ABSTRACT
Introduction@#Folkloric claims have surrounded essential oils, including their enhancement of learning and memory through inhalational exposure. Few studies in humans have shown a benefit in cognition, albeit incremental. However, this benefit may not be entirely attributable to the essential oil aroma but may be confounded by psychological associations. We investigated rosemary, peppermint, lemon, and coffee aromas in a learning and memory model of Drosophila melanogaster to eliminate this confounder.@*Methods@#We screened for concentrations of the four treatments that are non-stimulatory for altered locomotory behavior in the flies. At these concentrations, we determined if they were chemoneutral (i.e., neither chemoattractant nor chemorepellent) to the flies. Learning and memory of the flies exposed to these aromas were determined using an Aversive Phototaxis Suppression (APS) assay.@*Results@#The aromas of rosemary, peppermint, and lemon that did not elicit altered mobility in the flies were from dilute essential oil solutions that ranged from 0.2 to 0.5% v/v; whereas for the aroma in coffee, it was at a higher concentration of 7.5% m/v. At these concentrations, the aromas used were found to be chemoneutral towards the flies. We observed no improvement in both learning and memory in the four aromas tested. While a significant reduction (p < 0.05) in learning was observed when flies were treated with the aromas of rosemary, peppermint, and coffee, a significant reduction (p < 0.05) in memory was only observed in the peppermint aroma treatment.@*Conclusion@#This study demonstrated that in the absence of psychological association, the four aromas do not enhance learning and memory
Subject(s)
Drosophila melanogaster , Learning , Memory , Rosmarinus , Mentha piperita , Citrus , CoffeaABSTRACT
Background@#Consumption and abuse of alcohol remains a significant cause of concern worldwide. Furthermore, there is evidence of the association between chronic alcohol use and reduced life expectancy. @*Objectives@#To study the effects of Garcinia binucao extract (GBE) supplementation on lifespan of Drosophila melanogaster, in the presence or absence of chronic alcohol exposure.@*Methods@#D. melanogaster was mass cultured and given GBE supplementation in high (1 mg/mL) and low (200 µg/mL) sublethal doses. D. melanogaster flies were divided into groups - with and without chronic alcohol exposure, and their respective lifespans were monitored. @*Results@#In D. melanogaster without alcohol exposure, mean lifespan was highest in the control flies (38.15 days), followed by high-dose GBE (34.42 days), low-dose GBE (33.24 days), and DMSO (22.29 days). In D. melanogaster chronically exposed to alcohol, the longest mean lifespan was observed in flies treated with high-dose GBE (33.80 days), followed by low-dose GBE (33.63 days), the DMSO group (30.30), and the control group (29.65 days), but the differences were not statistically significant. Comparing groups with and without chronic alcohol exposure, the mean lifespan of the control group chronically exposed to alcohol significantly decreased by 9.51 days (p < 0.05). In GBE treatment groups, mean lifespan significantly decreased by 0.82 days in high-dose set-up (p < 0.05), and significantly increased by 0.39 days in the low-dose set-up (p < 0.05) upon chronic alcohol exposure. @*Conclusion@#Garcinia binucao extract supplementation ameliorated the observed reduction in lifespan of Drosophila melanogaster chronically exposed to alcohol.
Subject(s)
Drosophila melanogaster , LongevityABSTRACT
Females increase aggression for mating opportunities and for acquiring reproductive resources. Although the close relationship between female aggression and mating status is widely appreciated, whether and how female aggression is regulated by mating-related cues remains poorly understood. Here we report an interesting observation that Drosophila virgin females initiate high-frequency attacks toward mated females. We identify 11-cis-vaccenyl acetate (cVA), a male-derived pheromone transferred to females during mating, which promotes virgin female aggression. We subsequently reveal a cVA-responsive neural circuit consisting of four orders of neurons, including Or67d, DA1, aSP-g, and pC1 neurons, that mediate cVA-induced virgin female aggression. We also determine that aSP-g neurons release acetylcholine (ACh) to excite pC1 neurons via the nicotinic ACh receptor nAChRα7. Together, beyond revealing cVA as a mating-related inducer of virgin female aggression, our results identify a neural circuit linking the chemosensory perception of mating-related cues to aggressive behavior in Drosophila females.
Subject(s)
Animals , Male , Female , Drosophila/physiology , Drosophila Proteins/physiology , Cues , Sexual Behavior, Animal/physiology , Aggression/physiology , Drosophila melanogaster/physiologyABSTRACT
Introdução: embora o câncer seja um dos maiores problemas de saúde pública enfrentados mundialmente, diversas substâncias presentes no meio, como os fármacos, não estão muito bem elucidadas sobre seu possível potencial carcinogênico. Entre eles, estão os benzodiazepínicos, fármacos que possuem crescente aumento do consumo desde o século XX e, principalmente, na segunda década do século XXI, por suas ações ansiolíticas, sedativas e anticonvulsivantes. Objetivo: avaliar o efeito carcinogênico do bromazepam por meio do teste para detecção de tumores epiteliais (ETT) em Drosophila melanogaster. Metodologia: para realização do ETT foram utilizadas duas linhagens mutantes de D. melanogaster: wts (fêmeas) e mwh (machos). As larvas descendentes desse cruzamento foram tratadas isoladamente com cinco concentrações de bromazepam, sendo elas: 0,0375; 0,075; 0,15; 0,30 e 0,60 mM. A Doxorrubicina foi utilizada como controle positivo e a água ultrapura como controle negativo. Após tratamento, coleta e armazenamento, as moscas foram analisadas, identificando-se as frequências tumorais, por região corporal, em cada concentração testada. Resultados: o bromazepam não apresentou efeito carcinogênico em nenhuma das concentrações experimentadas neste estudo, não havendo diferença estatisticamente significativa nas frequências tumorais observadas nos indivíduos tratados com bromazepam quando comparadas à frequência obtida nos indivíduos tratados com o controle negativo. Conclusão: Nas presentes condições experimentais, o bromazepam não apresentou atividade carcinogênica, no entanto, há a necessidade de novos estudos, com diferentes metodologias e diferentes organismos testes, para a maior compreensão da ação do bromazepam no organismo.
Introduction: although cancer is one of the biggest public health problems faced worldwide, several substances present in the environment, such as drugs are not very well understood about its possible carcinogenic potential. Among them are benzodiazepines, drugs that have increased their consumption since the 20th century and, mainly, in the second decade of the 21st century, due to their anxiolytic, sedative and anticonvulsant actions. Objective: Evaluate the carcinogenic effect of bromazepam through the test to detect epithelial tumor clones (ETT) in Drosophila melanogaster. Methodology: to perform the ETT, two mutant strains of D. melanogaster were used: wts (female) and mwh (male). The descending larves of this cross were treated separately with five concentrations of bromazepam, namely: 0.0375; 0.075; 0.15; 0.30 and 0.60 mM. Doxorubicin was used as a positive control and ultrapure water as a negative control. After treatment, collection and storage, the flies were analyzed, identifying the tumor frequencies, by body region, at each concentration tested. Results: bromazepam did not have a carcinogenic effect at any of the concentrations experienced in this study, with no statistically significant difference in tumor frequencies observed in individuals treated with bromazepam when compared to the frequency obtained in individuals treated with the negative control. Conclusion: In the present experimental conditions, bromazepam did not show carcinogenic activity, however, there is a need for further studies with different methodologies and different test organisms to better understand the action of bromazepam in the body.
Subject(s)
Animals , Male , Female , Bromazepam , Carcinoma , Drosophila melanogaster , Carcinogenesis , Larva , EpitheliumABSTRACT
Introdução: a superexpressão da COX-2 relaciona-se com o aumento da produção de fatores de crescimento vascular e como consequência, com o desenvolvimento tumoral. O Firocoxib é um anti-inflamatório não esteroidal utilizado para inflamação associada à osteoartrite em cães. É o inibidor mais seletivo da COX-2, reduzindo eficientemente a ação desta enzima. Estudos indicam os benefícios do Firocoxib na terapia antineoplásica. Objetivo: este trabalho objetivou avaliar o efeito modulador do Firocoxib sobre a ação da doxorrubicina (DXR) por meio do teste de tumores epiteliais (ETT) em Drosophila melanogaster. Metodologia: foram preparadas três concentrações de Firocoxib: 2,5; 5 e 10 mg/mL, utilizadas isoladamente e em associação à doxorrubicina. O tratamento ocorreu com larvas de D. melanogaster descendentes do cruzamento de fêmeas wts/TM3 com machos mwh/mwh. Resultados: os resultados sugerem que o Firocoxib possui atividade moduladora sobre a ação carcinogênica da DXR, pois houve redução significativa nas frequências tumorais dos indivíduos tratados com diferentes concentrações de Firocoxib em cotratamento com a doxorrubicina quando comparadas à frequência tumoral do controle positivo. Conclusão: conclui-se que, nas presentes condições experimentais, o Firocoxib reduziu a frequência de tumores induzidos pela doxorrubicina em D. melanogaster.
Introduction: COX-2 overexpression is related to increased production of vascular growth factors and, as a consequence, to tumor development. Firocoxib is a non-steroidal anti-inflammatory drug used for inflammation associated with osteoarthritis in dogs. It is the most selective inhibitor of COX-2, efficiently reducing the action of this enzyme. Studies indicate the benefits of Firocoxib in anticancer therapy. Objective: this study aimed to evaluate the modulatory effect of Firocoxib on the action of doxorubicin (DXR) by means of the epithelial tumor test (ETT) in Drosophila melanogaster. Methodology: three concentrations of Firocoxib were prepared: 2.5; 5 and 10 mg/mL, used alone and in association with doxorubicin. The treatment occurred with D. melanogaster larvae descended from the crossing of wts/TM3 females with mwh/mwh males. Results: the results suggest that Firocoxib has modulating activity on the carcinogenic action of DXR, as there was a significant reduction in tumor frequencies in individuals treated with different concentrations of Firocoxib in co-treatment with doxorubicin when compared to the tumor frequency of the positive control. Conclusion: it is concluded that, under the present experimental conditions, Firocoxib reduced the frequency of tumors induced by doxorubicin in D. melanogaster.
Subject(s)
Animals , Doxorubicin , Drosophila melanogaster , Chemical Compounds , Evaluation StudyABSTRACT
Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.
Subject(s)
Animals , Humans , Dopa Decarboxylase/genetics , Dopamine/biosynthesis , Drosophila melanogaster/genetics , Escherichia coli/metabolism , Metabolic EngineeringABSTRACT
Introdução: óleos essenciais são muito utilizados para fins terapêuticos e nas últimas décadas esse interesse tem aumentado exponencialmente devido aos avanços nas terapias naturais. Neste contexto, destaca-se o óleo essencial do limão Taiti, um produto com grande potencial terapêutico e farmacológico. Objetivo: este trabalho objetivou avaliar o efeito carcinogênico e anticarcinogênico do limão Taiti (Citrus latifolia), por meio do teste para detecção de clones de tumores epiteliais em células somáticas de Drosophila melanogaster. Metodologia: larvas de terceiro estágio, do cruzamento entre fêmeas virgens wts/TM3, Sb1 com machos mwh/mwh, foram tratadas cronicamente com diferentes concentrações do óleo essencial do limão Taiti (1,5; 3,0; 6,0 µL) isoladamente e associadas à doxorrubicina (DXR), também foram incluídos dois controles, um negativo (Tween 80 1%) e um positivo (DXR 0,4 mM). Resultados: os resultados revelaram que os indivíduos tratados apenas com as concentrações isoladas do óleo essencial do limão Taiti não apresentaram frequências significativas de tumores quando comparadas ao controle negativo (p>0,05). Contudo, as concentrações associadas apresentaram efeito modulador sobre os danos induzidos pela DXR, pois houve redução significativa (p<0,05) na frequência de tumores, quando comparadas ao controle positivo. Conclusão: o presente trabalho, em condição experimental, atesta que o óleo essencial de limão Taiti (Citrus latifolia) não apresenta ação carcinogênica e sim ação anticarcinogênica em Drosophila melanogaster.
Introduction: essential oils are widely used for therapeutic purposes and in recent decades this interest has increased exponentially due to advances in natural therapies. In this context, the essential oil of lemon Taiti stands out, a product with great therapeutic and pharmacological potential. Objective: this study aimed to evaluate the carcinogenic and anticarcinogenic effect of Tahiti lemon (Citrus latifolia) by testing for clones of epithelial tumors in Drosophila melanogaster somatic cells. Methodology: third stage larvae from crossbreeding wts / TM3, Sb1 virgin females with mwh / mwh males were chronically treated with different concentrations of Taiti lemon essential oil (1.5; 3.0; 6.0 µL) alone and doxorubicin-associated (DXR), two controls were also included, one negative (Tween 80 1%) and one positive (0.4 mM DXR). Results: the results revealed that the individuals treated only with the isolated concentrations of Taiti lemon essential oil did not present significant tumor frequencies when compared to the negative control (p> 0.05). However, the associated concentrations had a modulating effect on DXR-induced damage, since there was a significant reduction (p <0.05) in the frequency of tumors when compared to the positive control. Conclusion: the present work, under experimental condition, attests that Taiti lemon essential oil (Citrus latifolia) did not show carcinogenic action but anticarcinogenic action in Drosophila melanogaster.
Subject(s)
Humans , Animals , Male , Female , Oils, Volatile , Citrus , Diptera , Drosophila melanogaster , Larva , Neoplasms , AntioxidantsABSTRACT
In most insects, polyunsaturated fatty acids (PUFAs) are mainly polyunsaturated fatty acids with a carbon-chain length less than 18 carbon atoms, hardly any long-chain polyunsaturated fatty acids such as C20 and C22 that are more valuable and bioactive. This study, by using Drosophila melanogaster (Fruit fly) as a model organism, optimized the Δ6-fatty acid elongase enzyme Elovl5 gene from mice and transferred it to fruit flies for expression. Vectors containing Elovl5 gene were successfully injected into drosophila embryo through the microscopic injection. There were enhanced green fluorescent proteins expressed in the whole developmental stage of Drosophila be means of fluorescence microscope. At the same time, expression of Elovl5 gene significantly contributed to the transformation of fruit flies C18-polyunsaturated fatty acids in the body towards the biosynthesis of longer-chain polyunsaturated fatty acids. The transgenic fruit fly model rich in long-chain polyunsaturated fatty acids such as C20 and C22 were obtained, providing a basis for further research on biosynthesis of polyunsaturated fatty acids in fruit flies.
Subject(s)
Animals , Mice , Acetyltransferases/genetics , Drosophila melanogaster/genetics , Fatty Acid Elongases/metabolism , Fatty Acids/genetics , Gene Transfer TechniquesABSTRACT
In this research, we studied the formation of Drosophila melanogaster FADD (Fas-associated death domain-containing protein) amyloid fiber and its influence on signal transduction in IMD (Immune deficiency) signaling pathway to better understand the regulation mechanism of Drosophila innate immune signaling pathway, which will provide reference for the immune regulation in other species. First, we purified dFADD protein expressed in Escherichia coli and performed Sulfur flavin T binding and transmission electron microscopy to identify the dFADD amyloid fibers formed in vitro. Then we investigated the formation of dFADD polymers in S2 cells using SDD-AGE and confocal microscope. We also constructed dFADD mutants to find out which domain is essential to fiber formation and its effect on IMD signal transduction. Our results revealed that dFADD could be polymerized to form amyloid fiber polymers in vitro and inside the cells. Formation of fibers relies on DED (Death-effector domain) domain of dFADD, since DED domain-deleted mutant existed as a monomer. Dual luciferase reporter assay showed that intact DED domain was required for the induction of downstream antimicrobial peptides, indicating that fiber formation was the key to IMD signal transduction. Our study revealed the role of dFADD in mediating the cascade between IMD and Dredd in the IMD signaling pathway by forming amyloid fibers, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.
Subject(s)
Animals , Drosophila Proteins , Allergy and Immunology , Drosophila melanogaster , Allergy and Immunology , Fas-Associated Death Domain Protein , Allergy and Immunology , Immunity, Innate , Allergy and Immunology , Signal TransductionABSTRACT
BACKGROUND: Tauopathies, a class of neurodegenerative diseases that includes Alzheimer's disease (AD), are characterized by the deposition of neurofibrillary tangles composed of hyperphosphorylated tau protein in the human brain. As abnormal alterations in histone acetylation and methylation show a cause and effect relationship with AD, we investigated the role of several Jumonji domain-containing histone demethylase (JHDM) genes, which have yet to be studied in AD pathology. METHODS: To examine alterations of several JHDM genes in AD pathology, we performed bioinformatics analyses of JHDM gene expression profiles in brain tissue samples from deceased AD patients. Furthermore, to investigate the possible relationship between alterations in JHDM gene expression profiles and AD pathology in vivo, we examined whether tissue-specific downregulation of JHDM Drosophila homologs (kdm) can affect tauR406W-induced neurotoxicity using transgenic flies containing the UAS-Gal4 binary system. RESULTS: The expression levels of JHDM1A, JHDM2A/2B, and JHDM3A/3B were significantly higher in postmortem brain tissue from patients with AD than from non-demented controls, whereas JHDM1B mRNA levels were downregulated in the brains of patients with AD. Using transgenic flies, we revealed that knockdown of kdm2 (homolog to human JHDM1), kdm3 (homolog to human JHDM2), kdm4a (homolog to human JHDM3A), or kdm4b (homolog to human JHDM3B) genes in the eye ameliorated the tauR406W-engendered defects, resulting in less severe phenotypes. However, kdm4a knockdown in the central nervous system uniquely ameliorated tauR406W-induced locomotion defects by restoring heterochromatin. CONCLUSION: Our results suggest that downregulation of kdm4a expression may be a potential therapeutic target in AD.
Subject(s)
Humans , Acetylation , Alzheimer Disease , Brain , Central Nervous System , Computational Biology , Diptera , Down-Regulation , Drosophila melanogaster , Drosophila , Heterochromatin , Histones , Locomotion , Methylation , Neurodegenerative Diseases , Neurofibrillary Tangles , Pathology , Phenotype , RNA, Messenger , tau Proteins , Tauopathies , TranscriptomeABSTRACT
Aging is the most basic life feature of organisms. It is a phenomenon of dysfunction of cells,tissues and organs under the influence of external environment and internal factors during the growth of organisms. During the process,many cellular signaling pathways and biologically active substances,such as insulin/insulin-like growth factor( IGF)-1 signaling( IIS) pathway,apoptotic signaling pathway,mTOR signaling pathway,AMPK pathway,sirtuin pathway,deacetylases have been found to be closely related to the molecular mechanism of aging. Modern studies have indicated that anti-aging natural compounds can cause great side effects,while delaying aging and even inducing another disease,which is against with the purpose of delaying aging and achieving healthy aging. Therefore,the researches of anti-aging traditional Chinese medicines with fewer side effects are extremely important. Based on the different mechanisms and theories of aging,many traditional Chinese medicines have been discovered to be related to anti-aging. As one of the most important model organisms,Drosophila melanogaster has been widely used in studies of aging process in recent years. In this paper,we reviewed three important signaling pathways related to aging,such as insulin/insulin-like growth factor( IGF)-1 signaling( IIS) pathway,m TOR pathway,AMPK pathway,and screened out anti-aging traditional Chinese medicines based on D. melanogaster in recent years.
Subject(s)
Animals , Aging , Drosophila melanogaster , Insulin , Longevity , Medicine, Chinese Traditional , Signal TransductionABSTRACT
Stroke is a leading cause of death worldwide. Up to one thousand potential drugs or interventions have been developed to treat stroke, out of which ~160 have gone on to clinical trials. However, none of them has been successful. New insights into the molecular and cellular mechanisms of ischemia-induced injury are needed for discovering new therapeutic targets. Recently, Drosophila has been used to uncover new hypoxia-related genes. In this study, we describe an efficient and reliable assay with a sophisticated apparatus for studying the effects of oxygen deprivation on flies. Using this assay, wild-type flies were exposed to an anoxic environment for varying lengths of time, then the cumulative death rate and mobility recovery were systematically analyzed. We found that anoxia for over one hour caused lethality. The cumulative death rate on day 5 after anoxia was linearly and positively correlated with the duration of anoxia, and reached 50% when the duration was 2.5 h-3 h. We also found that the mobility recovery in normoxia was slow, as the climbing ability remained largely unchanged 4 h-6 h after 2.5-h of anoxia. We suggest that 2.5 h-3 h of anoxia and 4 h-6 h of recovery before mobility analysis are appropriate for future use of the anoxia assay.
Subject(s)
Animals , Behavior, Animal , Disease Models, Animal , Drosophila melanogaster , HypoxiaABSTRACT
BACKGROUND/OBJECTIVES: The aim of this study was to evaluate the biological and sleep-promoting effects of combined γ-aminobutyric acid (GABA) and 5-hydroxytryptophan (5-HTP) using caffeine-induced sleepless fruit flies, ICR mice, and Sprague-Dawley rats. MATERIALS/METHODS: Video-tracking analysis was applied to investigate behavioral changes of Drosophila melanogaster. Pentobarbital-induced sleep test and electroencephalogram (EEG) patterns were used for analysis of sleep latency, duration, and quantity and quality of sleep in vertebrate models. RESULTS: Administration of combined GABA/5-HTP could significantly reverse the caffeine induced total distance of flies (P < 0.001). Also, individually administered and combined GABA/5-HTP significantly increased the total sleeping time in the caffeine-induced sleepless ICR mice (P < 0.001). In the caffeine-induced sleepless SD-rats, combined GABA/5-HTP showed significant differences in sleep quality between individual amino acid administrations (P < 0.05). CONCLUSIONS: Taken together, we identified inhibitory effects of combined GABA/5-HTP in locomotor activity, sleep quantity and quality in caffeine-induced sleepless models, indicating that combined GABA/5-HTP may be effective in patients with insomnia by providing sufficient sleep.
Subject(s)
Animals , Humans , Mice , 5-Hydroxytryptophan , Amino Acids , Caffeine , Diptera , Drosophila melanogaster , Electroencephalography , Fruit , gamma-Aminobutyric Acid , Mice, Inbred ICR , Motor Activity , Rats, Sprague-Dawley , Sleep Initiation and Maintenance Disorders , VertebratesABSTRACT
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/metabolismABSTRACT
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)
Magnetic Resonance Spectroscopy/methods , Sodium-Calcium Exchanger/analysis , Peptides , Drosophila melanogaster/classification , Membrane ProteinsABSTRACT
Studies have suggested that total energy intake and diet composition affect lifespan and ageing. A high-fat diet induces oxidative stress and affects the development of diseases. In contrast, antioxidants are capable of reducing its harmful effects. Yerba mate beverages are an important source of antioxidants, but there is scarce knowledge about their effects on suppressing fat accumulation. Here, we investigated the compounds present in yerba mate extracts and assessed their effects on Drosophila melanogaster given a high cholesterol diet. LS-ESI-MS analysis showed the presence of matesaponins, phenolic compounds and methylxanthines in all of the examined extracts. In Drosophila, under extract treatment conditions, the mean lifespan was significantly extended from 38 to 43 days, there was an increase in the ability to support induced stress and decrease in lipid peroxidation products. Moreover, yerba mate extracts recovered the glutathione S-transferases (GST) activity and reduced the cholesterol level. Taken together, our results support that extracts can extend lifespan by reducing the detrimental effect of a high-fat diet in D. melanogaster, and this outcome can be associated with the compound content in the extracts. This study improves the understanding of natural interventions that reduce stress-induced oxidative damage, which is fundamental in promoting healthy ageing.
Subject(s)
Animals , Plant Extracts/pharmacology , Oxidative Stress/drug effects , Ilex paraguariensis/chemistry , Drosophila melanogaster/physiology , Longevity/drug effects , Antioxidants/pharmacology , Oxidative Stress/physiology , Drosophila melanogaster/growth & development , Diet, High-Fat , Longevity/physiologyABSTRACT
O consumo de medicamentos está entre as principais causas de desenvolvimento do câncer. Faz-se, então, necessário investigar os efeitos de drogas amplamente consumidas no mundo, como é o caso do Viagra® (Citrato de Sildenafila - CS). O objetivo do presente estudo foi avaliar o efeito modulador do CS na carcinogênese induzida pela Doxorrubicina (DXR), por meio do Teste para Detecção de Clones de Tumores Epiteliais em Drosophila melanogaster. Foram utilizadas duas linhagens mutantes do organismo teste (wts e mwh) no intuito de obter larvas heterozigotas wts +/+ mwh, que foram tratadas com três concentrações de CS: 10 mM, 20 mM e 40 mM, isoladamente e em associação com a DXR. Constatou-se que o CS não exerceu efeito carcinogênico nas concentrações isoladamente avaliadas. Quando em associação com a DXR, propiciou efeito redutor sobre as frequências dos tumores induzidos por ela, conferindo papel anticarcinogênico ao CS. Acredita-se que este efeito seja devido a uma supra-regulação da via de apoptose dependente de caspase e ao impedimento do crescimento celular promovido pelo fármaco utilizado. As evidências obtidas comprovam o efeito protetor tumoral atribuído ao Viagra® nas condições experimentais propostas.(AU)
Drug consumption is among the leading causes of cancer development. Therefore, it is necessary to investigate the effects of drugs widely consumed in the world, as the case of Viagra® (Sildenafil Citrate - SC). The objective of the study was to evaluate the modulatory effect of SC on the carcinogenicity induced by Doxorubicin (DXR) by the Test for Detection of Epithelial Tumor Clones in Drosophila melanogaster. Two mutant lines (wts and mwh) were used in order to obtain heterozygous wts+/+mwh larvae, which were treated with three concentrations of CS: 10 mM, 20 mM and 40 mM, alone and in combination with doxorubicin. We verified that SC had no carcinogenic effect at the concentrations evaluated alone. In association with DXR, it was provided a reduced effect of tumor-induced, conferring anticarcinogenic role to SC. It is believed that this effect is due to up-regulation of the caspase-dependent apoptosis and the impediment of cell growth promoted by the drug used. Evidence obtained confirms the tumor protective effect attributed to Viagra® under experimental conditions. (AU)
Subject(s)
Anticarcinogenic Agents , Doxorubicin , Drosophila melanogaster , Neoplasms , Sildenafil CitrateABSTRACT
Introdução: o óleo de copaíba é o composto extraído do tronco da copaibeira (Copaifera sp.) que tem sido utilizado na medicina popular desde a chegada dos portugueses ao Brasil. Objetivo: o objetivo deste estudo foi avaliar possíveis efeitos carcinogênicos e/ ou anticarcinogênicos do óleo de copaíba (Copaifera officinalis L.), por meio do teste para detecção de clones de tumores epiteliais (warts) em Drosophila melanogaster. Metodologia: foram preparadas três soluções de óleo de copaíba, nas proporções de 0,5%, 1% e 2%. Nessas soluções foram cultivadas Drosophilas melanogaster expostas simultaneamente à doxorrubicina na concentração de 0,4 mM, agente conhecidamente cancerígeno, também utilizado como controle positivo na presente pesquisa. Para controle negativo foi utilizado Tween 80 (1%). O tratamento foi realizado com todas as larvas descendentes do cruzamento de fêmeas wts/ TM3 com machos mwh/mwh. Resultados: o óleo de copaíba apresentou atividade carcinogênica quando utilizado isoladamente na concentração 2%, visto que houve aumento estatisticamente significativo (p ≤ 0,05) na frequência de tumores em comparação com o controle negativo. Além disso, evidenciou-se potencialização do efeito carcinogênico da doxorrubicina nas concentrações 0,5% e 1%, uma vez que houve um aumento estatisticamente significativo (p ≤ 0,05) na frequência de tumores nessas concentrações, quando associadas à DXR, em comparação com o controle positivo. Conclusão: os resultados evidenciaram o efeito carcinogênico isolado do óleo de copaíba, bem como seu efeito potencializador quando associado à doxorrubicina.
Introduction: the copaiba oil is a compound extracted from the trunk of the copaiba tree (Copaifera sp.) that has been used in popular medicine since the arrival of the Portuguese in Brazil. Purpose: this study aimed to evaluate the possible carcinogenic and/ or anticarcinogenic effects of the copaiba oil (Copaifera officinalis L.) through the test for detection of epithelial tumor clones (warts) in Drosophila melanogaster. Methodology: three solutions of copaiba oil were prepared in the proportions 0,5%, 1% and 2%. In these solutions were cultivated Drosophilas melanogaster previously exposed to doxorubicin at a 0.4 mM concentration, admittedly carcinogenic agent, which was also used for positive control in the present research. For negative control Tween 80 (1%) was used. The treatment was performed on all larvae descendant of the crossing of females wts/TM3 with males mwh/mwh. Results: the results showed that the copaiba oil presented carcinogenic activity when used in isolation at the concentration of 2%, seeing that there was a statistically significant increase (p ≤ 0,05) in tumor frequency in comparison to the negative control. Moreover, there was a potentiation of doxorubicin's carcinogenic effect at concentrations 0,5% and 1%, since there was a statistically significant increase (p ≤ 0,05) of tumor frequency in these concentrations, when associated to DXR, in comparison to the positive control. Conclusion: the results evidenced the isolated carcinogenic effect of copaiba oil, as well as its potentiating effect when associated with doxorubicin
Subject(s)
Animals , Male , Female , Plants, Medicinal , Doxorubicin , Drosophila melanogaster , DipteraABSTRACT
Introdução: O uso de plantas como inseticidas podem substituir muitos produtos sintéticos por apresentarem baixa toxicidade para os animais e biodegradação no ambiente. A espécie Lantana montevidensis (Spreng.) Briq. (Verbenaceae), chumbinho, é um vegetal subarbustivo rico de compostos secundários para a sua defesa. O modelo experimental com Drosophila melanogaster tem sido usada para estudos, por apresentar vantagens, pois tem fácil manuseio e rápida taxa de reprodução. Objetivo: Avaliar a composição química e efeito inseticida do óleo essencial (OE) de L. montevidensis contra D. melanogaster. Metodos: O OE das folhas secas provenientes do Crato-CE, Brasil, foi extraído por hidrodestilação e foi analisado por CG/EMS para identificação dos constituintes. No ensaio foi avaliada a mortalidade e geotaxia negativa das moscas, em que elas foram expostas a diferentes concentrações do OE (3-30,5 µg/mL do óleo essencial/ar). Um total de vinte moscas adultas foram submetidas à diapausa por meio de resfriamento e em seguidas foram colocadas em frascos de 300 mL, cuja parte inferior havia papel filtro impregnado com 1 mL de sacarose a 20 por cento em água destilada. Na parte superior (tampa) foi afixado um papel filtro para a aplicação do produto a ser testado, nesse caso o OELM. As leituras foram realizadas a cada 3, 6, 12 e 24 h. Resultados: Os resultados mostraram que o OE apresentou o ß-Cariofileno (34,96 por cento), o Germacreno D (25,49 por cento) e o Biciclogermacreno (9, 78 por cento) como componentes majoritários, no ensaio inseticida houve uma CL50 na concentração de 15,14 µg/mL em 3 h de exposição, e houve um efeito significativo nos danos do aparelho locomotor das moscas.mConclusão: Este estudo fornece evidências que esse OE apresenta-se como um potencial bioinseticida(AU)
Introducción: el uso de plantas como insecticidas puede sustituir muchos productos sintéticos porque tienen baja toxicidad para los animales y son fácilmente biodegradables en el medio ambiente. La especie Lantana montevidensis (Spreng.) Briq. (Verbenaceae), Chumbinho, es una planta sub-arbustiva rica en metabolitos secundarios para su defensa. El modelo experimental en Drosophila melanogaster se ha utilizado para varios tipos de estudios, presenta algunas ventajas como: fácil manejo y tasa de reproducción rápida. Objetivo: evaluar la composición química y el efecto insecticida de aceite esencial (OE) de L. montevidensis contra las D. melanogaster. Métodos: el OE de hojas secas del Crato-CE, Brasil, se extrajo por hidrodestilación y se analizó por GC/EMS para identificar los constituyentes. En el test se evaluó la mortalidad y geotaxia negativas de las moscas, donde fueron expuestos a diferentes concentraciones de OE (3-30,5 µg/mL de aceite esencial/aire). Un total de veinte moscas adultas se sometieron a diapausa por enfriamiento y seguido se colocaron en botellas de 300 mL, la parte inferior contenido papel de filtro impregnado con 1 mL de 20 por ciento de sacarosa en agua destilada. En la parte superior (tapa) se haya colocado un papel de filtro para la aplicación del producto a ensayar, la OELM. Las lecturas se realizaron cada 3, 6, 12 y 24 h. Resultados: Los resultados mostraron que el OE presenta la ß-cariofileno (34,96 por ciento), el germacreno D (25,49 por ciento) y bicyclogermacrene (9,78 por ciento) como componentes principales en el ensayo insecticida había una CL50 de la concentración de 15,14 µg/mL en 3 h de exposición, y había un un efecto significativo en el daño del aparato locomotor de las moscas. Conclusión: este estudio proporciona evidencia de que este OE o se presenta como un biopesticida potencial(AU
Introduction: The use of plants as insecticides may be an alternative to many synthetic products because their toxicity to animals is low and they are easily biodegradable in the environment. The species Lantana montevidensis (Spreng.) Briq. (Verbenaceae), chumbinho, is a subshrub rich in secondary metabolites it uses for its own defence. The experimental model for Drosophila melanogaster has been used in several types of studies. Some of its advantages are its easy management and its fast rate of reproduction. Objective: Evaluate the chemical composition and insecticidal effect of the essential oil (EO) of L. montevidensis against D. melanogaster. Method: EO of dry leaves from Crato-CE, Brazil, was extracted by hydrodistillation and analyzed by GC/EMS to identify its constituents. As part of the test, evaluation was conducted or the mortality and negative geotaxis of the flies, to attain which they were exposed to different concentrations of the EO (3-30.5 µg/mL essential oil/air). A total 20 adult flies were subjected to cold diapause and placed in 300 mL bottles with filter paper in their lower section impregnated with 1 mL of 20 percent saccharose in distilled water. Filter paper was also attached to the upper section (lid) to apply the test product OELM. Readings were taken every 3, 6, 12 and 24 h. Results: Results showed that the principal components of the EO are ?-caryophyllene (34.96 percent), germacrene D (25.49 percent) and bicyclogermacrene (9.78 percent). In the insecticide test there was a CL50 for the concentration of 15.14 µg/ml in 3 h exposure, and significant damage to the locomotor system of the flies. Conclusion: The study provides evidence that the EO analyzed is a potential biopesticide(AU)
Subject(s)
Animals , Oils, Volatile , Lantana/toxicity , Drosophila melanogaster , InsecticidesABSTRACT
α-Synuclein (α-Syn) is a small presynaptic protein and its mutant forms (e.g. A53T) are known to be directly associated with Parkinson's disease (PD). Pathophysiological mechanisms underlying α-Syn-mediated neurodegeneration in PD still remain to be explored. However, several studies strongly support that overexpression of mutant α-Syn causes reduced release of dopamine (DA) in the brain, and contributes to motor deficits in PD. Using a favorable genetic model Drosophila larva, we examined whether reduced DA release is enough to induce key PD symptoms (i.e. locomotion deficiency and DA neurodegeneration), mimicking a PD gene α-Syn. In order to reduce DA release, we expressed electrical knockout (EKO) gene in DA neurons, which is known to make neurons hypo-excitable. EKO led to a decrease in a DA neuronal marker signal (i.e., TH – tyrosine hydroxylase) and locomotion deficits in Drosophila larva. In contrast, acute and prolonged exposure to blue light (BL, 470 nm) was sufficient to activate channelrhodopsin 2 (ChR2) and rescue PD symptoms caused by both α-Syn and EKO. We believe this is for the first time to confirm that locomotion defects by a genetic PD factor such as α-Syn can be rescued by increasing DA neuronal excitability with an optogenetic approach. Our findings strongly support that PD is a failure of DA synaptic transmission, which can be rescued by optogenetic activation of ChR2.