RESUMO
Pleurotus ostreatus (P. ostreatus) and Lentinus subnudus (L. subnudus) have been used by the locals for the management of Alzheimer's disease (AD) but with scant scientific sources. The aim of this study is to assess the neuroprotective properties of P. ostreatus and L. subnudus using transgenic Drosophila melanogaster flies (TDMF). The activity of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), as well as the antioxidant status of TDMF raised on a diet supplemented with P. ostreatus and L. subnudus were determined. The flies were raised on a diet devoid of supplements or supplemented with P. ostreatus or L. subnudus (1% and 5% inclusion) for 7 days. Afterward, AChE and BChE activities, as well as catalase and total thiol level, were determined. The reactive oxygen species (ROS) and malondialdehyde (MDA) levels were also determined in the flies raised on a diet devoid of supplement and on supplemented diets. Meanwhile, flies raised on P. ostreatus- and L. subnudus-supplemented diets exhibited a significant reduction in the activity of AChE and BChE in comparison with the controls. Also, supplemented diets significantly (p < 0.05) enhance catalase activity and improve total thiol level, while ROS and MDA levels were observed to be reduced in all the flies raised on the supplemented diets in comparison with the controls. In summary, reduction in the activity of AChE and BChE, as well as improved antioxidant status in TDMF, could be some of the mechanisms through which P. ostreatus and L. subnudus exhibit anti-AD properties. Nevertheless, L. subnudus exhibits a better neuroprotective effect than P. ostreatus.
Assuntos
Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Dieta , Drosophila melanogaster/enzimologia , Lentinula , Pleurotus , Animais , Animais Geneticamente Modificados , Antioxidantes/metabolismo , Fármacos Neuroprotetores/farmacologiaRESUMO
Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Proteína Fosfatase 2/fisiologia , Proteínas Quinases S6 Ribossômicas/fisiologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Ceramidas/farmacologia , Modelos Animais de Doenças , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Mutação com Ganho de Função , Técnicas de Silenciamento de Genes , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Mutação de Sentido Incorreto , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
OBJECTIVE: The purpose of the study was to generate information on the safety of probiotics, thus the study objectives were to evaluate the effects of Yoba® on basic physiochemical properties. The study assessed male w1118 Drosophila melanogaster which were provided food supplemented with Yoba® at 1%, 3%, 6%, and 12% on motor function, total protein, catalase activity, and hydrogen peroxide scavenging activity and lifespan. RESULTS: Yoba® at high concentration (≥ 6%) increased locomotor activity in Drosophila melanogaster, however, total protein, catalase, and hydrogen peroxide scavenging activity were significantly higher at 1% Yoba® compared to 3%, 6%, and 12% Yoba®. Yoba consumed at 1% was associated with greater physiological benefits in Drosophila melanogaster. Findings in the study offer a rationale for the consumption of Yoba® at 1% in humans as is currently being promoted by the Yoba for Life consortium, however, high concentrations of Yoba® would disrupt physiological function as shown by this study.
Assuntos
Drosophila melanogaster/microbiologia , Lacticaseibacillus rhamnosus/fisiologia , Animais , Catalase/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Sequestradores de Radicais Livres/farmacologia , Peróxido de Hidrogênio/metabolismo , Longevidade , Masculino , Atividade Motora/efeitos dos fármacosRESUMO
(-)-α-Bisabolol (BISA) is a sesquiterpene alcohol, which has several recognized biological activities, including anti-inflammatory, anti-irritant, and antibacterial properties. In the present study, we investigated the influence of BISA (5, 25, and 250 µmol/L) on rotenone (500 µmol/L)-induced toxicity in Drosophila melanogaster for 7 days. BISA supplementation significantly decreased rotenone-induced mortality and locomotor deficits. The loss of motor function induced by rotenone correlated with a significant change in stress response factors; it decreased thiol levels, inhibited mitochondria complex I, and increased the mRNA expression of antioxidant marker proteins such as superoxide dismutase (SOD), catalase (CAT), and the keap1 gene product. Taken together, our findings indicate that the toxicity of rotenone is likely due to the direct inhibition of complex I activity, resulting in a high level of oxidative stress. Dietary supplementation with BISA affected the expression of SOD mRNA only at a concentration of 250 µmol/L, and did not affect any other parameter measured. Our results showed a protective effect of BISA on rotenone-induced mortality and locomotor deficits in Drosophila; this effect did not correlate with mitochondrial complex I activity, but may be related to the antioxidant protection afforded by eliminating superoxide generated as a result of rotenone-induced mitochondrial dysfunction.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Rotenona/toxicidade , Sesquiterpenos/farmacologia , Animais , Catalase/genética , Catalase/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Complexo I de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Sesquiterpenos Monocíclicos , Atividade Motora/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Análise de SobrevidaRESUMO
Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.
Assuntos
Ciclo do Ácido Cítrico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , Isocitrato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Transmissão Sináptica , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Drosophila melanogaster/ultraestrutura , Ácidos Cetoglutáricos/metabolismo , Larva/metabolismo , Mitocôndrias/ultraestrutura , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ligação Proteica , Domínios Proteicos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptotagminas/química , Sinaptotagminas/metabolismoRESUMO
Most biodiesel currently in use consists of fatty acid methyl esters (FAMEs) produced by transesterification of plant oils with methanol. To reduce competition with food supplies, it would be desirable to directly produce biodiesel in microorganisms. To date, the most effective pathway for the production of biodiesel in bacteria yields fatty acid ethyl esters (FAEEs) at up to ~1.5 g/L. A much simpler route to biodiesel produces FAMEs by direct S-adenosyl-L-methionine (SAM) dependent methylation of free fatty acids, but FAME production by this route has been limited to only ~16 mg/L. Here we employ an alternative, broad spectrum methyltransferase, Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). By introducing DmJHAMT in E. coli engineered to produce medium chain fatty acids and overproduce SAM, we obtain medium chain FAMEs at titers of 0.56 g/L, a 35-fold increase over titers previously achieved. Although considerable improvements will be needed for viable bacterial production of FAMEs and FAEEs for biofuels, it may be easier to optimize and transport the FAME production pathway to other microorganisms because it involves fewer enzymes.
Assuntos
Biocombustíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Escherichia coli/metabolismo , Metiltransferases/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ensaios Enzimáticos/métodos , Escherichia coli/genética , Ácidos Graxos/metabolismo , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Metilação , Metiltransferases/genética , Reprodutibilidade dos Testes , S-Adenosilmetionina/metabolismoRESUMO
We developed a Drosophila model of T2D in which high sugar (HS) feeding leads to insulin resistance. In this model, adipose TG storage is protective against fatty acid toxicity and diabetes. Initial biochemical and gene expression studies suggested that deficiency in CoA might underlie reduced TG synthesis in animals during chronic HS feeding. Focusing on the Drosophila fat body (FB), which is specialized for TG storage and lipolysis, we undertook a series of experiments to test the hypothesis that CoA could protect against the deleterious effects of caloric overload. Quantitative metabolomics revealed a reduction in substrate availability for CoA synthesis in the face of an HS diet. Further reducing CoA synthetic capacity by expressing FB-specific RNAi targeting pantothenate kinase (PK orfumble) or phosphopantothenoylcysteine synthase (PPCS) exacerbated HS-diet-induced accumulation of FFAs. Dietary supplementation with pantothenic acid (vitamin B5, a precursor of CoA) was able to ameliorate HS-diet-induced FFA accumulation and hyperglycemia while increasing TG synthesis. Taken together, our data support a model where free CoA is required to support fatty acid esterification and to protect against the toxicity of HS diets.
Assuntos
Coenzima A/metabolismo , Drosophila melanogaster/metabolismo , Ingestão de Energia , Animais , Carboidratos da Dieta/efeitos adversos , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Ingestão de Energia/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , FenótipoRESUMO
As a survival strategy to environmental water deficits, desiccation-tolerant organisms are commonly known for their ability to recruit stress-protective biomolecules such as trehalose. We have previously reported the pivotal role of trehalose in larval desiccation tolerance in Drosophila melanogaster. Trehalose has emerged as a versatile molecule, serving mainly as energy source in insects and also being a stress protectant. While several recent reports have revealed the unconventional role of trehalose in scavenging reactive oxygen species in yeast and plants, this aspect has not received much attention in animals. We examined the status of desiccation-induced generation of reactive oxygen species in D. melanogaster larvae and the possible involvement of trehalose in ameliorating the harmful consequences thereof. Insect trehalose synthesis is governed by the enzyme trehalose 6-phosphate synthase 1 (TPS1). Using the ubiquitous da-GAL4-driven expression of the dTps1-RNAi transgene, we generated dTps1-downregulated Drosophila larvae possessing depleted levels of dTps1 transcripts. This resulted in the inability of the larvae for trehalose synthesis, thereby allowing us to elucidate the significance of trehalose in the regulation of desiccation-responsive redox homeostasis. Furthermore, the results from molecular genetics studies, biochemical assays, electron spin resonance analyses and a simple, non-invasive method of whole larval live imaging suggested that trehalose in collaboration with superoxide dismutase (SOD) is involved in the maintenance of redox state in D. melanogaster.
Assuntos
Drosophila melanogaster/enzimologia , Glucosiltransferases/metabolismo , Estresse Oxidativo , Trealose/metabolismo , Animais , Dessecação , Regulação para Baixo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glucosiltransferases/genética , Larva/enzimologia , Larva/genética , Larva/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Trealose/genética , Água/metabolismoRESUMO
Plant phenolics are a group of important secondary metabolites that are toxic to many animals and insects if ingested at high concentrations. Because most insects consume plant phenolics daily, they have likely evolved the capacity to detoxify these compounds. Here, we used Drosophila melanogaster, Bombyx mori and Helicoverpa armigera as models to study the metabolism of plant phenolics by prophenoloxidases. We found that insect foreguts release prophenoloxidases into the lumen, and that the survival of prophenoloxidase-deletion mutants was impaired when fed several plant phenolics and tea extracts. Using l-DOPA as a model substrate, biochemical assays in large Lepidopteran insects demonstrated that low levels of l-DOPA are rapidly metabolized into intermediates by phenoloxidases. Feeding with excess l-DOPA showed that the metabolic intermediate 5,6-dihydroxyindole reached the hindgut either by passing directly through the midgut, or by transport through the hemolymph. In the hindgut, 5,6-dihydroxyindole was further oxidized by prophenoloxidases. Intermediates exerted no toxicity in the hemocoel or midgut. These results show that plant phenolics are not toxic to insects unless prophenoloxidase genes are lost or the levels of phenolics exceed the catalytic activity of the gut prophenoloxidases.
Assuntos
Bombyx/enzimologia , Catecol Oxidase/genética , Drosophila melanogaster/enzimologia , Precursores Enzimáticos/genética , Proteínas de Insetos/genética , Lepidópteros/enzimologia , Desintoxicação Metabólica Fase I/genética , Fenóis/metabolismo , Animais , Biotransformação , Bombyx/genética , Bombyx/metabolismo , Catecol Oxidase/deficiência , Drosophila melanogaster/genética , Precursores Enzimáticos/deficiência , Deleção de Genes , Expressão Gênica , Hemolinfa/metabolismo , Indóis/metabolismo , Proteínas de Insetos/deficiência , Mucosa Intestinal/metabolismo , Lepidópteros/genética , Levodopa/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/metabolismo , Plantas/químicaRESUMO
Exposure to chlorpyrifos (CPF) poses several harmful effects to human and animal health. The present study investigated the influence of diphenyl diselenide (DPDS) on CPF-induced toxicity in Drosophila melanogaster. Firstly, the time course lethality response of virgin flies (2- to 3-day-old) to CPF (0.075-0.6µg/g) and DPDP (5-40µmol/kg) in the diet for 28 consecutive days were investigated. Subsequently, the protective effect of DPDS (10, 20 and 40µmol/kg) on CPF (0.15µg/g)-induced mortality, locomotor deficits, neurotoxicity and oxidative stress was assessed in a co-exposure paradigm for 7 days. Results showed that CPF exposure significantly decreased the percent live flies in a time- and concentration-dependent manner, whereas the percent live flies with DPDS treatment was not statistically different from control following 28 days of treatment. In the co-exposure study, CPF significantly increased flies mortality while the survivors exhibited significant locomotor deficits with decreased acetylcholinesterase (AChE) activity. Dietary supplementation with DPDS was associated with marked decrease in mortality, improvement in locomotor activity and restoration of AChE activity in CPF-exposed flies. Moreover, CPF exposure significantly decreased catalase and glutathione-S-transferase activities, total thiol level with concomitant significant elevation in the levels of reactive oxygen species and thiobarbituric acid reactive substances in the head and body regions of the treated flies. Dietary supplementation with DPDS significantly improved the antioxidant status and prevented CPF-induced oxidative stress, thus demonstrating the protective effect of DPDS in CPF-treated flies.
Assuntos
Derivados de Benzeno/farmacologia , Clorpirifos/toxicidade , Drosophila melanogaster/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Acetilcolinesterase/metabolismo , Animais , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Catalase/metabolismo , Drosophila melanogaster/enzimologia , Cabeça , Locomoção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismo , Análise de Sobrevida , Fatores de TempoRESUMO
Heterologous expression of the Drosophila melanogaster multi-substrate deoxyribonucleoside kinase (Dm-dNK) increases the sensitivity of cancer cells to several cytotoxic nucleoside analogs. Thus, it may be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize this potential suicide gene, we constructed two retroviral vectors that enabled the expression of Dm-dNK in cancer cells. One vector harbored the wildtype enzyme that localized to the nucleus. The other vector harbored a mitochondrial localized mutant enzyme that was constructed by deleting the nuclear localization signal and fusing it to a mitochondrial import signal of cytochrome c oxidase. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant viruses. The sensitivity and bystander cell killing in the presence of pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)2'deoxyuridine and 1-ß-D-arabinofuranosylthymine were investigated. Tanshinone IIA is a constituent of Danshen; a traditional Chinese medicine used in the treatment of cardiovascular diseases. This study also looked at the influence of Tanshinone IIA on the bystander effect and the underlying mechanisms. We showed that sensitivity of the osteosarcoma cell line to the nucleoside analogs and the efficiency of bystander cell killing were independent of the subcellular localization of Dm-dNK. The enhanced effect of tanshinone IIA on the bystander effect was related to the increased expression of Cx43 and Cx26.
Assuntos
Abietanos/administração & dosagem , Terapia Genética , Osteossarcoma/tratamento farmacológico , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Efeito Espectador , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Conexina 26 , Conexinas , Desoxiuridina/administração & dosagem , Desoxiuridina/análogos & derivados , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Galactose/administração & dosagem , Galactose/análogos & derivados , Vetores Genéticos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Sinais de Localização Nuclear/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Retroviridae/genética , Timina/administração & dosagem , Timina/análogos & derivadosRESUMO
We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.
Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/enzimologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/metabolismo , Glutationa Transferase/antagonistas & inibidores , Animais , Proteínas de Drosophila/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaios Enzimáticos , Corantes Fluorescentes/química , Glutationa/química , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Most of our knowledge on protein tyrosine phosphatases (PTPs) is derived from human pathologies and mouse knockout models. These models largely correlate well with human disease phenotypes, but can be ambiguous due to compensatory mechanisms introduced by paralogous genes. Here we present the analysis of the PTP complement of the fruit fly and the complementary view that PTP studies in Drosophila will accelerate our understanding of PTPs in physiological and pathological conditions. With only 44 PTP genes, Drosophila represents a streamlined version of the human complement. Our integrated analysis places the Drosophila PTPs into evolutionary and functional contexts, thereby providing a platform for the exploitation of the fly for PTP research and the transfer of knowledge onto other model systems.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Família Multigênica , Proteínas Tirosina Fosfatases/genética , Animais , Proteínas de Drosophila/classificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Evolução Molecular , Humanos , Camundongos , Mutação , Filogenia , Proteínas Tirosina Fosfatases/classificação , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Various molecular forms of acetylcholinesterase (AChE) have been characterized in insects. Post-translational modification is known to be a major mechanism for the molecular diversity of insect AChE. However, multiple forms of Drosophila melanogaster AChE (DmAChE) were recently suggested to be generated via alternative splicing (Kim and Lee, 2013). To confirm alternative splicing as the mechanism for generating the soluble form of DmAChE, we generated a transgenic fly strain carrying the cDNA of DmAChE gene (Dm_ace) that predominantly expressed a single transcript variant encoding the membrane-anchored dimer. 3' RACE (rapid amplification of cDNA ends) and western blotting were performed to compare Dm_ace transcript variants and DmAChE forms between wild-type and transgenic strains. Various Dm_ace transcripts and DmAChE molecular forms were observed in wild-type flies, whereas the transgenic fly predominantly expressed Dm_ace transcript variant encoding the membrane-anchored dimer. This supports alternative splicing as the major determinant in the generation of multiple forms of DmAChE. In addition, treatment with DDVP as a chemical stress induced the expression of the Dm_ace splice variant without the glycosylphosphatidylinositol anchor site in a dose-dependent manner and, accordingly, the soluble form of DmAChE in wild-type flies. In contrast, little soluble DmAChE was expressed in the transgenic fly upon exposure to DDVP. DDVP bioassays revealed that transgenic flies, which were unable to express a sufficient amount of soluble monomeric DmAChE, were more sensitive to DDVP compared to wild-type flies, suggesting that the soluble monomer may exert non-neuronal functions, such as chemical defense against xenobiotics.
Assuntos
Acetilcolinesterase/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Acetilcolinesterase/genética , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Inibidores da Colinesterase/farmacologia , DNA Complementar , Diclorvós/farmacologia , InseticidasRESUMO
The toxicity of potassium ferrocyanide (PFC) and protective effects of 2,4-dinitrophenol (DNP) under PFC treatment were tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with PFC at concentrations of 1.0 mM and mixtures with DNP in concentrations of 0.50 and 1.25 mM, either alone or in combination with 1.0 mM PFC. Food supplementation with PFC decreased larvae viability or pupation height, whereas when larvae were fed by PFC and DNP combination the decrease was less pronounced. Larval exposure to PFC and mixtures of DNP and PFC lowered activities of aconitase. Larval treatment with PFC resulted in higher carbonyl protein, uric acid, and low molecular mass thiols content and higher activity of thioredoxin reductase in adult flies, while DNP in mixtures with PFC relieved these effects. Furthermore, treatment with PFC/DNP mixtures resulted in higher activities of superoxide dismutase and glutathione-S-transferase. It is proposed that PFC toxicity is mainly related to the cyanide and iron ions, released during its decomposition. The potential mechanisms of protective DNP effects against PFC toxicity are discussed.
Assuntos
2,4-Dinitrofenol/farmacologia , Antídotos/toxicidade , Antioxidantes/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Ferrocianetos/toxicidade , Desacopladores/farmacologia , 2,4-Dinitrofenol/administração & dosagem , Ração Animal/análise , Animais , Antídotos/administração & dosagem , Dieta , Suplementos Nutricionais/análise , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Ferrocianetos/administração & dosagem , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/crescimento & desenvolvimento , Larva/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Pupa/efeitos dos fármacos , Pupa/enzimologia , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , Desacopladores/administração & dosagem , Desacopladores/metabolismoRESUMO
Superoxide dismutase (SOD, EC 1.15.1.1) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned cDNA encoding SOD activated with copper/zinc (CuZn SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of CuZn SOD was 692bp and had a 465bp open reading frame encoding 154 amino acids. The deduced amino acid sequence of B. calyciflorus CuZn SOD showed 63.87%, 60.00%, 59.74% and 48.89% similarity with the CuZn SOD of the Ctenopharyn godonidella, Schistosoma japonicum, Drosophila melanogaster and Caenorhabditis elegans, respectively. The phylogenetic tree constructed based on the amino acid sequences of CuZn SODs from B. calyciflorus and other organisms revealed that rotifer is closely related to nematode. Analysis of the expression of CuZn SOD under different temperatures (15, 30 and 37°C) revealed that its expression was enhanced 4.2-fold (p<0.001) at 30°C after 2h, however, the lower temperature (15°C) promoted CuZn SOD transiently (4.1-fold, p<0.001) and then the expression of CuZn SOD decreased to normal level (p>0.05). When exposed to H2O2 (0.1mM), CuZn SOD, manganese superoxide dismutase (Mn SOD) and catalase (CAT) gene were upregulated, and in addition, the mRNA expression of CuZn SOD gene was induced instantaneously after exposure to vitamin E. It indicates that the CuZn SOD gene would be an important gene in response to oxidative and temperature stress.
Assuntos
Rotíferos/enzimologia , Rotíferos/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Carpas/genética , Catalase/genética , Clonagem Molecular , DNA Complementar , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Peróxido de Hidrogênio , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Schistosoma japonicum/enzimologia , Schistosoma japonicum/genética , Análise de Sequência de DNA , Superóxido Dismutase/química , Temperatura , Vitamina ERESUMO
Curcumin is a polyphenolic bioactive compound in turmeric. We examined if antioxidant effects of curcumin are associated with lifespan extension in Drosophila. In this experiment, females and males of Drosophila were fed diets either containing no curcumin (C0) or supplemented with curcumin at 0.5 (C1) and 1.0 (C2) mg/g of diet. The levels of malondialdehyde (MDA), enzyme activity of superoxide dismutase (SOD), and expression of seven age-related genes in females and males were analyzed. We found that C1 and C2 increased mean lifespan by 6.2 % and 25.8 % in females, and by 15.5 % and 12.6 % in males, respectively. Meanwhile, C1 and C2 significantly decreased MDA levels and increased SOD activity in both genders. Diets C1 in females and C2 in males are effective in extending mean lifespan and improving levels of two physiological and biochemical measures related to aging in Drosophila. Lifespan extension of curcumin in Drosophila was associated with the up-regulation of Mn-SOD and CuZn-SOD genes, and the down-regulation of dInR, ATTD, Def, CecB, and DptB genes. The present results suggest that curcumin increases mean lifespan of Drosophila via regulating gene expression of the key enzyme SOD and reducing accumulation of MDA and lipid peroxidation. This study provided new insights for understanding the anti-aging mechanism of curcumin in Drosophila.
Assuntos
Envelhecimento/genética , Curcumina/farmacologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Longevidade/genética , RNA/genética , Superóxido Dismutase/genética , Envelhecimento/efeitos dos fármacos , Animais , Cromatografia Líquida de Alta Pressão , Suplementos Nutricionais , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/genética , Longevidade/efeitos dos fármacos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Superóxido Dismutase/biossínteseRESUMO
Superoxide dismutase 1 (SOD1), a critical enzyme against oxidative stress, is implicated in aging and degenerative diseases. We previously showed that a nutraceutical containing freeze-dried açai pulp promotes survival of flies fed a high-fat diet or sod1 knockdown flies fed a standard diet. Here, we investigated the effect of açai supplementation initiated at the early or late young adulthood on lifespan, physiological function, and oxidative damage in sod1 knockdown flies. We found that Açai supplementation extended lifespan even when started at the age of 10 days, which is the time shortly before the mortality rate of flies accelerated. Life-long açai supplementation increased lifetime reproductive output in sod1 knockdown flies. Our molecular studies indicate that açai supplementation reduced the protein levels of genes involved in oxidative stress response, cellular growth, and nutrient metabolism. Açai supplementation also affected the protein levels of ribosomal proteins. In addition, açai supplementation decreased the transcript levels of genes involved in oxidative stress response and gluconeogenesis, while increasing the transcript levels of mitochondrial biogenesis genes. Moreover, açai supplementation reduced the level of 4-hydroxynonenal-protein adducts, a lipid peroxidation marker. Our findings suggest that açai supplementation promotes healthy aging in sod1-deficient flies partly through reducing oxidative damage, and modulating nutrient metabolism and oxidative stress response pathways. Our findings provide a foundation to further evaluate the viability of using açai as an effective dietary intervention to promote healthy aging and alleviate symptoms of diseases with a high level of oxidative stress.
Assuntos
Envelhecimento/fisiologia , Arecaceae , Suplementos Nutricionais , Drosophila melanogaster/genética , Frutas , Estresse Oxidativo/fisiologia , Extratos Vegetais/farmacologia , Animais , Dieta , Drosophila melanogaster/enzimologia , Liofilização , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Estresse Oxidativo , S-Nitrosoglutationa/toxicidade , Aconitato Hidratase/metabolismo , Animais , Biomarcadores/metabolismo , Catalase/metabolismo , Meios de Cultura/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Ativação Enzimática , Comportamento Alimentar/efeitos dos fármacos , Feminino , Glutationa Transferase/metabolismo , Isocitrato Desidrogenase/metabolismo , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/metabolismo , Masculino , Nitritos/metabolismo , Carbonilação Proteica , Pupa/efeitos dos fármacos , Pupa/enzimologia , Pupa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , S-Nitrosoglutationa/administração & dosagem , Fatores de TempoRESUMO
Classic galactosemia is a genetic disorder that results from profound loss of galactose-1P-uridylyltransferase (GALT). Affected infants experience a rapid escalation of potentially lethal acute symptoms following exposure to milk. Dietary restriction of galactose prevents or resolves the acute sequelae; however, many patients experience profound long-term complications. Despite decades of research, the mechanisms that underlie pathophysiology in classic galactosemia remain unclear. Recently, we developed a Drosophila melanogaster model of classic galactosemia and demonstrated that, like patients, GALT-null Drosophila succumb in development if exposed to galactose but live if maintained on a galactose-restricted diet. Prior models of experimental galactosemia have implicated a possible association between galactose exposure and oxidative stress. Here we describe application of our fly genetic model of galactosemia to the question of whether oxidative stress contributes to the acute galactose sensitivity of GALT-null animals. Our first approach tested the impact of pro- and antioxidant food supplements on the survival of GALT-null and control larvae. We observed a clear pattern: the oxidants paraquat and DMSO each had a negative impact on the survival of mutant but not control animals exposed to galactose, and the antioxidants vitamin C and α-mangostin each had the opposite effect. Biochemical markers also confirmed that galactose and paraquat synergistically increased oxidative stress on all cohorts tested but, interestingly, the mutant animals showed a decreased response relative to controls. Finally, we tested the expression levels of two transcripts responsive to oxidative stress, GSTD6 and GSTE7, in mutant and control larvae exposed to galactose and found that both genes were induced, one by more than 40-fold. Combined, these results implicate oxidative stress and response as contributing factors in the acute galactose sensitivity of GALT-null Drosophila and, by extension, suggest that reactive oxygen species might also contribute to the acute pathophysiology in classic galactosemia.