RESUMO
Essential oils extracted from plant sources along with their biologically active components may have negative effects on insects. Diallyl trisulfide (DAT) is an active component of garlic essential oil, and it exhibits multi-targeted activity against many organisms. Previously we reported that DAT induces male infertility and leads to apyrene and eupyrene sperm dysfunction in Sitotroga cerealella. In this study, we conducted an analysis of testis-specific RNA-Seq data and identified 449 downregulated genes and 60 upregulated genes in the DAT group compared to the control group. The downregulated genes were significantly enriched in the ubiquitin-proteasome pathway. Furthermore, DAT caused a significant reduction in mRNA expression of proteasome regulatory subunit particles required for ATP-dependent degradation of ubiquitinated proteins as well as decreased the expression profile of proteasome core particles, including ß1, ß2, and ß5. Sperm physiological analysis showed that DAT decreased the chymotrypsin-like activity of the 20S proteasome and formed aggresomes in spermatozoa. Overall, our findings suggest that DAT impairs the testis proteasome, ultimately causing male infertility characterized by oligoasthenoteratospermia due to disruption in sperm proteasome assembly in S. cerealella.
Assuntos
Infertilidade Masculina , Mariposas , Humanos , Animais , Masculino , Complexo de Endopeptidases do Proteassoma , Sêmen , Infertilidade Masculina/genética , UbiquitinasRESUMO
The structural plasticity of dendritic spines plays a critical role in NMDA-induced long-term potentiation (LTP) in the brain. The small GTPases RhoA and Ras are considered key regulators of spine morphology and enlargement. However, the regulatory interaction between RhoA and Ras underlying NMDA-induced spine enlargement is largely unknown. In this study, we found that Rho-kinase/ROCK, an effector of RhoA, phosphorylated SynGAP1 (a synaptic Ras-GTPase activating protein) at Ser842 and increased its interaction with 14-3-3ζ, thereby activating Ras-ERK signaling in a reconstitution system in HeLa cells. We also found that the stimulation of NMDA receptor by glycine treatment for LTP induction stimulated SynGAP1 phosphorylation, Ras-ERK activation, spine enlargement and SynGAP1 delocalization from the spines in striatal neurons, and these effects were prevented by Rho-kinase inhibition. Rho-kinase-mediated phosphorylation of SynGAP1 appeared to increase its dissociation from PSD95, a postsynaptic scaffolding protein located at postsynaptic density, by forming a complex with 14-3-3ζ. These results suggest that Rho-kinase phosphorylates SynGAP1 at Ser842, thereby activating the Ras-ERK pathway for NMDA-induced morphological changes in dendritic spines.
Assuntos
Espinhas Dendríticas , Potenciação de Longa Duração , Proteínas Ativadoras de ras GTPase , Proteínas 14-3-3/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , N-Metilaspartato/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Triacylglycerol lipase (TGL) is an essential lipid metabolism enzyme that also plays a critical role in energy metabolism; however, how it regulates other life processes is unknown. To investigate the functional role of TGL in moth reproduction, males Sitotroga cerealella were used as a model. The TGL gene was cloned and analysed. The results showed that the open reading frame of TGL was 1968 bp long and contained three conserved regions. TGL gene expression was higher in the larval and early adult stages than in the pupal stage, with the highest levels observed in the fat body, testis and accessory glands during the early adult stage. Moreover, after TGL in male adults was silenced through RNAi, the protein content in male accessory glands remained unchanged, and the spermatophore transferred into females mated with TGL-silenced males became small and empty; meanwhile, the number of apyrene sperm in the spermatophore was significantly reduced due to the reduction of apyrene sperm in males, which eventually led to the significant reduction of egg-laying amount. All of the findings suggest that TGL regulates the amount of sperm in male moths as well as the morphology and quality of spermatophores transferred to females after mating with treated males, implying that TGL is critical for Sitotroga cerealella's reproductive process.
Assuntos
Lipase/fisiologia , Reprodução , Testículo/metabolismo , Animais , Larva/metabolismo , Masculino , MariposasRESUMO
This study investigated the effects of diallyl trisulfide (DAT), an active substance from garlic essential oil, on the metabolism of the main energy substances of pre- and postmating males of Sitotroga cerealella. Males at 12 h postemergence were fumigated with DAT at a concentration (LC10 = 0.010 µL/L) in a glass jar for 7 h. The main energy metabolites from pre- and postmating males were determined, including protein, triglyceride, glycogen, total soluble sugar, trehalose, and trehalase. The contents of total protein and total soluble sugar and the trehalase activity of premating males were significantly increased following DAT treatment, whereas the contents of protein from the accessory gland, triglyceride, glycogen, and trehalose were significantly decreased after treatment. Additionally, after mating, the total protein and soluble sugar contents were significantly increased and the glycogen content was significantly decreased in the treatment group relative to the levels in controls, but there was no significant difference observed in triglyceride, accessory gland proteins, trehalose content, or trehalase activity between the treatment and control groups. Furthermore, the changes in the main energy substances between pre- and postmating in males after the DAT treatment (∆DAT) were smaller than those in the control group (∆CK). This result indicated that DAT can accelerate the rate of metabolism in males at LC10, leading to the accumulation of greater levels of total soluble sugar to support life activities and to the increased synthesis of proteins to resist an adverse environment.
RESUMO
Dopamine (DA) activates mitogen-activated protein kinase (MAPK) via protein kinase A (PKA)/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R) in the nucleus accumbens (NAc), thereby regulating reward-related behavior. However, how MAPK regulates reward-related learning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we perform proteomic analysis using affinity beads coated with cyclic AMP response element binding protein (CREB)-binding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identify more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We find that MAPK phosphorylates Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impairs cocaine-induced place preference, which is rescued by Npas4-wild-type (WT), but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Expressão Gênica/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Cocaína/farmacologia , Dopamina/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Proteômica/métodos , Receptores de Dopamina D1/metabolismo , Recompensa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
The Angoumois grain moth, Sitotroga cerealella (Olivier, 1789) (Lepidoptera: Gelechiidae), is primarily a pest of stored products, that feeds inside the grain as larvae inducing significant economic loss in various stored commodities. Our previous studies proved that garlic essential oil and its active substances inhibit oviposition in moths. To further explore the effect on reproductive behavior and accurately interpret the results in terms of effective control of the moth population, moths were treated with diallyl trisulfide (DATS), an active substance of garlic essential oil, at a dose of 0.015 µL/L in air (LC20, sub-lethal concentration). The results showed that fecundity and the proportion of viable eggs significantly decreased when the moths were treated with LC20 DATS. Furthermore, female circadian mating rhythms and calling periodicity changed significantly after treatment. Compared with controls, the peak in mating occurred approximately 1 h earlier on the first day after DATS treatment, while it was significantly later on days 2 and 3. Moreover, mating frequency declined in presence of DATS compared with the controls. The percentage of females engaging in calling behavior decreased significantly with time, to less than 50%, 2 days after treatment, while a high calling percentage (>80%) was recorded for control moths on all 4 days. In addition, DATS had an inhibitory effect on the mating duration of S. cerealella. Moreover, a significant reduction was observed in the amount of sex pheromones extracted 8 h and 9 h after treatment. Our findings suggested that DATS has the potential to manipulate the moth population at LC20 and would be an efficient alternative to synthetic insecticides for the control of pests having low toxicity to non-target organisms and ecosystems.
RESUMO
To further explore the structure activity relationships (SARs) of our previously discovered antifungal lead compound (1), a series of biphenyl imidazole analogues were designed, synthesized and evaluated for their in vitro antifungal activity. Many of the synthesized compounds showed excellent activity against Candida albicans and Candida tropicalis. Among these compounds, 2-F substituted analogue 12m displayed the most remarkable in vitro activity against C. albicans, C. neoformans, A. fumigatus and fluconazole-resistant C. alb. strains, which is superior or comparable to the activity of the reference drugs fluconazole and itraconazole. Notably, the compound 12m exhibited low inhibition profiles for various human cytochrome P450 isoforms and showed low toxicity to mammalian A549 cells and U87 cells. The SARs and binding mode established in this study will be useful for further lead optimization.
Assuntos
Antifúngicos/síntese química , Desenho de Fármacos , Imidazóis/química , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Sítios de Ligação , Compostos de Bifenilo/química , Candida albicans/efeitos dos fármacos , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Humanos , Imidazóis/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Relação Estrutura-AtividadeRESUMO
To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compound 1, a series of benzoheterocycle analogues were designed, synthesized and evaluated for their in vitro antifungal activity. The most promising compounds 13s and 14a exhibited excellent antifungal activity against C. albicans, C. neoformans, A. fumigatus and fluconazole-resistant C. albicans strains, that was superior or comparable to those of the reference drugs fluconazole and voriconazole. GC-MS analyses suggested that the novel compound 13s might have a similar mechanism to fluconazole by inhibiting fungal lanosterol 14α-demethylase (CYP51). Furthermore, compounds 13s and 14a exhibited low inhibition profiles for various human cytochrome P450 isoforms as well as excellent blood plasma stability.
Assuntos
Antifúngicos/síntese química , Desenho de Fármacos , Proteínas Fúngicas/metabolismo , Esterol 14-Desmetilase/metabolismo , Triazóis/química , Antifúngicos/química , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Sítios de Ligação , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Domínio Catalítico , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Estabilidade de Medicamentos , Fluconazol/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Esterol 14-Desmetilase/química , Esteróis/análise , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. Here, the authors offer in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer's disease.
Assuntos
Segmento Inicial do Axônio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Cones de Crescimento/metabolismo , Neuritos/metabolismo , Proteínas rho de Ligação ao GTP/genética , Animais , Segmento Inicial do Axônio/ultraestrutura , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Comunicação Celular , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Cones de Crescimento/ultraestrutura , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Neuritos/ultraestrutura , Neurogênese/genética , Imagem Óptica , Optogenética , Cultura Primária de Células , Transporte Proteico , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compounds (1), a series of aromatic heterocyclic derivatives were designed, synthesized and evaluated for in vitro antifungal activity. Many of the target compounds showed good inhibitory activity against Candida albicans and Cryptococcus neoformans. In particular, the isoxazole nuclei were more suited for improving the activity against Aspergillus spp. Among these compounds, 2-F substituted analogues 23g and 23h displayed the most remarkable in vitro activity against Candida spp., C. neoformans, A. fumigatus and fluconazole-resistant C.alb. strains, which is superior or comparable to the activity of the reference drugs fluconazole and voriconazole. Notably, the compounds 23g and 23h exhibited low inhibition profiles for various isoforms of human cytochrome P450 and excellent blood plasma stability.