambda-cyhalothrin, and the benzoylphenylurea derivative lufenuron, whereas cross-resistance to spinosad was below two-fold. The W-4Km strain was selected with lambda-cyhalothrin to establish the lambda-cyhalothrin-resistant W-1Klamda strain (35-fold resistant to lambda-cyhalothrin). The synergistic activity of the esterase inhibitor DEF with lambda-cyhalothrin and the increase in esterase activity in the W-1Klamda strain suggests that esterases may be involved in the development of resistance to this insecticide. Our results showed that resistance to malathion may confer some degree of cross-resistance to insecticides currently approved for the control of Mediterranean fruit fly in citrus crops (lambda-cyhalothrin, lufenuron, and methyl-chlorpyrifos). Especially relevant is the case of lambda-cyhalothrin, because we have shown that resistance to this insecticide can rapidly evolve to levels that may compromise its effectiveness in the field.

Release of Reduced Inorganic Selenium Species into Waters by the Green Fresh Water Algae Chlorella vulgaris

The common green fresh water algae Chlorella vulgaris was exposed to starting concentrations of 10 μg/L selenium in the form of selenate, selenite, or selenocyanate (SeCN(-)) for nine days in 10% Bold's basal medium. Uptake of selenate was more pronounced than that of selenite, and there was very little uptake of selenocyanate. Upon uptake of selenate, significant quantities of selenite and selenocyanate were produced by the algae and released back into the growth medium; no selenocyanate was released after selenite uptake. Release of the reduced metabolites after selenate exposure appeared to coincide with increasing esterase activity in solution, indicating that cell death (lysis) was the primary emission pathway. This is the first observation of biotic formation of selenocyanate and its release into waters from a nonindustrial source. The potential environmental implications of this laboratory observation are discussed with respect to the fate of selenium in impacted aquatic systems, the ecotoxicology of selenium bioaccumulation, and the interpretation of environmental selenium speciation data generated, using methods incapable of positively identifying reduced inorganic selenium species, such as selenocyanate.

Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae)

A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.

Biochemical mechanisms of insecticide resistance in the diamondback moth (DBM), Plutella xylostella L. (Lepidopterata: Yponomeutidae), in the Sydney region, Australia

AbstractFollowing the detection of resistant diamondback moth (DBM) populations to synthetic pyrethroid, organophosphorus and indoxacarb insecticides in the Sydney Basin, a study of the major biochemical mechanisms was conducted to determine the type of resistance in these populations. The activity of cytochrome P450 monooxygenases increased two‐ to sixfold when compared with the susceptible strain. Up to a 1.9‐fold increase in esterase activity in resistant strains compared with the susceptible strain was observed. In vitro inhibition studies showed that profenofos, methamidophos and chlorpyrifos strongly inhibited the esterases while permethrin and esfenvalerate resulted in less than 30% inhibition. Qualitative analysis of the esterases using native polyacrylamide gel electrophoresis showed four bands in both the susceptible and resistant individuals with more intense staining in the resistant individuals. The development of these bands was inhibited by methamidophos and chlorpyrifos pretreatment of the protein extract while permethrin and esfenvalerate did not exhibit this effect. Glutathione S‐transferase (GST) activity was significantly higher in two field populations compared with the remaining populations. Overall, the study showed that the mechanisms of insecticide resistance in the DBM populations in the area studied were due to cytochrome P450 monooxygenases, esterase and GSTs, and possibly other non‐metabolic mechanisms that were not investigated in the present study.

Effect of adenosine triphosphate on changes of fatty acids in harvested litchi fruit infected by Peronophythora litchii

Recent investigations have shown that disease development of harvested horticultural crops may be attributed to a limited availability of energy or low energy production. In this study, litchi fruit were treated with 1.0 mM adenosine triphosphate (ATP) or 0.5 mM 2,4-dinitrophenol (DNP) and then half of the ATP-treated fruit were inoculated with Peronophythora litchii. The composition and contents of fatty acids (FAs) and esterase activity in litchi fruit during storage were investigated. Free fatty acids (FFAs) in all fruit increased over storage, especially in the P. litchii-inoculated fruit. In particular, the content of saturated FAs increased faster than unsaturated FAs. In polar lipids (PL), a decrease in the amount of C18:3 and an increase in the amount of C16:0 or C18:0 was found during storage, while the proportions of C16:0, C18:0 and C18:1 in neutral lipid (NL) gradually increased but the proportions of C18:3 decreased during storage. The proportion of C18:2 increased within the first four days and then decreased. Exogenous ATP treatment suppressed the release of FFAs and increased the contents of each FA in PL, indicating a slower hydrolysis of lipids. ATP treatment also delayed the increase in the proportion of C18:0 in NL. Further analysis showed that the double bond index (DBI) of litchi fruit decreased in all fractions of FAs and ATP treatment can slow the decrease in DBI. In addition, lower esterase enzyme activity was detected in all ATP-treated fruit. Treatment with DNP (a respiration uncoupler) increased esterase activity. P. litchii-inoculated fruit after ATP treatment also exhibited similar trends in delaying the release of FFAs. Enhanced disease resistance of litchi fruit by ATP could involve the levels of FAs and esterase activity.

Effect of sex, ovariectomy and female sex hormones on neutral cholesterol ester hydrolase in rat liver.

The effects of sex, ovariectomy and female sex hormones on neutral cholesterol ester hydrolase activity were investigated in rat liver cytoplasm. Hydrolase activity was determined at pH 7.45 with an acetone dispersed cholesterol oleate substrate preparation. Neutral cholesterol esterase activity was higher in male than in female rats and the ovariectomy decreased the enzyme activity. The administration of only estradiol or combined with progesterone to ovariectomized rats for three consecutive days resulted in an increase in esterase activity to the level observed in intact animals. By contrast, the only progesterone resulted in no significant changes of activity. In intact female rats, the short-term treatments of gonadal hormones did not produce any alteration on cholesterol esterase. Examination of the effects of a single dose of estradiol and progesterone for intervals between 1 and 24 h showed that both hormones promoted early and opposite effects on neutral ester hydrolase. Activity was decreased in 3-4 h by the estradiol and was markedly increased throughout 3-4-6 h by the progesterone injection. In vitro, the two female sex hormones behaved as potent inhibitors of the enzyme, competing with the substrate for the active centre of the hydrolase.

Changes of Oxidase and Hydrolase Activities in Pecan Leaves Elicited by Black Pecan Aphid (Hemiptera: Aphididae) Feeding

The black pecan aphid, Melanocallis caryaefoliae (Davis) (Hemiptera: Aphididae), is a foliar feeder of pecan, Carya illinoinensis (Wangenh.) K. Koch (Juglandaceae). The pest causes chlorosis of leaflet lamina, physiological damage to foliage and trees, and commonly limits the profitability of commercial pecan orchard enterprises. However, key aspects of this host-pest interaction are poorly understood. We report here the effects of M. caryaefoliae feeding on the foliar activity of oxidative (i.e., catalase, lipoxygenase [LOX]-1 and 3, and peroxidase) and hydrolytic (i.e., esterase) enzymes in relation to the degree of aphid resistance among pecan varieties. The 2-yr study showed that M. caryaefoliae-infested foliage exhibited elevated peroxidase activity only in susceptible ('Desirable', 'Sumner', and 'Schley'), but not in resistant ('Cape Fear', 'Gloria Grande', and 'Money Maker') genotypes. Susceptible genotypes also exhibited more severe leaf chlorosis in response to M. caryaefoliae feeding than the resistant genotypes; however, the aphid feeding did not influence catalase or esterase activity in all varieties, except the increase of esterase activity in Desirable and Gloria Grande. Melanocallis caryaefoliae feeding also influences activity of two lipoxygenase isozymes, with LOX3 being more frequently induced than LOX1. Foliar LOX3 activity was more frequently induced by M. caryaefoliae feeding in the moderately resistant 'Oconee' and highly resistant Money Maker and Cape Fear than in the susceptible genotypes. Therefore, the elevation of peroxidase is likely to be associated with aphid susceptibility and contributed to the severe leaf chlorosis, whereas the increase of LOX3 activity might be associated with aphid resistance in pecan. These findings contribute to our understanding of the etiology of M. caryaefoliae-elicited leaf chlorosis on pecan foliage. Such information may also be used to develop enzyme markers for identifying black pecan aphid resistance and/or susceptibility in pecan germplasm.

Nanoparticles Containing Anti-inflammatory Agents as Chemotherapy Adjuvants II: Role of Plasma Esterases in Drug Release

The pre-administration of the anti-inflammatory drugs dexamethasone (DEX) and cortisone acetate reduces toxicity and enhances efficacy of anticancer agents in murine models and in human clinical trials (1–5). We previously reported on the formulation of the lipophilic dexamethasone palmitate ester (DEX-P) in nanoparticles (NPs) employing a microemulsion template engineering technique to achieve tumor-specific delivery of dexamethasone (6). The nanoparticles exhibited significantly enhanced stealth properties as indicated by reduced macrophage uptake and decreased adsorption of opsonin proteins in in vitro assays (6). Unexpectedly, preliminary biodistribution studies of NPs containing [3H]-DEX-P in tumor-bearing mice showed that the radiolabel was cleared from the circulation rapidly and exhibited high liver uptake. Our previous in vitro release studies demonstrated that rapid release of the radiolabel from the NPs was observed when 10% mouse plasma was used as the medium, while nominal release was observed in phosphate-buffered saline (PBS) buffer (6). Esterolysis of NP-associated DEX-P was presumed to be the main cause for the rapid drug release in plasma, as most of the released radioactivity was in the form of DEX and not DEX-P. High degradation rates of ester prodrugs in rodent plasma has been attributed to increased esterase activity, while only minimal degradation in human plasma has been observed (7–9). Based on our observation of the release of [3H]-DEX from NPs in mouse plasma, we studied the release of DEX from nanoparticles in various plasma sources as a guide for the design of future in vivo experiments.

The Effect of Insecticide Synergists on the Response of Scabies Mites to Pyrethroid Acaricides

BackgroundPermethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways.Methodology/Principal FindingsTo determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites.ConclusionsThese findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites.

Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign

Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occurring esterases. The design was based on a previously developed strategy [G. Höst, L.G. Mårtensson, B.H. Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, Biochim. Biophys. Acta 1764 (2006) 1601–1606.], in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with k cat / K M = 625 (± 38) M −1 s −1. It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with k cat / K M = 101,700 (± 4800) M −1 s −1, which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable K M values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV), but for V121A/V143A/T200A no K M could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, k cat / K M is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.