RESUMO
The attraction of anthropophilic mosquitoes to human host cues, such as body odour and carbon dioxide, gradually increases during adult maturation. This acquisition of host-seeking behaviour correlates with age-dependent changes in odorant receptor (OR) transcript abundance and sensitivity of olfactory sensory neurons (OSNs). One OR gene of the human malaria vector, Anopheles coluzzii, AcolOR39, is significantly downregulated in mature females, and a cognate ligand of AcolOR39, sulcatone, a major component of human emanations, mediates the observed behavioural inhibition of newly emerged (teneral) females to human body odour. Knockout of AcolOR39, using CRISPR-Cas9 mutagenesis, selectively abolished sulcatone detection in OSNs, housed in trichoid sensilla. However, knockout of AcolOR39 altered neither the response rate nor the flight behaviour of teneral females in a wind tunnel, indicating the involvement of other genes, and thus a redundancy, in regulating the acquisition of host seeking in mosquitoes.
Assuntos
Anopheles , Malária , Receptores Odorantes , Animais , Feminino , Humanos , Condutos Olfatórios/metabolismo , Anopheles/genética , Odor Corporal , Mosquitos Vetores/genética , Odorantes , Receptores Odorantes/genética , Receptores Odorantes/metabolismoRESUMO
In Culex quinquefasciatus, CquiOR91 is the ortholog of 2 larvae-specific odorant receptors (ORs) from Anopheles gambiae (Agam\Or40, previously shown to respond to several odorant ligands including the broad-spectrum repellent N,N-diethyl-3-methylbenzamide, DEET) and Aedes aegypti (Aaeg\Or40). When we cloned full-length CquiOR91 from a Culex quinquefasciatus larval head RNA sample, we found 2 alleles of this OR, differing at 9 residues. Functional analysis using the Xenopus oocyte expression system and 2-electrode voltage clamp electrophysiology revealed one allele (CquiOR91.1) to be nonfunctional, whereas the other allele (CquiOR91.2) was functional. Receptors formed by CquiOR91.2 and Cqui\Orco responded to (-)-fenchone, (+)-fenchone, and DEET, similar to what has been reported for Agam\Or40. We also identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal, with safranal being the most potent. To explore possible reasons for the lack of function for CquiOR91.1, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 polymorphic residue positions was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Eight of the 9 mutant versions of CquiOR91.2 formed functional receptors, responding to (-)-fenchone. Only the CquiOR91.2 Y183C mutant was nonfunctional. The reverse mutation (C183Y) conferred function on CquiOR91.1 , which became responsive to (-)-fenchone and safranal. These results indicate that the "defect" in CquiOR91.1 that prevents function is the cysteine at position 183.
Assuntos
Culicidae/química , Proteínas de Insetos/genética , Receptores Odorantes/genética , Alelos , Animais , Canfanos , DEET/metabolismo , Proteínas de Insetos/metabolismo , Ligantes , Mutação , Norbornanos/metabolismo , Subunidades Proteicas , Receptores Odorantes/metabolismoRESUMO
Mosquitoes rely mainly on the sense of smell to decipher their environment and locate suitable food sources, hosts for blood feeding and oviposition sites. The molecular bases of olfaction involve multigenic families of olfactory proteins that have evolved to interact with a narrow set of odorants that are critical for survival. Understanding the complex interplay between diversified repertoires of olfactory proteins and ecologically-relevant odorant signals, which elicit important behaviors, is fundamental for the design of novel control strategies targeting the sense of smell of disease vector mosquitoes. Previously, large multigene families of odorant receptor and ionotropic receptor proteins, as well as a subset of odorant-binding proteins have been shown to mediate the selectivity and sensitivity of the mosquito olfactory system. In this study, we identify a mosquito-specific antennal protein (MSAP) gene as a novel molecular actor of odorant reception. MSAP is highly conserved across mosquito species and is transcribed at an extremely high level in female antennae. In order to understand its role in the mosquito olfactory system, we generated knockout mutant lines in Anopheles gambiae, and performed comparative analysis of behavioral and physiological responses to human-associated odorants. We found that MSAP promotes female mosquito attraction to human odor and enhances the sensitivity of the antennae to a variety of odorants. These findings suggest that MSAP is an important component of the mosquito olfactory system, which until now has gone completely unnoticed.
Assuntos
Anopheles , Malária , Receptores Odorantes , Humanos , Feminino , Animais , Odorantes , Anopheles/metabolismo , Mosquitos Vetores/genética , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Olfato/fisiologiaRESUMO
The fine structure of heparan sulfate (HS), the glycosaminoglycan polysaccharide component of cell surface and extracellular matrix HS proteoglycans, coordinates the complex cell signalling processes that control homeostasis and drive development in multicellular animals. In addition, HS is involved in the infection of mammals by viruses, bacteria and parasites. The current detection limit for fluorescently labelled HS disaccharides (low femtomole; 10-15 mol), has effectively hampered investigations of HS composition in small, functionally-relevant populations of cells and tissues that may illuminate the structural requirements for infection and other biochemical processes. Here, an ultra-high sensitivity method is described that utilises a combination of reverse-phase HPLC, with tetraoctylammonium bromide (TOAB) as the ion-pairing reagent and laser-induced fluorescence detection of BODIPY-FL-labelled disaccharides. The method provides an unparalleled increase in the sensitivity of detection by â¼six orders of magnitude, enabling detection in the zeptomolar range (â¼10-21 moles; <1000 labelled molecules). This facilitates determination of HS disaccharide compositional analysis from minute samples of selected tissues, as demonstrated by analysis of HS isolated from the midguts of Anopheles gambiae mosquitoes that was achieved without approaching the limit of detection.
Assuntos
Culicidae , Dissacarídeos , Animais , Dissacarídeos/análise , Dissacarídeos/química , Cromatografia Líquida de Alta Pressão/métodos , Heparitina Sulfato/análise , Heparitina Sulfato/química , MamíferosRESUMO
All living things speak chemistry. The challenge is to reveal the vocabulary, the odorants that enable communication across phylogenies and to translate them to physiological, behavioral, and ecological function. Olfactory receptors (ORs) interface animals with airborne odorants. Expression in heterologous cells makes it possible to interrogate single ORs and to identify cognate ligands. The cosmopolitan, anthropophilic strain of the vinegar fly Drosophila melanogaster depends on human resources and housing for survival. Curiously, humans sense the pheromone (Z)-4-undecenal (Z4-11Al) released by single fly females. A screening of all human ORs shows that the most highly expressed OR10A6 is tuned to Z4-11Al. Females of an ancestral African fly strain release a blend of Z4-11Al and Z4-9Al that produces a different aroma, which is how we distinguish these fly strains by nose. That flies and humans sense Z4-11Al via dedicated ORs shows how convergent evolution shapes communication channels between vertebrate and invertebrate animals.
RESUMO
Oviposition attractants are environmental cues that allow Culex gravid female mosquitoes to locate suitable sites for egg-laying and, therefore, may be exploited for environmentally friendly strategies for controlling mosquito populations. Naturally occurring skatole has been identified as an oviposition attractant for the Southern House mosquito, Culex quinquefasciatus. Previously, we identified in Cx. quinquefasciatus female antennae an olfactory receptor neuron (ORN) highly sensitive to skatole and an odorant-binding protein involved in the detection of this semiochemical. Here, we describe the characterization of an odorant receptor (OR), CquiOR10, which is narrowly tuned to skatole when expressed in the Xenopus oocyte system. Odorant-induced response profiles generated by heterologously expressed CquiOR10 suggest that this OR is expressed in the mosquito ORN sensitive to skatole. However, geranylacetone, which stimulates the antennal ORN, was not detected by CquiOR10-expressing oocytes, thus raising interesting questions about reception of oviposition attractants in mosquitoes.
Assuntos
Culex/efeitos dos fármacos , Culex/metabolismo , Oviposição/efeitos dos fármacos , Receptores Odorantes/metabolismo , Escatol/farmacologia , Animais , Sinais (Psicologia) , Culex/fisiologia , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Odorant-binding proteins (OBPs) were discovered almost three decades ago, but there is still considerable debate regarding their role(s) in insect olfaction, particularly due to our inability to knockdown OBPs and demonstrate their direct phenotypic effects. By using RNA interference (RNAi), we reduced transcription of a major OBP gene, CquiOBP1, in the antennae of the Southern house mosquito, Culex quinquefasciatus. Previously, we had demonstrated that the mosquito oviposition pheromone (MOP) binds to CquiOBP1, which is expressed in MOP-sensitive sensilla. Antennae of RNAi-treated mosquitoes showed significantly lower electrophysiological responses to known mosquito oviposition attractants than the antennae of water-injected, control mosquitoes. While electroantennogram (EAG) responses to MOP, skatole, and indole were reduced in the knockdowns, there was no significant difference in the EAG responses from RNAi-treated and water-injected mosquito antennae to nonanal at all doses tested. These data suggest that CquiOBP1 is involved in the reception of some oviposition attractants, and that high levels of OBPs expression are essential for the sensitivity of the insect's olfactory system.
Assuntos
Oviposição/fisiologia , Feromônios/fisiologia , Receptores Odorantes/metabolismo , Animais , Culicidae/genética , Eletrocardiografia , Técnicas de Silenciamento de Genes , Interferência de RNA , Receptores Odorantes/antagonistas & inibidores , Receptores Odorantes/genéticaRESUMO
Infectious diseases affecting livestock and human health that involve vector-borne pathogens are a global problem, unrestricted by borders or boundaries, which may be exacerbated by changing global climate. Thus, the availability of effective tools for control of pathogen vectors is of the utmost importance. The aim of this article is to review, selectively, current knowledge of the chemical ecology of pathogen vectors that affect livestock and human health in the developed and developing world, based on key note lectures presented in a symposium on "The Chemical Ecology of Disease Vectors" at the 25th Annual ISCE meeting in Neuchatel, Switzerland. The focus is on the deployment of semiochemicals for monitoring and control strategies, and discusses briefly future directions that such research should proceed along, bearing in mind the environmental challenges associated with climate change that we will face during the 21st century.
Assuntos
Doenças Transmissíveis/transmissão , Doenças Transmissíveis/veterinária , Vetores de Doenças , Animais , Mudança Climática , Controle de Doenças Transmissíveis/métodos , HumanosRESUMO
Tsetse flies are the principal insect vectors of African trypanosomes-sleeping sickness in humans and Nagana in cattle. One of the tsetse fly species, Glossina morsitans morsitans, is host to the parasite, Trypanosoma brucei, a major cause of African trypanosomiasis. Precise details of the life cycle have yet to be established, but the parasite life cycle involves crossing the insect peritrophic matrix (PM). The PM consists of the polysaccharide chitin, several hundred proteins, and both glycosamino- and galactosaminoglycan (GAG) polysaccharides. Owing to the technical challenges of detecting small amounts of GAG polysaccharides, their conclusive identification and composition have not been possible until now. Following removal of PMs from the insects and the application of heparinases (bacterial lyase enzymes that are specific for heparan sulphate (HS) GAG polysaccharides), dot blots with a HS-specific antibody showed heparan sulphate proteoglycans (HSPGs) to be present, consistent with Glossina morsitans morsitans genome analysis, as well as the likely expression of the HSPGs syndecan and perlecan. Exhaustive HS digestion with heparinases, fluorescent labeling of the resulting disaccharides with BODIPY fluorophore, and separation by strong anion exchange chromatography then demonstrated the presence of HS for the first time and provided the disaccharide composition. There were no significant differences in the type of disaccharide species present between genders or between ages (24 vs. 48 h post emergence), although the HS from female flies was more heavily sulphated overall. Significant differences, which may relate to differences in infection between genders or ages, were evident, however, in overall levels of 2-O-sulphation between sexes and, for females, between 24 and 48 h post-emergence, implying a change in expression or activity for the 2-O-sulphotransferase enzyme. The presence of significant quantities of disaccharides containing the monosaccharide GlcNAc6S contrasts with previous findings in Drosophila melanogaster and suggests subtle differences in HS fine structure between species of the Diptera.
RESUMO
Signal inactivation is a crucial step in the dynamic of olfactory process and involves various Odorant-Degrading Enzymes. In the silkworm Bombyx mori, one of the best models for studying olfaction in insects, the involvement of an antennal-specific aldehyde oxidase in the degradation of the sex pheromone component bombykal has been demonstrated over the three past decades by biochemical studies. However, the corresponding enzyme has never been characterized at the molecular level. Bioinformatic screening of B. mori genome and molecular approaches have been used to isolate several candidate sequences of aldehyde oxidases. Two interesting antennal-expressed genes have been further characterized and their putative functions are discussed in regard to their respective expression pattern and to our knowledge on aldehyde oxidase properties. Interestingly, one gene appeared as specifically expressed in the antennae of B. mori and associated in males with the bombykal-sensitive sensilla, strongly suggesting that it could encode for the previously biochemically characterized enzyme.
Assuntos
Aldeído Oxidase/genética , Bombyx/enzimologia , Genes de Insetos , Feromônios/metabolismo , Olfato/genética , Aldeído Oxidase/química , Aldeído Oxidase/classificação , Sequência de Aminoácidos , Animais , Bombyx/genética , Feminino , Genoma de Inseto/genética , Masculino , Dados de Sequência Molecular , FilogeniaRESUMO
The recent Zika virus (ZIKV) outbreak, which mainly affected Brazil and neighbouring states, demonstrated the paucity of information concerning the epidemiology of several flaviruses, but also highlighted the lack of available agents with which to treat such emerging diseases. Here, we show that heparin, a widely used anticoagulant, while exerting a modest inhibitory effect on Zika Virus replication, fully prevents virus-induced cell death of human neural progenitor cells (NPCs).
Assuntos
Efeito Citopatogênico Viral/efeitos dos fármacos , Heparina/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/virologia , Zika virus/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Humanos , Células-Tronco Neurais/patologia , Zika virus/patogenicidadeRESUMO
Vanadium compounds have been explored as therapy of diabetes, and most studies have focussed on insulin mimetic effects, i.e. reducing hyperglycemia by improving glucose sensitivity and thus glucose uptake in sensitive tissues. We have recently shown that bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)oxidovanadium(IV), VO(dmpp)2, has promising effects when compared to another vanadium compound, bis(maltolato)oxidovanadium(IV), BMOV, and insulin itself, in isolated adipocytes and in vivo in Goto-Kakizaki (GK) rats, an animal model of hereditary type 2 diabetes (T2D).We now have investigated in GK rats whether VO(dmpp)2 also modulates another important defect in T2D, impaired insulin secretion. VO(dmpp)2, but not BMOV, stimulated insulin secretion from isolated GK rat pancreatic islets at high, 16.7mM, but not at lownormal, 3.3 mM, glucose concentration. Mechanistic studies demonstrate that the insulin releasing effect of VO(dmpp)2 is due to its interaction with several steps in the stimulus-secretion coupling for glucose, including islet glucose metabolism and K-ATP channels, L-type Ca2+ channels, modulation by protein kinases A and C, as well as the exocytotic machinery. In conclusion, VO(dmpp)2 exhibits properties of interest for treatment of the insulin secretory defect in T2D, in addition to its well-described insulin mimetic activity.
Assuntos
Complexos de Coordenação/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Técnicas de Cultura de TecidosRESUMO
Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by intestinal cells and stimulates insulin secretion from the pancreas in a glucose-dependent manner. Exogenously supplied GLP-1 analogues are used in the treatment of type 2 diabetes. An anti-diabetic effect of Lactobacillus in lowering plasma glucose levels and its use as a vehicle for delivery of protein and antibody fragments has been shown previously. The aim of this study was to employ lactobacilli as a vehicle for in situ production and delivery of GLP-1 analogue to normalize blood glucose level in diabetic GK (Goto-Kakizaki) rats. In this study, we designed pentameric GLP-1 (5×GLP-1) analogues which were both expressed in a secreted form and anchored to the surface of lactobacilli. Intestinal trypsin sites were introduced within 5×GLP-1, leading to digestion of the pentamer into an active monomeric form. The E. coli-produced 5×GLP-1 peptides delivered by intestinal intubation to GK rats resulted in a significant improvement of glycemic control demonstrated by an intraperitoneal glucose tolerance test. Meanwhile, the purified 5×GLP-1 (trypsin-digested) from the Lactobacillus cultures stimulated insulin secretion from HIT-T15 cells, similar to the E. coli-produced 5×GLP-1 peptides. When delivered by gavage to GK rats, non-expressor L. paracasei significantly lowered the blood glucose level but 5×GLP-1 expression did not provide an additional anti-diabetic effect, possibly due to the low levels produced. Our results indicate that lactobacilli themselves might be used as an alternative treatment method for type 2 diabetes, but further work is needed to increase the expression level of GLP-1 by lactobacilli in order to obtain a significant insulinotropic effect in vivo.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Lactobacillus/metabolismo , Recombinação Genética , Administração Oral , Animais , Glicemia/metabolismo , Western Blotting , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/patologia , Escherichia coli/metabolismo , Comportamento Alimentar , Teste de Tolerância a Glucose , Injeções Subcutâneas , Insulina/metabolismo , Secreção de Insulina , Masculino , Mesocricetus , Biblioteca de Peptídeos , Ratos , Recombinação Genética/genética , Reprodutibilidade dos Testes , Transformação Genética , Tripsina/metabolismoRESUMO
The body louse, Pediculus humanus humanus, is an obligate blood-feeding ectoparasite and an important insect vector that mediates the transmission of diseases to humans. The analysis of the body louse genome revealed a drastic reduction of the chemosensory gene repertoires when compared to other insects, suggesting specific olfactory adaptations to host specialization and permanent parasitic lifestyle. Here, we present for the first time functional evidence for the role of odorant receptors (ORs) in this insect, with the objective to gain insight into the chemical ecology of this vector. We identified seven putative full-length ORs, in addition to the odorant receptor co-receptor (Orco), and expressed four of them in the Xenopus laevis oocytes system. When screened with a panel of ecologically-relevant odorants, PhumOR2 responded to a narrow set of compounds. At the behavior level, both head and body lice were repelled by the physiologically-active chemicals. This study presents the first evidence of the OR pathway being functional in lice and identifies PhumOR2 as a sensitive receptor of natural repellents that could be used to develop novel efficient molecules to control these insects.
Assuntos
Repelentes de Insetos/farmacologia , Pediculus/efeitos dos fármacos , Pediculus/genética , Receptores Odorantes/genética , Olfato , Animais , Fenômenos Eletrofisiológicos , Feminino , Insetos Vetores , Oócitos , Xenopus laevisRESUMO
Nerve recovery following injury is usually incomplete, leaving functional deficits. Our aim was to investigate the neural changes in pro-angiogenic, pro-inflammatory and apoptotic factors during and after chronic nerve compression (CNC). Nerve function was impaired after CNC and was progressively restored after nerve decompression, while nerve blood flow was elevated. While the expression of the pro-inflammatory and pro-angiogenic cytokines IL-6, TNF-α and VEGF-A was high during and after CNC, we observed that inhibition of VEGF-A receptors strongly counteracted the angiogenic response induced by the ex vivo CNC. Activation of the pro-survival transcription factor nuclear factor-kappa B (NF-κB) increased during CNC, returning to control levels after nerve decompression. After nerve decompression, the downregulation of Mdm2 correlated well with an increased expression of pro-apoptotic transcription factor p53. All together, we bring novel evidence that CNC activates transcription factors such as NF-κB and p53, which are key effectors of the cellular stress response, suggesting a neuroprotective process associated with an increased VEGF-A-mediated neurotrophic effect. Our results highlight the role of pro-angiogenic and pro-inflammatory cytokines during CNC that are reinforced by increasing neurotrophic capacity during recovery to promote nerve regeneration.
Assuntos
Neovascularização Fisiológica , Síndromes de Compressão Nervosa/fisiopatologia , Fatores de Crescimento Neural/metabolismo , Recuperação de Função Fisiológica , Nervo Isquiático/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Masculino , NF-kappa B/metabolismo , Síndromes de Compressão Nervosa/metabolismo , Síndromes de Compressão Nervosa/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ratos Wistar , Fluxo Sanguíneo Regional , Nervo Isquiático/irrigação sanguínea , Nervo Isquiático/patologia , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Protein kinase C (PKC) is a family of protein kinases controlling protein phosphorylation and playing important roles in the regulation of metabolism. We have investigated expression levels of PKC isoforms in pancreatic islets and liver of diabetic Goto-Kakizaki (GK) rats with and without insulin treatment to evaluate their association with glucose homeostasis. mRNA and protein expression levels of PKC isoforms were assessed in pancreatic islets and liver of Wistar rats and GK rats with or without insulin treatment. PKCα and PKCζ mRNA expressions were down-regulated in islets of GK compared with Wistar rats. PKCα and phosphorylated PKCα (p-PKCα) protein expressions were decreased in islets of GK compared with insulin-treated GK and Wistar rats. PKCζ protein expression in islets was reduced in GK and insulin-treated GK compared with Wistar rats, but p-PKCζ was decreased only in GK rats. Islet PKCε mRNA and protein expressions were lower in GK compared with insulin-treated GK and Wistar rats. In liver, PKCδ and PKCζ mRNA expressions were decreased in both GK and insulin-treated GK compared with Wistar rats. Hepatic PKCζ protein expression was diminished in both GK rats with and without insulin treatment compared with Wistar rats. Hepatic PKCε mRNA expression was down-regulated in insulin-treated GK compared with GK and Wistar rats. PKCα, PKCε, and p-PKCζ expressions were secondary to hyperglycaemia in GK rat islets. Hepatic PKCδ and PKCζ mRNA expressions were primarily linked to hyperglycaemia. Additionally, hepatic PKCε mRNA expression could be under control of insulin.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Animais , Glicemia , Modelos Animais de Doenças , Glucose/metabolismo , Insulina/metabolismo , Isoenzimas , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Antennae-specific odorant-degrading enzymes (ODEs) are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes--the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs). Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW), Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13-16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13-16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.
Assuntos
Aldeído Oxidase/metabolismo , Antenas de Artrópodes/enzimologia , Mariposas/enzimologia , 1-Propanol/metabolismo , Aldeído Oxidase/antagonistas & inibidores , Aldeído Oxidase/química , Aldeído Oxidase/genética , Sequência de Aminoácidos , Animais , Antenas de Artrópodes/efeitos dos fármacos , Bombyx/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Perfilação da Expressão Gênica , Temperatura Alta , Masculino , Dados de Sequência Molecular , Peso Molecular , Mariposas/genética , Especificidade de Órgãos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Olfato/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacosRESUMO
The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx-CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occurring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents.
Assuntos
Antenas de Artrópodes/metabolismo , Culex/genética , Receptores Odorantes/genética , Animais , Culex/metabolismo , Eugenol/metabolismo , Feminino , Masculino , Feromônios/metabolismo , Filogenia , Receptores Odorantes/metabolismo , Terminologia como Assunto , XenopusRESUMO
Most studies of chronic nerve compression focus on large nerve function in painful conditions, and only few studies have assessed potential changes in the function of small nerve fibers during chronic nerve compression and recovery from compression. Cutaneous pressure-induced vasodilation is a neurovascular phenomenon that relies on small neuropeptidergic fibers controlling the cutaneous microvasculature. We aimed to characterize potential changes in function of these small fibers and/or in cutaneous microvascular function following short-term (1-month) and long-term (6-month) nerve compression and after release of compression (ie, potential recovery of function). A compressive tube was left on one sciatic nerve for 1 or 6 months and then removed for 1-month recovery in Wistar rats. Cutaneous vasodilator responses were measured by laser Doppler flowmetry in hind limb skin innervated by the injured nerve to assess neurovascular function. Nociceptive thermal and low mechanical thresholds were evaluated to assess small and large nerve fiber functions, respectively. Pressure-induced vasodilation was impaired following nerve compression and restored following nerve release; both impairment and restoration were strongly related to duration of compression. Small and large nerve fiber functions were less closely related to duration of compression. Our data therefore suggest that cutaneous pressure-induced vasodilation provides a non-invasive and mechanistic test of neurovascular function that gives direct information regarding extent and severity of damage during chronic nerve compression and recovery, and may ultimately provide a clinically useful tool in the evaluation of nerve injury such as carpal tunnel syndrome.
Assuntos
Síndromes de Compressão Nervosa/fisiopatologia , Nervo Isquiático/lesões , Neuropatia Ciática/fisiopatologia , Pele/irrigação sanguínea , Pele/inervação , Animais , Masculino , Medição da Dor , Nervos Periféricos/fisiopatologia , Ratos , Ratos Wistar , Nervo Isquiático/fisiopatologia , Pele/fisiopatologiaRESUMO
Odorant reception in insects is mediated by different families of olfactory proteins. Here we focus on the characterization of odorant-binding proteins (OBPs), "plus-C" odorant-binding proteins ("plus-C" OBPs), chemosensory proteins (CSPs) and sensory neuron membrane proteins (SNMPs) families from the Southern house mosquito, Culex quinquefasciatus, a vector of pathogens implicated in multiple human diseases. Using bioinformatics and molecular approaches, we have identified a diversity of genes in the genome of Culex quinquefasciatus and examined their expression profiles by RT-PCR and real-time quantitative PCR. Based on their high transcript enrichment in female antennae compared to non-olfactory tissues, we have identified twelve OBPs, two "plus-C" OBPs and two SNMPs that likely play important roles in odorant reception. Transcripts of two genes were clearly enriched in female antennae compared to male antennae, whereas other genes displayed relatively equivalent transcript levels in antennae of both sexes. Additionally, eight genes were found to be transcribed at very high levels in female antennae compared to CquiOR7, suggesting they might encode highly abundant olfactory proteins. Comparative analysis across different mosquito species revealed that olfactory genes of Culex quinquefasciatus are related to putative orthologs in other species, indicating that they might perform similar functions. Understanding how mosquitoes are able to detect ecologically relevant odorant cues might help designing better control strategies. We have identified olfactory genes from different families which are likely important in Culex quinquefasciatus behaviors, thus paving the way towards a better understanding of the diversity of proteins involved in the reception of semiochemicals in this species.