RESUMEN
Group III hybrid histidine kinases are fungal-specific proteins and part of the multistep phosphorelay, representing the initial part of the high osmolarity glycerol (HOG) pathway. TcsC, the corresponding kinase in Aspergillus fumigatus, was expected to be a cytosolic protein but is targeted to the nucleus. Activation of TcsC by the antifungal fludioxonil has lethal consequences for the fungus. The agent triggers a fast and TcsC-dependent activation of SakA and later on a redistribution of TcsC to the cytoplasm. High osmolarity also activates TcsC, which then exits the nucleus or concentrates in spot-like, intra-nuclear structures. The sequence corresponding to the N-terminal 208 amino acids of TcsC lacks detectable domains. Its loss renders TcsC cytosolic and non-responsive to hyperosmotic stress, but it has no impact on the antifungal activity of fludioxonil. A point mutation in one of the three putative nuclear localization sequences, which are present in the N-terminus, prevents the nuclear localization of TcsC, but not its ability to respond to hyperosmotic stress. Hence, this striking intracellular localization is no prerequisite for the role of TcsC in the adaptive response to hyperosmotic stress, instead, TcsC proteins that are present in the nuclei seem to modulate the cell wall composition of hyphae, which takes place in the absence of stress. The results of the present study underline that the spatiotemporal dynamics of the individual components of the multistep phosphorelay is a crucial feature of this unique signaling hub. IMPORTANCE: Signaling pathways enable pathogens, such as Aspergillus fumigatus, to respond to a changing environment. The TcsC protein is the major sensor of the high osmolarity glycerol (HOG) pathway of A. fumigatus and it is also the target of certain antifungals. Insights in its function are therefore relevant for the pathogenicity and new therapeutic treatment options. TcsC was expected to be cytoplasmic, but we detected it in the nucleus and showed that it translocates to the cytoplasm upon activation. We have identified the motif that guides TcsC to the nucleus. An exchange of a single amino acid in this motif prevents a nuclear localization, but this nuclear targeting is no prerequisite for the TcsC-mediated stress response. Loss of the N-terminal 208 amino acids prevents the nuclear localization and renders TcsC unable to respond to hyperosmotic stress demonstrating that this part of the protein is of crucial importance.
Asunto(s)
Aspergillus fumigatus , Núcleo Celular , Dioxoles , Proteínas Fúngicas , Histidina Quinasa , Pirroles , Aspergillus fumigatus/genética , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/efectos de los fármacos , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/química , Núcleo Celular/metabolismo , Pirroles/farmacología , Pirroles/metabolismo , Dioxoles/farmacología , Dioxoles/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Antifúngicos/farmacología , Antifúngicos/metabolismo , Presión Osmótica , Citoplasma/metabolismo , Transporte de Proteínas , Regulación Fúngica de la Expresión Génica , Concentración Osmolar , Transducción de SeñalRESUMEN
Lignans are widely distributed plant secondary metabolites that have received attention for their benefits to human health. Sesamin is a furofran lignan that is conventionally extracted from Sesamum seeds and shows anti-oxidant and anti-inflammatory activities in the human liver. Sesamin is biosynthesized by the Sesamum-specific enzyme CYP81Q1, and the natural sources of sesamin are annual plants that are at risk from climate change. In contrast, Forsythia species are widely distributed perennial woody plants that highly accumulate the precursor lignan pinoresinol. To sustainably supply sesamin, we developed a transformation method for Forsythia leaf explants and generated transgenic Forsythia plants that heterologously expressed the CYP81Q1 gene. High-performance liquid chromatography (HPLC) and LC-mass spectrometry analyses detected sesamin and its intermediate piperitol in the leaves of two independent transgenic lines of F. intermedia and F. koreana. We also detected the accumulation of sesamin and piperitol in their vegetatively propagated descendants, demonstrating the stable and efficient production of these lignans. These results indicate that CYP81Q1-transgenic Forsythia plants are promising prototypes to produce diverse lignans and provide an important strategy for the cost-effective and scalable production of lignans.
Asunto(s)
Forsythia , Lignanos , Sesamum , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Dioxoles/metabolismo , Forsythia/genética , Forsythia/metabolismo , Humanos , Lignanos/química , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Sesamum/metabolismoRESUMEN
Sesamolin is one of the major active compounds found in sesame seeds (Sesamum indicum L.) that are commonly and increasingly used as an ingredient in cuisines and various food products. The compound has been reported to have several pharmaceutical activities such as antioxidant, antimicrobial, neuroprotective, and anticancer. However, the toxicological profile of sesamolin does not currently include developmental toxicity. In this study, we assessed sesamolin toxicity to embryonic development of zebrafish by exposure for 72 h at concentrations ranging from 10 to 50 µM. The evaluation revealed that sesamolin did not affect survival and hatching rates. However, it did induce embryo malformations and reduced embryonic heart rates in a dose-dependent manner. By qRT-PCR analysis, it downregulated the expression of oxidative stress-related genes, including superoxide dismutase 1 (sod1), catalase (cat), and glutathione S-transferase pi 2 (gstp2). Alkaline phosphatase staining of embryos revealed that sesamolin inhibited the development of subintestinal vessels, and hemoglobin staining revealed a negative impact on embryonic erythropoiesis. These findings showed that sesamolin affected genes related to angiogenesis and erythropoiesis. The risks of sesamolin to embryonic development found in this study may imply similar effects in humans and other mammals.
Asunto(s)
Embrión no Mamífero , Pez Cebra , Animales , Dioxoles/metabolismo , Dioxoles/farmacología , Mamíferos , Estrés Oxidativo , Pez Cebra/metabolismoRESUMEN
Sesamin and episesamin are the main lignans found in refined sesame oil and have been reported to exert various health benefits. However, the health benefits of these lignans and their molecular mechanisms have not been fully understood. This study evaluated the effects of sesamin, episesamin, and their metabolites on the nuclear bile acid receptor, farnesoid X receptor (FXR, NR1H4), which regulate gene expression involved in bile acid metabolism and gluconeogenesis. By using two different cell-based luciferase reporter assay systems, we found that sesamin, sesamin metabolites, and some episesamin metabolites inhibited FXR activation driven by a bile acid and a synthesized agonist, and it is suggested that these compounds exert their antagonist activity by competing with the FXR agonists on the ligand-binding domain. Sesamin and its major metabolite SC-1 suppressed the expression of several gluconeogenesis-related genes governed by FXR in HepG2 cells but did not affect the expression level of CYP7A1, the rate-limiting enzyme for bile acid synthesis. Dietary sesamin supplementation (AIN-93G supplemented with 0.5% sesamin) led to the decreased hepatic expression of several gluconeogenesis-related genes and reduced blood glucose levels in mice, without adverse effects on bile acid metabolism. These results shed light on the health benefits of taking sesamin and episesamin.
Asunto(s)
Gluconeogénesis , Lignanos , Animales , Dioxoles/química , Dioxoles/metabolismo , Dioxoles/farmacología , Gluconeogénesis/genética , Lignanos/química , Lignanos/metabolismo , Lignanos/farmacología , Hígado/metabolismo , RatonesRESUMEN
Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. IMPORTANCE There are more than 9,000 polyfluorinated compounds developed for commercial use, some negatively impacting human health, and they are generally considered to be resistant to biodegradation. Only a limited number of studies have identified microbes with enzymes sufficiently reactive to defluorinate difluoromethylene carbon groups. The present study examined one important group of commercial fluorinated chemicals and showed its rapid defluorination by a bacterium and its key enzyme, a Rieske dioxygenase. Rieske dioxygenases are common in environmental bacteria, and those closely resembling toluene dioxygenase from Pseudomonas putida F1 are candidates for biodegradative defluorination of the common 2,2-fluoro-1,3-benzodioxole (DFBD) moiety.
Asunto(s)
Dioxoles/metabolismo , Pseudomonas putida/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Dioxoles/química , Halogenación , Oxigenasas/genética , Oxigenasas/metabolismo , Pseudomonas putida/química , Pseudomonas putida/enzimología , Pseudomonas putida/genéticaRESUMEN
Sesamum spp. (sesame) are known to accumulate a variety of lignans in a lineage-specific manner. In cultivated sesame (Sesamum indicum), (+)-sesamin, (+)-sesamolin and (+)-sesaminol triglucoside are the three major lignans found richly in the seeds. A recent study demonstrated that SiCYP92B14 is a pivotal enzyme that allocates the substrate (+)-sesamin to two products, (+)-sesamolin and (+)-sesaminol, through multiple reaction schemes including oxidative rearrangement of α-oxy-substituted aryl groups (ORA). In contrast, it remains unclear whether (+)-sesamin in wild sesame undergoes oxidation reactions as in S. indicum and how, if at all, the ratio of the co-products is tailored at the molecular level. Here, we functionally characterised SrCYP92B14 as a SiCYP92B14 orthologue from a wild sesame, Sesamum radiatum, in which we revealed accumulation of the (+)-sesaminol derivatives (+)-sesangolin and its novel structural isomer (+)-7´-episesantalin. Intriguingly, SrCYP92B14 predominantly produced (+)-sesaminol either through ORA or direct oxidation on the aromatic ring, while a relatively low but detectable level of (+)-sesamolin was produced. Amino acid substitution analysis suggested that residues in the putative distal helix and the neighbouring heme propionate of CYP92B14 affect the ratios of its co-products. These data collectively show that the bimodal oxidation mechanism of (+)-sesamin might be widespread across Sesamum spp., and that CYP92B14 is likely to be a key enzyme in shaping the ratio of (+)-sesaminol- and (+)-sesamolin-derived lignans from the biochemical and evolutionary perspectives.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Dioxoles/metabolismo , Lignanos/metabolismo , Sesamum/enzimología , Secuencia de Aminoácidos , Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/genética , Dioxoles/química , Furanos/química , Furanos/metabolismo , Glucósidos/química , Glucósidos/metabolismo , Lignanos/química , Modelos Moleculares , Oxidación-Reducción , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/química , Semillas/enzimología , Semillas/genética , Alineación de Secuencia , Sesamum/química , Sesamum/genéticaRESUMEN
Brasiliamides are a class of piperazine-containing alkaloids produced by Penicillium brasilianum with a range of pharmaceutical activities. The mechanism of brasiliamide biosynthesis, including piperazine ring formation and multiple tailoring modifications, still remains unclear. In this study, the biosynthetic gene cluster of brasiliamides, brs, was identified from the marine-derived fungal strain Penicillium brasilianum WZXY-M122-9. Deletion of a histone deacetylase-encoding gene using a CRISPR/Cas9 gene editing system led to the production of a new compound, namely brasiliamide I (1). The brs-encoded single-module nonribosomal peptide synthetase (NRPS) BrsA is involved in the formation of the piperazine skeleton of brasiliamides. Full-length BrsA protein (113.6 kDa) was purified, and reconstitution of enzymatic activity in vitro confirmed that BrsA stereoselectively accepts L-phenylalanine as the substrate. Multiple deletion of tailoring genes and analysis of purified proteins in vitro enabled us to propose a brasiliamide biosynthetic pathway. In the tailoring steps, an α-ketoglutarate (KG)-dependent nonheme iron dioxygenase, BrsJ, was identified to catalyze piperazine ring cleavage during biosynthesis of brasiliamide A (2). KEY POINTS: The gene cluster encoding brasiliamide biosynthesis, brs, is identified. Deletion of a histone deacetylase-encoding gene produces brasiliamide I. BrsA catalyzes brasiliamide piperazine skeleton formation. BrsJ catalyzes piperazine ring cleavage to produce brasiliamide A. Graphical abstract.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Dioxoles/metabolismo , Proteínas Fúngicas/metabolismo , Péptido Sintasas/metabolismo , Piperazina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Vías Biosintéticas/genética , Catálisis , Dioxoles/química , Dioxoles/aislamiento & purificación , Proteínas Fúngicas/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Estructura Molecular , Familia de Multigenes , Mutación , Penicillium/genética , Penicillium/metabolismo , Péptido Sintasas/genética , Piperazina/química , Piperazina/aislamiento & purificaciónRESUMEN
Pregnane X receptor (PXR) as a ligand dependent transcription factor, is capable of regulating gene expression of cytochromes P450 and transporters involved in xenobiotic/drug metabolism and elimination. Due to the species differences in the regulatory specificity of PXR, gene regulation should not be extrapolated from mammal to fish without research data.The aim of present study was to investigate the effect of 27 natural products on PXR, CYP3A30 and MDR1 genes in channel catfish (Ietalurus punetaus) kidney cells (CC-K). The results showed that bisdemethoxycurcumin, glycyrrhetnic acid, rotenone, artemisinin, dihydroartemisinin, ligustilide and matrine strongly induced the mRNA levels of PXR. Additionally, the up-regulation of CYP3A30 gene ran parallel with PXR gene after the treatment of demethoxycurcumin, glycyrrhetnic acid, artemisinin, matrine, baicalein, schisantherin A, ligustilide, and dihydroartemisinin. Moreover, we found that natural products schisandrin A, schisandrin B, schisandrol A, and schisandrol B significantly up-regulated the mRNA level of MDR1 gene.Our work with a view to provide experimental data support for further research, which will make for the rational application of natural products in channel catfish, such as to avoid adverse herb-drug interactions or accelerating the residue elimination of chemical medicine.
Asunto(s)
Productos Biológicos/farmacología , Biotransformación/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Productos Biológicos/metabolismo , Línea Celular , Ciclooctanos/metabolismo , Ciclooctanos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Dioxoles/metabolismo , Dioxoles/farmacología , Ictaluridae , Lignanos/metabolismo , Lignanos/farmacología , Compuestos Policíclicos/metabolismo , Compuestos Policíclicos/farmacología , Receptor X de Pregnano/metabolismoRESUMEN
Schisantherin A is an active ingredient originating from Schisandra chinensis (Turcz.) which has hepatoprotective and anti-oxidation activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on male Sprague Dawley rats of Schisantherin A were tested, respectively. The metabolites of Schisantherin A were identified using ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). Based on the method, 60 metabolites were successfully identified and structurally characterized including 48 phase-I and 12 phase-II metabolites. Among the metabolites, 45 metabolites were reported for the first time. Moreover, 56 and eight metabolites were detected in urine and bile and 19 metabolites were identified in rats' plasma. It demonstrated that hepatic and extra-hepatic metabolic pathways were both involved in Schisantherin A biotransformation in rats. Five in vitro metabolites were structurally characterized for the first time. The results indicated that the metabolic pathways mainly include oxidation, reduction, methylation, and conjugation with glucuronide, taurine, glucose, and glutathione groups. This study provides a practical strategy for rapidly screening and identifying metabolites, and the results provide basic data for future pharmacological and toxicology studies of Schisantherin A and other lignin ingredients.
Asunto(s)
Ciclooctanos/análisis , Ciclooctanos/metabolismo , Dioxoles/análisis , Dioxoles/metabolismo , Evaluación Preclínica de Medicamentos , Lignanos/análisis , Lignanos/metabolismo , Metaboloma , Espectrometría de Masas en Tándem , Animales , Cromatografía Líquida de Alta Presión , Ciclooctanos/química , Dioxoles/química , Iones , Lignanos/química , Masculino , Redes y Vías Metabólicas , Metabolómica , Oxidación-Reducción , Ratas Sprague-DawleyRESUMEN
The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.
Asunto(s)
Dioxoles/metabolismo , Antagonistas de los Receptores de Endotelina/administración & dosificación , Colorantes Fluorescentes/administración & dosificación , Infarto del Miocardio/metabolismo , Receptores de Endotelina/metabolismo , Animales , Crioultramicrotomía , Dioxoles/química , Modelos Animales de Enfermedad , Antagonistas de los Receptores de Endotelina/análisis , Antagonistas de los Receptores de Endotelina/química , Femenino , Colorantes Fluorescentes/análisis , Inmunohistoquímica , Indoles/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/diagnóstico por imagen , Neovascularización Fisiológica , Imagen Óptica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismoRESUMEN
Production of chrysanthemum (Dendranthema grandiflora) in greenhouses often requires intensive pesticide use, which raises serious concerns over food safety and human health. This study investigated uptake, translocation and residue dissipation of typical fungicides (metalaxyl-M and fludioxonil) and insecticides (cyantraniliprole and thiamethoxam) in greenhouse chrysanthemum when applied in soils. Chrysanthemum plants could absorb these pesticides from soils via roots to various degrees, and bioconcentration factors (BCFLS) were positively correlated with lipophilicity (log Kow) of pesticides. Highly lipophilic fludioxonil (log Kowâ¯=â¯4.12) had the greatest BCFLS (2.96⯱â¯0.41â¯gâ¯g-1), whereas hydrophilic thiamethoxam (log Kowâ¯=â¯-0.13) had the lowest (0.09⯱â¯0.03â¯gâ¯g-1). Translocation factors (TF) from roots to shoots followed the order of TFleafâ¯>â¯TFstemâ¯>â¯TFflower. Metalaxyl-M and cyantraniliprole with medium lipophilicity (log Kow of 1.71 and 2.02, respectively) and hydrophilic thiamethoxam showed relatively strong translocation potentials with TF values in the range of 0.29-0.81, 0.36-2.74 and 0.30-1.03, respectively. Dissipation kinetics in chrysanthemum flowers followed the first-order with a half-life of 21.7, 5.5, 10.0 or 8.2 days for metalaxyl-M, fludioxonil, cyantraniliprole and thiamethoxam, respectively. Final residues of these four pesticides, including clothianidin (a primary toxic metabolite of thiamethoxam), in all chrysanthemum flower samples were below the maximum residue limit (MRL) values 21 days after two soil applications each at the recommended dose (i.e., 3.2, 2.1, 4.3 and 4.3â¯kgâ¯ha-1, respectively). However, when doubling the recommended dose, the metabolite clothianidin remained at concentrations greater than the MRL, despite that thiamethoxam concentration was lower than the MRL value. This study provided valuable insights on the uptake and residues of metalaxyl-M, fludioxonil, cyantraniliprole and thiamethoxam (including its metabolite clothianidin) in greenhouse chrysanthemum production, and could help better assess food safety risks of chrysanthemum contamination by parent pesticides and their metabolites.
Asunto(s)
Chrysanthemum/metabolismo , Plaguicidas/metabolismo , Alanina/análogos & derivados , Alanina/metabolismo , Dioxoles/metabolismo , Fungicidas Industriales/análisis , Guanidinas , Humanos , Insecticidas/análisis , Neonicotinoides , Residuos de Plaguicidas/análisis , Pirazoles/metabolismo , Pirroles/metabolismo , Suelo/química , Contaminantes del Suelo/análisis , Tiametoxam/metabolismo , Tiazoles , ortoaminobenzoatos/metabolismoRESUMEN
Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid-related plant specialized metabolites. (+)-Sesamin and (+)-sesamolin are major hydrophobic lignans, whereas (+)-sesaminol primarily accumulates as a water-soluble sesaminol triglucoside (STG) with a sugar chain branched via ß1â2 and ß1â6-O-glucosidic linkages [i.e. (+)-sesaminol 2-O-ß-d-glucosyl-(1â2)-O-ß-d-glucoside-(1â6)-O-ß-d-glucoside]. We previously reported that the 2-O-glucosylation of (+)-sesaminol aglycon and ß1â6-O-glucosylation of (+)-sesaminol 2-O-ß-d-glucoside (SMG) are mediated by UDP-sugar-dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the ß1â2-O-glucosylation of SMG and (+)-sesaminol 2-O-ß-d-glucosyl-(1â6)-O-ß-d-glucoside [termed SDG(ß1â6)]. UGT94AG1 was phylogenetically related to glycoside-specific glycosyltransferases (GGTs) and co-ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG-deficient sesame mutant that predominantly accumulates SDG(ß1â6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar-sugar ß1â6-O-glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two-hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane-associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane-bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main ß-O-glucosylation sequence of STG biosynthesis is ß1â2-O-glucosylation of SMG by UGT94AG1 followed by UGT94AA2-mediated ß1â6-O-glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio-selectivity of sequential glucosylations catalyzed by GGTs.
Asunto(s)
Vías Biosintéticas , Glucósidos/metabolismo , Glicosiltransferasas/metabolismo , Lignanos/metabolismo , Sesamum/enzimología , Catálisis , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Dioxoles/metabolismo , Furanos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glicosiltransferasas/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/química , Semillas/enzimología , Semillas/genética , Sesamum/química , Sesamum/genéticaRESUMEN
BACKGROUND Osteoporosis is a common osteopathy, resulting in fractures, especially in elder people. Sesamin has many pharmacological effects, including supplying calcium. However, how sesamin might prevent osteoporosis is still under study. MATERIAL AND METHODS Bone marrow stromal cells (BMSCs) extracted from rat femur were induced for osteoblastic differentiation. Cell proliferation, alkaline phosphatase (ALP), osterix (OSX), SRY-box 9 (SOX9), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), ß-catenin, low density lipoprotein receptor-related protein 5 (LRP5), and glycogen synthase kinase-3ß (GSK-3ß) levels in BMSCs were detected in the presence or absence of sesamin (1 µM or 10 µM). In addition, FH535 (1 µM) was used to silence Wnt/ß-catenin in vitro. Ovariectomized (OVX) rats were established and intragastrically administrated sesamin (80 mg/kg), and then the rat bones were analyzed by micro-computed tomography. Osteocalcin and collagen type I were measured in the rat femurs. RESULTS Sesamin had no influence on BMSC proliferation. Higher sesamin concentration promoted Wnt/ß-catenin activity and enhanced more expressions of ALP, OSX, SOX9, RUNX2, and OCN, gradually and significantly (P<0.05). Silencing Wnt/ß-catenin weakened the enhancement on RUNX2 and OCN expression. Sesamin (80 mg/kg) promoted bone structure in ovariectomized rats, and significantly enhanced osteocalcin and collage type I expression (P<0.05). CONCLUSIONS Sesamin promoted osteoblastic differentiation of rat BMSCs by regulating the Wnt/ß-catenin pathway, and improved rat bone structure. Sesamin could have therapeutic and preventive effects on osteoporosis.
Asunto(s)
Dioxoles/farmacología , Lignanos/farmacología , Osteoblastos/efectos de los fármacos , Osteoporosis/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Huesos/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , China , Colágeno Tipo I/metabolismo , Dioxoles/metabolismo , Femenino , Lignanos/metabolismo , Células Madre Mesenquimatosas , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Osteoporosis/prevención & control , Ovariectomía , Ratas , Ratas Sprague-Dawley , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.
Asunto(s)
Proteínas Portadoras/metabolismo , Dioxoles/metabolismo , Lignanos/metabolismo , Desarrollo de la Planta , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Dioxoles/química , Germinación , Lignanos/química , Plantas Modificadas Genéticamente , Espectroscopía de Protones por Resonancia Magnética , Semillas/crecimiento & desarrolloRESUMEN
AIMS: Hippocampal oxidative stress and apoptosis of CA1 neurons play significant roles in the pathophysiology of diabetes-associated cognitive decline (DACD). The present study was aimed to elucidate the putative effects of sesamin, a major lignan of sesame seed, against DACD, and possible involvement of anti-oxidative and anti-apoptotic mechanisms. MAIN METHODS: Fifty adult male Wistar rats were randomly divided into control, control-sesamin (30â¯mg/kg/day), diabetic, diabetic-sesamin (30â¯mg/kg/day), and diabetic-insulin (6â¯IU/rat/day) groups. Diabetic rats were treated with sesamin (P.O.) or insulin (S.C.) for eight consecutive weeks. Cognitive performance was evaluated in a Morris Water Maze (MWM) test; in addition, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) concentrations were assayed in the hippocampus using assay kits. Moreover, hematoxylin-eosin (HE), TUNEL, and immunohistochemistry (IHC) stainings were conducted to evaluate histological changes, the apoptosis status and expression of pro- and anti-apoptotic proteins in the hippocampal CA1 neurons, respectively. KEY FINDINGS: The results showed that diabetes reduced the spatial cognitive ability in MWM, which was accompanied by decrease in SOD, CAT, and GPx activities and increase in MDA level in the hippocampus. Additionally, diabetes resulted in neuronal loss, enhanced apoptotic index, elevated the expression of pro-apoptotic Bax protein, and decreased the expression of anti-apoptotic Bcl-2 protein in the hippocampal CA1 neurons. Interestingly, sesamin treatment improved all the above-mentioned deficits of diabetes at a comparable level with insulin therapy. SIGNIFICANCE: The results suggest that sesamin could be a promising potential therapeutic agent against DACD, possibly through its intertwined anti-hyperglycemic, anti-oxidative, and anti-apoptotic properties.
Asunto(s)
Disfunción Cognitiva/tratamiento farmacológico , Complicaciones de la Diabetes/tratamiento farmacológico , Dioxoles/farmacología , Lignanos/farmacología , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Dioxoles/metabolismo , Glutatión Peroxidasa/metabolismo , Hipocampo/metabolismo , Insulina/farmacología , Lignanos/metabolismo , Masculino , Malondialdehído/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Sesamin, a representative sesame lignan, has health-promoting activities. Sesamin is converted into catechol derivatives and further into their glucuronides or sulfates in vivo, whereas the biological activities of sesamin metabolites remain unclear. We examined the inhibitory effects of sesamin metabolites on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse macrophage-like J774.1 cells and found that a monocatechol derivative SC1, (7α,7'α,8α,8'α)-3,4-dihydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane, has a much higher activity than sesamin and other metabolites. The inhibitory effects of SC1 glucuronides were time-dependently enhanced, associated with the intracellular accumulation of SC1 and the methylated form. SC1 glucuronides and SC1 attenuated the expression of inducible NO synthase (iNOS) and upstream interferon-ß (IFN-ß) in the LPS-stimulated macrophages. The inhibitory effects of SC1 glucuronides against NO production were canceled by the ß-glucuronidase inhibitor and enhanced by the catechol-O-methyltransferase inhibitor. Our results suggest that SC1 glucuronides exert the anti-inflammatory effects by inhibiting the IFN-ß/iNOS signaling through macrophage-mediated deconjugation.
Asunto(s)
Antiinflamatorios , Catecoles/farmacología , Dioxoles/farmacología , Glucurónidos/farmacología , Interferón beta/antagonistas & inhibidores , Lignanos/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Animales , Catecol O-Metiltransferasa/metabolismo , Catecoles/química , Catecoles/metabolismo , Línea Celular , Sistema Enzimático del Citocromo P-450/metabolismo , Dioxoles/metabolismo , Glucuronidasa/metabolismo , Glucurónidos/química , Lignanos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Estructura MolecularRESUMEN
The mesenchymal stem cell (MSC) is a potential strategy in the pretreatment of traumatic acute lung injury (ALI), a disease that causes inflammation and oxidative stress. This study aimed to investigate whether MSC-exosomal microRNA-124-3p (miR-124-3p) affects traumatic ALI. Initially, a traumatic ALI rat model was established using the weight-drop method. Then, exosomes were obtained from MSCs of Sprague-Dawley rats, which were injected into the traumatic ALI rats. We found that miR-124-3p was abundantly-expressed in MSCs-derived exosomes and could directly target purinergic receptor P2X ligand-gated ion channel 7 (P2X7), which was overexpressed in traumatic ALI rats. After that, a loss- and gain-of-function study was performed in MSCs and traumatic ALI rats to investigate the role of miR-124-3p and P2X7 in traumatic ALI. MSC-derived exosomal miR-124-3p or silenced P2X7 was observed to increase the survival rate of traumatic ALI rats and enhance the glutathione/superoxide dismutase activity in their lung tissues. However, the wet/dry weight of lung tissues, activity of methylenedioxyamphetamine and H2O2, and levels of inflammatory factors (TNF-a, IL-6, and IL-8) were reduced. Similarly, the numbers of total cells, macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid were also reduced when treated with exosomal miR-124-3p or silenced P2X7. In conclusion, the results provide evidence that miR-124-3p transferred by MSC-derived exosomes inhibited P2X7 expression, thus improving oxidative stress injury and suppressing inflammatory response in traumatic ALI, highlighting a potential pretreatment for traumatic ALI.
Asunto(s)
Lesión Pulmonar Aguda/terapia , Exosomas/genética , Células Madre Mesenquimatosas/citología , MicroARNs/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Dioxoles/metabolismo , Modelos Animales de Enfermedad , Peróxido de Hidrógeno/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , MicroARNs/genética , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
WHAT IS KNOWN AND OBJECTIVES: Tacrolimus is widely used for kidney transplantation in children. However, the narrow therapeutic window and considerable interindividual and intraindividual variabilities make tacrolimus untoward to design an optimum dosage for paediatric personalized medicine. Our research aims to establish the tacrolimus population pharmacokinetics (PPK) of Chinese paediatric kidney transplantation patients and to distinguish covariates impacting variabilities. METHODS: Chinese paediatric kidney transplantation patients treated with tacrolimus between January 2014 and April 2018 from Children's Hospital of Fudan University were retrospectively analysed. A total of 51 Chinese paediatric kidney transplantation patients were analysed using non-linear mixed effects modelling (NONMEM). The effects of population characteristics, biological features and drug combination were assessed. The final PPK model was evaluated using visual inspection of routine diagnostic plots and the internal validation method of bootstrap. RESULTS: Our data met the condition of a one-compartment model, and the final model was CL/F = 32.7 × (WT/70)0.75 × (1 - WZ × 0.341) × (HGB/97)-0.508 ; V/F = 1890 × (WT/70) × (POD/57)0.816 , where WT, WZ, HGB and POD were weight, Wuzhi capsule (extracted from schisandra sphenanthera, whose primary efficient constituents are schisantherin A, schisandrol B, schisandrin etc, and often used to treat drug-induced hepatitis in Chinese organ transplant patients), haemoglobin and post-transplant day, respectively. WHAT IS NEW AND CONCLUSION: The tacrolimus PPK model in Chinese paediatric kidney transplantation patients was developed, and Wuzhi capsule and haemoglobin influence tacrolimus elimination in paediatric kidney transplantation patients.
Asunto(s)
Cápsulas/administración & dosificación , Medicamentos Herbarios Chinos/administración & dosificación , Hemoglobinas/administración & dosificación , Tacrolimus/farmacocinética , Adolescente , Pueblo Asiatico , Niño , Preescolar , Ciclooctanos/metabolismo , Dioxoles/metabolismo , Femenino , Humanos , Inmunosupresores/farmacocinética , Trasplante de Riñón/métodos , Lignanos/metabolismo , Masculino , Modelos Biológicos , Compuestos Policíclicos/metabolismo , Estudios RetrospectivosRESUMEN
OBJECTIVE: Sesamin is a major lignan constituent of sesame and possesses various health-promoting effects. Previous studies have demonstrated that sesamin extends the lifespan of Drosophila and Caenorhabditis elegans and corrects oxidative damage-related tissue dysfunction in mammals. To understand its anti-aging effects, we aimed to determine whether sesamin restores tissue function hampered by oxidative damage and suppresses several aging-related phenotypes using Drosophila senescence-accelerated models. MATERIALS AND METHODS: We elucidated the anti-aging effects of sesamin on several aging-related phenotypes in the muscle, brain and midgut using the senescence-accelerated models (Sod1n1 mutant and Sod1-depleted flies) by immunostaining experiments. We determined the expression levels of several anti-oxidative and DNA repair genes using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). We also identified the metabolite of sesamin in Drosophila by LC-MS/MS. RESULTS: We confirmed that sesamin (0.35 and 2 mg/ml) extended the lifespan of the fly models. As observed in mammals, it can be absorbed and metabolized by Drosophila adults. The sesamin feeding suppressed the age-dependent impairment of locomotor activity and inhibited the accumulation of reactive oxygen species (ROS) in their bodies. Sesamin delayed the age-dependent accumulation of damaged proteins in the muscle, partially suppressed the loss of dopaminergic neurons in adult brains displaying ROS accumulation, and suppressed the accumulation of DNA damage and hyperproliferation of intestinal stem cells. Four antioxidative genes and two DNA repair genes were simultaneously upregulated in sesamin-fed adults. CONCLUSIONS: These observations represent the first direct evidence of the anti-aging effects of sesamin at the individual level. We propose that sesamin exerts anti-aging effects in the muscles, brain and midgut by inducing antioxidative and DNA repair genes, resulting in extended lifespan in flies.
Asunto(s)
Envejecimiento/efectos de los fármacos , Antioxidantes/farmacología , Dioxoles/farmacología , Modelos Animales de Enfermedad , Drosophila melanogaster , Intestinos , Lignanos/farmacología , Longevidad , Envejecimiento/genética , Animales , Antioxidantes/análisis , Antioxidantes/metabolismo , Células Cultivadas , Cromatografía Liquida , Dioxoles/análisis , Dioxoles/metabolismo , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Intestinos/efectos de los fármacos , Lignanos/análisis , Lignanos/metabolismo , Músculos/efectos de los fármacos , Músculos/metabolismo , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/metabolismo , Fenotipo , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Espectrometría de Masas en TándemRESUMEN
Sesamin is known to have various biological effects. Although several metabolites of sesamin have been identified, its metabolism by phase II enzymes remains unclear, because usually its sulfo- and glucurono-conjugates in plasma and urine are analyzed after sulfatase/ß-glucuronidase treatment. In this study, the metabolites of sesamin in rats administrated with sesamin (100 mg/kg b.w.) were analyzed without sulfatase/ß-glucuronidase treatment. Two sulfate conjugates of sesamin monocatechol (SC-1) were detected in the liver and plasma. Their Cmax values were 5- and 10-times higher than that of sesamin itself. The Vmax/Km values for sulfate conjugation in the cytosol fraction of human liver were 1.7-times larger than that in the cytosol fraction of rat liver, suggesting that sulfate conjugation also occurs in human liver. The recombinant human proteins SULT1A1, 1A3, 1B1, and 1E1 expressed in Saccharomyces cerevisiae cells produced sulfate conjugates effectively. Our results could help revealing the mechanism of physiological effects of sesamin.