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1.
Insect Mol Biol ; 23(5): 539-49, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24698447

RESUMEN

Uridine diphosphate UDP-glycosyltransferases (UGTs) are detoxification enzymes widely distributed within living organisms. They are involved in the biotransformation of various lipophilic endogenous compounds and xenobiotics, including odorants. Several UGTs have been reported in the olfactory organs of mammals and involved in olfactory processing and detoxification within the olfactory mucosa but, in insects, this enzyme family is still poorly studied. Despite recent transcriptomic analyses, the diversity of antennal UGTs in insects has not been investigated. To date, only three UGT cDNAs have been shown to be expressed in insect olfactory organs. In the present study, we report the identification of eleven putative UGTs expressed in the antennae of the model pest insect Spodoptera littoralis. Phylogenetic analysis revealed that these UGTs belong to five different families, highlighting their structural diversity. In addition, two genes, UGT40R3 and UGT46A6, were either specifically expressed or overexpressed in the antennae, suggesting specific roles in this sensory organ. Exposure of male moths to the sex pheromone and to a plant odorant differentially downregulated the transcription levels of these two genes, revealing for the first time the regulation of insect UGTs by odorant exposure. Moreover, the specific antennal gene UGT46A6 was upregulated by insecticide topical application on antennae, suggesting its role in the protection of the olfactory organ towards xenobiotics. This work highlights the structural and functional diversity of UGTs within this highly specialized tissue.


Asunto(s)
Antenas de Artrópodos/enzimología , Glicosiltransferasas/genética , Spodoptera/enzimología , Spodoptera/genética , Uridina Difosfato/genética , Xenobióticos/metabolismo , Secuencia de Aminoácidos , Animales , Etiquetas de Secuencia Expresada/química , Femenino , Regulación de la Expresión Génica , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Cinética , Masculino , Datos de Secuencia Molecular , Odorantes , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Spodoptera/metabolismo , Uridina Difosfato/química , Uridina Difosfato/metabolismo
2.
Insect Mol Biol ; 21(6): 568-80, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22984814

RESUMEN

Cytochrome P450 enzymes (P450s) are involved in many physiological functions in insects, such as the metabolism of signal molecules, adaptation to host plants and insecticide resistance. Several P450s have been reported in the olfactory organs of insects, the antennae, and have been proposed to play a role in odorant processing and/or xenobiotic metabolism. Despite recent transcriptomic analyses in several species, the diversity of antennal P450s in insects has not yet been investigated. Here, we report the identification of 37 putative P450s expressed in the antennae of the pest moth Spodoptera littoralis, as well as the characterization of a redox partner, cytochrome P450 reductase (CPR). Phylogenetic analysis revealed that S. littoralis P450s belong to four clades defined by their conservation with vertebrate P450s and their cellular localization. Interestingly, the CYP3 and CYP4 clans, which have been described to be mainly involved in the metabolism of plant compounds and xenobiotics, were largely predominant. More surprisingly, two P450s related to ecdysteroid metabolism were also identified. Expression patterns in adult and larval tissues were studied. Eight P450s appeared to be specific to the chemosensory organs, ie the antennae and proboscis, suggesting a specific role in odorant and tastant processing. Moreover, exposure of males to a plant odorant down-regulated the transcript level of CPR, revealing for the first time the regulation of this gene by odorants within insect antennae. This work suggests that the antennae of insects are a key site for P450-mediated metabolism of a large range of exogenous and endogenous molecules.


Asunto(s)
Antenas de Artrópodos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Insectos/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Spodoptera/enzimología , Animales , Secuencia de Bases , Femenino , Larva/metabolismo , Masculino , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Olfato
3.
Insect Mol Biol ; 19(1): 87-97, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20002215

RESUMEN

Recent studies have suggested that pheromone-degrading enzymes belonging to the carboxylesterase family could play a role in the dynamics of the olfactory response to acetate sex pheromones in insects. Bioinformatic analyses of a male antennal expressed sequence tag library allowed the identification of 19 putative esterase genes expressed in the antennae of the moth Spodoptera littoralis. Phylogenetic analysis revealed that these genes belong to different insect esterase clades, defined by their putative cellular localization and substrate preferences. Interestingly, two of the 19 genes appeared to be antennal specific, suggesting a specific role in olfactory processing. This high esterase diversity suggested that the antennae are the location for intense esterase-based metabolism, against potentially a large range of exogenous and endogenous molecules.


Asunto(s)
Carboxilesterasa/metabolismo , Proteínas de Insectos/metabolismo , Neuronas Receptoras Olfatorias/enzimología , Spodoptera/enzimología , Animales , Carboxilesterasa/genética , Femenino , Proteínas de Insectos/genética , Larva/enzimología , Masculino , Filogenia , Reacción en Cadena de la Polimerasa , Pupa/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Caracteres Sexuales , Spodoptera/genética , Spodoptera/crecimiento & desarrollo
4.
Insect Mol Biol ; 18(2): 213-22, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19191929

RESUMEN

The molecular characterization of post-receptor actors involved in insect olfactory transduction has yet to be understood. We have investigated the presence of a Transient Receptor Potential (TRP) channel in the peripheral olfactory system of the moth Spodoptera littoralis. A cDNA encoding a Lepidopteran TRP channel (TRPgamma) was identified by analysis of a male-antennal EST database and subsequently cloned by RACE PCR. In adult males, the TRPgamma transcript was detected in antennae, at the base of olfactory sensilla. Moreover, TRPgamma was observed in antennae in both pupal and adult stages. This work is the first step in understanding the involvement of TRPgamma in signalling pathways involved in the development and function of the insect olfactory system.


Asunto(s)
Estructuras Animales/metabolismo , Vías Olfatorias/metabolismo , Transducción de Señal , Spodoptera/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/genética
5.
Insect Mol Biol ; 16(1): 73-81, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17257210

RESUMEN

Rapid degradation of odours after interaction with olfactory receptors is a critical step of the signal reception process. However, the implied mechanisms are still largely unknown in vertebrates as well as in insects. Involvement of odourant-degrading enzymes in odourant degradation within the antennae has been shown in some insect species and, in particular, esterases could play a key role in degradation of sex pheromones from Lepidoptera. Using a PCR-based strategy, we isolated cDNAs encoding two new esterases from two moths which used acetates as pheromone compounds: the Egyptian armyworm Spodoptera littoralis and the Mediterranean corn borer Sesamia nonagrioides. In antennae, both transcripts were clearly restricted to olfactory sensilla, suggesting their involvement in the degradation of odourant acetate components.


Asunto(s)
Esterasas/genética , Esterasas/metabolismo , Mariposas Nocturnas/enzimología , Mariposas Nocturnas/genética , Odorantes , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Regulación Enzimológica de la Expresión Génica , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Masculino , Datos de Secuencia Molecular , Filogenia , Órganos de los Sentidos/metabolismo , Órganos de los Sentidos/ultraestructura
6.
Insect Mol Biol ; 15(2): 137-45, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16640724

RESUMEN

Circadian rhythms are generated by endogenous circadian clocks, organized in central and peripheral clocks. An antennal peripheral clock has been demonstrated to be necessary and sufficient to generate Drosophila olfactory rhythms in response to food odours. As moth pheromonal communication has been demonstrated to follow daily rhythms, we thus investigated the occurence of a putative antennal clock in the noctuid Mamestra brassicae. From moth antennae, we isolated two full-length cDNAs encoding clock genes, period and cryptochrome, which appeared to be expressed throughout the body. In the antennae, expression of both transcripts was restricted to cells that likely represent olfactory sensory neurones. Our results suggest the occurence of a putative antennal clock that could participate in the pheromonal communication rhythms observed in vivo.


Asunto(s)
Ritmo Circadiano/genética , Flavoproteínas/genética , Mariposas Nocturnas/fisiología , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Criptocromos , Proteínas de Drosophila , Femenino , Expresión Génica , Genes de Insecto , Masculino , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Neuronas Receptoras Olfatorias/fisiología , Proteínas Circadianas Period , Feromonas , Alineación de Secuencia , Análisis de Secuencia de ADN , Olfato/fisiología
7.
Biochem Biophys Res Commun ; 332(1): 4-10, 2005 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-15896291

RESUMEN

Signal termination is a crucial step in the dynamic of the olfactory process. It involves different classes of odorant-degrading enzymes. Whereas aldehyde oxidase enzymatic activities have been demonstrated in insect antennae by previous biochemical studies, the corresponding enzymes have never been characterized at the molecular level. In the cabbage armyworm Mamestra brassicae, we isolated for the first time an aldehyde oxidase partial cDNA specifically expressed in chemosensory organs, with the strongest expression in antennae of both sexes. In these organs, expression was restricted to the olfactory sensilla. Our results suggest that the corresponding enzyme could degrade aldehyde odorant compounds, such as pheromones or plant's volatiles.


Asunto(s)
Aldehído Oxidasa/química , Aldehído Oxidasa/metabolismo , Células Quimiorreceptoras/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Mariposas Nocturnas/enzimología , Olfato/fisiología , Aldehído Oxidasa/análisis , Aldehído Oxidasa/genética , Aldehídos/metabolismo , Secuencia de Aminoácidos , Animales , Hombres , Datos de Secuencia Molecular , Especificidad de Órganos , Homología de Secuencia de Aminoácido , Distribución Tisular , Mujeres
8.
Insect Mol Biol ; 11(3): 273-81, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12000647

RESUMEN

The involvement of cytochrome P450 enzymes in olfaction was demonstrated in vertebrates some time ago. In insects these enzymes are well known for their role in insecticide resistance, but the involvement of P450 in pheromone degradation was only recently demonstrated. Using a PCR strategy, we have isolated two cDNAs from the antennae of the cabbage armyworm Mamestra brassicae - CYP4L4 and CYP4S4 - which encode microsomal P450s. CYP4S4 expression is restricted to the antennae, whereas CYP4L4 is also found in the proboscis and legs. Moreover, the two genes are strongly expressed in one type of sensory unit of the antennae - the sensilla trichodea - which are tuned to the detection of odourants. The putative function of the corresponding enzymes is discussed with regard to their respective expression patterns.


Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Mariposas Nocturnas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting/métodos , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Distribución Tisular
9.
Biochem Biophys Res Commun ; 282(1): 314-20, 2001 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-11264009

RESUMEN

Biochemical studies on ecdysteroid metabolism in arthropods suggest that aldoketoreductase enzymes (AKRs) may be involved in this pathway, but very few molecular data are available on these oxidoreductases in invertebrates. Looking for such enzymes in the crayfish Orconectes limosus, we have used a PCR strategy with primers deduced from a recent insect 3beta-reductase sequence, and from mammalian 5beta-reductase sequences. A full-length cDNA, corresponding to a putative AKR, was isolated from crayfish antennal gland. This cDNA contains an open-reading frame of 1008 bp, encoding a predicted protein of 336 amino acids. Northern blots indicated a restricted expression of the transcript in the antennal glands, quite constant during the molting cycle, and in situ hybridization demonstrated a strong expression of the transcript in the labyrinth. This is to date the first member of the AKRs superfamily characterized in a crustacean species, and the putative function of the corresponding enzyme is discussed.


Asunto(s)
Oxidorreductasas de Alcohol/genética , Astacoidea/genética , Expresión Génica , Aldehído Reductasa , Aldo-Ceto Reductasas , Secuencia de Aminoácidos , Animales , Astacoidea/anatomía & histología , Astacoidea/enzimología , Secuencia de Bases , Northern Blotting , ADN Complementario , Hibridación in Situ , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido
10.
Biochem Biophys Res Commun ; 273(3): 1132-7, 2000 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-10891384

RESUMEN

A novel cytochrome P450 was isolated from Drosophila melanogaster by PCR strategy with primers deduced from the crayfish Orconectes limosus CYP4C15 sequence, which is supposed to be involved in ecdysteroid biosynthesis. The full-length cDNA contains a 1980 bp open reading frame encoding a predicted protein of 574 amino acids and was designated CYP4G15. The corresponding gene is located at 10C1 on the X chromosome. The presence of a N-terminal segment mainly hydrophobic indicated that the corresponding enzyme is probably microsomal. In situ hybridization demonstrated predominant expression of CYP4G15 in the brain of third larval instar and Northern-blots showed no overexpression in insecticide resistant strain. This is the first indication of a specific P450 expressed in the central nervous system of Drosophila, and the putative function of the corresponding enzyme is discussed.


Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Drosophila melanogaster/genética , Sistema Nervioso/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Enzimático del Citocromo P-450/química , Familia 4 del Citocromo P450 , ADN Complementario , Proteínas de Drosophila , Drosophila melanogaster/enzimología , Datos de Secuencia Molecular , Filogenia
11.
Insect Biochem Mol Biol ; 28(11): 815-8, 1998 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9818382

RESUMEN

Two cDNA clones encoding pheromone binding proteins (PBPs) were isolated from antennal cDNA of Mamestra brassicae by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends-PCR (RACE-PCR) performed with specific primers deduced from the N-terminal sequences of two PBPs previously reported. The deduced protein sequences of the two PBPs showed a strong relationship between primary structures and functional properties of the corresponding mature proteins.


Asunto(s)
Proteínas Portadoras/química , ADN Complementario/química , Proteínas de Insectos/genética , Mariposas Nocturnas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Insectos/química , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido
12.
Eur J Biochem ; 258(2): 768-74, 1998 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-9874246

RESUMEN

A cDNA clone encoding a general odorant-binding protein (GOBP2) was isolated from antennal RNA of Mamestra brassicae by reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA encoding the GOBP2 was further used for bacterial expression. Most of the recombinant GOBP2 (>90%) was found to be insoluble. Purification under denaturing conditions consisted of solubilisation of inclusion bodies, affinity chromatography, refolding and gel filtration. The refolded rGOBP2 was cross-reactive with a serum raised against the GOBP2 of the Lepidoptera Antheraea polyphemus. The purified refolded rGOBP2 was further characterised by native PAGE, IEF, N-terminal sequencing, and two-dimensional NMR. A functional characterisation of the rGOBP2 was carried out by testing its ability to bind pheromone compounds. The yields of production and purification fulfil the requirements of structural studies.


Asunto(s)
Insectos/química , Receptores Odorantes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Feromonas/metabolismo , Unión Proteica/fisiología , Pliegue de Proteína , ARN Mensajero/genética , Receptores Odorantes/química , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido
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