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

BACKGROUND: Drastic 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. METHODS: In 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. RESULTS: The 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 ß-esterase was similar or lower than that in the NO strain. CONCLUSIONS: Aedes 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.

Impact of deltamethrin selection on kdr mutations and insecticide detoxifying enzymes in Aedes aegypti from Mexico.

BACKGROUND: Insecticide resistance is a serious problem for vector control programmes worldwide. Resistance is commonly attributed to mutations at the insecticide's target site or increased activity of detoxification enzymes. METHODS: We determined the knockdown concentration (KC50) and lethal concentration (LC50) of deltamethrin in six natural populations of adult Aedes aegypti from southeastern Mexico. These populations were then selected over five generations using the LC50 from the preceding generation that underwent selection, and the heritability of deltamethrin resistance was quantified. For each generation, we also determined the frequency of the kdr alleles L410, I1016 and C1534, and the levels of activity of three enzyme families (α- and ß-esterases, mixed-function oxidases and glutathione S-transferases (GST)) associated with insecticide detoxification. RESULTS: There was an increase in KC50 and LC50 values in the subsequent generations of selection with deltamethrin (FS5vs FS0). According to the resistance ratios (RRs), we detected increases in LC50 ranging from 1.5 to 5.6 times the values of the parental generation and in KC50 ranging from 1.3-3.8 times the values of the parental generation. Triple homozygous mutant individuals (tri-locus, LL/II/CC) were present in the parental generations and increased in frequency after selection. The frequency of L410 increased from 1.18-fold to 2.63-fold after selection with deltamethrin (FS5vs FS0) in the populations analyzed; for I1016 an increase between 1.19-fold to 2.79-fold was observed, and C1534 was fixed in all populations after deltamethrin selection. Enzymatic activity varied significantly over the generations of selection. However, only α- esterase activity remained elevated in multiple populations after five generations of deltamethrin selection. We observed an increase in the mean activity levels of GSTs in two of the six populations analyzed. CONCLUSIONS: The high levels of resistance and their association with high frequencies of kdr mutations (V410L, V1016I and F1534C) obtained through artificial selection, suggest an important role of these mutations in conferring resistance to deltamethrin. We highlight the need to implement strategies that involve the monitoring of kdr frequencies in insecticide resistance monitoring and management programmes.

Esterase activity and interaction of human hemoglobin with diclofenac sodium: A spectroscopic and molecular docking study.

To get an idea about the pharmacokinetics and pharmacodynamics, it is important to study the drug-protein interaction. Therefore, herein, we studied the interaction of diclofenac sodium (DIC) with human hemoglobin. The binding study of nonsteroidal antiinflammatory drug, DIC with human hemoglobin (HHB) was done by utilizing fluorescence, UV-visible, time-resolved fluorescence and far-UV circular dichroism spectroscopy (CD). Various thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) were also calculated. CD results showed that DIC induces secondary structure change in HHB. Fluorescence resonance energy transfer was also performed. Additionally, it was also observed that DIC inhibits the esterase-like enzymatic activity of HHB via competitive inhibition.

Antimicrobial capacity of ultrasound and ozone for enhancing bacterial safety on inoculated shredded green cabbage (Brassica oleracea var. capitata).

The high frequency and incidence of foodborne outbreaks related to fresh vegetables consumption is a major public health concern and an economic burden worldwide. This study evaluated the effect of individual and combined application of ultrasound (40 kHz, 100 W) and ozone on the inactivation of foodborne Escherichia coli and Salmonella, as well as their impact on cabbage color and vitamin C content. Plate count, scanning electron microscopy (SEM), and flow cytometry (FCM) following single or double staining with carboxyfluorescein diacetate and (or) propidium iodide were used to determine bacterial inactivation parameters, such as cell culturability, membrane integrity, intracellular enzyme activity, and injured and dead cells. The results of FCM and SEM showed that ultrasound treatment affected bacteria mainly by acting on the cell membrane and inactivating intracellular esterase, which resulted in bacterial death. Furthermore, when combined with ozone at 1.5 mg/L, the maximum reduction of bacterial populations was observed at 8 min with no damage on the surface of treated leaves. Therefore, fresh products sanitization using a combination of ultrasound and ozone has the potential to be an alternative for maintaining the color and vitamin C content of green cabbage.

O-hexyl O-2,5-dichlorophenyl phosphoramidate as a substrate for domestic and sea bird serum A-esterases: Hydrolysis levels, Cu2+- and Zn2+-dependence and stereoselectivity.

O-Hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy in hens. It has been used as a tool to identify new A-esterase activities in animal tissues. This study shows the EDTA-resistant, Cu2+- and Zn2+-dependent hydrolysis of racemic HDCP in domestic and sea bird serum using UV/Vis spectrophotometry and chiral chromatography. The results clearly show a significant (p ˂ 0.05) Cu2+- and Zn2+-dependent HDCP hydrolysis in the serum of all bird species versus EDTA, except for the Zn2+-dependent HDCPase activity from Yucatecan quail serum. The ratio of Cu2+/Zn2+ hydrolysis varied between 1 and 7 (intraspecies) and 15.6 (interspecies). EDTA affected the Cu2+- and Zn2+-dependent HDCPase activity in the range of 37-95% and 40-50%, respectively. HDCP hydrolysis activated by Cu2+ was significantly (p ˂ 0.05) stereoselective (R-(+)-HDCP ˃ S-(-)-HDCP) in chicken and sea bird serum. Its R-(+)-HDCP/S-(-)-HDCP ratios were 6.8 and 1.6-2.8, respectively. EDTA-resistant and zinc-dependent HDCP hydrolysis were not stereospecific in all bird sera tested. The present ex vivo study reinforces the idea that bird sera have HDCPase activity that is sensitive to divalent metals, resistant to EDTA and possibly associated with the protein albumin.

The role of SIPK signaling pathway in antioxidant activity and programmed cell death of tobacco cells after exposure to cadmium.

Cadmium (Cd) toxicity induces oxidative burst and leads to programmed cell death (PCD) in plant cells. The role of salicylic acid-induced protein kinase (SIPK) signaling pathway in Cd-induced oxidative stress was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Barley 21). The cells were pretreated with 40 µM PD98059 (inhibitor of MAPKK) and then exposed to 50 µM Cd for 24 h. The percentages of cell viability, apoptosis, necrosis, and the content of reactive oxygen species (ROS) were monitored by flow cytometry. Expression of PCD related gene (Hsr203J) and the contents of certain signaling molecules were measured as well. The results showed that Cd increased the expression of SIPK, Hsr203J, and CAT genes, the activities of catalase and caspase-3-like enzymes. Addition of PD98059 inhibitor reduced the expression of Hsr203J and CAT genes, decreased CAT activity, but increased ROS and SA contents, and caspase-3-like activity and apoptosis rate. The highest apoptosis level was accompanied by the highest level of Hsr203J gene expression. From the results it can be suggested that upon treatment of tobacco cells with Cd, internal SA content increased and induced the SIPK signaling pathway, thereby inhibited the antioxidant system and led to PCD.

Assessment of mouse strain differences in baseline esterase activities and toxic response to sarin.

Genetics likely play a role in various responses to nerve agent (NA) exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses. This study uses different mouse strains to identify if mouse strain differences in sarin exposure exist. In Experiment 1, basal levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CE) were measured in different strains of naïve mice to account for potential pharmacokinetic determinants of individual differences. In Experiment 2, median lethal dose (MLD) levels were estimated in 8 inbred mouse strains following subcutaneous (s.c.) administration of sarin. Few strain or sex differences in esterase activity levels were observed, with the exception of erythrocyte AChE activity in the C57BL/6J strain. Both sex and strain differences in toxicity were observed, with the most resistant strains being the BALB/cByJ and FVB/NJ strains and the most sensitive strain being the DBA/2J strain. These findings can be expanded to explore pathways involved in NA response, which may provide an avenue to develop therapeutics for preventing and treating the damaging effects of NA exposure.

Molecular dynamics simulation as a tool for assessment of drug binding property of human serum albumin.

Human serum albumin (HSA) is a major plasma protein and binding of drugs with this plasma protein has a great importance. It possess esterase activity which can cleave the drugs containing ester bond and thus, can regulate the effect of drugs. Till date no systematic study has been done to analyse binding of such drugs and to compare the results with the drugs which do not have ester bond. Therefore, in the present study two different categories-ester and non-ester drugs have been considered to analyse their interaction with HSA at two principle drug binding sites using molecular modelling tools. It is observed that the drugs irrespective of ester or non-ester nature prefer either Sudlow site I or II by hydrogen bond and hydrophobic interactions. The information obtained from the study can assist to study pharmacokinetics of the drugs and that in turn will help in noval drug discoveries.

Establishment of a multi-acaricide resistant reference tick strain (IVRI-V) of Rhipicephalus microplus.

Tick-borne diseases is a global threat and tick resistance to commonly used acaricides is a growing problem, thus calling for improved resistance monitoring tools. To aid in monitoring of resistance in field tick populations, a resistant colony of Rhipicephalus microplus was characterized with the aim to establish a reference multi-acaricide resistant tick strain. Using a standardized adult immersion test, the Lethal Concentration(LC)50 values for deltamethrin, cypermethrin, fenvalerate and diazinon against the laboratory selected resistant tick (LSRT) strain were determined as 306.7 ppm, 2776.9 ppm, 30262.1 ppm and 9458.7 ppm. Relative to the susceptible IVRI-I tick strain, the LSRT strain showed 4.78- and 5.84-fold increases in activity of esterases, a 6-fold increase for monooxygenases and a 2.24 fold increase for glutathione S-transferase. In the acetylcholinesterase 2 gene, 22 single nucleotide polymorphisms (SNPs) were identified in the LSRT strain. Four of these SNPs lead to amino acid substitutions and were consistently found in resistant field populations in India. A C190A mutation in the domain II S4-5 linker region of sodium channel gene resulting in a L64I amino acid substitution was recorded in the LSRT strain. Monitorable indicators for the maintenance of the strain, designated as the reference IVRI-V tick strain and representing the first established multi-acaricide resistant tick strain in India, were identified.

Effect of selenium on Penaeus monodon and Perna viridis: Enzyme activities and histopathological responses.

The study was carried out to evaluate enzyme activities and histopathological changes due to the effect of acute and chronic definitive toxicity of selenium (Se) on the post larvae (PL) of giant tiger shrimp (Penaeus monodon), and green mussel (Perna viridis). The 96-h Median Lethal concentration (LC50) for the PL of shrimp was 3.36 mg L-1 and the chronic value for the long-term survival endpoint in a 21-d exposure was 0.10 mg L-1. The green mussel 96-h LC50 was 28.41 mg L-1 and the chronic value for the long-term survival endpoint in a 30-d exposure was 3.06 mg L-1. Native polyacrylamide gel electrophoresis revealed altered diverse isoforms of esterase, superoxide dismutase and malate dehydrogenase activities in the PL of shrimp and green mussel exposed to sublethal concentration of Se. Cellular anomalies such as deformation and fusion of corneal cells, detachment of corneal cells from cornea facet and increased space between ommatidia were observed in the compound eye of PL of shrimp exposed to Se for 21-d. Shrinkage and clumping of mucous gland, degenerative changes in phenol gland, and ciliated epithelium were observed in the foot of green mussel exposed to Se for 30-d. This study shows that cellular anomalies in the compound eye of PL of P. monodon and foot tissues of P. viridis described would affect the vision of shrimp and byssus thread formation in green mussel.

Ligand binding to the FA3-FA4 cleft inhibits the esterase-like activity of human serum albumin.

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of Ks, k+2, and k+2/Ks obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k+3 << k+2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.

Neurologic dysfunction and genotoxicity induced by low levels of chlorpyrifos.

Chlorpyrifos (CPF) is an organophosphorus cholinesterase inhibitor widely used as an insecticide. Neuro and genotoxicity of this agent were evaluated following daily subcutaneous injections at 0.1, 1 and 10mg/kg or its vehicle to laboratory rats during one week, at the end of which somatosensory evoked potentials (SEP) and power spectrum of the electroencephalogram (EEGp) were recorded under urethane anesthesia. In another group of conscious animals, auditory startle reflex (ASR) was evaluated followed, after euthanasia, with measurements of plasma B-esterases, and genotoxicity with the alkaline comet assay (ACA) at the same CPF doses. The results indicated a CPF dose related inhibition of B-esterases. Enhanced inhibition of the ASR by a subthreshold pre-pulse was observed at all doses and ACA showed a significant higher DNA damage than vehicle controls in animals exposed to 10mg/kg CPF. A trend to higher frequencies of EEGp and an increase in amplitude of the first negative wave of the SEP were found at all doses. The first positive wave of the SEP decreased at the CPF dose of 10mg/kg. In summary, a shift to higher EEG frequencies and alterations of somatosensory and auditory input to the central nervous system were sensitive manifestations of CPF toxicity, associated with depression of B-esterases. The changes in electrical activity of the cerebral cortex and DNA damage observed at doses that do not elicit overt toxicity may be useful in the detection of CPF exposure before clinical signs appear.

Esterase metabolism of cholinesterase inhibitors using rat liver in vitro.

A variety of chemicals, such as organophosphate (OP) and carbamate pesticides, nerve agents, and industrial chemicals, inhibit acetylcholinesterase (AChE) leading to overstimulation of the cholinergic nervous system. The resultant neurotoxicity is similar across mammalian species; however, the relative potencies of the chemicals across and within species depend in part on chemical-specific metabolic and detoxification processes. Carboxylesterases and A-esterases (paraoxonases, PON) are two enzymatic detoxification pathways that have been widely studied. We used an in vitro system to measure esterase-dependent detoxification of 15 AChE inhibitors. The target enzyme AChE served as a bioassay of inhibitor concentration following incubation with detoxifying tissue. Concentration-inhibition curves were determined for the inhibitor in the presence of buffer (no liver), rat liver plus calcium (to stimulate PONs and thereby measure both PON and carboxylesterase), and rat liver plus EGTA (to inhibit calcium-dependent PONs, measuring carboxylesterase activity). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (chlorpyrifos oxon, paraoxon, methyl paraoxon, malaoxon) were in agreement with the literature, serving to support the use of this assay. Only a few other inhibitors showed slight or a trend towards detoxification via carboxylesterases or PONs (mevinphos, aldicarb, oxamyl). There was no apparent PON- or carboxylesterase-mediated detoxification of the remaining inhibitors (carbofuran, chlorfenvinphos, dicrotophos, fenamiphos, methamidophos, methomyl, monocrotophos, phosphamidon), suggesting that the influence of esterases on these chemicals is minimal. Thus, generalizations regarding these metabolic pathways may not be appropriate. As with other aspects of AChE inhibitors, their metabolic patterns appear to be chemical-specific.

Effects of rotenone from derris crude extract on esterase enzyme mechanism in the beet armyworm, Spodoptera exiqua (Hubner).

This research was to elucidate the toxicity and esterase enzyme mechanism of derris crude acssst (Derris elliptica Benth) in the beet armyworm (Spodoptera exigua Hubner) by larvae dipping method. Derris crude extract was extracted by Soxhlet's apparatus with ethanoL solvent. The ethanolic derris crude extract was diluted into 5 concentrations to test the toxicity value. The result showed that the toxicity dramatically increased when the concentration of substance and the time exposure were increased. Toxicity in terms of LC50 value against 2nd instar larvae indicated that ca. 69.15 (r2 = 0.98), 51.32 (r2 = 0.99) and 46.60 (r2 = 0.98) ppm at 24, 48 and 72 hours, respectively. The in vivo study with Yang et al. (2004); Visetson and Milne (2001) modified method, revealed that the derris crude extract inhibited esterase enzyme activities ca. 0.75-1.07 fold without a change in the protein concentration. This result indicated that, derris crude extract had efficacy as an insecticide, therefore it had the potential to be another insecticide alternative in S. exiqua control. Moreover, some separate experiments indicated that this extract was safe to human and the environment.

Carnosic acid protects neuronal HT22 Cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners.

In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.

Malondialdehyde, a lipid-derived aldehyde alters the reactivity of Cys34 and the esterase activity of serum albumin.

Malondialdehyde (MDA), one of the key end products of lipid oxidation is elevated in a variety of diseases. It is now well established that MDA can modify proteins in vivo. This paper describes the effects of modification of albumin by MDA and peroxidized linolenic acid on the reactivity of Cys34, a crucial residue conferring antioxidant properties. BSA (10 mg/ml) was incubated with MDA (1 mM) for 72 h in phosphate buffer (100 mM, pH 7.4). BSA was also incubated for three days with lipid samples, which have already undergone peroxidation for 2, 5, 7, and 9 days respectively. The reactivity of Cys34 after modification was monitored using cystamine and 5,5'-dithiobis(2-nitro benzoic acid) (DTNB). The Kobs for the reaction was found to be different between native and MDA modified protein clearly indicating that modification affects the reactivity of Cys34. The individual rate constant (K1) for reaction with DTNB varied significantly between albumin and modified albumin suggesting that loss in reactivity was due to changes at Cys34. However, (K2), the rate constant for reaction of protein with cystamine, determined from a plot of Kobs versus cystamine concentration did not change. This study further shows that modification results in significant loss of the esterase like activity of albumin. Since albumin plays a crucial role in the antioxidant defence due to its abundance (approximately 0.6 mM) in serum, these findings have implications in disease states where increased levels of MDA and oxidative stress drastically may affect the antioxidant capacity of serum.

Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis.

Random mutagenesis followed by a filter-based screening assay has been used to identify a mutant of human class 1 aldehyde dehydrogenase (ALDH1) that was no longer inhibited by Mg(2+) ions but was activated in their presence. Several mutants possessed double, triple, and quadruple amino acid substitutions with a total of seven different residues being altered, but each had a common T244S change. This point mutation proved to be responsible for the Mg(2+) ion activation. An ALDH1 T244S mutant was recombinantly expressed and was used for mechanistic studies. Mg(2+) ions have been shown to increase the rate of deacylation. Consistent with the rate-limiting step for ALDH1 being changed from coenzyme dissociation to deacylation was finding that chloroacetaldehyde was oxidized more rapidly than acetaldehyde. Furthermore, Mg(2+) ions only in the presence of NAD(H) increased the rate of hydrolysis of p-nitrophenyl acetate showing that the metal only affects the binary complex. Though the rate-limiting step for the T244S mutant was different from that of the native enzyme, the catalytic efficiency of the mutant was just 20% that of the native enzyme. The basis for the change in the rate-limiting step appears to be related to NAD(+) binding. Using the structure of a sheep class 1 ALDH, it was possible to deduce that the interaction between the side chain of T244 and its neighboring residues with the nicotinamide ring of NAD(+) were an essential determinant in the catalytic action of ALDH1.

Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block.

A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).

Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions.

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions.

Biochemical studies on different field strains of the pink bollworm Pectinophora gossypiella (Saund.) (Lepidoptera: Gelechiidae) in Egypt.

The specific activity of the esterases and the corresponding total proteins of the diapaused pink bollworm, Pectinophora gossypiella larvae taken from dry bolls which had been left in the cotton fields of the growing seasons of 2000&2001 were measured in four field strains representing the four Governorates: Alexandria, Al-Behaira , Kafr El-Shaikh, and Al-Gharbia. Parallel to these larvae the last generation of the pink bollworm moths trapped from Al-Behaira fields at the end of the same seasons were included in the comparative study. Besides, the susceptible strain larvae and moths from the Lab. of Bollworms Department at the Institute of Plant Protection, Cairo were used as a reference for comparing levels of resistance in the four bollworm strains representing the four Governorates and its impact on proteins and esterases types and activity. Gel scanning with respective standard molecular weights revealed variation in protein bands number and molecular weight type among the four tested field strains. Similarly, the sites of esterases on the SDS-PAGE proved high level of resistance reflected in appearance of isozymes, especially in Al-Gharbia field strain. Generally, Alexandria field strain showed the least level of resistance due to the limited area of cotton fields and the less use of insecticides in Alexandria fields. It is suggested that SDS-PAGE technique can be a biomarker for detection of susceptibility levels or resistance in the field strains, which should lead to a change in the currently applied chemicals. All field strains which had exposed annually to successive insecticide applications revealed an increase of protein bands and a decrease of esterases of lower molecular weight than those in the susceptible strain. Chemical insecticides are known to be able to act as inducers for the biosynthesis of cellular proteins or as inhibitors of enzymes; so, the molecular biochemical explanation of variations among the tested field strains can be attributed to either variation or similarity in the background exposure to the chemical control programs.