摘要
Proteínas de membrana estão envolvidas em processos fisiológicos essenciais como, por exemplo, a manutenção do equilíbrio iônico e sinalização intracelular. No entanto, apesar do envolvimento em inúmeros processos fisiológicos e de grande interesse farmacêutico, o estudo estrutural de proteínas de membrana ainda é um processo custoso e muito mais complexo do que o estudo estrutural de proteínas solúveis. Os trocadores de Na+/Ca2+ são proteínas de membrana que atuam na manutenção da homeostase de Ca2+ intracelular e estão envolvidos em processos patológicos como doenças cardíacas. Estes trocadores estão presentes em diversas espécies de mamíferos (NCX) e insetos, por exemplo, na mosca Drosophila melanogaster (CALX). A topologia destas proteínas é constituída de dois domínios. O domínio transmembranar, que contém dois segmentos de 5 hélices transmembranares (TMH) e é responsável por promover o transporte específico de íons Ca2+ e Na+ através da membrana, e o domínio citoplasmático, responsável por regular a atividade do trocador. O domínio citoplasmático consiste de uma alça que contém dois domínios sensores de Ca2+ intracelular (CBD1 e CBD2). Trabalhos mostraram que o trocador CALX é inibido pela ligação de Ca em CBD1, enquanto que trocadores NCX são ativados. As regiões citosólicas que conectam CBD1 e CBD2 à TMH5 e TMH6 são conservadas e ainda não foram caracterizadas estruturalmente. Adjacente à TMH5 há um segmento anfipático, denominado exchanger inhibitory peptide (XIP), que está envolvido no mecanismo de regulação do trocador. Na ausência de dados estruturais do CALX completo, o estudo de TMH5-XIP poderá aumentar a compreensão sobre a estrutura e o funcionamento do trocador. A construção TMH5-XIP foi fusionada à MBP no N-terminal e a uma sequência de 8 histidinas no C-terminal. Apesar da expressão da proteína de fusão ter sido bem sucedida, problemas de precipitação e ineficiência durante a clivagem da conexão com a MBP impediram a conclusão dos estudos estruturais. Logo, uma construção menor, contendo apenas a região equivalente ao XIP, foi estudada por espectroscopia de RMN em solução e dicroísmo circular. XIP forma uma 310-hélice a baixa temperatura, 7 oC, que se desestabiliza a maior temperatura, 27 oC. Estes dados permitem a formulação de hipóteses sobre o papel de XIP no mecanismo de regulação do domínio transmembranar de CALX
Membrane proteins are involved in essential physiological processes such as maintenance of the ionic balance and intracellular signaling. However, despite their role in numerous physiological processes of well-recognized pharmaceutical relevance, structural studies of membrane proteins remain being more complex than structural studies of globular proteins. Na+/Ca2+ exchangers (NCX) are membrane proteins that play essential roles in the maintenance of the intracellular Ca2+ homeostasis. Not surprisingly, the NCXs are involved in pathologies such as heart diseases. These exchangers are present in several species of mammals (NCX) and insects, for example, in the fly Drosophila melanogaster (CALX). The topology of these proteins consists of a transmembrane and a hydrophilic domain. The transmembrane domain corresponds to two segments of 5 transmembrane helices (TMH) forming a 10-helix bundle that is responsible for the specific transport of Ca2+ and Na+ across the cellular membrane. The hydrophilic domain is composed of a large cytoplasmic loop, which is associated with the regulation of the ion exchange activity of the transmembrane domain. The loop contains two Ca2+-sensors domains, CBD1 and CBD2, and uncharacterized regions. Studies showed that Ca2+ binding to CBD1 inhibits the CALX, whereas it activates the NCX. The juxtamembrane cytosolic regions linking the CBD1 and CBD2 domains to the TMH5 and TMH6, respectively, are highly conserved but have not yet been structurally characterized. The segment near TMH5 is amphipathic, and it is also called exchanger inhibitory peptide (XIP). In the absence of a three-dimensional structure of the complete CALX, the study of TMH5-XIP may contribute to our understanding of the structure and operation of the exchanger. In order to study TMH5-XIP, it was fused to an MBP tag at the N-terminus, and to a sequence of 8 histidines at the C-terminus. Although the expression of the fusion protein was successful, precipitation and inefficient MBP-tag cleavage prevented the isolation of pure TMH5-XIP for structural studies. Hence, a smaller construct, containing only the region equivalent to XIP, was studied by NMR spectroscopy in solution and circular dichroism. The structure assumed by XIP in solution is temperature dependent, being intrinsically disordered at 27 C or a 310-helix at 7 C, respectively. These findings allowed us to infer how XIP could participate in the CALX regulation mechanism
Subject(s)
Sodium-Calcium Exchanger/analysis , Magnetic Resonance Spectroscopy/methods , Drosophila melanogaster/metabolism摘要
Introdução: a utilização de plantas como recurso terapêutico é uma prática bastante antiga, e desde então tem sido alvo de estudos. Neste contexto existe o jambolão (Syzygiumcumini) que é uma planta pertencente à família Mirtaceae. as folhas possuem substâncias com ação antidiabética, exercendo função hipoglicemiante, mimetizando as ações da insulina, regulando os níveis glicêmicos. Objetivo: avaliara atividade antioxidante das folhas de Syzygiumcumini e os efeitos desta infusão em um modelo experimental de dieta enriquecida com altas concentrações de glicose em Drosophila melanogaster. Métodos: para verificar a atividade antioxidante do jambolão utilizou-se o método DPPH (2,2-difenil-1picrilhidrazila) segundo BrandWillians et al. (1995). As Drosophilas foram separadas por sexo, pesadas e tratadas com infusão de folhas de jambolão com exceção do controle, por três dias e no quarto dia foram sacrificadas e pesadas para análises bioquímicas de GlicosePAP Liquiform triglicerídeos enzimático (Labtest). Resultado e Discussão: em relação aos machos, o efeito do jambolão foi extremamente significativo como fator protetor na dieta com 20% de sacarose e também na dieta com 30% de sacarose. Conclusão: este estudo demonstrou que a exposição a uma dieta rica em carboidratos foi prejudicial à D. melanogaster e que o chá de Syzygiumcumini (jambolão) teve efeito positivo nos parâmetros de glicose e triglicérides, validando este modelo de invertebrado como ferramenta para a investigação da Diabetes Melitus.
Introduction: the use of plants as a therapeutic resource is a very ancient practice, and has since been the subject of studies. In this context there is jambolan (Syzygiumcumini) whichis a plant belonging to the family Mirtaceae. The leave shave substance swith anti diabetic action, acting hypoglycemic function, mimicking the actions of insulin, regulating blood glucose levels. Objective: the aim of this study was to evaluate the antioxidant activityof the leaves of Syzygium cumini and the effects of this infusion on a diet enriched experimental model with high glucose concentrations in Drosophila melanogaster. Methods: to verify jambolan the antioxidant used the DPPH (2,2-diphenyl-1picrilhidrazila) second Brand-Williams et al. (1995). The Drosophila were separated by sex, weighed and treated with infusion jambolan leaves except for the control, for three days and on the fourth day were sacrificed and weighed to biochemistry Glucose PAP Liquiform enzymatic triglycerides (Labtest). Results and discussion: regarding males, jambolan effect was highly significant as a protective factor in the diet with 20% sucroseandalso in the diet with 30% sucrose. Conclusion: this study demonstrated that exposure to a high-carbohydrate diet was harmful to D. melanogaster and the Syzygiumtea cumini (jambolan) had a positive effecton glucose and triglycerides parameters, validating this invertebrate model as a tool for the investigation of Diabetes mellitus.
Subject(s)
Animals , Male , Female , Syzygium/analysis , Syzygium/adverse effects , Diabetes Mellitus/metabolism , Drosophila melanogaster/drug effects , Drosophila melanogaster/metabolism , Hypoglycemic Agents/therapeutic use , Analysis of Variance , Models, Animal , Invertebrates , Antioxidants/chemistry摘要
The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.
Subject(s)
Animals , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Fucosyltransferases/metabolism , Guanosine Diphosphate Fucose/metabolism , Guanosine Diphosphate Mannose/metabolism , Receptors, Notch/metabolism , Wings, Animal/metabolism , Alleles , Drosophila Proteins/genetics , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/metabolism , Endocytosis/genetics , Fucosyltransferases/genetics , Guanosine Diphosphate Fucose/genetics , Guanosine Diphosphate Mannose/genetics , Immunohistochemistry , In Situ Hybridization , Intracellular Signaling Peptides and Proteins/genetics , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation/genetics , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Receptors, Notch/genetics , Signal Transduction , Wings, Animal/anatomy & histology摘要
In Drosophila melanogaster, dosage compensation occurs through hypertranscription of sex-linked genes in males. The hypertranscription involves acetylation of histone 4 at lysine 16 (H4K16) on amale X-chromosome, brought about by a histone acetyltransferase encoded by the dosage compensation gene, males absent on the first (mof). We report a phenomenon in the strain In(1)B(M2)(reinverted) of D. melanogaster where the global structure of the male X-chromosome can be altered at the third instar larval stage through a 4-h cold shock at 12+/-1 degrees C. We show that the cold shock results in a transient hyperacetylation of H4K16 and an increased expression of MOF. Control proteins H4 acetylated at lysine 5, and the dosage compensation gene msl-2, do not show any change in expression after cold shock. Cytology of the male X-chromosome at different time points during cold shock and recovery, suggests that the hyperacetylation of H4 at lysine 16 causes the X-chromosome to corkscrew into itself, thereby achieving the cold-induced change in the higher order structure of the male polytene X-chromosome. Our studies suggest a role for H4K16 in maintaining the structure of the male X-chromosome in Drosophila.
Subject(s)
Acetylation , Animals , Cell Nucleus/metabolism , Cold Temperature , Drosophila melanogaster/metabolism , Female , Histones/metabolism , Immunoblotting , Larva/metabolism , Lysine/metabolism , Male , X Chromosome/metabolism摘要
Juxtacrine signaling is intercellular communication, in which the receptor of the signal (typically a protein) as well as the ligand (also typically a protein, responsible for the activation of the receptor) are anchored in the plasma membranes, so that in this type of signaling the activation of the receptor depends on direct contact between the membranes of the cells involved. Juxtacrine signaling is present in many important cellular events of several organisms, especially in the development process. We propose a generic formal model (a modeling framework) for juxtacrine signaling systems that is a class of discrete dynamic systems. It possesses desirable characteristics in a good modeling framework, such as: a) structural similarity with biological models, b) capacity of operating in different scales of time, and c) capacity of explicitly treating both the events and molecular elements that occur in the membrane, and those that occur in the intracellular environment and that are involved in the juxtacrine signaling process. We have implemented this framework and used it to develop a new three-level discrete model for the neurogenic network and its participation in neuroblast segregation. This paper presents the details of this framework and its current status.
Subject(s)
Animals , Models, Biological , Drosophila Proteins/metabolism , RNA, Messenger/genetics , Software , Signal Transduction , Stem Cells/cytology , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Neurons/cytology , Organogenesis , RNA, Messenger/metabolism , Receptors, Notch/metabolism摘要
In a number of animal species it has been shown that exposure to low levels of stress at a young age has a positive effect on stress resistance later in life, and on longevity. The positive effects have been attributed to the activation of defence/cleaning systems (heat shock proteins (Hsps), antioxidases, DNA repair) or to effects of a changed metabolic rate, or both. We investigated the effect of mild stress exposures early in life on Hsp70 synthesis after a harder stress exposure later in life in five isofemale lines of Drosophila melanogaster. Female flies were either exposed to repeated bouts of mild heat stress (3 h at 34 degrees C) at a young age (days 2, 4 and 6 post-eclosion) or held under standard laboratory conditions. At 16 and 32 days of adult age, respectively, flies were exposed to a high-temperature treatment known to induce Hsp70 in the investigated species (1 h at 37 degrees C). Thereafter, the inducible Hsp70 levels were measured. Our data show a tendency towards increased Hsp70 synthesis with increased age for both 'mild stress' and 'no stress' flies. Moreover, the results show that flies exposed to mild stress at a young age synthesized more Hsp70 upon induction, compared to control flies, and that this difference was accentuated at 32 days compared to 16 days of age. Thus, bouts of mild heat stress at a young age impact on the physiological stress response system later in life. This may be caused by an increased ability to react to future stresses. Alternatively, the mild stress exposure at a young age may actually have caused cellular damages increasing the need for Hsp70 levels after stress exposure later in life. The importance of an Hsp70 upregulation (throughout life) in explaining the phenomenon of hormesis is discussed, together with alternative hypotheses, and suggestions for further studies.
Subject(s)
Animals , Drosophila melanogaster/metabolism , Female , HSP70 Heat-Shock Proteins/biosynthesis , Heat Stress Disorders/metabolism摘要
Experiments were conducted by using nicotine (plant extract) for its toxic effects on Drosophila melanogaster, LC50 estimated is 2.9552 microl/100 ml. Studies revealed that nicotine affects adult emergence of males and females (sex-ratio) of mutant form (Yellow) of Drosophila melanogaster.
Subject(s)
Animals , Drosophila melanogaster/metabolism , Mutation/genetics , Nicotine/toxicity , Reproduction/drug effects , Sex Ratio摘要
The UAS/GAL4 ectopic expression system is widely used in Drosophila melanogaster for the overexpression of transgenes. This system operates under the assumption that the yeast transcription factor, GAL4, is inactive in D. melanogaster. Thus, GAL4 can be expressed under the control of D. melanogaster -specific promoters with little effect upon the organism. We have shown that expression of GAL4 in the developing eye under the control of the glass multiple reporter (GMR) promoter element does have an effect on eye development. Although GMR-GAL4 heterozygotes appear normal when raised at 25 degrees C, the homozygotes have a highly disorganized ommatidial array. In addition, the levels of apoptosis in the third-instar larval eye imaginal disc (where GAL4 is expressed) are slightly higher in GMR-GAL4 heterozygotes, and much higher in GMR-GAL4 homozygotes when compared to wild type discs. The morphological eye defects caused by GMR-GAL4 are significantly enhanced when flies are raised at 29 degrees C (presumably due to the higher activity of GAL4 at this temperature); however, the levels of apoptosis appear to be similar at these two temperatures. Taken together, these data suggest that GAL4 can have adverse effects on D. melanogaster development, especially at high expression levels. In addition, GAL4 appears to induce apoptosis even in the absence of any visible morphological defects. Thus, despite the benefits of the UAS/GAL4 ectopic expression system, one must use caution in the design and interpretation of experiments