RESUMEN
Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.
Asunto(s)
Aedes/efectos de los fármacos , Anopheles/efectos de los fármacos , Proteínas de Insectos/antagonistas & inhibidores , Repelentes de Insectos/farmacología , Receptores Odorantes/antagonistas & inhibidores , Compuestos Orgánicos Volátiles/farmacología , Aedes/fisiología , Aldehídos/química , Aldehídos/farmacología , Animales , Anopheles/fisiología , Monoterpenos Bicíclicos/química , Monoterpenos Bicíclicos/farmacología , Unión Competitiva , Cinamatos/química , Cinamatos/farmacología , Cimenos/química , Cimenos/farmacología , DEET/química , DEET/farmacología , Relación Dosis-Respuesta a Droga , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Repelentes de Insectos/química , Cinética , Monoterpenos/química , Monoterpenos/farmacología , Mosquitos Vectores/efectos de los fármacos , Mosquitos Vectores/fisiología , Odorantes/análisis , Unión Proteica , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Olfato/fisiología , Relación Estructura-Actividad , Compuestos Orgánicos Volátiles/químicaRESUMEN
The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca(2+)-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET.
Asunto(s)
Anopheles/fisiología , Repelentes de Insectos , Receptores Odorantes/metabolismo , Animales , Anopheles/metabolismoRESUMEN
In this study, we have deleted the lef8 gene of the baculovirus BmNPV, which encodes one of the viral RNA polymerase subunits, in order to create a knockout bacmid, Δlef8, directing cytopathology-free single-cell infections for gene transduction and recombinant protein production. However, while removal of the complete lef8 ORF produced the expected phenotype, it also affected the function of the closely linked essential gene orf40, thus hampering the mutant bacmid rescue in cultured Bombyx cells expressing recombinant LEF8. Subsequently, we determined that several diverse sequences can substitute for the orf40 5'-upstream sequences that were removed by the deletion of the lef8 gene and also showed that neither a physical linkage nor expression of the two relevant genes under native promoter control is a prerequisite for a fully functional virus. Based on these findings, we generated a rescue-competent lef8-null vector, which contained a heterologous promoter-driven orf40. This lef8-deficient vector, which produces productive infections and progeny virus lacking lef8 in deficiency-complementing cells expressing LEF8, could be used as the basis for an alternative to current silkmoth transduction systems.
Asunto(s)
Bombyx/virología , ARN Polimerasas Dirigidas por ADN/genética , Eliminación de Gen , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Transducción Genética , Proteínas Virales/genética , Animales , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Viral de la Expresión Génica , Proteínas Virales/metabolismoRESUMEN
Differential regulation at the level of transcription provides a means for controlling gene expression in eukaryotes, especially during development. Insect model systems have been extensively used to decipher the molecular basis of such regulatory cascades, and one of the oldest such model systems is the regulation of chorion gene expression during ovarian follicle maturation. Recent experimental and technological advances have shed new light onto the system, allowing us to revisit it. Thus, in this review we try to summarize almost 40 years' worth of studies on chorion gene regulation while-by comparing Bombyx mori and Drosophila melanogaster models-attempting to present a comprehensive, unified model of the various regulatory aspects of choriogenesis that takes into account the evolutionary conservation and divergence of the underlying mechanisms.
Asunto(s)
Bombyx/genética , Drosophila melanogaster/genética , Proteínas del Huevo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/genética , Animales , Evolución Biológica , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Corion/crecimiento & desarrollo , Corion/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas del Huevo/metabolismo , Proteínas de Insectos/metabolismo , Pupa/crecimiento & desarrollo , Pupa/metabolismoRESUMEN
Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases.
Asunto(s)
Anopheles/química , Proteínas de Insectos/química , Lipocalinas/química , Multimerización de Proteína , Animales , Anopheles/genética , Anopheles/metabolismo , Antenas de Artrópodos/química , Antenas de Artrópodos/metabolismo , Cristalografía por Rayos X , Femenino , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.
Asunto(s)
Anopheles/metabolismo , Receptores Odorantes/metabolismo , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Dimerización , Colorantes Fluorescentes/farmacología , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptores Odorantes/química , Receptores Odorantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensilos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies.
Asunto(s)
Baculoviridae/metabolismo , Movimiento Celular/fisiología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Células de Schwann/fisiología , Animales , Animales Recién Nacidos , Antígeno CD146/química , Antígeno CD146/metabolismo , Movimiento Celular/genética , Células Cultivadas , Cromatografía de Afinidad/métodos , Técnicas de Cocultivo/métodos , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Humanos , Insectos , Ratones , Vaina de Mielina/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/química , Molécula L1 de Adhesión de Célula Nerviosa/genética , Prosencéfalo/citología , Prosencéfalo/fisiología , Ratas , Ratas Wistar , Nervio Ciático/citología , Transducción Genética/métodosRESUMEN
Successful embryonic development of parasitoid wasps in lepidopteran hosts is achieved through co-injection of polydna viruses whose gene products are thought to target the immune responses of the host. One gene product of the endosymbiont bracovirus of the parasitic wasp Cotesia rubecula, CrV1, has been reported to inhibit the immune responses of its endoparasitized lepidopteran host through interference with the haematocyte cytoskeletal structure. Here we establish that CcV1, the Cotesia congregata bracovirus orthologue of CrV1, is also uptaken by lepidopteran haemocytes and haemocyte-like established cell lines, but we also report on a different function of CcV1, which is highly relevant to the inhibition of the host immune responses and is based on its direct interaction with the pattern recognition molecule hemolin. Recombinant CcV1 inhibits hemolin functions, such as lipopolysaccharide binding and bacterial agglutination as well as bacterial phagocytosis by haemocytes and haemocyte-like cell lines, producing functional phenotypes equivalent to those observed to arise from RNAi-based inhibition of hemolin gene expression. Finally, we show that CcV1 and hemolin colocalize on the membrane surface of hemolin-expressing cells, a finding suggesting that CcV1 may be uptaken by haemocytes and inhibit haemocyte function as a result of its interaction with membrane-anchored hemolin.
Asunto(s)
Bombyx/inmunología , Terapia de Inmunosupresión , Proteínas de Insectos/antagonistas & inhibidores , Polydnaviridae/inmunología , Avispas/virología , Aglutinación , Animales , Membrana Celular/química , Hemocitos/inmunología , Inmunoglobulinas/inmunología , Proteínas de Insectos/inmunología , Microscopía Fluorescente , Unión Proteica , Proteínas Virales/análisisRESUMEN
At the heart of the odor recognition process in all animals are G-protein-coupled receptors, which are seven-transmembrane domain proteins that initiate G-protein-mediated signaling cascades when activated by their ligands. Odorant receptors (ORs) are a large, diverse family of proteins with some 80 members in the mosquito Anopheles gambiae. With the assumption that more sensilla on female antennae are tuned to human odors than on male antennae, comparison of specific OR mRNA levels in male and female antennae can provide an indication as to which receptors may be stimulated by host odors. We have used RT PCR and quantitative real-time PCR (qRT PCR) to investigate sex-biased expression levels of 80 A. gambiae ORs in male and female antennae and maxillary palps. On the basis of prevalence of expression in female antennae and on a strong female relative to male expression bias we identified a short list of ORs that are likely involved in host odor recognition by female mosquitoes.
Asunto(s)
Anopheles/metabolismo , Interacciones Huésped-Parásitos/fisiología , Odorantes , Receptores Odorantes/metabolismo , Caracteres Sexuales , Animales , Femenino , Perfilación de la Expresión Génica , Humanos , Malaria/transmisión , Masculino , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although dibenzoylhydrazine-type non-steroidal ecdysone agonists such as methoxyfenozide (RH-2485) have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, cell lines derived from the polyphagous noctuid pest Spodoptera exigua (Se4 cells) were selected for continuous growth in the presence of high concentrations of 20-hydroxyecdysone (20E) or methoxyfenozide. Here we describe an analysis of ecdysteroid receptor signaling in the ecdysteroid-resistant Se4 cell lines. In contrast to other ecdysteroid-resistant cell lines described in literature, our data support the existence of a normal functioning ecdysteroid receptor complex in the resistant Se4 cell lines: (1) using a recombinant BmNPV baculovirus as a transduction tool, activation of an ecdysone-responsive luciferase cassette was demonstrated; (2) the early gene HR3 is constitutively expressed in the resistant cell lines that are grown in the presence of 20E or methoxyfenozide. Quantitative RT-PCR experiments indicated that expression levels of SeEcR mRNA were comparable among sensitive and resistant cell lines. Sequencing of PCR fragments also revealed the presence of SeEcR mRNA with a wild-type ligand-binding domain in resistant cells. Finally, a possible role for the gene FTZ-F1, whose expression correlates with the absence of circulating ecdysteroids during insect development, in the resistance mechanism was investigated. However, it was observed that FTZ-F1, in contrast to what is observed during insect development, is constitutively expressed in Se4 cells and that its expression is not regulated by the addition of ecdysteroid. It is proposed that the resistance mechanism in Se4 cells resides at the coupling between the conserved hierarchical cascade of early and early-late gene expression and the differentiation program in the Se4 cell line. The use of insect cell lines for the investigation of resistance against dibenzoylhydrazine ecdysone agonists and their relevance for uncovering resistance mechanisms in insects during pest control programs is discussed.
Asunto(s)
Ecdisteroides/metabolismo , Receptores de Esteroides/metabolismo , Transducción de Señal , Spodoptera/metabolismo , Factor Esteroidogénico 1/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Expresión Génica , Genes Reporteros , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/metabolismo , Receptores de Esteroides/genética , Spodoptera/genética , Factor Esteroidogénico 1/genética , Factores de Transcripción/metabolismoRESUMEN
During insect development, ecdysteroids and juvenile hormones (JHs) interact to regulate larval growth, metamorphosis and reproduction but the molecular mechanisms by which both hormones influence each other's activity remain unknown. Because of their ease of use and straightforward genetic manipulation, insect cell lines often have been used to clarify the actions and interactions of hormones at the molecular level. Here we report on the use of two insect culture cell lines, Drosophila melanogaster S2 and Bombyx mori Bm5 cells, to investigate two molecular processes in which ecdysteroids and JH have been shown to interact: (1) direct modulation of the activity of the ecdysteroid receptor transcription complex and (2) interference at the level of induction of the primary gene E75. Our data do not support JH analogs (JHAs) acting through the above processes: 'antagonism' of ecdysteroid receptor activity by JHAs correlated with cytotoxicity and induction of E75 expression by JHAs was not demonstrated. However, we confirm previous studies in which it was observed that methoprene can partially reverse the growth inhibition by 20E in S2 cells (but not Bm5 cells). Therefore, the molecular mechanism by which both hormones influence each other's activity to regulate cell growth in S2 cells remains unknown.
Asunto(s)
Bombyx/citología , Bombyx/efectos de los fármacos , Drosophila melanogaster/citología , Drosophila melanogaster/efectos de los fármacos , Hormonas Juveniles/agonistas , Hormonas Juveniles/antagonistas & inhibidores , Receptores de Esteroides/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila , Humanos , Insecticidas/farmacología , Metopreno/farmacología , Fenilcarbamatos/farmacología , Isoformas de Proteínas , Receptores de Esteroides/química , Factores de Transcripción/metabolismoRESUMEN
There is increasing interest in the development of effective mosquito repellents of natural origin to reduce transmission of diseases such as malaria and yellow fever. To achieve this we have employed an in vitro competition assay involving odorant-binding proteins (OBPs) of the malaria mosquito, Anopheles gambiae, with a predominantly female expression bias to identify plant essential oils (EOs) containing bioactive compounds that target mosquito olfactory function. EOs and their fractions capable of binding to such OBPs displayed repellence against female mosquitoes in a laboratory repellent assay. Repellent EOs were subjected to gas chromatographic analysis linked to antennogram (EAG) recordings from female A. gambiae to identify the biologically active constituents. Among these compounds cumin alcohol, carvacrol, ethyl cinnamate and butyl cinnamate proved as effective as DEET at an equivalent dose in the repellent assay, and combinations of carvacrol with either butyl cinnamate or cumin alcohol proved to be significantly more effective than DEET in the assay. When tested as spatial repellents in experimental shelters housing sleeping humans in northern Nigeria a binary mixture of carvacrol plus cumin alcohol caused mosquitoes to leave shelters in significantly higher numbers to those induced by DEET in female Anopheles spp. and in numbers equivalent to that of DEET in Culex spp. mosquitoes. These findings indicate an approach for the identification of biologically active molecules of natural origin serving as repellents for mosquitoes.
Asunto(s)
Anopheles , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Insectos , Repelentes de Insectos/farmacología , Receptores Odorantes , Caracteres Sexuales , Animales , Anopheles/genética , Anopheles/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Receptores Odorantes/biosíntesis , Receptores Odorantes/genéticaRESUMEN
In this work we report a fast and efficient virtual screening protocol for discovery of novel bioinspired synthetic mosquito repellents with lower volatility and, in all likelihood, increased protection time as compared with their plant-derived parental compounds. Our screening protocol comprises two filtering steps. The first filter is based on the shape and chemical similarity to known plant-derived repellents, whereas the second filter is based on the predicted similarity of the ligand's binding mode to the Anopheles gambiae odorant binding protein (AgamOBP1) relative to that of DEET and Icaridin to the same OBP. Using this protocol, a chemical library containing 42,755 synthetic molecules was screened in silico and sixteen selected compounds were tested for their affinity to AgamOBP1 in vitro and repellence against A. gambiae female mosquitoes using a warm-body repellent assay. One of them showed DEET-like repellence (91%) but with significantly lower volatility (2.84â¯×â¯10-6â¯mmHg) than either DEET (1.35â¯×â¯10-3â¯mmHg) or its parental cuminic acid (3.08â¯×â¯10-3â¯mmHg), and four other compounds were found to exhibit repellent indices between 69 and 79%. Overall, a correlation was not evident between repellence and OBP-binding strength. In contrast, a correlation between binding mode and repellence was found.
Asunto(s)
Descubrimiento de Drogas/métodos , Repelentes de Insectos/análisis , Receptores Odorantes/agonistas , Animales , Culicidae , Femenino , Cobayas , Ligandos , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas PequeñasRESUMEN
Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.
Asunto(s)
Bombyx/crecimiento & desarrollo , Folículo Ovárico/crecimiento & desarrollo , Prostaglandinas/fisiología , Transducción de Señal , Animales , Aspirina/farmacología , Bombyx/efectos de los fármacos , Bombyx/metabolismo , AMP Cíclico/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Ecdisona/agonistas , Femenino , Homeostasis , Hidrazinas/farmacología , Indometacina/farmacología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/metabolismo , Prostaglandinas/farmacología , Vitelogénesis/efectos de los fármacosRESUMEN
Insect olfactory receptors (ORs) are heteromeric ligand-gated cation channels composed of a common olfactory receptor subunit (ORco) and a variable subunit (ORx) of as yet unknown structures and undetermined stoichiometries. In this study, we examined the allosteric modulation exerted on Anopheles gambiae heteromeric ORx/ORco olfactory receptors in vitro by a specific class of ORco agonists (OAs) comprising ORcoRAM2 and VUAA1. High OA concentrations produced stronger functional responses in cells expressing heteromeric receptor channels relative to cells expressing ORco alone. These OA-induced responses of ORx/ORco channels were also notably much stronger than those obtained upon administration of ORx-specific ligands to the same receptors. Most importantly, small concentrations of OAs were found to act as strong potentiators of ORx/ORco function, increasing dramatically both the efficacy and potency of ORx-specific odorants. These results suggest that insect heteromeric ORs are highly dynamic complexes adopting different conformations that change in a concerted fashion as a result of the interplay between the subunits of the oligomeric assemblies, and that allosteric modulation may constitute an important element in the modulation and fining tuning of olfactory reception function.
RESUMEN
Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.
Asunto(s)
Técnicas Químicas Combinatorias , Ecdisterona/agonistas , Ecdisterona/antagonistas & inhibidores , Extractos Vegetales/farmacología , Animales , Bombyx/citología , Línea Celular , Fluorescencia , Genes Reporteros , Hidrazinas/químicaRESUMEN
Baculovirus vectors constitute important tools for therapeutic protein production and mammalian cell transduction for gene therapy applications. A prerequisite for such applications is that the cell lines in which baculoviruses are propagated be maintained in serum-free media that are devoid of potential human pathogens. However, in serum-free media, the performance of baculovirus-based systems can be significantly reduced. In this report, we show that silkmoth-derived host cell lines for the Bombyx mori-nuclear polyhedrosis virus (BmNPV) that are transformed with the gene for the promoting protein (PP), a silkmoth-derived secreted factor containing a lipid-binding domain, display enhanced susceptibility to BmNPV infection and enhanced budded virus productivity in serum-free media. For transformed silkmoth cells maintained in serum-free media, the rate of BmNPV entry is enhanced by two orders of magnitude relative to the untransformed cells, while the rate of budded virus production is increased five-fold. The infectivity-enhancing effect can be also conferred to normal cells grown in serum-free media by addition of conditioned media from the transformed cells, which contain the secreted recombinant PP. Thus, PP substitutes for serum factors whose presence facilitates baculovirus entry into the cells. However, the effects of silkmoth-derived PP may be specific to the BmNPV-silkmoth system since little or no changes in viral infectivity are obtained by PP expression in Trichoplusia ni-derived High-Fivetrade mark cells grown in serum-free media and infected with a different baculovirus (AcNPV).
Asunto(s)
Bombyx/metabolismo , Transformación Celular Viral , Medio de Cultivo Libre de Suero/farmacología , Proteínas de Insectos/metabolismo , Nucleopoliedrovirus/fisiología , Replicación Viral/efectos de los fármacos , Animales , Baculoviridae/genética , Bombyx/citología , Bombyx/virología , Técnicas de Cultivo de Célula , Línea Celular Transformada , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero/química , Susceptibilidad a Enfermedades/virología , Cuerpo Adiposo/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
The silkmoth chorion was studied extensively by F.C. Kafatos' group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis.
Asunto(s)
Bombyx/genética , Corion , Genoma de los Insectos , Animales , Bombyx/embriología , Mapeo Cromosómico , Estructuras Cromosómicas , Cromosomas Artificiales Bacterianos , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Anotación de Secuencia MolecularRESUMEN
Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig, and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as "middle", and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization, and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins.
Asunto(s)
Bombyx/genética , Corion , Sitios de Carácter Cuantitativo , Animales , Bombyx/metabolismo , Biología Computacional/métodos , Proteínas del Huevo , Cáscara de Huevo , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica , Biblioteca de Genes , Orden Génico , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Proteoma , Proteómica/métodos , Transcripción GenéticaRESUMEN
We have cloned and functionally characterized a novel protein, BmVMP30, which is synthesized by the cells of the follicular epithelium of the ovarian follicles of the domesticated silkworm Bombyx mori, secreted from them and associated with the vitelline membrane. BmVMP30 is a 30 kDa protein that bears limited structural features reminiscent of other insect vitelline membrane proteins. Although BmVMP30 does not share pronounced similarities or signature motifs with other reported proteins, its temporal and spatial expression and its behavior throughout oogenesis suggest that it is a novel member of the insect vitelline membrane protein family. The protein is expressed exclusively in the cells of the follicular epithelium during stages -15 to -1 of vitellogenesis, secreted from them and, ultimately, localized at the junction between the oocyte and the eggshell, where the vitelline membrane is located. Treatment of follicles with an antisense oligonucleotide that encompasses the translation initiation codon results in the production of an N-terminally truncated protein and disruption of the integrity of the follicular epithelium. Antisense oligonucleotide treatment, however, has no effect on the implementation of the developmental program that directs the autonomous progression of ovarian follicles through the last stages of vitellogenesis and choriogenesis.