RESUMO
Peptidic messengers constitute a highly diversified group of intercellular messengers widely distributed in nature that regulate a great number of physiological processes in Metazoa. Being crucial for life, it seem that they have appeared in the ancestral group from which Metazoa evolved, and were highly conserved along the evolutionary process. Peptides act mainly through G-protein coupled receptors (GPCRs), a family of transmembrane molecules. GPCRs are also widely distributed in nature being present in metazoan, but also in Choanoflagellata and Fungi. Among GPCRs, the Allatotropin/Orexin (AT/Ox) family is particularly characterized by the presence of the DRW motif in the second intracellular loop (IC Loop 2), and seems to be present in Cnidaria, Placozoa and in Bilateria, suggesting that it was present in the common ancestor of Metazoa. Looking for the evolutionary history of this GPCRs we searched for corresponding sequences in public databases. Our results suggest that AT/Ox receptors were highly conserved along evolutionary process, and that they are characterized by the presence of the E/DRWYAI motif at the IC Loop 2. Phylogenetic analyses show that AT/Ox family of receptors reflects evolutionary relationships that agree with current phylogenetic understanding in Actinopterygii and Sauropsida, including also the largely discussed position of Testudines.
Assuntos
Hormônios de Inseto/genética , Neuropeptídeos/genética , Orexinas/genética , Animais , Evolução Biológica , Classificação/métodos , Cnidários/classificação , Cnidários/genética , Bases de Dados Genéticas , Evolução Molecular , Hormônios de Inseto/metabolismo , Neuropeptídeos/metabolismo , Orexinas/metabolismo , Filogenia , Placozoa/classificação , Placozoa/genética , Receptores Acoplados a Proteínas G/genética , Análise de Sequência de DNA , Vertebrados/genéticaRESUMO
Ecdysis is a vital process for insects, during which they shed the old cuticle in order to emerge as the following developmental stage. Given its relevance for survival and reproduction, ecdysis is tightly regulated by peptidic hormones that conform an interrelated neuromodulatory network. This network was studied in species that undergo a complete metamorphosis, but not in hemimetabola. In a recent work, we demonstrated that orcokinin neuropeptides are essential for ecdysis to occur in the kissing bug Rhodnius prolixus. Here we performed gene silencing, quantitative PCR and in vitro treatments in order to study the interrelationships between RhoprOKs and hormones such as ecdysis triggering hormone, corazonin, eclosion hormone, crustacean cardioactive peptide and ecdysone. Our results suggest that RhoprOKs directly or indirectly regulate the expression of other genes. Whereas RhoprOKA is centrally involved in the regulation of gene expression, RhoprOKB is implicated in processes related to midgut physiology. Therefore, we propose that the different transcripts encoded in RhoprOK gene could integrate signaling cues, in order to coordinate the nutritional state with development and ecdysis. Given the emerging data that point to OKs as important factors for survival and reproduction, they could be candidates in the search for new insect management strategies based on neuroendocrine targets.
Assuntos
Neuropeptídeos/fisiologia , Rhodnius/fisiologia , Animais , Inativação Gênica , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Muda/genética , Muda/fisiologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Rhodnius/genéticaRESUMO
Insect growth is punctuated by molts, during which the animal produces a new exoskeleton. The molt culminates in ecdysis, an ordered sequence of behaviors that causes the old cuticle to be shed. This sequence is activated by Ecdysis triggering hormone (ETH), which acts on the CNS to activate neurons that produce neuropeptides implicated in ecdysis, including Eclosion hormone (EH), Crustacean cardioactive peptide (CCAP) and Bursicon. Despite more than 40â years of research on ecdysis, our understanding of the precise roles of these neurohormones remains rudimentary. Of particular interest is EH; although it is known to upregulate ETH release, other roles for EH have remained elusive. We isolated an Eh null mutant in Drosophila and used it to investigate the role of EH in larval ecdysis. We found that null mutant animals invariably died at around the time of ecdysis, revealing an essential role in its control. Further analyses showed that these animals failed to express the preparatory behavior of pre-ecdysis while directly expressing the motor program of ecdysis. Although ETH release could not be detected, the lack of pre-ecdysis could not be rescued by injections of ETH, suggesting that EH is required within the CNS for ETH to trigger the normal ecdysial sequence. Using a genetically encoded calcium probe, we showed that EH configured the response of the CNS to ETH. These findings show that EH plays an essential role in the Drosophila CNS in the control of ecdysis, in addition to its known role in the periphery of triggering ETH release.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Hormônios de Inseto/genética , Muda , Alelos , Animais , Comportamento Animal , Hemizigoto , Injeções , Hormônios de Inseto/metabolismo , Larva/crescimento & desenvolvimento , Mutação/genética , Neurônios/metabolismo , Neuropeptídeos/metabolismoRESUMO
Bombyx mori BmHRP28 and BmPSI, which belong to the family of RNA-binding proteins, have been identified binding to the female-specific exon 4 of the sex-determining gene Bmdsx pre-mRNA. However, the relationships between BmHRP28 and BmPSI still remain unclear. In this study, we carried out yeast two-hybrid (Y2H) and co-immunoprecipitation (Co-IP) analyses to address them. Y2H analysis showed that there was little or no direct binding between the BmHRP28 and BmPSI proteins. Also, the Co-IP experiments revealed that BmHRP28 and BmPSI coexisted in a multiprotein complex. Our results suggested that BmHRP28 and BmPSI form a muliprotein complex to regulate the splicing of Bmdsx pre-mRNA, but are not directly bound to each other. In an effort to find other regulatory factors in the multiprotein complex, we constructed a silkworm Y2H cDNA library of male early embryo. By Y2H screening, we identified an RNA-binding protein BmSPX, a putative component of the spliceosome, binding to BmPSI. These results indicated that BmHRP28 and BmPSI make up a spliceosome complex to regulate Bmdsx splicing and that BmSPX is another potential protein involved in this process. Our study provides some clues to better understand the mechanism of sex determination in the silkworm.
Assuntos
Bombyx/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Hormônios de Inseto/genética , Proteínas de Insetos/genética , Proteínas de Ligação a RNA/genética , Processos de Determinação Sexual , Processamento Alternativo , Sequência de Aminoácidos , Animais , Bombyx/crescimento & desenvolvimento , Embrião não Mamífero , Epistasia Genética , Éxons , Feminino , Biblioteca Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Hormônios de Inseto/metabolismo , Proteínas de Insetos/metabolismo , Masculino , Dados de Sequência Molecular , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Objetivo. Examinar la investigación hecha en México sobre los determinantes sociales de la salud (DSS) durante el periodo 2005-2012 con base en la caracterización del sistema nacional de investigación en salud y la producción científica sobre este tema. Material y métodos. Análisis en dos etapas: revisión documental de fuentes oficiales sobre investigación en salud en México y búsqueda sistemática de literatura sobre DSS. Resultados. Los DSS fueron mencionados en el Programa de Acción Específico de Investigación en Salud 2007-2012, pero no figuran en las estrategias y objetivos; en su lugar, se enfatizan primordialmente aspectos de infraestructura y administrativos. En el periodo se publicaron 145 artículos sobre DSS, cuyas temáticas más abordadas fueron "condiciones de salud", "sistemas de salud" y "nutrición y obesidad". Conclusiones. A pesar de que existe investigación en México sobre DSS, la instrumentación de esos hallazgos en políticas de salud no se ha implementado. El Programa Sectorial de Salud 2013-2018 representa una ventana de oportunidad para posicionar resultados de investigación que promuevan políticas de equidad en salud.
Objective. To examine the research on social determinants of health (SDH) produced in Mexico during the period 2005-2012, based on the characterization of the national health research system and the scientific production on this topic. Materials and methods. Two-stage analyses: Review of Mexican documents and official sources on health research and systematic bibliographic review of the literature on SDH. Results. Although SDH were mentioned in the Specific Action Plan for Health Research 2007-2012, they are not implemented in strategies and goals, as the emphasis is put mostly in infrastructure and administrative aspects of research. In the period studied, 145 articles were published on SDH topics such as health conditions, health systems and nutrition and obesity. Conclusions. In spite of the availability of research on SDH in Mexico, the operationalization of such findings into health policies has not been possible. The current Sectorial Program on Health 2013-2018 represents a window of opportunity to position research findings that promote health equity policies.
Assuntos
Animais , Proteínas de Drosophila , Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Hormônios de Inseto/genética , Junção Neuromuscular/fisiologia , Sinapses/fisiologia , Sinapses/ultraestrutura , Proteínas Supressoras de Tumor , Axônios , Drosophila/genética , Potenciais Evocados , Genes de Insetos , Hormônios de Inseto/biossíntese , Microscopia Eletrônica , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Músculos/inervação , Mutagênese , Junção Neuromuscular/ultraestrutura , Transmissão SinápticaRESUMO
Mechanisms coordinating cell-cell interaction have appeared early in evolution. Allatotropin (AT), a neuropeptide isolated based on its ability to stimulate the synthesis of juvenile hormones (JHs) in insects has also been found in other invertebrate phyla. Despite this function, AT has proved to be myotropic. In the present study we analyze its expression in two groups of Turbellaria (Catenulida, Macrostomida), and its probable relationship with muscle tissue. The results show the presence of an AT-like peptide in the free living turbellaria analyzed. The analysis of the expression of the peptide together with phalloidin, suggests a functional relationship between the peptide and muscle tissue, showing that it could be acting as a myoregulator. The finding of immunoreactive fibers associated with sensory organs like ciliated pits in Catenulida and eyes in Macrostomida makes probable that AT could play a role in the physiological mechanisms controlling circadian activities. Furthermore, the existence of AT in several phyla of Protostomata suggests that this peptide could be a synapomorphic feature of this group. Indeed, the presence in organisms that do not undergo metamorphosis, could be signaling that it was first involved in myotropic activities, being the stimulation of the synthesis of JHs a secondary function acquired by the phylum Arthropoda.
Assuntos
Hormônios de Inseto/metabolismo , Músculos/metabolismo , Neuropeptídeos/metabolismo , Faloidina/metabolismo , Turbelários/metabolismo , Animais , Regulação da Expressão Gênica , Hormônios de Inseto/genética , Hormônios Juvenis/metabolismo , Músculos/fisiologia , Neuropeptídeos/genética , Faloidina/genética , Turbelários/citologia , Turbelários/genéticaRESUMO
We show a straightforward workflow combining homology search in Rhodnius prolixus genome sequence with cloning by rapid amplification of cDNA ends and mass spectrometry. We have identified 32 genes and their transcripts that encode a number of neuropeptide precursors leading to 194 putative peptides. We validated by mass spectrometry 82 of those predicted neuropeptides in the brain of R. prolixus to achieve the first comprehensive genomic, transcriptomic and neuropeptidomic analysis of an insect disease vector. Comparisons of available insect neuropeptide sequences revealed that the R. prolixus genome contains most of the conserved neuropeptides in insects, many of them displaying specific features at the sequence level. Some gene families reported here are identified for the first time in the order Hemiptera, a highly biodiverse group of insects that includes many human, animal and plant disease agents.
Assuntos
Hormônios de Inseto/genética , Neuropeptídeos/genética , Precursores de Proteínas/genética , Rhodnius/genética , Sequência de Aminoácidos , Animais , Química Encefálica , Doença de Chagas/transmissão , Feminino , Genoma de Inseto , Hormônios de Inseto/análise , Proteínas de Insetos/genética , Insetos Vetores/genética , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Família Multigênica , Neuropeptídeos/análise , Neuropeptídeos/classificação , Precursores de Proteínas/análise , Rhodnius/químicaRESUMO
Attacin, a bactericidal small protein is produced by the giant silk moth Hyalophora cecropia. This paper deals with our efforts to clone the attacin cDNA in a bacterial vector to express it in Escherichia coli and produce the protein in sufficient amount, for further studies. We chose two inducible expression vector/bacterial cell systems: pPL-lambda/N99cI+ cells which is able to be induced by nalidixic acid, and pET3d/BL21(DE3) cells carrying a T7 RNA polymerase gene which is IPTG-inducible. After cloning in the pPL-lambda system and under no addition of the inducer, isolated transformants carried this plasmid with at least 2 concurrent deletions that drastically affected attacin expression, even though attacin gene seems to be intact as deduced by its PCR amplification. It was concluded that basal attacin expression occurred in this system and bacterial growth was limited. Plasmid deletions may have emerged by selection pressure as a way to avoid bactericidal expression and allow bacteria survival. The second cloning attempt was done in pET3d vector/BL21 cells, that should not express the cloned sequence (they lack T7 RNA polymerase gene). Transformed BL21 cells gave 3 recombinant plasmids, 2 of them presented a C deletion that generated an early stop signal in the attacin coding region. The third clone, pET-ATT18, carrying an intact gene, was transferred to BL21(DE3)-IPTG inducible cells in order to be expressed. Attacin was undetectable in stained gels or by Western blot analysis. However, expression was visualized in grown cells after 30 min of IPTG induction and 5 min of [35S]-methionine labeling, as a 22.5 kDa protein band by using gel electrophoresis and fluorography. This low level of expression drastically affected bacterial growth. Considering that attacin has no lytic activity, these results suggest that this molecule should block bacterial growth directly at the cytoplasm by an unknown mechanism, since no signal peptide coding sequence was incorporated in this gene construction, precluding periplasmic or external destination of this protein.