Functional analysis of UDP-glycosyltransferase genes conferring indoxacarb resistance in Spodoptera litura.

UDP-glycosyltransferase (UGT) is the major detoxification enzymes of phase II involved in xenobiotics metabolism, which potentially mediates the formation of insect resistance. Previous transcriptome sequencing studies have found that several UGT genes were upregulated in indoxacarb resistant strains of Spodoptera litura, but whether these UGT genes were involved in indoxacarb resistance and their functions in resistance were unclear. In this study, the UGTs inhibitor, 5-nitrouracil, enhanced the toxicity of indoxacarb against S. litura, preliminarily suggesting that UGTs were participated in indoxacarb resistance. Two UGT genes, UGT33J17 and UGT41D10 were upregulated in the resistant strains and could be induced by indoxacarb. Alignment of UGT protein sequences revealed two conserved donor-binding regions with several key residues that interact with catalytic sites and sugar donors. Further molecular modeling and docking analysis indicated that two UGT proteins were able to stably bind indoxacarb and N-decarbomethoxylated metabolite (DCJW). Furthermore, knockdown of UGT33J17 and UGT41D10 decreased viability of Spli-221 cells and enhanced susceptibility of larvae to indoxacarb. Transgenic overexpression of these genes reduced the toxicity of indoxacarb in Drosophila melanogaster. This work revealed that upregulation of UGT genes significantly contributes to indoxacarb resistance in S. litura, and is of great significance for the development of integrated and sustainable management strategies for resistant pests in the field.

Heterologous investigation of metabotropic and ionotropic odorant receptors in ab3A neurons of Drosophila melanogaster.

In insects, antennal ionotropic receptors (IRs) and odorant receptors (ORs) are among the main sensors of olfactory cues. To functionally characterize the subunits from these receptors, the use of ab3A neurons from transgenic Drosophila melanogaster represented one of the most powerful tools, allowing the identification of ligands (deorphanization) and decrypting their pharmacological properties. However, further investigation is needed to shed light on possible metabotropic functionalities behind insect olfactory receptors and test potentials from the up-to-now-used empty neuronal systems to express subunits belonging to variegate receptor classes. In this project, we adopted the most updated system of Drosophila ab3A empty neurons to test various olfactory receptors, ranging from human ORs working as metabotropic G-protein coupled receptors to insect ionotropic IRs and ORs. Testing transgenic Drosophila expressing human ORs into ab3A neurons by single sensillum recording did not result in an OR response to ligands, but it rather re-established neuronal spiking from the empty neurons. When transgenic D. melanogaster expressed ionotropic IRs and ORs, both heterologous and cis-expressed IRs were non-functional, but the Drosophila suzukii OR19A1 subunit responded to a wide asset of ligands, distinguishing phasic or tonic compound-dependent effects. Despite the use of Drosophila ab3A neurons to test the activation of some metabotropic and ionotropic receptor subunits resulted non-functional, this study deorphanized a key OR of D. suzukii demonstrating its binding to alcohols, ketones, terpenes, and esters.

CGG repeat expansion in NOTCH2NLC causes mitochondrial dysfunction and progressive neurodegeneration in Drosophila model.

Neuronal intranuclear inclusion disease (NIID) is a neuromuscular/neurodegenerative disease caused by the expansion of CGG repeats in the 5' untranslated region (UTR) of the NOTCH2NLC gene. These repeats can be translated into a polyglycine-containing protein, uN2CpolyG, which forms protein inclusions and is toxic in cell models, albeit through an unknown mechanism. Here, we established a transgenic Drosophila model expressing uN2CpolyG in multiple systems, which resulted in progressive neuronal cell loss, locomotor deficiency, and shortened lifespan. Interestingly, electron microscopy revealed mitochondrial swelling both in transgenic flies and in muscle biopsies of individuals with NIID. Immunofluorescence and immunoelectron microscopy showed colocalization of uN2CpolyG with mitochondria in cell and patient samples, while biochemical analysis revealed that uN2CpolyG interacted with a mitochondrial RNA binding protein, LRPPRC (leucine-rich pentatricopeptide repeat motif-containing protein). Furthermore, RNA sequencing (RNA-seq) analysis and functional assays showed down-regulated mitochondrial oxidative phosphorylation in uN2CpolyG-expressing flies and NIID muscle biopsies. Finally, idebenone treatment restored mitochondrial function and alleviated neurodegenerative phenotypes in transgenic flies. Overall, these results indicate that transgenic flies expressing uN2CpolyG recapitulate key features of NIID and that reversing mitochondrial dysfunction might provide a potential therapeutic approach for this disorder.

Pan-neuronal expression of human mutant huntingtin protein in Drosophila impairs immune response of hemocytes.

Huntington's disease (HD) is a late-onset; progressive, dominantly inherited neurological disorder marked by an abnormal expansion of polyglutamine (poly Q) repeats in Huntingtin (HTT) protein. The pathological effects of mutant Huntingtin (mHTT) are not restricted to the nervous system but systemic abnormalities including immune dysregulation have been evidenced in clinical and experimental settings of HD. Indeed, mHTT is ubiquitously expressed and could induce cellular toxicity by directly acting on immune cells. However, it is still unclear if selective expression of mHTT exon1 in neurons could induce immune responses and hemocytes' function. In the present study, we intended to monitor perturbations in the hemocytes' population and their physiological functions in Drosophila, caused by pan-neuronal expression of mHTT protein. A measure of hemocyte count and their physiological activities caused by pan-neuronal expression of mHTT protein highlighted the extent of immune dysregulation occurring with disease progression. We found that pan-neuronal expression of mHTT significantly alters crystal cells and plasmatocyte count in larvae and adults with disease progression. Interestingly, plasmatocytes isolated from diseased conditions exhibit a gradual decline in phagocytic activity ex vivo at progressive stages of the disease as compared to age-matched control groups. In addition, diseased flies displayed elevated reactive oxygen species (ROS) in circulating plasmatocytes at the larval stage and in sessile plasmatocytes of hematopoietic pockets at terminal stages of disease. These findings strongly implicate that neuronal expression of mHTT alone is sufficient to induce non-cell-autonomous immune dysregulation in vivo.

DNT1 Downregulation and Increased Ethanol Sensitivity in Transgenic Drosophila Models of Alzheimer's Disease.

Two major pathological hallmarks of Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles of hyperphosphorylated tau. Aggregation of amyloid-ß (Aß) is considered as the primary insult in AD. However, failure in treatments based on targetingAß without considering the pathologic tau and close correlation between pathological tau and cognitive decline highlighted the crucial role of tau in AD. Loss of synaptic plasticity and cognitive decline, partly due to decrease in Brain Derived Neurotrophic Factor (BDNF), are other hallmarks of AD. Aß and tau downregulate BDNF at both transcriptional and translational levels. The aim of this research was to study the expression levels of Drosophila Neuroteophin 1 (DNT1), as an orthologue of BDNF, in flies expressing Aß42 or tauR406W. Levels of DNT1 were determined using quantitative real time PCR. Behavioral and Biochemical investigations were also performed in parallel. Our results showed that there is a significant decrease in the levels of DNT1 expression in Aß42 or tauR406W expressing flies. Interestingly, a significant increase was observed in sensitivity to ethanol in both transgenic flies. Rise in Reactive Oxygen Species (ROS) levels was also detected. We concluded that both Aß and pathological tau exert their toxic effect on DNT1 expression, ROS production, and response to ethanol, independently. Interestingly, pathological tau showed higher impact on the ROS production compared to Aß. It seems that Aß42 and tauR406W transgenic flies are proper models to investigate the interplay between BDNF and oxidative stress, and also to assess the mechanism underlying behavioral response to ethanol.

A SUMO1-Derived Peptide Targeting SUMO-Interacting Motif Inhibits α-Synuclein Aggregation.

The accumulation of α-synuclein amyloid fibrils in the brain is linked to Parkinson's disease and other synucleinopathies. The intermediate species in the early aggregation phase of α-synuclein are involved in the emergence of amyloid toxicity and considered to be the most neurotoxic. The N-terminal region flanking the non-amyloid-ß component domain of α-synuclein has been implicated in modulating its aggregation. Herein, we report the development of a SUMO1-derived peptide inhibitor (SUMO1(15-55)), which targets two SUMO-interacting motifs (SIMs) within this aggregation-regulating region and suppresses α-synuclein aggregation. Molecular modeling, site-directed mutagenesis, and binding studies are used to elucidate the mode of interaction, namely, via the binding of either of the two SIM sequences on α-synuclein to a putative hydrophobic binding groove on SUMO1(15-55). Subsequent studies show that SUMO1(15-55) also reduces α-synuclein-induced cytotoxicity in cell-based and Drosophila disease models.

Co-Expression of a Homologous Cytochrome P450 Reductase Is Required for In Vivo Validation of the Tetranychus urticae CYP392A16-Based Abamectin Resistance in Drosophila.

Overexpression of the cytochrome P450 monooxygenase CYP392A16 has been previously associated with abamectin resistance using transcriptional analysis in the two-spotted spider mite Tetranychus urticae, an important pest species worldwide; however, this association has not been functionally validated in vivo despite the demonstrated ability of CYP392A16 to metabolize abamectin in vitro. We expressed CYP392A16 in vivo via a Gal4 transcription activator protein/Upstream Activating Sequence (GAL4/UAS) system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. We demonstrated that CYP392A16 expression confers statistically significant abamectin resistance in toxicity bioassays in Drosophila only when its homologous redox partner, cytochrome P450 reductase (TuCPR), is co-expressed in transgenic flies. Our study shows that the Drosophila model can be further improved, to facilitate the functional analysis of insecticide resistance mechanisms acting alone or in combination.

An interplay between immune response and neurodegenerative disease progression: An assessment using Drosophila as a model.

Neurodegeneration, the slow and progressive loss of neurons in the central nervous system has become a major challenge to public health worldwide particularly with elderly people. Until recently, the brain and immune system were studied exclusively, independent of each other representing two distinct systems. Recent studies ensue crosstalk between these two systems to maintain homeostasis. Though the progressive loss of specific neuronal subsets is a hallmark of neurodegenerative disease, emerging evidences indicate that immune response also plays a critical role in disease progression. Due to conservation of mechanisms that govern neural development and innate immune activation in flies and humans, and availability of powerful genetic tools, the fruit fly Drosophila melanogaster is one of the best model organisms to investigate the immune response in neurodegenerative disease. Owing to significant homology between human and Drosophila immune system and recent reports on interplay between immune system and neurodegenerative disease progression, the main focus of the review is to develop a comprehensive understanding of how neuro-immune interactions contribute to neurodegeneration using Drosophila as a model system.

Functional characterization of CYP6A51, a cytochrome P450 associated with pyrethroid resistance in the Mediterranean fruit fly Ceratitis capitata.

Overexpression of the cytochrome P450 monooxygenase CYP6A51 has been previously associated with pyrethroid resistance in the Mediterranean fruit fly (medfly) Ceratitis capitata, an important pest species worldwide; however, this association has not been functionally validated. We expressed CYP6A51 gene in Escherichia coli and produced a functional enzyme with preference for the chemiluminescent substrate Luciferin-ME EGE. In vitro metabolism assays revealed that CYP6A51 is capable of metabolizing two insecticides that share the same mode of action, λ-cyhalothrin and deltamethrin, whereas no metabolism or substrate depletion was observed in the presence of spinosad or malathion. We further expressed CYP6A51 in vivo via a GAL4/UAS system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. Toxicity bioassays indicated that CYP6A51 confers knock-down resistance to both λ-cyhalothrin and deltamethrin. Detection of CYP6A51 - associated pyrethroid resistance in field populations may be important for efficient Insecticide Resistance Management (IRM) strategies.

Wnt2 overexpression protects PINK1B9 transgenic flies by improving mitochondrial function / 医学研究生学报

Objective PINK1 and Parkin are directly invoveled in the regulation and maintenance of mitochondrial functional morphology. We aim to explore the effect of Wnt2 overexpression on PINK1B9 Mutant Drosophila and its mechanism in this study. Methods The GAL4-UAS system was used to construct the normal control flies（W1118/ + ；MHC-GAL4/+）, PINK1B9 transgenic Drosophila model flies（UAS-PINK1B9 /y；MHC-GAL4 / +；Parkinson's disease model of Drosophila melanogaster), the Wnt2 overexpression flies（UAS-PINK1B9 /y；MHC-GAL4 / Wnt2 OE） and the Wnt2 RNAi flies（UAS-PINK1B9 /y；MHC-GAL4 /Wnt2）. On the 5th day, the abnormal wings phenotype rate and flying rate of flies were observed. The contents of Ndufs3 proteins were detected by Western blot. The mRNA expression levels of PGC-1α, Nrf1 and TFAM related to mitochondrial metabolism and synthesis were detected by real-time fluorescence quantitative PCR. The morphology of mitochondria was observed by electron microscopy. Complex I and Complex II function was detected by high-resolution mitochondrial respiratory system. Results Compared with the normal control flies, PINK1B9 transgenic Drosophila model flies showed increased abnormal wings phenotype rate([1.87±0.06]% vs [68.79±0.70]%), decreased flying rate([ 97.51±0.52)% vs(3.95±0.53)%], and the differences were statistically significant(P<0.05); Compared with PINK1B9 transgenic Drosophila model flies, the Wnt2 RNAi flies showed decreased abnormal wings phenotype rate[(10.14±1.72)%], increased flying rate([41.83±2.57]%)(P<0.05). Compared with the normal control flies, PINK1B9 transgenic Drosophila model flies showed decreased expression levels of PGC-1α and Nrf1,Ndufs3 proteins, Complex I and Complex II(P<0.05);On the contrary, the Wnt2 RNAi flies showed increased trends compared with PINK1B9 transgenic Drosophila model flies(P<0.05). Conclusion Overexpression of Wnt2 protects PINK1B9 transgenic Drosophila models, which is related to the improvement of mitochondrial function.

Design, Synthesis of N-phenethyl Cinnamide Derivatives and Their Biological Activities for the Treatment of Alzheimer's Disease: Antioxidant, Beta-amyloid Disaggregating and Rescue Effects on Memory Loss.

Gx-50 is a bioactive compound for the treatment of Alzheimer's disease (AD) found in Sichuan pepper (Zanthoxylum bungeanum). In order to find a stronger anti-AD lead compound, 20 gx-50 (1⁻20) analogs have been designed and synthesized, and their molecular structures were determined based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis, as well as comparison with literature data. Compounds 1⁻20 were evaluated for their anti-AD potential by using DPPH radical scavenging assay for considering their anti-oxidant activity, thioflavin T (ThT) fluorescence assay for considering the inhibitory or disaggregate potency of Aß, and transgenic Drosophila model assay for evaluating their rescue effect on memory loss. Finally, compound 13 was determined as a promising anti-AD candidate.

Rescue effect of Drp1 gene over -expression on drosophila models of Parkinson's disease: a mechanism research by iTRAQ-based proteomic analysis / 中华神经医学杂志

Objective To investigate the rescue effect of Drp1 gene over-expression on drosophila models of Parkinson's disease (PD) and their specific mechanism using iTRAQ-based proteomic technology. Methods The drosophilae from 3 groups, control group, PD group (PINK1 mutant), and rescue group (PINK1 mutant+Drp1 over-expression) were cultured at routine ways. The wing shape and movement ability of drosophilae were observed, and the percentages of drosophilae having abnormal wings and normal flight were calculated. The proteomic changes were determined by iTRAQ technology; the functions and signaling pathways of the differential expressed proteins were analyzed by GO and KEGG pathway enrichment analyses. Results Percentage of drosophilae having abnormal wings in the rescue group (2.60％±0.47％) was significantly decreased as compared with that in the PD group (82.40％±12.47％, P<0.05), and the percentage of drosophilae having normal flight in the rescue group (89.70％±7.76％) was significantly increased as compared with that in the PD group (3.30％±1.69％, P<0.05). A total of 3630 proteins were identified using iTRAQ; 282 differential expressed proteins between the PD group and control group were detected, which mainly were iron ion related proteins; 170 differential expressed proteins between the PD group and rescue group were detected, which mainly were zinc ion related proteins. GO and KEGG pathway enrichment analyses on these co-differential expressed proteins revealed that 21％ proteins had metal ion binding activity, especially zinc ion. Conclusion Abnormal iron homeostasis, especially zinc homeostasis, participates in PD pathophysiological processes and over-expressing Drp1 rescued PD processes.

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site.

Drosophila photoreceptors respond to oscillating light of high frequency (∼100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca2+-dependent process. We further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trpS936A), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trpS936D transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. Here we extend these findings by showing that dark-adapted trpS936A flies reveal light-induced current (LIC) with short latency relative to trpWT or trpS936D flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics.

Nortriptyline inhibits aggregation and neurotoxicity of alpha-synuclein by enhancing reconfiguration of the monomeric form.

The pathology of Parkinson's disease and other synucleinopathies is characterized by the formation of intracellular inclusions comprised primarily of misfolded, fibrillar α-synuclein (α-syn). One strategy to slow disease progression is to prevent the misfolding and aggregation of its native monomeric form. Here we present findings that support the contention that the tricyclic antidepressant compound nortriptyline (NOR) has disease-modifying potential for synucleinopathies. Findings from in vitro aggregation and kinetics assays support the view that NOR inhibits aggregation of α-syn by directly binding to the soluble, monomeric form, and by enhancing reconfiguration of the monomer, inhibits formation of toxic conformations of the protein. We go on to demonstrate that NOR inhibits the accumulation, aggregation and neurotoxicity of α-syn in multiple cell and animal models. These findings suggest that NOR, a compound with established safety and efficacy for treatment of depression, may slow progression of α-syn pathology by directly binding to soluble, native, α-syn, thereby inhibiting pathological aggregation and preserving its normal functions.

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust, Locusta migratoria.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.

4-Hydroxyisophthalic acid from Decalepis hamiltonii rescues the neurobehavioral deficit in transgenic Drosophila model of taupathies.

Oxidative stress is one of the major etiological factors implicated in pathogenesis of neurodegenerative diseases. Since neurons are more sensitive to oxidative damage there is an increasing interest in developing novel antioxidant therapies, especially herbal preparations due to their safety profile and high efficiency. In this regard, the neuroprotective potential of a novel antioxidant compound, 4-hydroxyisophthalic acid (4-HIPA) isolated from aqueous extract of Decalepis hamiltonii roots was examined using transgenic Drosophila model of taupathy expressing wild-type and mutant forms of 2N4R isoform of human microtubule associated protein tau (MAPT). Taupathy model flies showed cognitive deficits in olfactory memory and deteriorated circadian rhythm of locomotory activities. Administration of 0.1 mg/ml 4-HIPA, markedly enhanced their olfactory memory performance and restored circadian rhythmicity of the transgenic flies locomotory behavior to the normal range. The mechanism of action that underlies 4-HIPA neuroprotection involves enhancement in efficiency of cellular antioxidant defense system by means of elevation in antioxidant enzyme activities and attenuation of oxidative stress. The molecule could positively affect the activity of neurotransmitter enzymes, which in turn enhances neuronal function and ameliorates the Tau-induced neurobehavioral deficits. Our findings showed that 4-HIPA can be considered as a suitable therapeutic candidate for drug development towards treatment of neurodegenerative disorders.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration.

The ε4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing ε3 and ε4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity.

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate.

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (kcat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (kcat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance. The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.

Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response.

Protein misfolding neurodegenerative diseases arise through neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer's disease, Huntington's disease, Parkinson's disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding-induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion-like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion-induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease.

Carboxylesterase-mediated insecticide resistance: Quantitative increase induces broader metabolic resistance than qualitative change.

Carboxylesterases are mainly involved in the mediation of metabolic resistance of many insects to organophosphate (OP) insecticides. Carboxylesterases underwent two divergent evolutionary events: (1) quantitative mechanism characterized by the overproduction of carboxylesterase protein; and (2) qualitative mechanism caused by changes in enzymatic properties because of mutation from glycine/alanine to aspartate at the 151 site (G/A151D) or from tryptophan to leucine at the 271 site (W271L), following the numbering of Drosophila melanogaster AChE. Qualitative mechanism has been observed in few species. However, whether this carboxylesterase mutation mechanism is prevalent in insects remains unclear. In this study, wild-type, G/A151D and W271L mutant carboxylesterases from Culex pipiens and Aphis gossypii were subjected to germline transformation and then transferred to D. melanogaster. These germlines were ubiquitously expressed as induced by tub-Gal4. In carboxylesterase activity assay, the introduced mutant carboxylesterase did not enhance the overall carboxylesterase activity of flies. This result indicated that G/A151D or W271L mutation disrupted the original activities of the enzyme. Less than 1.5-fold OP resistance was only observed in flies expressing A. gossypii mutant carboxylesterases compared with those expressing A. gossypii wild-type carboxylesterase. However, transgenic flies universally showed low resistance to OP insecticides compared with non-transgenic flies. The flies expressing A. gossypii W271L mutant esterase exhibited 1.5-fold resistance to deltamethrin, a pyrethroid insecticide compared with non-transgenic flies. The present transgenic Drosophila system potentially showed that a quantitative increase in carboxylesterases induced broader resistance of insects to insecticides than a qualitative change.