The Electrical Response of the Planarian Ocellus

The planarian ocellar potential (OP), an action potential evoked from the planarian ocellus by a light flash, was recorded with microelectrodes. OP amplitude, latency, and peak delay varied as a function of stimulus intensity and state of adaptation in a manner similar to the responses of other photoreceptors. Changes in the OP that occurred with different directions of incident light are described and attributed to screening effects of the ocellar pigment cells. The temperature coefficient (Q₁₀) of OP latency was 1.5; latency decreased continuously as temperature was increased to destructive levels. The energy of activation of the rate of OP formation was calculated to approximate 10 kcal. These findings suggest dependence of OP latency on ionic diffusion and of OP formation on a biocatalytic process.     

The skeletal muscle Wnt pathway may modulate insulin resistance and muscle development in a diet-induced obese rat model

The Wnt signaling pathway is involved in lipid metabolism and obesity development. Skeletal muscle, a pivotal tissue for metabolism, is regulated by the Wnt signaling. However, little is known of this pathway's involvement in insulin sensitivity and myogenesis in animals. The current study focused on the potential role of Wnt signaling in insulin sensitivity and myogenic events and its further impact on intramuscular fat accumulation. Obesity resistant (OR) and obesity prone (OP) rats were fed a high-fat (HF, 45% kcal fat) diet for 13 weeks. Body weight and circulating triglyceride (TG) were measured and gastrocnemius muscle was collected for analysis of gene expression and protein amount. OP rats had higher body weight and blood TG than OR, and our study demonstrated that the skeletal muscle of OR and OP rats had different levels of β-catenin, which also corresponded to the expression of Wnt downstream genes. The expression of insulin receptor substrate (IRS) was significantly lower in OP than OR skeletal muscle, as was the protein amount of phosphorylated Akt, myocyte enhancer factor-2 (MEF2), and GLUT4. Expression of Myogenic regulatory factor (Myf) 5 and Myf3 (MyoD) were decreased significantly in OP skeletal muscle when compared to OR. Additionally, intramuscular fat was higher in OP than in OR rats. Thus, we propose that the differential Wnt signaling in the skeletal muscle of OR and OP rats is highly likely associated with the differences in insulin sensitivity and myogenic capability in these two strains.     

Expression of osteopontin in Meckel’s cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis

 The localization of osteopontin (OP) was examined in Meckel’s cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix. Accepted: 26 May 1998     

Effect of optical panel distance in a photobioreactor for nutrient removal and cultivation of microalgae

The aim of present investigation is to assess the effect of illumination area and distance of an optical panel (OP) on the growth of microalgae with simultaneous removal of nutrients. Four different distances of OPs were used i.e. 225 mm OP (Run 1), 150 mm OP (Run 2), 112.5 mm OP (Run 3) and 90 mm distance OP (Run 4) in the photobioreactor (PBR) operations. The PBR was equipped with an OP and operated with a total volume of 37-l. A batch culture was prepared using an initial cell concentration of 1.12 ± 0.05 g l^?1. The experiments were conducted at neutral pH (7.2 ± 0.3) under dark and light cycles of 8 and 16 h, respectively. The diffuse light intensity was recorded as 91, 93, 95 and 98 % for Run 1, Run 2, Run 3 and Run 4, respectively, at a 300 mm depth level in the PBR. The chlorophyll content per cell was found to be an average of 701, 721, 785 and 808 fg for Run 1, Run 2, Run 3 and Run 4, respectively. High chlorophyll content per cell and diffuse light intensity was observed for Run 4 compared to other fractions. However, Run 3 sample possessed approximately 10.51 % higher biomass growth along with 15.22 % chemical oxygen demand, 18.15 % total nitrogen 2.37 % NH₄-N, 15.1 % total phosphorus and 15.05 % PO₄-P more removal than the Run 1. These findings suggest that in terms of economic consideration and efficiency of nutrients removal, Run 3 is found to be more effective than other samples. Moreover, an enhanced growth of Chlorella vulgaris could further be utilized as a potential biodiesel source.     

Omeprazole treatment elicits contrasting responses to salt stress in two basil genotypes

Omeprazole (OP) has been shown to act as a plant growth regulator and enhances tolerance to salt stress. In this study, two Ocimum basilicum genotypes were tested for their responses to OP under salt stress. The two genotypes, Napoletano (NAP) a salt sensitive genotype, and Genovese (GEN) a salt tolerant genotype, had contrasting responses to OP treatment. NAP demonstrated increases in terms of growth (+36%) and salt tolerance (+19%) upon treatment while GEN had a growth increase (+35%) and OP enhanced sensitivity to salt stress (?13%). OP treatment also had an effect on the post‐harvest behaviour of these two genotypes by increasing NAP shelf life while decreasing GEN shelf life. The contrasting responses to OP in these two genotypes has provided insight into the role of this molecule in mediating growth and adaptation to stress and, more importantly, into the complexity of the mechanisms mediating these processes.     

Biochemical monitoring of acetylcholinesterase sensitivity to organophosphorus insecticides in glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) and smoke-tree sharpshooter H. lacerta Fowler

The glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) is a new pest to California agriculture. It is the principal vector of several plant pathogenic diseases, particularly Pierce's Disease in grapevines, and oleander leaf scorch. A microplate-based assay is described that measures the sensitivity of acetylcholinesterase (AChE) activity to inhibition by organophosphorus (OP) insecticides in this important pest. The technique provides users with an accurate measure of the efficacy of OP binding to this target site, and is a valuable tool for monitoring field populations of the insect to determine whether the use of OP insecticides has selected for resistant individuals. The technique will also measure AChE sensitivity in the smoke-tree sharpshooter, H. lacerta Fowler. This species is native to California and is regarded only as a minor pest. Both inhibition and kinetic measurements for the AChE enzymes in these sharpshooters demonstrate the close phylogenetic relationships between the two species.     

Paraoxonase (PON 1) as a biomarker of susceptibility for organophosphate toxicity

Paraoxonase (PON1) is an A-esterase capable of hydrolysing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and plasma, and among animal species significant differences exist, with birds and rabbits displaying very low and high activity, respectively. Human PON1 has two polymorphisms in the coding region (Q192R and L55M) and five polymorphisms in the promoter region. The Q192R polymorphism imparts different catalytic activity toward some OP substrates, while the polymorphism at position -108 (C/T) is the major contributor to differences in the level of PON1 expression. Animal studies have shown that PON1 is an important determinant of OP toxicity, with animal species with a low PON1 activity having an increased sensitivity to OPs. Administration of exogenous PON1 to rats or mice protects them from the toxicity of OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not paraoxon. In vitro assayed catalytic efficiencies of purified PON₁₉₂ isoforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Low PON1 activity may also contribute to the higher sensitivity of newborns to OP toxicity.     

Adrenal responses to chronic and acute water stress in Japanese quail Coturnix japonica

Water deprivation (WD) resulted in increased serum osmotic pressure (OP) and decreased body weight (WB); adrenal aldosterone content did not change. Adrenal corticosterone content tended to be elevated during early WD, indicating a stress response, but tended to decrease after seven days of WD, suggesting adrenal fatigue. During water restriction (WR), after the period of weight loss, adrenal corticosterone content and serum OP were elevated. As the birds began to gain weight, aldosterone levels did not change but adrenal corticosterone content and serum OP approached control values, suggesting that the birds were beginning to adapt to the WR. Adrenal sensitivity to ACTH was indicated by the elevated adrenal aldosterone and corticosterone content after ACTH injection.     

Effect of Salinity on the Photosynthesis of the Halophyte Salicornia rubra and Distichlis stricta

The photosynthesis and biomass production of two contrasting species, Salicornia rubra and Distichlis stricta from the same habitat, were studied under different light intensities and osmotic conditions. Application of NaCl increased the biomass and succulence of Salicornia and decreased that of Distichlis. Ethylene glycol, EG mol. wt. 62.07, and 1:1 NaCl-EG killed Salicornia and affected Distichlis adversely. The CO₂ uptake of both the species increased with increasing light intensity and was strongly influenced by the application of NaCl. All concentrations of NaCl stimulated the CO₂ uptake of Salicornia, but decreased that of Distichlis. NaCl significantly decreased the light compensation point of Salicornia but had no effect on that of Distichlis. The CO₂ efflux of the two species at 4.52 klx was not significantly different. The rate of CO₂ uptake of both the species declined with time at all levels of treatment. Salicornia had a more rapid decline in CO₂ uptake in absence of NaCl, whereas that of Distichlis was relatively unaffected by it. Although the tissue Na concentration of the two species increased with increasing NaCl concentration in the medium, the increase in Salicornia was 3.0 to 5.5 times greater than that of Distichlis. Increasing NaCl concentration decreased the chlorophyll concentration of Salicornia and increased that of Distichlis. This resulted in higher ratios of photosynthesis/chlorophyll concentration in Salicornia as against lower ratios in Distichlis. The tissue OP (osmotic potential) of both the species decreased with decreasing OP of the medium. In Salicornia the tissue OP decrease was concurrent with an increase in the CO₂ uptake, whereas the CO₂ uptake of Distichlis declined with decreasing OP of the medium despite a decrease in its tissue OP. It is concluded that Salicornia requires NaCl for its normal development, whereas Distichlis is adversely affected by it. Both species however, are better adapted to osmotic stress due to natural osmotic agents like NaCl, than to the osmotic stress due to artificial osmotic agents like ethylene glycol.     

MRI study of pharyngeal airway changes during stimulation of the hypoglossal nerve branches in rats.

The medial branch (Med) of the hypoglossal nerve innervates the tongue protrudor muscles, whereas the lateral branch (Lat) innervates tongue retractor muscles. Our previous finding that pharyngeal airflow increased during either selective Med stimulation or whole hypoglossal nerve (WHL) stimulation (coactivation of protrudor and retractor muscles) led us to examine how WHL, Med, or Lat stimulation affected tongue movements and nasopharyngeal (NP) and oropharyngeal (OP) airway volume. Electrical stimulation of either WHL, Med, or Lat nerves was performed in anesthetized, tracheotomized rats while magnetic resonance images of the NP and OP were acquired (slice thickness 0.5 mm, in-plane resolution 0.25 mm). NP and OP volume was greater during WHL and Med stimulation vs. no stimulation (P < 0.05). Ventral tongue depression (measured in the midsagittal images) and OP volume were greater during Med stimulation than during WHL stimulation (P < 0.05). Lat stimulation did not alter NP volume (P = 0.39). Our finding that either WHL or Med stimulation dilates the NP and OP airways sheds new light on the control of pharyngeal airway caliber by extrinsic tongue muscles and may lead to new treatments for patients with obstructive sleep apnea.     

Inner retinal photoreceptors (IRPs) in mammals and teleost fish.

Research over the past decade has provided overwhelming evidence that photoreception in the vertebrate eye is not confined to the rods and cones. The discovery of non-rod, non-cone ocular photoreceptors in mammals and fish arose from quite different lines of investigation. In transgenic mice entirely lacking functional rod and cone photoreceptors a range of responses to light, including the regulation of the circadian system and a pupillary light reflex, are preserved. Electrophysiological and imaging approaches were then able to characterise a coupled plexus of directly light sensitive ganglion cells. Most recently action spectroscopy has shown that a novel 'blue-light' sensitive photopigment based upon opsin/vitamin A (OP480) mediates these responses to light. Several candidate genes have emerged for OP480, with melanopsin being by far the strongest. A definitive link, however, between this gene and OP480 has still to be established. In contrast to the mammals, the discovery of inner retinal photoreceptors (IRPs) in fish started with the discovery of a new gene family (VA opsin). The teleost VA opsins form functional photopigments and are expressed in several different types of inner retinal neuron, including retinal horizontal cells. Recent studies have investigated the electrical properties of these photosensitive neurones, but their light-sensing role remains a matter of speculation. Thus the study of IRP is developing along quite separate lines. In the mammals the research is directed towards a molecular identification of the photopigment (OP480) and its cascade, whilst in fish the major effort is directed towards identifying a role for these novel photoreceptors using physiological approaches. The discovery of IRPs in the vertebrates tells us that despite 150 years of research, we still have much to learn about how the eye processes light.     

Why specific anti‐integrase antibodies from HIV‐infected patients can efficiently hydrolyze 21‐mer oligopeptide corresponding to antigenic determinant of human myelin basic protein

Human immunodeficiency virus‐infected patients possess anti‐integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti‐myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti‐MBP and anti‐IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti‐IN abzymes efficiently hydrolyze a 21‐mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti‐MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG‐mediated and IgM‐mediated proteolysis of OP21 by anti‐IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti‐IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti‐IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non‐cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease‐like abzymes against different proteins, anti‐IN abzymes possess serine, thiol, acidic, and metal‐dependent protease activities. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of Diet on Preference and Intake of Sucrose in Obese Prone and Resistant Rats

Increased orosensory stimulation from palatable diets and decreased feedback from gut signals have been proposed as contributing factors to obesity development. Whether altered taste functions associated with obesity are common traits or acquired deficits to environmental factors, such as a high-energy (HE)-diet, however, is not clear. To address this, we examined preference and sensitivity of increasing concentrations of sucrose solutions in rats prone (OP) and resistant (OR) to obesity during chow and HE feeding and measured lingual gene expression of the sweet taste receptor T1R3. When chow-fed, OP rats exhibited reduced preference and acceptance of dilute sucrose solutions, sham-fed less sucrose compared to OR rats, and had reduced lingual T1R3 gene expression. HE-feeding abrogated differences in sucrose preference and intake and lingual T1R3 expression between phenotypes. Despite similar sucrose intakes however, OP rats consumed significantly more total calories during 48-h two-bottle testing compared to OR rats. The results demonstrate that OP rats have an innate deficit for sweet taste detection, as illustrated by a reduction in sensitivity to sweets and reduced T1R3 gene expression; however their hyperphagia and subsequent obesity during HE-feeding is most likely not due to altered consumption of sweets.     

Opsins and mammalian photoentrainment

Research over the past decade has provided overwhelming evidence that photoreception in the vertebrate eye is not confined to the rod and cone photoreceptors. It appears that photoreceptor cells within the inner retina provide irradiance information to a wide variety of different photosensory tasks including photoentrainment, pupillary constriction and masking behaviour. Action spectra in mice lacking all rod and cone photoreceptors ( rd/rd cl) have demonstrated the existence of a previously uncharacterised, opsin/vitamin-A-based photopigment with peak sensitivity at 479 nm (opsin photopigment/OP(479)). The review addresses the question: has the gene encoding OP(479) already been isolated, and if not, what type of gene should we be seeking and where in the eye might this gene be expressed? On the basis of available data, the gene that encodes OP(479) remains unidentified, and two broad possibilities exist. On the assumption that OP(479) will be like all of the other vertebrate photopigments (ocular and extraocular) and share a close phylogenetic relationship based upon amino acid identity and a conserved genomic structure, then the gene encoding OP(479) has yet to be isolated. Alternatively, there may have been a separate line of photopigment evolution in the vertebrates that has given rise to the melanopsin family. If true then the mammalian melanopsin gene may encode OP(479). Only when melanopsin and other candidates for OP(479) have been functionally expressed, and shown to encode a photopigment that matches the action spectrum of OP(479), can firm conclusions about the identity of the non-rod, non-cone ocular photoreceptor of mammals be made.     

Dissociation between the activity of the right middle frontal gyrus and the middle temporal gyrus in processing semantic priming

The aim of this event-related functional magnetic resonance imaging (fMRI) study was to test whether the right middle frontal gyrus (MFG) and middle temporal gyrus (MTG) would show differential sensitivity to the effect of prime-target association strength on repetition priming. In the experimental condition (RP), the target occurred after repetitive presentation of the prime within an oddball design. In the control condition (CTR), the target followed a single presentation of the prime with equal probability of the target as in RP. To manipulate semantic overlap between the prime and the target both conditions (RP and CTR) employed either the onomatopoeia "oink" as the prime and the referent "pig" as the target (OP) or vice-versa (PO) since semantic overlap was previously shown to be greater in OP. The results showed that the left MTG was sensitive to release of adaptation while both the right MTG and MFG were sensitive to sequence regularity extraction and its verification. However, dissociated activity between OP and PO was revealed in RP only in the right MFG. Specifically, target "pig" (OP) and the physically equivalent target in CTR elicited comparable deactivations whereas target "oink" (PO) elicited less inhibited response in RP than in CTR. This interaction in the right MFG was explained by integrating these effects into a competition model between perceptual and conceptual effects in priming processing.     

Biochemical responses as early and reliable biomarkers of organophosphate and carbamate pesticides intoxication: A systematic literature review

Inhibition of cholinesterase (ChE) activity has been long considered as the main diagnostic method of organophosphate (OP) and carbamate pesticides poisoning; however, it has been shown that ChE activity may also be altered due to exposure to other non-organophosphorus toxicants and variety of different medical conditions. Hence, to avoid misdiagnosis, we aimed to systematically review available documents to look for additional biomarkers of OP and carbamate poisoning. The electronic databases in addition to Google scholar were searched for eligible articles on March 2022 using “organophosphate,” “carbamate,” and “biomarker” including all their similar terms. After collecting the relevant documents, the data were extracted and described qualitatively. In total, data of 66 articles from 51 human and 15 animal studies were extracted. Findings demonstrated that enzymes such as β-glucuronidase, neuropathy target esterase, amylase, and lipase, in addition to hematological indicators such as CBC, CRP, lactate dehydrogenase, and CPK have high sensitivity and accuracy in the diagnosis of OP poisoning. Findings suggest that using various markers for diagnosis of OP intoxication is helpful for appropriate management, and early identifying the patients at risk of death. The suggested biomarkers also help to avoid misdiagnosis of OP poisoning with other similar conditions.     

Growth characteristics of ultrahigh-density microalgal cultures

The physiological characteristics of cultures of very high cell mass (e.g. 10 g cell mass/L), termed “ultrahigh cell density cultures” is reviewed. A close relationship was found between the length of the optical path (OP) in flat-plate reactors and the optimal cell density of the culture as well as its areal (g m⁻² day⁻¹) productivity. Cell-growth inhibition (GI) unfolds, as culture density surpasses a certain threshold. If it is constantly relieved, a 1.0 cm OP reactor could produceca. 50% more than reactors with longer OP,e.g. 5 or 10 cm. This unique effect, discovered by Hu et al. [3], is explained in terms of the relationships between the frequency of the light-dark cycle (L-D cycle), cells undergo in their travel between the light and dark volumes in the reactor, and the turnover time of the photosynthetic center (PC). In long OP reactors (5 cm and above) the L-D cycle time may be orders of magnitude longer than the PC turnover time, resulting in a light regime in which the cells are exposed along the L-D cycle, to long, wasteful dark periods. In contrast, in reactors with an OP ofca. 1.0 cm, the L-D cycle frequency approaches the PC turnover time resulting in a significant reduction of the wasteful dark exposure time, thereby inducing a surge in photosynthetic efficiency. Presently, the major difficulty in mass cultivation of ultrahigh-density culture (UHDC) concerns cell grwoth inhibition in the culture, the exact nature of which is awaiting detailed investigation.     

神经细胞尼氏体和神经髓鞘的双重组合染色法与应用

探讨了显示实验动物脊髓组织中的神经尼氏体和神经髓鞘等组织成分的双重组合染色法,通过分别选用孔雀石绿(Malachite green)和橙黄G、磷钨酸(Orange G,Phosphotungstic acid)组合染色(简称MG-OP法)。已能够显示狗脊髓神经尼氏体呈绿色,细胞核呈黄色。神经纤维髓鞘轴突呈黄色,神经膜和结缔组织纤维呈绿色,背景呈淡黄色。MG-OP法克服了原法成分单一,色彩效果差,所建立的双重组合染色法,对比清晰,色彩鲜艳,方法简便的较好染色方法。     

Improved Electrochemical Analysis of Neuropathy Target Esterase Activity by a Tyrosinase Carbon Paste Electrode Modified by 1-Methoxyphenazine Methosulfate

A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N=2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r=0.990, n=7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.