RESUMO
PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 (PECT1) regulates phosphatidylethanolamine biosynthesis and controls the phosphatidylethanolamine:phosphatidylcholine ratio in Arabidopsis thaliana Previous studies have suggested that PECT1 regulates flowering time by modulating the interaction between phosphatidylcholine and FLOWERING LOCUS T (FT), a florigen, in the shoot apical meristem (SAM). Here, we show that knockdown of PECT1 by artificial microRNA in the SAM (pFD::amiR-PECT1) accelerated flowering under inductive and even non-inductive conditions, in which FT transcription is almost absent, and in ft-10 twin sister of ft-1 double mutants under both conditions. Transcriptome analyses suggested that PECT1 affects flowering by regulating SHORT VEGETATIVE PHASE (SVP) and GIBBERELLIN 20 OXIDASE 2 (GA20ox2). SVP misexpression in the SAM suppressed the early flowering of pFD::amiR-PECT1 plants. pFD::amiR-PECT1 plants showed increased gibberellin (GA) levels in the SAM, concomitant with the reduction of REPRESSOR OF GA1-3 levels. Consistent with this, GA treatment had little effect on flowering time of pFD::amiR-PECT1 plants and the GA antagonist paclobutrazol strongly affected flowering in these plants. Together, these results suggest that PECT1 also regulates flowering time through a florigen-independent pathway, modulating SVP expression and thus regulating GA production.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Florígeno/metabolismo , Flores/fisiologia , Nucleotidiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Meristema/metabolismo , Oxigenases de Função Mista/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genéticaRESUMO
The present study aims to explore the probable anti-adipogenesis effect of Dictyopteris divaricata (D. divaricata) in 3T3-L1 preadipocytes by regulating heme oxygenase-1 (HO-1). The extract of D. divaricata retarded lipid accretion and decreased triglyceride (TG) content in 3T3-L1 adipocytes but increased free glycerol levels. Treatment with the extract inhibited lipogenesis by inhibiting protein expressions of fatty acid synthase (FAS) and lipoprotein lipase (LPL), whereas lipolysis increased by activating phosphorylation of hormone-sensitive lipase (p-HSL) and AMP-activated protein kinase (p-AMPK). The extract inhibited adipocyte differentiation of 3T3-L1 preadipocytes through down-regulating adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1). This is attributed to the triggering of Wnt/ß-catenin signaling. In addition, this study found that treatment with the extract activated HO-1 expression. Pharmacological approaches revealed that treatment with Zinc Protoporphyrin (ZnPP), an HO-1 inhibitor, resulted in an increase in lipid accumulation and a decrease in free glycerol levels. Finally, three adipogenic transcription factors, such as PPARγ, C/EBPα, and SREBP1, restored their expression in the presence of ZnPP. Analysis of chemical constituents revealed that the extract of D. divaricata is rich in 1,4-benzenediol, 7-tetradecenal, fucosterol, and n-hexadecanoic acid, which are known to have multiple pharmacological properties.
Assuntos
Adipogenia , Phaeophyceae , Animais , Camundongos , Lipólise , Células 3T3-L1 , Heme Oxigenase-1/metabolismo , PPAR gama/metabolismo , Glicerol/farmacologia , Glicerol/metabolismo , Diferenciação Celular , Adipócitos , Proteína alfa Estimuladora de Ligação a CCAAT , Fatores de Transcrição/metabolismo , Lipídeos/farmacologiaRESUMO
White adipose tissue (WAT) is a key regulator of systemic energy metabolism, and impaired WAT plasticity characterized by enlargement of preexisting adipocytes associates with WAT dysfunction, obesity, and metabolic complications. However, the mechanisms that retain proper adipose tissue plasticity required for metabolic fitness are unclear. Here, we comprehensively showed that adipocyte-specific DNA methylation, manifested in enhancers and CTCF sites, directs distal enhancer-mediated transcriptomic features required to conserve metabolic functions of white adipocytes. Particularly, genetic ablation of adipocyte Dnmt1, the major methylation writer, led to increased adiposity characterized by increased adipocyte hypertrophy along with reduced expansion of adipocyte precursors (APs). These effects of Dnmt1 deficiency provoked systemic hyperlipidemia and impaired energy metabolism both in lean and obese mice. Mechanistically, Dnmt1 deficiency abrogated mitochondrial bioenergetics by inhibiting mitochondrial fission and promoted aberrant lipid metabolism in adipocytes, rendering adipocyte hypertrophy and WAT dysfunction. Dnmt1-dependent DNA methylation prevented aberrant CTCF binding and, in turn, sustained the proper chromosome architecture to permit interactions between enhancer and dynamin-1-like protein gene Dnm1l (Drp1) in adipocytes. Also, adipose DNMT1 expression inversely correlated with adiposity and markers of metabolic health but positively correlated with AP-specific markers in obese human subjects. Thus, these findings support strategies utilizing Dnmt1 action on mitochondrial bioenergetics in adipocytes to combat obesity and related metabolic pathology.
Assuntos
Adipócitos/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Epigênese Genética , Dinâmica Mitocondrial , Adipócitos/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Adiposidade , Animais , Fator de Ligação a CCCTC/metabolismo , Estruturas Cromossômicas , DNA (Citosina-5-)-Metiltransferase 1/deficiência , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Dinaminas/genética , Dinaminas/metabolismo , Metabolismo Energético , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Metabolismo dos Lipídeos , Camundongos , Mitocôndrias/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Angiotensin I-converting enzyme (ACE) is an important blood pressure regulator. In this study, we aimed to investigate the ACE-inhibitory effects of meroterpenoids isolated from the brown alga, Sargassum macrocarpum, and the molecular mechanisms underlying ACE inhibition. Four fractions of S. macrocarpum were prepared using hexane, chloroform, ethyl acetate, and water as solvents and analyzed for their potential ACE-inhibitory effects. The chloroform fraction showed the strongest ACE-inhibitory effect, with an IC50 value of 0.18 mg/mL. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, were isolated from the chloroform fraction. Meroterpenoids isolated from S. macrocarpum had IC50 values of 0.44, 0.37, and 0.14 mM. The molecular docking study revealed that the ACE-inhibitory effect of the isolated meroterpenoids was mainly attributed to Zn-ion, hydrogen bonds, pi-anion, and pi-alkyl interactions between the meroterpenoids and ACE. These results suggest that S. macrocarpum could be a potential raw material for manufacturing antihypertensive nutraceutical ingredients.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Sargassum , Inibidores da Enzima Conversora de Angiotensina/química , Simulação de Acoplamento Molecular , Sargassum/química , Peptidil Dipeptidase A/química , ClorofórmioRESUMO
Sargassum horneri is a seaweed species with diverse bioactivities. However, its antifibrotic effects during nasal polyp (NP) formation are not clearly understood. Therefore, we investigated the inhibitory effect of S. horneri on fibrosis progression in NP-derived fibroblasts (NPDFs) and NP tissues ex vivo. NPDFs were stimulated with TGF-ß1 in the presence or absence of S. horneri ethanol extract (SHE). The extracellular matrix (ECM) protein production levels, myofibroblast differentiation (α-smooth muscle actin, α-SMA), and phosphorylation of Smad 2/3 and -ERK in TGF-ß1-stimulated NPDFs were investigated using western blotting. Further, the contractile activity of SHE was assessed by performing a collagen gel contraction assay. The expression levels of collagen-1, fibronectin, and α-SMA were investigated in NP organ cultures treated with SHE. TGF-ß1 stimulated ECM protein expression, myofibroblast differentiation, and collagen contractile activity while these were attenuated by pretreatment with SHE. We also found antifibrotic effect of SHE on ex vivo NP tissues. The antifibrotic effects of SHE were modulated through the attenuation of Smad 2/3 and ERK signaling pathways in TGF-ß1-stimulated NPDFs. In conclusion, SHE inhibited ECM protein accumulation and myofibroblast differentiation during NP remodeling. Thus, SHE may be helpful as a treatment for NP recurrence after endoscopic sinus surgery.
RESUMO
Oxidative stress-induced neuronal cell loss is considered to be the major mechanism underlying the pathogenesis of neurodegenerative diseases, which could be induced by a high concentration of glutamate. In this study, sargachromenol (SC) was isolated from a marine brown seaweed Sargassum horneri (S. horneri) and its neuroprotective effects against glutamate-induced oxidative stress in HT22 cells were investigated. An MTT assay was applied to assess the cytotoxicity of the SC, and the efficacies of SC were determined by flow cytometry, an analysis of ROS production, quantitative Real-Time PCR, and the Western blot assay. Our results showed that the pretreatment of SC reduced glutamate-induced apoptosis in HT22 cells via inhibiting the sub-G1 population, DNA fragmentation, and nuclear condensation, as well as up-regulating anti-apoptotic protein (Bcl-2) and down-regulating apoptotic proteins (Bax, p53, cleaved-PARP, caspase-3, caspase-9, and cytochrome c). Additionally, SC attenuated glutamate-induced oxidative stress by suppressing mitogen-activated protein kinases (MAPKs;ERK, JNK, and p38) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling (IκBα and NF-κB p65), while activating nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling (Nrf2; HO-1, and NQO-1). Our results suggest that SC could be used as a pharmacological candidate for the prevention and treatment of neurodegenerative diseases.
Assuntos
Heme Oxigenase-1 , Sargassum , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Sargassum/metabolismo , Ácido Glutâmico/toxicidade , Ácido Glutâmico/metabolismo , Estresse Oxidativo , Morte Celular , Transdução de SinaisRESUMO
Ecklonia maxima is a brown seaweed, which is abundantly distributed in South Africa. This study investigated an efficient approach using high-performance centrifugal partition chromatography (HPCPC), which has been successfully developed for the isolation and purification of phlorotannins, eckmaxol, and dieckol from the ethyl acetate fraction of E. maxima (EEM). We evaluated EEM for its inhibitory effect against lipopolysaccharide (LPS)-induced inflammatory responses in zebrafish embryos. The separation of eckmaxol and dieckol from samples of EEM using HPCPC was found to be of high purity and yield under an optimal solvent system composed of n-hexane:ethyl acetate:methanol:water (2:7:3:7, v/v/v/v). To evaluate the anti-inflammatory efficacy of EEM containing active compounds, zebrafish embryos exposed to LPS were compared with and without EEM treatment for nitric oxide (NO) production, reactive oxygen species (ROS) generation, and cell death two days after fertilization. These evaluations indicate that EEM alleviated inflammation by inhibiting cell death, ROS, and NO generation induced by LPS treatment. According to these results, eckmaxol and dieckol isolated from brown seaweed E. maxima could be considered effective anti-inflammatory agents as pharmaceutical and functional food ingredients.
Assuntos
Phaeophyceae , Alga Marinha , Animais , Anti-Inflamatórios/farmacologia , Cromatografia Líquida , Lipopolissacarídeos/farmacologia , Óxido Nítrico/metabolismo , Phaeophyceae/química , Espécies Reativas de Oxigênio/metabolismo , Alga Marinha/metabolismo , África do Sul , Peixe-Zebra/metabolismoRESUMO
The aim of this study was to assess the potential hypertensive effects of the IGTGIPGIW peptide purified from Hippocampus abdominalis alcalase hydrolysate (HA) for application in the functional food industry. We investigated the antihypertensive effects of IGTGIPGIW in vitro by assessing nitric oxide production in EA.hy926 endothelial cells, which is a major factor affecting vasorelaxation. The potential vasorelaxation effect was evaluated using 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, a fluorescent stain. IGTGIPGIW significantly increased the expression of endothelial-derived relaxing factors, including endothelial nitric oxide synthase and protein kinase B, in EA.hy926 cells. Furthermore, oral administration of IGTGIPGIW significantly lowered the systolic blood pressure (183.60 ± 1.34 mmHg) and rapidly recovered the diastolic blood pressure (143.50 ± 5.55 mmHg) in the spontaneously hypertensive rat model in vivo. Our results demonstrate the antihypertensive activity of the IGTGIPGIW peptide purified from H. abdominalis and indicate its suitability for application in the functional food industry.
Assuntos
Anti-Hipertensivos , Óxido Nítrico Sintase Tipo III , Smegmamorpha , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea , Células Endoteliais , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos SHRRESUMO
Accumulating evidence suggests that subcutaneous and visceral adipose tissues are differentially associated with metabolic disorders. In obesity, subcutaneous adipose tissue is beneficial for metabolic homeostasis because of repressed inflammation. However, the underlying mechanism remains unclear. Here, we demonstrate that γ-aminobutyric acid (GABA) sensitivity is crucial in determining fat depot-selective adipose tissue macrophage (ATM) infiltration in obesity. In diet-induced obesity, GABA reduced monocyte migration in subcutaneous inguinal adipose tissue (IAT), but not in visceral epididymal adipose tissue (EAT). Pharmacological modulation of the GABAB receptor affected the levels of ATM infiltration and adipose tissue inflammation in IAT, but not in EAT, and GABA administration ameliorated systemic insulin resistance and enhanced insulin-dependent glucose uptake in IAT, accompanied by lower inflammatory responses. Intriguingly, compared with adipose-derived stem cells (ADSCs) from EAT, IAT-ADSCs played key roles in mediating GABA responses that repressed ATM infiltration in high-fat diet-fed mice. These data suggest that selective GABA responses in IAT contribute to fat depot-selective suppression of inflammatory responses and protection from insulin resistance in obesity.
Assuntos
Tecido Adiposo/metabolismo , Inflamação/metabolismo , Obesidade/metabolismo , Células-Tronco/metabolismo , Tela Subcutânea/metabolismo , Ácido gama-Aminobutírico/metabolismo , Adipócitos/metabolismo , Adiposidade/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Humanos , Insulina/metabolismo , Gordura Intra-Abdominal/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.
Assuntos
Colesterol/análogos & derivados , Lipossomos/química , Fosfatidiletanolaminas/química , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Bevacizumab/biossíntese , Bevacizumab/genética , Células CHO , Colesterol/química , Colesterol/metabolismo , Cricetulus , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/genética , Cinética , Lipossomos/metabolismo , Fosfatidiletanolaminas/metabolismo , Plasmídeos/química , Eletricidade Estática , TransgenesRESUMO
Dryopteris crassirhizoma rhizomes are used as a traditional medicine in Asia. The EtOAc extract of these roots has shown potent xanthine oxidase (XO) inhibitory activity. However, the main phloroglucinols in D. crassirhizoma rhizomes have not been analyzed. Thus, we investigated the major constituents responsible for this effect. Bioassay-guided purification isolated four compounds: flavaspidic acid AP (1), flavaspidic acid AB (2), flavaspidic acid PB (3), and flavaspidic acid BB (4). Among these, 1 showed the most potent inhibitory activity with a half-maximal inhibitory concentration (IC50) value of 6.3 µM, similar to that of allopurinol (IC50 = 5.7 µM) and better than that of oxypurinol (IC50 = 43.1 µM), which are XO inhibitors. A comparative activity screen indicated that the acetyl group at C3 and C3' is crucial for XO inhibition. For example, 1 showed nearly 4-fold higher efficacy than 4 (IC50 = 20.9 µM). Representative inhibitors (1-4) in the rhizomes of D. crassirhizoma showed reversible and noncompetitive inhibition toward XO. Furthermore, the potent inhibitors were shown to be present in high quantities in the rhizomes by a UPLC-QTOF-MS analysis. Therefore, the rhizomes of D. crassirhizoma could be used to develop nutraceuticals and medicines for the treatment of gout.
Assuntos
Dryopteris/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/farmacologia , Xantina Oxidase/antagonistas & inibidores , Butirofenonas/química , Butirofenonas/farmacologia , Humanos , Hiperuricemia/tratamento farmacológico , Hiperuricemia/enzimologia , Rizoma/química , Xantina Oxidase/metabolismoRESUMO
BACKGROUND: Nitrogen is an essential nutrient in plants. Despite the importance of nitrogen for plant growth and agricultural productivity, signal transduction pathways in response to nitrate starvation have not been fully elucidated in plants. RESULTS: Gene expression analysis and ectopic expression were used to discover that many CC-type glutaredoxins (ROXYs) are differentially expressed in response to nitrate deprivation. A gain-of-function approach showed that ROXYs may play a role in nutrient sensing through the regulation of chlorophyll content, root hair growth, and transcription of nitrate-related genes such as NRT2.1 under low or high nitrate conditions. Reactive oxygen species (ROS) were produced in plant roots under nitrate starvation and H2O2 treatment differentially regulated the expression of the ROXYs, suggesting the involvement of ROS in signaling pathways under nitrate deficiency. CONCLUSION: This work adds to what is known about nitrogen sensing and signaling through the findings that the ROXYs and ROS are likely to be involved in the nitrate deprivation signaling pathway.
Assuntos
Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Glutarredoxinas/metabolismo , Nitratos/metabolismo , Nitrogênio/metabolismo , Transdução de Sinais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Glutarredoxinas/genética , Peróxido de Hidrogênio/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Plântula/fisiologiaRESUMO
Protein transport between organelles is an essential process in all eukaryotic cells and is mediated by the regulation of processes such as vesicle formation, transport, docking, and fusion. In animals, SCY1-LIKE2 (SCYL2) binds to clathrin and has been shown to play roles in trans-Golgi network-mediated clathrin-coated vesicle trafficking. Here, we demonstrate that SCYL2A and SCYL2B, which are Arabidopsis (Arabidopsis thaliana) homologs of animal SCYL2, are vital for plant cell growth and root hair development. Studies of the SCYL2 isoforms using multiple single or double loss-of-function alleles show that SCYL2B is involved in root hair development and that SCYL2A and SCYL2B are essential for plant growth and development and act redundantly in those processes. Quantitative reverse transcription-polymerase chain reaction and a ß-glucuronidase-aided promoter assay show that SCYL2A and SCYL2B are differentially expressed in various tissues. We also show that SCYL2 proteins localize to the Golgi, trans-Golgi network, and prevacuolar compartment and colocalize with Clathrin Heavy Chain1 (CHC1). Furthermore, bimolecular fluorescence complementation and coimmunoprecipitation data show that SCYL2B interacts with CHC1 and two Soluble NSF Attachment Protein Receptors (SNAREs): Vesicle Transport through t-SNARE Interaction11 (VTI11) and VTI12. Finally, we present evidence that the root hair tip localization of Cellulose Synthase-Like D3 is dependent on SCYL2B. These findings suggest the role of SCYL2 genes in plant cell developmental processes via clathrin-mediated vesicle membrane trafficking.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Desenvolvimento Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/genética , Cadeias Pesadas de Clatrina/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Qb-SNARE/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIß via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo dos Lipídeos/fisiologia , Células 3T3-L1 , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , CamundongosRESUMO
The response of shoots to phosphate (Pi) deficiency implicates long-distance communication between roots and shoots, but the participating components are poorly understood. We have studied the topology of the Arabidopsis (Arabidopsis thaliana) PHOSPHATE1 (PHO1) Pi exporter and defined the functions of its different domains in Pi homeostasis and signaling. The results indicate that the amino and carboxyl termini of PHO1 are both oriented toward the cytosol and that the protein spans the membrane twice in the EXS domain, resulting in a total of six transmembrane α-helices. Using transient expression in Nicotiana benthamiana leaf, we demonstrated that the EXS domain of PHO1 is essential for Pi export activity and proper localization to the Golgi and trans-Golgi network, although the EXS domain by itself cannot mediate Pi export. In contrast, removal of the amino-terminal hydrophilic SPX domain does not affect the Pi export capacity of the truncated PHO1 in N. benthamiana. While the Arabidopsis pho1 mutant has low shoot Pi and shows all the hallmarks associated with Pi deficiency, including poor shoot growth and overexpression of numerous Pi deficiency-responsive genes, expression of only the EXS domain of PHO1 in the roots of the pho1 mutant results in a remarkable improvement of shoot growth despite low shoot Pi. Transcriptomic analysis of pho1 expressing the EXS domain indicates an attenuation of the Pi signaling cascade and the up-regulation of genes involved in cell wall synthesis and the synthesis or response to several phytohormones in leaves as well as an altered expression of genes responsive to abscisic acid in roots.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfatos/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Transdução de Sinais , Nicotiana/genética , Rede trans-Golgi/metabolismoRESUMO
Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.
Assuntos
Cloroplastos/enzimologia , Flores/fisiologia , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Helianthus/enzimologia , Nicotiana/crescimento & desenvolvimento , Nicotiana/genética , Sementes/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Biomassa , Carotenoides/metabolismo , Clorofila/metabolismo , Cruzamentos Genéticos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/anatomia & histologia , Plantas Geneticamente Modificadas , Transporte Proteico , Frações Subcelulares/enzimologia , Taraxacum/genética , Taraxacum/crescimento & desenvolvimento , TransgenesRESUMO
The active constituents of Korean Papaver rhoeas bee pollen conferring neuraminidase inhibitory activities (H1N1, H3N2, and H5N1) were investigated. Six flavonoids and one alkaloid were isolated and characterized by nuclear magnetic resonance and mass spectrometry data. These included kaempferol-3-sophoroside (1), kaempferol-3-neohesperidoside (2), kaempferol-3-sambubioside (3), kaempferol-3-glucoside (4), quercetin-3-sophoroside (5), luteolin (6), and chelianthifoline (7). All compounds showed neuraminidase inhibitory activities with IC50 values ranging from 10.7 to 151.1 µM. The most potent neuraminidase inhibitor was luteolin, which was the dominant content in the ethyl acetate fraction. All tested compounds displayed noncompetitive inhibition of H3N2 neuraminidase. Furthermore, compounds 1-7 all reduced the severity of virally induced cytopathic effects as determined by the Madin-Darby canine kidney cell-based assay showing antiviral activity with IC50 values ranging from 10.7 to 33.4 µM (zanamivir: 58.3 µM). The active compounds were quantified by high-performance liquid chromatography, and the total amount of compounds 1-7 made up about 0.592 g/100 g bee pollen, contributing a rich resource of a natural antiviral product.
Assuntos
Antivirais/química , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Papaver/química , Pólen/química , Animais , Abelhas , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Three new nonenes, verrucanonenes AâC (1â3), were isolated from culture broth of marine-derived fungus Albifimbria verrucaria. These compounds were isolated using silica gel column chromatography, reversed-phase medium pressure liquid chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. Their structures were determined using a spectroscopic method. Cytotoxicities of these isolated compounds to A549, DU145, HCT116, and HT1080 cancer cell lines were assessed. Compounds 1â3 exhibited cytotoxicities to DU145 cancer cell line, with IC50 values of 23.4, 28.6, and 20.1 µM, respectively. Compound 2 decreased H1N1-induced cytopathic effects on MDCK cells in a dose-dependent manner.
Assuntos
Antineoplásicos , Antivirais , Humanos , Antivirais/farmacologia , Antivirais/isolamento & purificação , Antivirais/química , Linhagem Celular Tumoral , Animais , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Cães , Células Madin Darby de Rim Canino , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Ascomicetos/química , Concentração Inibidora 50 , Cromatografia Líquida de Alta Pressão , Estrutura Molecular , Relação Dose-Resposta a DrogaRESUMO
Ambient temperature affects flowering time in plants, and the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a crucial role in the response to changes in ambient temperature. SVP protein stability is regulated by the 26S proteasome pathway and decreases at high ambient temperature, but the details of SVP degradation are unclear. Here, we show that SVP degradation at high ambient temperature is mediated by the CULLIN3-RING E3 ubiquitin ligase (CRL3) complex in Arabidopsis thaliana. We identified a previously uncharacterized protein that interacts with SVP at high ambient temperature and contains a BTB/POZ domain. We named this protein LATE FLOWERING AT HIGH TEMPERATURE 1 (LFH1). Single mutants of LFH1 or CULLIN3A (CUL3A) showed late flowering specifically at 27°C. LFH1 protein levels increased at high ambient temperature. We found that LFH1 interacts with CUL3A in the cytoplasm and is important for SVP-CUL3A complex formation. Mutations in CUL3A and/or LFH1 led to increased SVP protein stability at high ambient temperature, suggesting that the CUL3-LFH1 complex functions in SVP degradation. Screening E2 ubiquitin-conjugating enzymes (UBCs) using RING-BOX PROTEIN 1 (RBX1), a component of the CRL3 complex, as bait identified UBC15. ubc15 mutants also showed late flowering at high ambient temperature. In vitro and in vivo ubiquitination assays using recombinant CUL3A, LFH1, RBX1, and UBC15 showed that SVP is highly ubiquitinated in an ATP-dependent manner. Collectively, these results indicate that the degradation of SVP at high ambient temperature is mediated by a CRL3 complex comprising CUL3A, LFH1, and UBC15.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ubiquitina-Proteína Ligases , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ligases/metabolismo , Temperatura , Ubiquitinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Obesity has emerged as a prominent risk factor for the development of malignant tumors. However, the existing literature on the role of adipocytes in the tumor microenvironment (TME) to elucidate the correlation between obesity and cancer remains insufficient. Here, we aim to investigate the formation of cancer-associated adipocytes (CAAs) and their contribution to tumor growth using mouse models harboring dysfunctional adipocytes. Specifically, we employ adipocyte-specific BECN1 KO (BaKO) mice, which exhibit lipodystrophy due to dysfunctional adipocytes. Our results reveal the activation of YAP/TAZ signaling in both CAAs and BECN1-deficient adipocytes, inducing adipocyte dedifferentiation and formation of a malignant TME. The additional deletion of YAP/TAZ from BaKO mice significantly restores the lipodystrophy and inflammatory phenotypes, leading to tumor regression. Furthermore, mice fed a high-fat diet (HFD) exhibit decreased BECN1 and increased YAP/TAZ expression in their adipose tissues. Treatment with the YAP/TAZ inhibitor, verteporfin, suppresses tumor progression in BaKO and HFD-fed mice, highlighting its efficacy against mice with metabolic dysregulation. Overall, our findings provide insights into the key mediators of CAA and their significance in developing a TME, thereby suggesting a viable approach targeting adipocyte homeostasis to suppress cancer growth.