RESUMO
Delayed cerebral ischemia (DCI) and vasospasm are two complications of subarachnoid hemorrhages (SAHs) which entail high risks of morbidity and mortality. However, it is unknown why only some patients who suffer SAHs will experience DCI and vasospasm. The purpose of this review is to describe the main genetic single nucleotide polymorphisms (SNPs) that have demonstrated a relationship with these complications. The SNP of the nitric oxide endothelial synthase (eNOS) has been related to the size and rupture of an aneurysm, as well as to DCI, vasospasm, and poor neurological outcome. The SNPs responsible for the asymmetric dimetilarginine and the high-mobility group box 1 have also been associated with DCI. An association between vasospasm and the SNPs of the eNOS, the haptoglobin, and the endothelin-1 receptor has been found. The SNPs of the angiotensin-converting enzyme have been related to DCI and poor neurological outcome. Studies on the SNPs of the Ryanodine Receptor yielded varying results regarding their association with vasospasm.
Assuntos
Isquemia Encefálica , Hemorragia Subaracnóidea , Vasoespasmo Intracraniano , Humanos , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/genética , Vasoespasmo Intracraniano/genética , Isquemia Encefálica/complicações , Isquemia Encefálica/genética , Infarto Cerebral/complicações , Polimorfismo de Nucleotídeo Único , Suscetibilidade a DoençasRESUMO
Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.
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Síndrome de Angelman/genética , Ácido Aspártico Proteases/genética , Proteínas de Drosophila/genética , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/fisiopatologia , Animais , Drosophila , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Proteômica , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação/genéticaRESUMO
SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes.
Assuntos
Evolução Biológica , Insetos/metabolismo , Proteína SUMO-1/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ecdisteroides/biossíntese , Evolução Molecular , Humanos , Insetos/classificação , Insetos/genética , Metamorfose Biológica/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenótipo , Filogenia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína SUMO-1/química , Proteína SUMO-1/genética , Alinhamento de Sequência , SumoilaçãoRESUMO
SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Dados de Sequência Molecular , Receptores Depuradores , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: To evaluate and predict factors influencing prognosis and/or clinical outcome at 6 months in patients with spontaneous subarachnoid haemorrhage, World Federation of Neurosurgical Societies (WFNS) grades iv and v. MATERIAL AND METHODS: This was a retrospective study of a consecutive series of 394 patients admitted to our hospital with clinical and radiological diagnosis of spontaneous subarachnoid haemorrhage, from 1 January 1999 to 30 June 2009. We selected 121 patients who met the criteria of being in WFNS grades iv or v before treatment; 3 patients were excluded due to loss of tracking. The outcome variable was assessed 6 months after the event using the Glasgow Outcome Scale. A P value<.05 was considered statistically significant. RESULTS: One hundred and twenty-one patients were included in the statistical analysis. The average age of the patients in the series was 54 years (14-92). Patients who had a mean Glasgow Coma Scale lower than 7 points (P<.0001), those who were grade v (P<.0001) in the pre-treatment WFNS scale and those with pupillary disorder (P=.002) had a worse clinical outcome. Likewise, those with associated intraparenchymal hematoma (P=.020) and those not receiving any treatment (P=.020) were also associated with a poor clinical outcome. These results were statistically significant. CONCLUSIONS: Patients admitted with a WFNS grade v and/or presenting pupil disorder and/or intraparenchymal hematoma were associated with worse clinical outcomes.
Assuntos
Hemorragia Subaracnóidea , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Índice de Gravidade de Doença , Hemorragia Subaracnóidea/classificação , Hemorragia Subaracnóidea/diagnóstico , Hemorragia Subaracnóidea/terapia , Adulto JovemRESUMO
Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteômica , ProteóliseRESUMO
BACKGROUND: Neurodegenerative diseases are progressive and irreversible and they can be initiated by mutations in specific genes. Spalt-like genes (Sall) encode transcription factors expressed in the central nervous system. In humans, SALL mutations are associated with hereditary syndromes characterized by mental retardation, sensorineural deafness and motoneuron problems, among others. Drosophila sall mutants exhibit severe neurodegeneration of the central nervous system at embryonic stage 16, which surprisingly reverts later in development at embryonic stage 17, suggesting a potential to recover from neurodegeneration. We hypothesize that this recovery is mediated by a reorganization of the transcriptome counteracting SALL lost. To identify genes associated to neurodegeneration and neuroprotection, we used mRNA-Seq to compare the transcriptome of Drosophila sall mutant and wild type embryos from neurodegeneration and reversal stages. RESULTS: Neurodegeneration stage is associated with transcriptional changes in 220 genes, of which only 5% were already described as relevant for neurodegeneration. Genes related to the groups of Redox, Lifespan/Aging and Mitochondrial diseases are significantly represented at this stage. By contrast, neurodegeneration reversal stage is associated with significant changes in 480 genes, including 424 not previously associated with neuroprotection. Immune response and Salt stress are the most represented groups at this stage. CONCLUSIONS: We identify new genes associated to neurodegeneration and neuroprotection by using an mRNA-Seq approach. The strong homology between Drosophila and human genes raises the possibility to unveil novel genes involved in neurodegeneration and neuroprotection also in humans.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Doenças Neurodegenerativas/genética , Transcriptoma , Animais , Biologia Computacional , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Análise de Sequência de RNARESUMO
The Spalt-like family of zinc finger transcription factors is conserved throughout evolution and is involved in fundamental processes during development and during embryonic stem cell maintenance. Although human SALL1 is modified by SUMO-1 in vitro, it is not known whether this post-translational modification plays a role in regulating the activity of this family of transcription factors. Here, we show that the Drosophila Spalt transcription factors are modified by sumoylation. This modification influences their nuclear localization and capacity to induce vein formation through the regulation of target genes during wing development. Furthermore, spalt genes interact genetically with the sumoylation machinery to repress vein formation in intervein regions and to attain the wing final size. Our results suggest a new level of regulation of Sall activity in vivo during animal development through post-translational modification by sumoylation. The evolutionary conservation of this family of transcription factors suggests a functional role for sumoylation in vertebrate Sall members.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/metabolismo , Animais , Linhagem Celular , Drosophila , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição/genética , Asas de Animais/crescimento & desenvolvimentoRESUMO
The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Proteína da Leucemia Promielocítica/metabolismo , Ligação Proteica , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismoRESUMO
Development is orchestrated through a complex interplay of multiple transcription factors. The comprehension of this interplay will help us to understand developmental processes. Here we analyze the relationship between two key transcription factors: CBX4, a member of the Polycomb Repressive Complex 1 (PRC1), and SALL1, a member of the Spalt-like family with important roles in embryogenesis and limb development. Both proteins localize to nuclear bodies and are modified by the small ubiquitin-like modifier (SUMO). Our results show that CBX4 and SALL1 interact in the nucleoplasm and that increased SALL1 expression reduces ubiquitination of CBX4, enhancing its stability. This is accompanied by an increase in the number and size of CBX4-containing Polycomb bodies, and by a greater repression of CBX4 target genes. Thus, our findings uncover a new way of SALL1-mediated regulation of Polycomb bodies through modulation of CBX4 stability, with consequences in the regulation of its target genes, which could have an impact in cell differentiation and development.
RESUMO
1. Inhibitors of intestinal glucosidases have been shown to improve glycaemic control in diabetic and obese humans and animals. In the present study, we have investigated the effect of 3 months treatment with acarbose on adiposity, food intake and the modulation of hypothalamic neuropeptide Y (NPY) in obese diabetic Wistar (WDF) rats and the possible correlation between changes in overall insulin sensitivity and the level of circulating adipokines, leptin and adiponectin. In addition, we investigated the effect of acarbose on adipocyte insulin signalling. 2. Mature male WDF rats were randomly distributed to one of three treatment groups (no acarbose or 20 or 40 mg of acarbose/100 g diet). After 3 months, blood glucose, cholesterol, triglyceride, insulin, leptin and adiponectin were analysed. Insulin signalling was determined in isolated adipocytes as the stimulation of mitogen-activated protein kinase (MAPK) and Akt phosphorylation; the level of hypothalamic NPY was assessed by immunohistochemistry. 3. Acarbose-treated rats had lower levels of blood glucose, cholesterol, triglyceride, insulin and leptin and an increase in adiponectin compared with untreated animals. There were no changes in bodyweight and adiposity. Stimulation of adipocyte MAPK activity by insulin was higher in rats treated with both doses of acarbose, whereas higher stimulation of Akt phosphorylation was observed with the highest dose of acarbose. Although food intake was not significantly reduced in rats treated with acarbose, the acarbose-treated rats had lower NPY expression in the arcuate nucleus. 4. We conclude that the improvement in overall insulin sensitivity in WDF rats after prolonged acarbose treatment is paralleled by increases in circulating adiponectin and adipocyte insulin responsiveness. Acarbose neither decreases food intake nor reverts obesity, but decreases leptin levels and the expression of the orexigenic NPY in the hypothalamus.
Assuntos
Acarbose/uso terapêutico , Adipócitos/efeitos dos fármacos , Adiponectina/sangue , Hipotálamo/efeitos dos fármacos , Insulina/metabolismo , Leptina/sangue , Neuropeptídeo Y/metabolismo , Acarbose/administração & dosagem , Adipócitos/metabolismo , Animais , Diabetes Mellitus , Relação Dose-Resposta a Droga , Esquema de Medicação , Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/uso terapêutico , Hipotálamo/metabolismo , Masculino , Neuropeptídeo Y/genética , Obesidade , Ratos , Ratos WistarRESUMO
Insulin resistance develops with ageing in humans and rodents. Here, we have studied the evolution of insulin sensitivity with ageing trying to discriminate the role of adiposity from that of ageing in this process. We performed oral glucose tolerance tests and determined overall and tissue-specific glucose utilization under euglycemic-hyper-insulinemic conditions in 3-, 8-, and 24-month-old rats fed ad libitum, and in 8- and 24-month-old rats after 3 months of calorie restriction. Body composition and adipocyte-derived cytokines such as leptin, resistin, and adiponectin were analyzed. Overall insulin sensitivity decreases with ageing. Calorie restriction improves global insulin sensitivity in 8- but not in 24-month-old rats. Insulin-stimulated glucose utilization in adipose tissues decreases in 8 months, while in oxidative muscles it reaches significance only in older rats. Calorie restriction restores adipose tissue insulin sensitivity only in 8-month-old rats and no changes are observed in muscles of 24-month-old rats. Resistin and leptin increase with ageing. Food restriction lowers resistin and increases adiponectin in 8-month-old rats and decreases leptin in both ages. Visceral and total fat increase with ageing and decrease after calorie restriction. We conclude that accretion of visceral fat plays a key role in the development of insulin resistance after sexual maturity, which is reversible by calorie restriction. With aging, accumulation of retroperitoneal and total body fat leads to impaired muscle glucose uptake and to a state of insulin resistance that is difficult to reverse.
Assuntos
Adiposidade/fisiologia , Envelhecimento/fisiologia , Privação de Alimentos/fisiologia , Resistência à Insulina , Adipócitos/metabolismo , Adiponectina/sangue , Animais , Biomarcadores/sangue , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Insulina/sangue , Leptina/sangue , Fígado/química , Masculino , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/análise , Ratos , Ratos Wistar , Resistina/sangueRESUMO
Key scientific discoveries have resulted from genetic studies of Drosophila melanogaster, using a multitude of transgenic fly strains, the majority of which are constructed in a genetic background containing mutations in the white gene. Here we report that white mutant flies from w1118 strain undergo retinal degeneration. We observed also that w1118 mutants have progressive loss of climbing ability, shortened life span, as well as impaired resistance to various forms of stress. Retinal degeneration was abolished by transgenic expression of mini-white+ in the white null background w1118 . We conclude that beyond the classical eye-color phenotype, mutations in Drosophila white gene could impair several biological functions affecting parameters like mobility, life span and stress tolerance. Consequently, we suggest caution and attentiveness during the interpretation of old experiments employing white mutant flies and when planning new ones, especially within the research field of neurodegeneration and neuroprotection. We also encourage that the use of w1118 strain as a wild-type control should be avoided.
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Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.
Assuntos
Ubiquitinas/metabolismo , Animais , Animais Geneticamente Modificados , Biotinilação , Linhagem Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Sumoilação , Ubiquitinas/genéticaRESUMO
Leptin modulates glucose homeostasis by acting as an insulin-sensitizing factor in most insulin target tissues. Nevertheless, insulin-dependent glucose uptake in white adipose tissue decreases after in vivo treatment with leptin. Moreover, elevated leptin concentrations inhibit insulin metabolic effects in adipocytes. Here we studied both, direct and centrally mediated effects of leptin on insulin signaling in rat adipocytes. Adipocyte incubation with low leptin concentrations did not modify the insulin stimulation of mitogen-activated protein kinase (MAPK). However, at elevated concentrations, leptin impaired insulin-stimulated MAPK activity, glycogen synthase kinase (GSK)3beta phosphorylation, and insulin receptor tyrosine phosphorylation without altering vanadate stimulation. An increase of suppressor of cytokine signaling-3 protein was also observed. Central administration of leptin decreased insulin effects on adipocyte MAPK and GSK3beta phosphorylation. In insulin-resistant aged rats with hyperleptinemia and central leptin resistance, insulin poorly stimulated MAPK and central leptin infusion did not further deteriorate adipocyte insulin responsiveness. Food restriction increased MAPK stimulation by insulin and restored the ability of centrally infused leptin to attenuate adipocyte insulin signaling in aged rats. We conclude that leptin can modulate, in an inhibitory manner, adipocyte insulin signaling by two different ways: as an autocrine signal and, indirectly, through neuroendocrine pathways. These mechanisms may be of relevance in situations of hyperleptinemia, such as aging and/or obesity.
Assuntos
Adipócitos/fisiologia , Insulina/farmacologia , Leptina/farmacologia , Adipócitos/efeitos dos fármacos , Envelhecimento , Animais , Dieta Redutora , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Ratos , Ratos Wistar , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Fatores de Transcrição/genéticaRESUMO
Animals have a determined species-specific body size that results from the combined action of hormones and signaling pathways regulating growth rate and duration. In Drosophila, the steroid hormone ecdysone controls developmental transitions, thereby regulating the duration of the growth period. Here we show that ecdysone promotes the growth of imaginal discs in mid-third instar larvae, since imaginal discs from larvae with reduced or no ecdysone synthesis are smaller than wild type due to smaller and fewer cells. We show that insulin-like peptides are produced and secreted normally in larvae with reduced ecdysone synthesis, and upstream components of insulin/insulin-like signaling are activated in their discs. Instead, ecdysone appears to regulate the growth of imaginal discs via Thor/4E-BP, a negative growth regulator downstream of the insulin/insulin-like growth factor/Tor pathways. Discs from larvae with reduced ecdysone synthesis have elevated levels of Thor, while mutations in Thor partially rescue their growth. The regulation of organ growth by ecdysone is evolutionarily conserved in hemimetabolous insects, as shown by our results obtained using Blattella germanica. In summary, our data provide new insights into the relationship between components of the insulin/insulin-like/Tor and ecdysone pathways in the control of organ growth.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Ecdisona/metabolismo , Discos Imaginais/crescimento & desenvolvimento , Somatomedinas/metabolismo , Animais , Tamanho Corporal/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Insulina/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Transdução de Sinais/fisiologiaRESUMO
In mammals, cholesterol is transformed into steroid hormones in the adrenal gland, the ovaries or the testes. The Scavenger Receptors Class B Type I (SR-BI) are membrane proteins that belong to the CD36 family and participate in the selective uptake of high density lipoprotein cholesteryl ester in the mammalian steroidogenic tissues. Fourteen members of the CD36 family have been identified in Diptera, although their expression patterns remain uncharacterized. Using in situ hybridization we have characterized the expression patterns of the fourteen SR-BIs in Drosophila melanogaster. We analyzed three different developmental larval stages prior to and during the peak of the insect steroid hormone ecdysone, which triggers the larval to pupal transition. We focused on the steroidogenic tissues, such as the prothoracic gland, the ovaries and the testes, and extended our analysis to non-steroidogenic tissues, such as the fat body, salivary glands, the gut, the gastric caeca or the central nervous system. Our results show highly regulated expression patterns, with three genes crq, pes and Snmp being upregulated in steroidogenic tissues at the onset of pupariation when steroidogenesis is crucial. This study underlines the importance of the transport of cholesterol and steroids in the process of ecdysone synthesis.
Assuntos
Antígenos CD36/genética , Ésteres do Colesterol/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Ecdisona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Lipoproteínas HDL/metabolismo , Animais , Transporte Biológico Ativo , Antígenos CD36/biossíntese , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Hibridização In Situ , Larva/crescimento & desenvolvimento , Larva/metabolismo , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Receptores de Feromônios/biossíntese , Receptores de Feromônios/genética , Receptores Depuradores/biossíntese , Receptores Depuradores/genéticaRESUMO
Sumoylation, the covalent attachment of the small ubiquitin-related modifier SUMO to target proteins, regulates different cellular processes, although its role in the control of development remains unclear. We studied the role of sumoylation during Drosophila development by using RNAi to reduce smt3 mRNA levels in specific tissues. smt3 knockdown in the prothoracic gland, which controls key developmental processes through the synthesis and release of ecdysteroids, caused a 4-fold prolongation of larval life and completely blocked the transition from larval to pupal stages. The reduced ecdysteroid titer of smt3 knockdown compared with wild-type larvae explains this phenotype. In fact, after dietary administration of exogenous 20-hydroxyecdysone, knockdown larvae formed pupal cases. The phenotype is not due to massive cell death or degeneration of the prothoracic glands at the time when puparium formation should occur. Knockdown cells show alterations in expression levels and/or the subcellular localisation of enzymes and transcription factors involved in the regulation of ecdysteroid synthesis. In addition, they present reduced intracellular channels and a reduced content of lipid droplets and cholesterol, which could explain the deficit in steroidogenesis. In summary, our study indicates that Smt3 is required for the ecdysteroid synthesis pathway at the time of puparium formation.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Ecdisteroides/biossíntese , Proteínas Repressoras/fisiologia , Animais , Núcleo Celular/metabolismo , Colesterol/biossíntese , Citoplasma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Ecdisterona/farmacologia , Larva/crescimento & desenvolvimento , Larva/fisiologia , Lipídeos/biossíntese , Metamorfose Biológica/fisiologia , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , Proteínas Repressoras/genética , Proteínas Modificadoras Pequenas Relacionadas à UbiquitinaRESUMO
Objetivo: Evaluar y predecir factores que influyan en el pronóstico y/o resultado clínico a los 6 meses de pacientes con hemorragia subaracnoidea espontánea, en grados iv y v de la World Federation of Neurosurgical Societies (WFNS). Material y métodos: Estudio retrospectivo de una serie consecutiva de 394 pacientes que ingresaron en nuestro hospital, con diagnóstico clínico y radiológico de hemorragia subaracnoidea espontánea, desde el 1 de enero de 1999 hasta el 30 de junio de 2009. Se seleccionaron 121 pacientes que reunían el criterio de estar en grado iv o v de la WFNS antes del tratamiento; se excluyeron 3 pacientes por pérdida de seguimiento. La variable resultado se evaluó a los 6 meses del evento mediante la Escala de Resultados de Glasgow. Se consideró un resultado estadísticamente significativo un valor de p < 0,05. Resultados: Ciento veintiún pacientes se incluyeron en el análisis estadístico. La edad media de la serie fue de 54 años (14-92). Los pacientes que presentaban una puntuación en la Escala de Coma de Glasgow media inferior a 7 puntos (p < 0,0001) y un grado de v (p < 0,0001) en la escala de la WFNS pretratamiento, así como los que presentaban trastorno pupilar (p = 0,002), mostraron un peor resultado clínico final; igualmente, los que asociaron hematoma intraparenquimatoso (p = 0,020) y aquellos a los que no se les efectuó ningún tipo de tratamiento (p = 0,020) también asociaron un mal resultado clínico final, siendo estos resultados estadísticamente significativos. Conclusiones: Los pacientes que ingresan con una gradación de v en la escala de la WFNS y/o presentan trastorno pupilar y/o hematoma intraparenquimatoso asocian un peor resultado clínico final
Objective: To evaluate and predict factors influencing prognosis and/or clinical outcome at 6 months in patients with spontaneous subarachnoid haemorrhage, World Federation of Neurosurgical Societies (WFNS) grades iv and v. Material and methods: This was a retrospective study of a consecutive series of 394 patients admitted to our hospital with clinical and radiological diagnosis of spontaneous subarachnoid haemorrhage, from 1 January 1999 to 30 June 2009. We selected 121 patients who met the criteria of being in WFNS grades iv or v before treatment; 3 patients were excluded due to loss of tracking. The outcome variable was assessed 6 months after the event using the Glasgow Outcome Scale. A P value < .05 was considered statistically significant. Results: One hundred and twenty-one patients were included in the statistical analysis. The average age of the patients in the series was 54 years (14-92). Patients who had a mean Glasgow Coma Scale lower than 7 points (P < .0001), those who were grade v (P < .0001) in the pre-treatment WFNS scale and those with pupillary disorder (P = .002) had a worse clinical outcome. Likewise, those with associated intraparenchymal hematoma (P = .020) and those not receiving any treatment (P = .020) were also associated with a poor clinical outcome. These results were statistically significant. Conclusions: Patients admitted with a WFNS grade v and/or presenting pupil disorder and/or intraparenchymal hematoma were associated with worse clinical outcomes