RESUMO
The cnidarian Hydra possesses remarkable regenerative capabilities which allow it to regrow lost or damaged body parts in a matter of days. Given that many key regulators of regeneration and development are evolutionarily conserved, Hydra is a valuable model system for studying the fundamental molecular mechanisms underlying these processes. In the past, kinase inhibitors have been useful tools for determining the role of conserved signaling pathways in Hydra regeneration and patterning. Here, we present a systematic screen of a commercially available panel of kinase inhibitors for their effects on Hydra regeneration. Isolated Hydra gastric segments were exposed to 5 µM of each kinase inhibitor and regeneration of the head and foot regions were scored over a period of 96 hours. Of the 80 kinase inhibitors tested, 28 compounds resulted in abnormal regeneration. We directed our focus to the checkpoint kinase 1 (Chk1) inhibitor, SB 218078, considering the role of Chk1 in G2 checkpoint regulation and the importance of G2-paused cells in Hydra regeneration. We found that Hydra exposed to SB 218078 were unable to regenerate the head and maintain head-specific structures. Furthermore, SB 218078-treated Hydra displayed a reduction in the relative proportion of epithelial cells; however, no differences were seen for interstitial stem cells or their derivatives. Lastly, exposure to SB 218078 appeared to have no impact on the level of mitosis or apoptosis. Overall, our study demonstrates the feasibility of kinase inhibitor screens for studying Hydra regeneration processes and highlights the possible role for Hydra Chk1 in head regeneration and maintenance.
Assuntos
Hydra , Animais , Quinase 1 do Ponto de Checagem , Células Epiteliais , Hydra/fisiologia , Transdução de Sinais , Células-TroncoRESUMO
BACKGROUND: The multiple endocrine neoplasia type I gene functions as a tumor suppressor gene in humans and mouse models. In Drosophila melanogaster, mutants of the menin gene (Mnn1) are hypersensitive to mutagens or gamma irradiation and have profound defects in the response to several stresses including heat shock, hypoxia, hyperosmolarity and oxidative stress. However, it is not known if the function of menin in the stress response contributes to genome stability. The objective of this study was to examine the role of menin in the control of the stress response and genome stability. METHODOLOGY/PRINCIPAL FINDINGS: Using a test of loss-of-heterozygosity, we show that Drosophila strains lacking a functional Mnn1 gene or expressing a Mnn1 dsRNA display increased genome instability in response to non-lethal heat shock or hypoxia treatments. This is also true for strains lacking all Hsp70 genes, implying that a precise control of the stress response is required for genome stability. While menin is required for Hsp70 expression, the results of epistatic studies indicate that the increase in genome instability observed in Mnn1 lack-of-function mutants cannot be accounted for by mis-expression of Hsp70. Therefore, menin may promote genome stability by controlling the expression of other stress-responsive genes. In agreement with this notion, gene profiling reveals that Mnn1 is required for sustained expression of all heat shock protein genes but is dispensable for early induction of the heat shock response. CONCLUSIONS/SIGNIFICANCE: Mutants of the Mnn1 gene are hypersensitive to several stresses and display increased genome instability when subjected to conditions, such as heat shock, generally regarded as non-genotoxic. In this report, we describe a role for menin as a global regulator of heat shock gene expression and critical factor in the maintenance of genome integrity. Therefore, menin links the stress response to the control of genome stability in Drosophila melanogaster.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Instabilidade Genômica , Resposta ao Choque Térmico/genética , Animais , Análise por Conglomerados , Drosophila melanogaster/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Genoma de Inseto/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Hipóxia , Perda de Heterozigosidade , Camundongos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNARESUMO
In Drosophila, the as yet uncloned heterochromatic locus flamenco (flam) controls mobilization of the endogenous retrovirus gypsy through the repeat-associated small interfering (rasi) RNA silencing pathway. Restrictive alleles (flamR) downregulate accumulation of gypsy transcripts in the somatic follicular epithelium of the ovary. In contrast, permissive alleles (flamP) are unable to repress gypsy. DIP1, the closest transcription unit to a flam-insertional mutation, was considered as a good candidate to be a gypsy regulator, since it encodes a dsRNA-binding protein. To further characterize the locus we analyzed P-induced flam mutants and generated new mutations by transposon mobilization. We show that flam is required somatically for morphogenesis of the follicular epithelium, the tissue where gypsy is repressed. This developmental activity is necessary to control gypsy and another retroelement, ZAM. We also show that flam is not DIP1, as none of the new permissive mutants affect the DIP1 coding sequence. In addition, two deletions removing DIP1 coding sequences do not affect any of the flamenco functions. Our results suggest that flamenco extends proximally to DIP1, spanning >130 kb of transposon-rich heterochromatin. We propose a model explaining the multiple functions of this large heterochromatic locus.
Assuntos
Caderinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/virologia , Genes de Insetos , Oogênese/genética , Retroviridae/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Teste de Complementação Genética , Heterocromatina/genética , Mutagênese Insercional , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genéticaRESUMO
A produção de proteínas microbianas sobre a casca de maracujá visando à produção de suplemento protéico para animais é uma alternativa encontrada para o grande desperdício desse resíduo agroindustrial. Através das isotermas de dessorção da casca do maracujá, foi verificado que as condições de umidade inicial para o crescimento de microrganismos neste resíduo por meio da fermentação semi-sólida deverá ser acima de 55 por cento (base úmida) e a faixa de umidade ideal para o armazenamento do resíduo em torno de 5,3 por cento (base úmida).
The microbial protein production on passion fruit peel shell as an animal protein supplement production is an alternative found out to avoid the large wastage of agri-industrial residues. Through the desorption isotherms of passion fruit peel, it is verified that for the microorganism growth by solid-state fermentation on this residue, the initial moisture content condition should be above 55 percent (wet basis). The ideal moisture content range for residue storage is around 5.3 percent (wet basis).
RESUMO
Menin, the product of the multiple endocrine neoplasia type I gene, has been implicated in several biological processes, including the control of gene expression and apoptosis, the modulation of mitogen-activated protein kinase pathways, and DNA damage sensing or repair. In this study, we have investigated the function of menin in the model organism Drosophila melanogaster. We show that Drosophila lines overexpressing menin or an RNA interference for this gene develop normally but are impaired in their response to several stresses, including heat shock, hypoxia, hyperosmolarity and oxidative stress. In the embryo subjected to heat shock, this impairment was characterized by a high degree of developmental arrest and lethality. The overexpression of menin enhanced the expression of HSP70 in embryos and interfered with its down-regulation during recovery at the normal temperature. In contrast, the inhibition of menin with RNA interference reduced the induction of HSP70 and blocked the activation of HSP23 upon heat shock, Menin was recruited to the Hsp70 promoter upon heat shock and menin overexpression stimulated the activity of this promoter in embryos. A 70-kDa inducible form of menin was expressed in response to heat shock, indicating that menin is also regulated in conditions of stress. The induction of HSP70 and HSP23 was markedly reduced or absent in mutant embryos harboring a deletion of the menin gene. These embryos, which did not express the heat shock-inducible form of menin, were also hypersensitive to various conditions of stress. These results suggest a novel role for menin in the control of the stress response and in processes associated with the maintenance of protein integrity.