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1.
BMC Genet ; 21(Suppl 2): 127, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33339510

RESUMO

BACKGROUND: Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. RESULTS: Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. CONCLUSIONS: We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.


Assuntos
Sistemas CRISPR-Cas , Mutação Puntual , Mutações Sintéticas Letais , Tephritidae/genética , Alelos , Sequência de Aminoácidos , Animais , Austrália , Aptidão Genética , Genótipo , Controle de Insetos , Fenótipo , Alinhamento de Sequência , Temperatura
2.
Dev Biol ; 399(2): 337-47, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25601451

RESUMO

Organizer activity, once thought to be restricted to vertebrates, has ancient origins. However, among non-bilaterians, it has only been subjected to detailed investigation during embryonic development of the sea anemone, Nematostella vectensis. As a step toward establishing the extent to which findings in Nematostella can be generalized across the large and diverse phylum Cnidaria, we examined the expression of some key organizer and gastrulation genes during the embryonic development of the coral Acropora millepora. Although anemones and corals both belong to the cnidarian class Anthozoa, the two lineages diverged during the Cambrian and the morphological development of Acropora differs in several important respects from that of Nematostella. While the expression patterns of the key genes brachyury, bmp2/4, chordin, goosecoid and forkhead are broadly similar, developmental differences between the two species enable novel observations, and new interpretations of their significance. Specifically, brachyury expression during the flattened prawnchip stage before gastrulation, a developmental peculiarity of Acropora, leads us to suggest that it is the key gene demarcating ectoderm from endoderm in Acropora, and by implication in other cnidarians, whereas previous studies in Nematostella proposed that forkhead plays this role. Other novel observations include the transient expression of Acropora forkhead in scattered ectodermal cells shortly after gastrulation, and in the developing mesenterial filaments, with no corresponding expression reported in Nematostella. In addition, the expression patterns of goosecoid and bmp2/4 confirm the fundamental bilaterality of the Anthozoa.


Assuntos
Antozoários/embriologia , Evolução Biológica , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Organizadores Embrionários/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Antozoários/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Ectoderma/embriologia , Ectoderma/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Goosecoid/metabolismo , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Especificidade da Espécie
3.
Cell Mol Life Sci ; 70(8): 1469-81, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23224429

RESUMO

Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Drosophila/genética , Inativação Gênica , RNA Polimerase Dependente de RNA/genética , Transgenes , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Fatores de Troca do Nucleotídeo Guanina/genética , MicroRNAs/genética , RNA Interferente Pequeno/genética , RNA Polimerase Dependente de RNA/metabolismo
4.
Dev Biol ; 372(1): 17-27, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23000359

RESUMO

The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants we identified the phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, we examined PtdInsP-binding of Pbl and the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pbl(ΔPH) mutants. Taken together, our results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mesoderma/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Movimento Celular , Drosophila/genética , Drosophila/metabolismo , Guanosina Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais
5.
Nucleic Acids Res ; 39(6): 2393-403, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21075793

RESUMO

The 3' untranslated regions (3'UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3'UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3'UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3'UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3'UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3'UTRs, as well as caution in the use of 3'UTR sequence probes to analyze gene expression.


Assuntos
Regiões 3' não Traduzidas , RNA não Traduzido/metabolismo , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário/genética , Éxons , Perfilação da Expressão Gênica , Humanos , Camundongos , Processamento Pós-Transcricional do RNA
6.
Dev Genes Evol ; 222(6): 361-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22945369

RESUMO

The Rho GTP exchange factor, Pebble (Pbl), long recognised as an essential activator of Rho during cytokinesis, also regulates mesoderm migration at gastrulation. Like other cell cycle components, pbl expression patterns broadly correlate with proliferative tissue. Surprisingly, in spite of its role in the early mesoderm, pbl is downregulated in the presumptive mesoderm before ventral furrow formation. Here, we show that this mesoderm-specific repression of pbl is dependent on the transcriptional repressor Snail (Sna). pbl repression was lost in sna mutants but was unaffected when Sna was ectopically expressed, showing that Sna is necessary, but not sufficient, for pbl repression. Using DamID, the first intron of pbl was identified as a Sna-binding region. Nine sites with the Sna-binding consensus motif CAGGT[GA] were identified in this intron. Mutating these to TAGGC[GA] abolished the ventral repression of pbl. Surprisingly, Sna-dependent repression of pbl was not essential for viability or fertility. Loss of repression did, however, increase the frequency of low-penetrance gastrulation defects. Consistent with this, expression of a pbl-GFP transgene in the presumptive mesoderm generated similar gastrulation defects. Finally, we show that a cluster of Snail-binding sites in the middle of the first intron of pbl orthologues is a conserved feature in the other 11 sequenced Drosophila species. We conclude that pbl levels are precisely regulated to ensure that there is enough protein available for its role in early mesoderm development but not so much as to inhibit the orderly progression of gastrulation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila/genética , Embrião não Mamífero/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Culicidae/embriologia , Culicidae/genética , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Gastrulação , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética
7.
J Cell Sci ; 123(Pt 13): 2179-89, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20516152

RESUMO

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.


Assuntos
Núcleo Celular/metabolismo , Citocinese/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Wnt/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Padronização Corporal/fisiologia , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Epistasia Genética , Proteínas Ativadoras de GTPase/genética , Genes Reporter , Humanos , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Asas de Animais/anatomia & histologia , Asas de Animais/fisiologia , Proteínas Wnt/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
8.
J Biol Chem ; 285(37): 28667-73, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20628062

RESUMO

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.


Assuntos
Citocinese/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Ativadoras de GTPase/genética , Cinesinas/genética , Cinesinas/metabolismo , Complexos Multiproteicos/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Fuso Acromático/genética
9.
Curr Biol ; 18(1): 25-9, 2008 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-18158242

RESUMO

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis [1-5] postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA [6], which subsequently recruits contractile-ring components such as actin, myosin, and Anillin [1-3]. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. Here, we report the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage [1-5], interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring [7-10]. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex.


Assuntos
Divisão Celular/fisiologia , Proteínas Contráteis/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/citologia , Proteínas Ativadoras de GTPase/fisiologia , Microtúbulos/metabolismo , Actomiosina/metabolismo , Animais , Proteínas Contráteis/análise , Proteínas Contráteis/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/análise , Proteínas de Drosophila/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Ativadoras de GTPase/análise , Proteínas Ativadoras de GTPase/metabolismo , Mapeamento de Interação de Proteínas , Fuso Acromático/metabolismo
10.
Trends Genet ; 23(5): 238-42, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17395332

RESUMO

The generation and analysis of mutants is central to studies of gene function in model organisms. Methods for random mutagenesis in Drosophila melanogaster have been available for many years, but an alternative approach--targeted mutagenesis using homologous recombination--has only recently been developed. This approach has the advantage of specificity, because genes of interest can be altered. One might expect with a gene-targeting approach that the frequency of background mutations would be minimal. Unfortunately, we have found that this is not the case. Although the possibility of background mutations arising during homologous-recombination-based gene targeting has been raised in the literature, it is not routinely taken into account when using this technique. Our experience suggests that it can be a considerable problem but that it has a relatively simple solution.


Assuntos
Drosophila melanogaster/genética , Marcação de Genes , Genes de Insetos , Recombinação Genética , Alelos , Animais , Modelos Genéticos , Mutagênese , Mutação , Proteômica/métodos
11.
IUBMB Life ; 62(4): 290-5, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20175154

RESUMO

Small GTPase pathways of the Ras superfamily are implicated in a wide range of signalling processes in animal cells. Small GTPases control pathways by acting as molecular switches. They are converted from an inactive GDP-bound form to an active GTP-bound form by GTP exchange factors (GEFs). The spatial and temporal regulation of GEFs is a major component of the regulation of small GTPases. Here we review the role of the Drosophila RhoGEF, Pebble (the Drosophila ortholog of mammalian ECT2). We discuss its roles in cytokinesis and cell migration, highlighting the diversity with which Rho family signalling pathways operate in biological systems.


Assuntos
Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais/fisiologia , Animais
12.
Transgenic Res ; 19(6): 1121-8, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20140643

RESUMO

Drosophila melanogaster, along with all insects and the vertebrates, lacks an RdRp gene. We created transgenic strains of Drosophila melanogaster in which the rrf-1 or ego-1 RdRp genes from C. elegans were placed under the control of the yeast GAL4 upstream activation sequence. Activation of the gene was performed by crossing these lines to flies carrying the GAL4 transgene under the control of various Drosophila enhancers. RT-PCR confirmed the successful expression of each RdRp gene. The resulting phenotypes indicated that introduction of the RdRp genes had no effect on D. melanogaster morphological development.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , RNA Polimerase Dependente de RNA/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Caenorhabditis elegans/metabolismo , DNA de Helmintos/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Expressão Gênica , Genes de Helmintos , Masculino , Morfogênese/genética , Morfogênese/fisiologia , Filogenia , Interferência de RNA , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
G3 (Bethesda) ; 10(12): 4459-4471, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33051260

RESUMO

Epigenetic silencing by Polycomb group (PcG) complexes can promote epithelial-mesenchymal transition (EMT) and stemness and is associated with malignancy of solid cancers. Here we report a role for Drosophila PcG repression in a partial EMT event that occurs during wing disc eversion, an early event during metamorphosis. In a screen for genes required for eversion we identified the PcG genes Sexcombs extra (Sce) and Sexcombs midleg (Scm) Depletion of Sce or Scm resulted in internalized wings and thoracic clefts, and loss of Sce inhibited the EMT of the peripodial epithelium and basement membrane breakdown, ex vivo. Targeted DamID (TaDa) using Dam-Pol II showed that Sce knockdown caused a genomic transcriptional response consistent with a shift toward a more stable epithelial fate. Surprisingly only 17 genes were significantly upregulated in Sce-depleted cells, including Abd-B, abd-A, caudal, and nubbin Each of these loci were enriched for Dam-Pc binding. Of the four genes, only Abd-B was robustly upregulated in cells lacking Sce expression. RNAi knockdown of all four genes could partly suppress the Sce RNAi eversion phenotype, though Abd-B had the strongest effect. Our results suggest that in the absence of continued PcG repression peripodial cells express genes such as Abd-B, which promote epithelial state and thereby disrupt eversion. Our results emphasize the important role that PcG suppression can play in maintaining cell states required for morphogenetic events throughout development and suggest that PcG repression of Hox genes may affect epithelial traits that could contribute to metastasis.


Assuntos
Proteínas de Drosophila , Drosophila , Proteínas do Grupo Polycomb , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Transição Epitelial-Mesenquimal/genética , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb/genética
14.
Dev Cell ; 4(1): 29-39, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12530961

RESUMO

The mechanism that positions the cytokinetic contractile ring is unknown, but derives from the spindle midzone. We show that an interaction between the Rho GTP exchange factor, Pebble, and the Rho family GTPase-activating protein, RacGAP50C, connects the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. We propose that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis.


Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Microtúbulos/metabolismo , Animais , Divisão Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Substâncias Macromoleculares , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Fuso Acromático , Técnicas do Sistema de Duplo-Híbrido , Asas de Animais/citologia , Asas de Animais/embriologia , Asas de Animais/metabolismo
15.
Kidney Int ; 76(4): 383-94, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19553913

RESUMO

Diabetic nephropathy is characterized by excessive extracellular matrix accumulation resulting in renal scarring and end-stage renal disease. Previous studies have suggested that transglutaminase type 2, by formation of its protein crosslink product epsilon-(gamma-glutamyl)lysine, alters extracellular matrix homeostasis, causing basement membrane thickening and expansion of the mesangium and interstitium. To determine whether transglutaminase inhibition can slow the progression of chronic experimental diabetic nephropathy over an extended treatment period, the inhibitor NTU281 was given to uninephrectomized streptozotocin-induced diabetic rats for up to 8 months. Effective transglutaminase inhibition significantly reversed the increased serum creatinine and albuminuria in the diabetic rats. These improvements were accompanied by a fivefold decrease in glomerulosclerosis and a sixfold reduction in tubulointerstitial scarring. This was associated with reductions in collagen IV accumulation by 4 months, along with reductions in collagens I and III by 8 months. This inhibition also decreased the number of myofibroblasts, suggesting that tissue transglutaminase may play a role in myofibroblast transformation. Our study suggests that transglutaminase inhibition ameliorates the progression of experimental diabetic nephropathy and can be considered for clinical application.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Transglutaminases/antagonistas & inibidores , Animais , Colágeno/metabolismo , Diabetes Mellitus Experimental , Nefropatias Diabéticas/prevenção & controle , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Fibroblastos/efeitos dos fármacos , Mesângio Glomerular/patologia , Túbulos Renais/patologia , Masculino , Ratos , Ratos Wistar , Estreptozocina , Resultado do Tratamento
16.
BMC Genomics ; 9: 540, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-19014561

RESUMO

BACKGROUND: Anthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development - when skeletogenesis is initiated, and symbionts are first acquired. RESULTS: Of 5081 unique peptide coding genes, 1084 were differentially expressed (P

Assuntos
Antozoários/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Sequência de Aminoácidos , Animais , Antozoários/crescimento & desenvolvimento , Calcificação Fisiológica/genética , Análise por Conglomerados , DNA Complementar/genética , Etiquetas de Sequências Expressas , Metamorfose Biológica/genética , Dados de Sequência Molecular , Simbiose/genética
17.
Trends Genet ; 21(12): 633-9, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16226338

RESUMO

Cnidarians are among the simplest extant animals; however EST analyses reveal that they have a remarkably high level of genetic complexity. In this article, we show that the full diversity of metazoan signaling pathways is represented in this phylum, as are antagonists previously known only in chordates. Many of the cnidarian ESTs match genes previously known only in non-animal kingdoms. At least some of these represent ancient genes lost by all bilaterians examined so far, rather than genes gained by recent lateral gene transfer.


Assuntos
Antozoários/genética , Genética Populacional , Animais , Proteínas de Bactérias/genética , Genes Duplicados , Proteínas de Choque Térmico/genética , Humanos , Filogenia
18.
Bioorg Med Chem Lett ; 18(20): 5559-62, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18812257

RESUMO

Dipeptide-based sulfonium peptidylmethylketones derived from 6-diazo-5-oxo-L-norleucine (DON) have been investigated as potential water-soluble inhibitors of extracellular transglutaminase. The lead compounds were prepared in four steps and exhibited potent activity against tissue transglutaminase.


Assuntos
Diazo-Oxo-Norleucina/química , Proteínas de Ligação ao GTP/antagonistas & inibidores , Transglutaminases/antagonistas & inibidores , Água/química , Domínio Catalítico , Química Farmacêutica/métodos , Desenho de Fármacos , Etanol/química , Humanos , Concentração Inibidora 50 , Cetonas/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Peptídeos/química , Proteína 2 Glutamina gama-Glutamiltransferase , Solubilidade , Espectrofotometria/métodos
19.
Biol Open ; 7(10)2018 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327366

RESUMO

Aneuploidy -- having an unbalanced genome - is poorly tolerated at the cellular and organismal level. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift which makes these cells sensitive to internal and environmental stresses. Using Drosophila as a model, we found that protein folding stress is exacerbated by redox stress that occurs in response to ongoing changes to ploidy (chromosomal instability, CIN). We also found that if de novo nucleotide synthesis is blocked, CIN cells are dependent on a high level of lysosome function to survive. Depletion of adenosine monophosphate (AMP) synthesis enzymes led to DNA damage in CIN cells, which showed elevated activity of the DNA repair enzyme activated poly(ADP ribose) polymerase (PARP). PARP activation causes depletion of its substrate, nicotinamide adenine dinucleotide (NAD+) and subsequent loss of Adenosine Tri-Phosphate (ATP), and we found that adding ATP or nicotinamide (a precursor in the synthesis of NAD+) could rescue the observed phenotypes. These findings provide ways to interpret, target and exploit aneuploidy, which has the potential to offer tumour-specific therapies.

20.
PLoS One ; 13(3): e0194003, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29518139

RESUMO

Netrin receptors of the DCC/NEO/UNC-40/Frazzled family have well established roles in cell migration and axon guidance but can also regulate epithelial features such as adhesion, polarity and adherens junction (AJ) stability. Previously, we have shown that overexpression of Drosophila Frazzled (Fra) in the peripodial epithelium (PE) inhibits wing disc eversion and also generates cellular protrusions typical of motile cells. Here, we tested whether the molecular pathways by which Fra inhibits eversion are distinct from those driving motility. We show that in disc proper (DP) epithelial cells Fra, in addition to inducing F-Actin rich protrusions, can affect localization of AJ components and columnar cell shape. We then show that these phenotypes have different requirements for the three conserved Fra cytoplasmic P-motifs and for downstream genes. The formation of protrusions required the P3 motif of Fra, as well as integrins (mys and mew), the Rac pathway (Rac1, wave and, arpc3) and myosin regulatory light chain (Sqh). In contrast, apico-basal cell shape change, which was accompanied by increased myosin phosphorylation, was critically dependent upon the P1 motif and was promoted by RhoGef2 but inhibited by Rac1. Fra also caused a loss of AJ proteins (DE-Cad and Arm) from basolateral regions of epithelial cells. This phenotype required all 3 P-motifs, and was dependent upon the polarity factor par6. par6 was not required for protrusions or cell shape change, but was required to block eversion suggesting that control of AJ components may underlie the ability of Fra to promote epithelial stability. The results imply that multiple molecular pathways act downstream of Fra in epithelial cells.


Assuntos
Caderinas/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Receptores de Netrina/fisiologia , Junções Aderentes/metabolismo , Motivos de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Ciclo Celular , Movimento Celular , Polaridade Celular , Forma Celular , Extensões da Superfície Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Discos Imaginais/citologia , Integrinas/fisiologia , Larva , Miosinas/metabolismo , Receptores de Netrina/química , Receptores de Netrina/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transgenes , Proteínas rac de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia
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