RESUMEN
Seed dormancy is an important agronomic trait under the control of complex genetic and environmental interactions, which have not been yet comprehensively understood. From the field screening of rice mutant library generated by a Ds transposable element, we identified a pre-harvest sprouting (PHS) mutant dor1. This mutant has a single insertion of Ds element at the second exon of OsDOR1 (LOC_Os03g20770), which encodes a novel seed-specific glycine-rich protein. This gene successfully complemented the PHS phenotype of dor1 mutant and its ectopic expression enhanced seed dormancy. Here, we demonstrated that OsDOR1 protein binds to the GA receptor protein, OsGID1 in rice protoplasts, and interrupts with the formation OsGID1-OsSLR1 complex in yeast cells. Co-expression of OsDOR1 with OsGID1 in rice protoplasts attenuated the GA-dependent degradation of OsSLR1, the key repressor of GA signaling. We showed the endogenous OsSLR1 protein level in the dor1 mutant seeds is significantly lower than that of wild type. The dor1 mutant featured a hypersensitive GA-response of α-amylase gene expression during seed germination. Based on these findings, we suggest that OsDOR1 is a novel negative player of GA signaling operated in the maintenance of seed dormancy. Our findings provide a novel source of PHS resistance.
Asunto(s)
Oryza , Latencia en las Plantas , Latencia en las Plantas/genética , Oryza/genética , Giberelinas/metabolismo , Semillas/genética , Glicina/metabolismoRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative motor disorder without an available therapeutic to halt the formation of Lewy bodies for preventing dopaminergic neuronal loss in the nigrostriatal pathway. Since oxidative-stress-mediated damage has been commonly reported as one of the main pathological mechanisms in PD, we assessed the efficacy of a novel NOX inhibitor from AptaBio Therapeutics (C-6) in dopaminergic cells and PD mouse models. The compound reduced the cytotoxicity and enhanced the cell viability at various concentrations against MPP+ and α-synuclein preformed fibrils (PFFs). Further, the levels of ROS and protein aggregation were significantly reduced at the optimal concentration (1 µM). Using two different mouse models, we gavaged C-6 at two different doses to the PD sign-displaying transgenic mice for 2 weeks and stereotaxically PFF-injected mice for 5 weeks. Our results demonstrated that both C-6-treated mouse models showed alleviated motor deficits in pole test, hindlimb clasping, crossbeam, rotarod, grooming, and nesting analyses. We also confirmed that the compound treatment reduced the levels of protein aggregation, along with phosphorylated-α-synuclein, in the striatum and ventral midbrain and further dopaminergic neuronal loss. Taken together, our results strongly suggest that NOX inhibition can be a potential therapeutic target for PD.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Ratones , Ratones Transgénicos , Degeneración Nerviosa/patología , Enfermedad de Parkinson/metabolismo , Agregado de Proteínas , alfa-Sinucleína/metabolismoRESUMEN
Drought and salinity are major important factors that restrain growth and productivity of rice. In plants, many really interesting new gene (RING) finger proteins have been reported to enhance drought and salt tolerance. However, their mode of action and interacting substrates are largely unknown. Here, we identified a new small RING-H2 type E3 ligase OsRF1, which is involved in the ABA and stress responses of rice. OsRF1 transcripts were highly induced by ABA, salt, or drought treatment. Upregulation of OsRF1 in transgenic rice conferred drought and salt tolerance and increased endogenous ABA levels. Consistent with this, faster transcriptional activation of key ABA biosynthetic genes, ZEP, NCED3, and ABA4, was observed in OsRF1-OE plants compared with wild type in response to drought stress. Yeast two-hybrid assay, BiFC, and co-immunoprecipitation analysis identified clade A PP2C proteins as direct interacting partners with OsRF1. In vitro ubiquitination assay indicated that OsRF1 exhibited E3 ligase activity, and that it targeted OsPP2C09 protein for ubiquitination and degradation. Cell-free degradation assay further showed that the OsPP2C09 protein is more rapidly degraded by ABA in the OsRF1-OE rice than in the wild type. The combined results suggested that OsRF1 is a positive player of stress responses by modulating protein stability of clade A PP2C proteins, negative regulators of ABA signaling.
RESUMEN
Acute myocarditis is an unpredictable heart disease that is caused by inflammation-associated cell death. Although viral infection and drug exposure are known to induce acute myocarditis, the molecular basis for its development remains undefined. Using proteomics and molecular analyses in myosin-induced rat experimental autoimmune myocarditis (EAM), we identified that elevated expression of aldolase 1A, retrogene 1 (Aldoart1) is critical to induce mitochondrial dysfunction and acute myocarditis development. Here, we demonstrate that cardiac cell death is associated with increased expressions of proapoptotic genes in addition to high levels of glucose, lactate, and triglyceride in metabolite profiling. The functional protein association network analysis also suggests that Aldoart1 upregulation correlates with high levels of dihydroxyacetone kinase and triglyceride. In H9c2 cardiac cells, lipopolysaccharides (LPS) or high glucose exposure significantly increases the cytochrome c release and the conversion of pro-caspase 3 into the cleaved form of caspase 3. We also found that LPS- or glucose-induced toxicities are almost completely reversed by siRNA-mediated knockdown of Aldoartl, which consequently increases cell viability. Together, our study strongly suggests that Aldoart1 may be involved in inducing mitochondrial apoptotic processes and can be a novel therapeutic target to prevent the onset of acute myocarditis or cardiac apoptosis.
Asunto(s)
Apoptosis/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Fructosa-Bifosfato Aldolasa/genética , Miocarditis/genética , Miocarditis/patología , Miocitos Cardíacos/patología , Animales , Modelos Animales de Enfermedad , Expresión Génica , Masculino , RatasRESUMEN
The objective of this study was to investigate the effects of the particle size of resveratrol (RSV)-loaded nanoparticles (NPs) on their solubility and stability and to optimize their preparation conditions for their solubility and stability. RSV-loaded NPs were prepared using chitosan and γ-poly(glutamic acid) (γ-PGA). Although the solubility and stability of RSV have been significantly increased using chitosan/γ-PGA nanoencapsulation, as the NP size decreased, the solubility increased, but the stability decreased. In order to understand the interrelationship of particle size, solubility, and stability, the target values of RSV solubility and ultraviolet (UV) stability for the aforementioned optimization were determined at two levels: solubility >153 µg/mL, UV stability >12 % (S153U12) and solubility >150 µg/mL, UV stability >18 % (S150U18). The S150U18-NPs (258 nm) showed a significantly higher UV stability and tyrosinase inhibition activity against UVA than S153U12-NPs (87 nm) (p < 0.01). Although insignificant, the S153U12-NPs exhibited higher solubility than the S150U18-NPs. In addition, the cellular antioxidant activity was significantly higher in the S153U12-NPs than the S150U18-NPs (p < 0.05). These results demonstrated that the solubility and stability of RSV-loaded NPs may be influenced by their particle size, which could be controlled by the chitosan and γ-PGA concentrations.
Asunto(s)
Antioxidantes/farmacología , Inhibidores Enzimáticos/farmacología , Monofenol Monooxigenasa/antagonistas & inhibidores , Nanopartículas/química , Rayos Ultravioleta , Quitosano/análogos & derivados , Quitosano/farmacología , Células Hep G2 , Humanos , Monofenol Monooxigenasa/metabolismo , Tamaño de la Partícula , Ácido Poliglutámico/análogos & derivados , Ácido Poliglutámico/farmacología , Resveratrol/farmacología , Solubilidad , Propiedades de SuperficieRESUMEN
KEY MESSAGE: PpCKX1 localizes to vacuoles and is dominantly expressed in the stem cells. PpCKX1 regulates developmental changes with increased growth of the rhizoid and enhances dehydration and salt tolerance. Cytokinins (CKs) are plant hormones that regulate plant development as well as many physiological processes, such as cell division, leaf senescence, control of shoot/root ratio, and reproductive competence. Cytokinin oxidases/dehydrogenases (CKXs) control CK concentrations by degradation, and thereby influence plant growth and development. In the moss Physcomitrella patens, an evolutionarily early divergent plant, we identified six putative CKXs that, by phylogenetic analysis, form a monophyletic clade. We also observed that ProPpCKX1:GUS is expressed specifically in the stem cells and surrounding cells and that CKX1 localizes to vacuoles, as indicated by Pro35S:PpCKX1-smGFP. Under normal growth conditions, overexpression of PpCKX1 caused many phenotypic changes at different developmental stages, and we suspected that increased growth of the rhizoid could affect those changes. In addition, we present evidence that the PpCKX1-overexpressor plants show enhanced dehydration and salt stress tolerance. Taken together, we suggest that PpCKX1 plays regulatory roles in development and adaptation to abiotic stresses in this evolutionarily early land plant species.
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Bryopsida/enzimología , Bryopsida/crecimiento & desarrollo , Oxidorreductasas/metabolismo , Tolerancia a la Sal , Bryopsida/genética , Citocininas/metabolismo , Deshidratación , Regulación de la Expresión Génica de las Plantas , Fenotipo , Filogenia , Plantas Modificadas Genéticamente , Estrés Salino/genética , Tolerancia a la Sal/genética , Células Madre/metabolismo , Vacuolas/metabolismoRESUMEN
Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have evolved the immune system to recognize and limit the growth of pathogens. Despite substantial progress in the study of plant immunity, the mechanism by which plants limit pathogen growth remains unclear. Here, we show that lignin accumulates in Arabidopsis leaves in response to incompatible interactions with bacterial pathogens in a manner dependent on Casparian strip membrane domain protein (CASP)-like proteins (CASPLs). CASPs are known to be the organizers of the lignin-based Casparian strip, which functions as a diffusion barrier in roots. The spread of invading avirulent pathogens is prevented by spatial restriction, which is disturbed by defects in lignin deposition. Moreover, the motility of pathogenic bacteria is negatively affected by lignin accumulation. These results suggest that the lignin-deposited structure functions as a physical barrier similar to the Casparian strip, trapping pathogens and thereby terminating their growth.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Infecciones Bacterianas/microbiología , Pared Celular/inmunología , Interacciones Huésped-Patógeno/inmunología , Lignina/metabolismo , Raíces de Plantas/inmunología , Arabidopsis/metabolismo , Arabidopsis/microbiología , Pared Celular/metabolismo , Pared Celular/microbiología , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiologíaRESUMEN
Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal.IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.
Asunto(s)
Proteínas Bacterianas/genética , Monofenol Monooxigenasa/genética , Pseudomonas fluorescens/metabolismo , Streptomyces antibioticus/genética , Proteínas Bacterianas/metabolismo , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Monofenol Monooxigenasa/metabolismo , Pseudomonas fluorescens/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces antibioticus/metabolismoRESUMEN
An inter-laboratory study was performed to evaluate the performance of a method developed for the quantification of enrofloxacin in chicken meat. Liquid-liquid extraction combined with a clean-up procedure based on solid-phase extraction followed by a liquid chromatography-tandem mass spectrometric method was used by three individual laboratories. All the investigated results of calibration curves and limits of quantification were within the acceptable range for regulatory testing of enrofloxacin. The three laboratories received blind a certified reference material to analyze in triplicate and assess using statistical analysis. From the results, no statistical differences were found between the laboratories in the precision of the method. Additionally, all the results of the z-score, which is an indication of fixed interval bias criteria for accuracy from the laboratories, fell within the allowable limits (±2σ). Based on this proficiency testing by inter-laboratory comparisons, the analytical method including the sample preparation step was proven to be applicable.
RESUMEN
Extremely low-frequency electromagnetic field (ELFEMF) can stimulate neural differentiation in human bone marrow-derived mesenchymal cells (hBM-MSCs), and this provides an opportunity for research on neurodegenerative diseases such as Alzheimer's disease (AD). Metallothionein-3 (MT3), an isoform of the metal-binding proteins, metallothioneins, involved in maintaining intracellular zinc (Zn) homeostasis and the deregulation of zinc homeostasis, has separately been implicated in AD. Here, we investigated the effect of ELFEMF-induced neural differentiation of hBM-MSCs on Zn-MT3 homeostatic interaction. Exposure to ELFEMF induced neural differentiation of hBM-MSCs, which was characterized by decreased proliferation and enhanced neural-like morphology. We observed expression of neuronal markers such as ß-tubulin3, pleiotrophin, and neurofilament-M at the mRNA level and MAP2 at the protein level. ELFEMF-induced neural differentiation correlated with decreased expression of metal-response element-transcription factor 1 and MT3, as well as decreased intracellular Zn concentration. In addition, upregulation of dihydropyrimidinase-related protein 2 was observed, but there was no change in γ-enolase expression. These data indicate a possible regulatory mechanism for MT3 during neural differentiation. Our findings provide considerable insight into molecular mechanisms involved in neural differentiation, which is useful for developing new treatments for neurodegenerative diseases. Bioelectromagnetics. 38:364-373, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Diferenciación Celular/efectos de la radiación , Campos Electromagnéticos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de la radiación , Metalotioneína/metabolismo , Neuronas/citología , Neuronas/efectos de la radiación , Regulación hacia Abajo/efectos de la radiación , Homeostasis/efectos de la radiación , Células Madre Mesenquimatosas/metabolismo , Metalotioneína/genéticaRESUMEN
Arabidopsis GDSL lipase 1 (GLIP1) has been shown to modulate systemic immunity through the regulation of ethylene signaling components. Here we demonstrate that the constitutive triple response mutant ctr1-1 requires GLIP1 for the ethylene response, gene expression, and pathogen resistance. The glip1-1 mutant was defective in induced resistance following primary inoculation of necrotrophic pathogens, whereas GLIP1-overexpressing plants showed resistance to multiple pathogens. Necrotrophic infection triggered the downregulation of EIN3 and the activation of ERF1 and SID2 in a GLIP1-dependent manner. These results suggest that GLIP1 positively and negatively regulates ethylene signaling, resulting in an ethylene-associated, necrotroph-induced immune response.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , Hidrolasas de Éster Carboxílico/fisiología , Resistencia a la Enfermedad , Etilenos/metabolismo , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN , Expresión Génica , Regulación de la Expresión Génica de las Plantas/inmunología , Interacciones Huésped-Patógeno , Transferasas Intramoleculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Pseudomonas syringae/fisiología , Análisis de Secuencia de ADN , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
To produce genistein in rice, the isoflavone synthase (IFS) genes, SpdIFS1 and SpdIFS2 were cloned from the Korean soybean cultivar, Sinpaldalkong II as it has a higher genistein content than other soybean varieties. SpdIFS1 and SpdIFS2 show a 99.6% and 98.2% identity at the nucleotide level and 99.4% and 97.9% identity at the amino acid level, respectively, with IFS1 and IFS2 from soybean (GenBank accession Nos. AF195798 and AF195819). Plant expression vectors were constructed harboring SpdIFS1 or SpdIFS2 under the control of a rice globulin promoter that directs seed specific expression, and used to transform two rice varieties, Heugnam, a black rice, and Nakdong, a normal rice cultivar without anthocyanin pigment. Because naringenin, the substrate of SpdIFS1 and SpdIFS2, is on the anthocyanin biosynthesis pathway, the relative production rate of genistein was compared between SpdIFS-expressing transgenic Heugnam and Nakdong. Southern blot analysis of eight of the resulting transgenic rice plants revealed that the T0 plants had one to three copies of the SpdIFS1 or SpdIFS2 gene. The highest level of genistein content found in rice seeds was 103 µg/g. These levels were about 30-fold higher in our transgenic rice lines than the genistein aglycon content of a non-leguminous IFS-expressing transgenic tobacco petal, equaling about 12% of total genistein content of Sinpaldalkong II. There were no significant differences found between the genistein content in Heugnam and Nakdong transgenic rice plants.
Asunto(s)
Endospermo/enzimología , Genisteína/metabolismo , Glycine max/genética , Oryza/enzimología , Oxigenasas/genética , Plantas Modificadas Genéticamente/metabolismo , Secuencia de Aminoácidos , Antocianinas/biosíntesis , Clonación Molecular , Datos de Secuencia Molecular , Oryza/embriología , Oryza/genética , SemillasRESUMEN
A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid-liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.
Asunto(s)
Aminas/química , Aminas/toxicidad , Compuestos Heterocíclicos/química , Mutágenos/análisis , Mutágenos/química , Urinálisis/métodos , Niño , Cromatografía Liquida , Exposición a Riesgos Ambientales/análisis , Humanos , Medición de Riesgo , Espectrometría de Masas en TándemRESUMEN
Ethylene is a key signal in the regulation of plant defense responses. It is required for the expression and function of GDSL LIPASE1 (GLIP1) in Arabidopsis (Arabidopsis thaliana), which plays an important role in plant immunity. Here, we explore molecular mechanisms underlying the relationship between GLIP1 and ethylene signaling by an epistatic analysis of ethylene response mutants and GLIP1-overexpressing (35S:GLIP1) plants. We show that GLIP1 expression is regulated by ethylene signaling components and, further, that GLIP1 expression or application of petiole exudates from 35S:GLIP1 plants affects ethylene signaling both positively and negatively, leading to ETHYLENE RESPONSE FACTOR1 activation and ETHYLENE INSENSITIVE3 (EIN3) down-regulation, respectively. Additionally, 35S:GLIP1 plants or their exudates increase the expression of the salicylic acid biosynthesis gene SALICYLIC ACID INDUCTION-DEFICIENT2, known to be inhibited by EIN3 and EIN3-LIKE1. These results suggest that GLIP1 regulates plant immunity through positive and negative feedback regulation of ethylene signaling, and this is mediated by its activity to accumulate a systemic signal(s) in the phloem. We propose a model explaining how GLIP1 regulates the fine-tuning of ethylene signaling and ethylene-salicylic acid cross talk.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Hidrolasas de Éster Carboxílico/metabolismo , Etilenos/metabolismo , Retroalimentación Fisiológica , Inmunidad de la Planta , Transducción de Señal/inmunología , Alternaria/fisiología , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Regulación hacia Abajo/genética , Epistasis Genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Modelos Biológicos , Mutación/genética , Fenotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Exudados de Plantas/metabolismo , Inmunidad de la Planta/genética , Unión Proteica , Ácido Salicílico/metabolismo , Transducción de Señal/genética , Regulación hacia Arriba/genéticaRESUMEN
Reactive oxygen species (ROS) and ethylene play an important role in determining the resistance or susceptibility of plants to pathogen attack. A previous study of the response of tobacco cultivar ( Nicotiana tabacum L. cv. Wisconsin 38) to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae (Ppn) showed that biphasic bursts of ROS and ethylene are positively associated with disease severity. The levels of ethylene and ROS might influence the susceptibility of plants to pathogens, with changing levels of metabolite related to disease resistance or susceptibility. In this study, to obtain more detailed information on the interaction of ROS and ethylene signaling related to resistance and/or susceptibility of plants to pathogen, Ppn-induced metabolic profiles from wild type (WT) and ethylene signaling-impaired transgenic plants that expressed Ein3 antisense (Ein3-AS) were compared using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Nonredundant mass ions (576 in ESI+ mode and 336 in ESI- mode) were selected, and 56 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Two-way hierarchical clustering analysis of the selected mass ions revealed that nicotine and phenylpropanoid-polyamine conjugates, such as caffeoyl-dihydrocaffeoyl-spermidine, dicaffeoyl-spermidine, caffeoyl-feruloyl-spermidine, and two bis(dihydrocaffeoyl)-spermine isomers, and their intermediates, such as arginine and putrecine, were present at lower levels in Ein3-AS transgenic plants during Ppn interaction than in WT, whereas galactolipid and oxidized free fatty acid levels were higher in Ein3-AS transgenic plants. Taken together, these results reveal a function for ethylene signaling in tobacco defense responses during Ppn interaction.
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Etilenos , Nicotiana/metabolismo , Nicotiana/parasitología , Phytophthora , Enfermedades de las Plantas/parasitología , Transducción de Señal , Resistencia a la Enfermedad , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/parasitología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Nicotiana/genéticaRESUMEN
A biphasic reactive oxygen species (ROS) production has previously been observed in tobacco at 1 and 48 h after inoculation with the hemibiotrophic compatible pathogen, Phytophthora parasitica var. nicotianae (Ppn). To characterize the response of tobacco to biphasically produced ROS concerning the propagation of Ppn, ultraperformance liquid chromatography-quadrupole-time of flight/ mass spectrometry (UPLC-Q-TOF/MS) based metabolic profiling combined with multivariate statistical analysis was performed. Among the nonredundant 355 mass ions in ESI+ mode and 345 mass ions in ESI- mode that were selected as significantly changed by Ppn inoculation (|p(corr)| > 0.6 on S-plot of orthogonal partial least-squares discriminant analysis (OPLS-DA), fold-change > 2, and p < 0.05 in the independent two-sample t test), 76 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Phenolic amino acids, phenylpropanoids, hydroxycinnamic acid amides, linoleic acid, linolenic acid, lysophospholipids, glycoglycerolipids, and trioxidized phospholipids were identified as having changed after Ppn inoculation. On the basis of their quantitative changes, the metabolic responses occurring at each phase of ROS production after Ppn inoculation were investigated in this study.
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Cromatografía Liquida/métodos , Metaboloma , Nicotiana/metabolismo , Espectrometría de Masas en Tándem/métodos , Aminoácidos/metabolismo , Lípidos/análisisRESUMEN
BACKGROUND: To investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM)-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders. RESULTS: By performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR), while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK)-1/2 and ribosomal protein S6 (rpS6) were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB)-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models. CONCLUSIONS: UPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or methods aimed at treating enigmatic myocarditis.
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Enfermedades Autoinmunes/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Miocarditis/metabolismo , Proteómica , Transducción de Señal/genética , Animales , Enfermedades Autoinmunes/patología , Modelos Animales de Enfermedad , Enterovirus/fisiología , Regulación de la Expresión Génica , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocarditis/patología , Ratas , Ratas Endogámicas Lew , Proteína S6 Ribosómica/metabolismo , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
OBJECTIVES: Rhus verniciflua Stokes (RVS), which has valuable medicinal properties, has for many years been prescribed for inflammation in east Asian medicine. Recent studies suggest that RVS has potent antioxidative, antitumor and anti-inflammatory properties. METHODS: In this study, the anti-inflammatory effects of RVS in vitro and in vivo were investigated. The ethanol extract from RVS was partitioned with different solvents in order of increasing polarity. KEY FINDINGS: Among the various extracts, the n-butanol extract displayed the most potent activity against nitric oxide and reactive oxygen species. The n-butanol extract also significantly regulates expression of nitric oxide synthase, which inhibits nitric oxide production at the transcriptional level in activated macrophages. Immunoblot analysis also showed that n-butanol extract suppresses the phosphorylation of extracellular signal-regulated kinase and Akt, suggesting that nitric oxide synthase suppression might be mediated via the extracellular signal-regulated kinase and Akt signaling pathways. This study also investigated whether n-butanol exerts an anti-inflammatory effect in an animal model. n-butanol extract significantly reduces carrageenan-induced mouse paw edema at 5 h. CONCLUSIONS: These results suggest that RVS could be a promising candidate agent for inflammation prevention and combination therapy with anti-inflammatory drugs.
Asunto(s)
Antiinflamatorios/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Óxido Nítrico/antagonistas & inhibidores , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Rhus , Animales , Carragenina , Células Cultivadas , Modelos Animales de Enfermedad , Edema/tratamiento farmacológico , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Fosforilación , Fitoterapia , Corteza de la Planta , Transducción de Señal/efectos de los fármacosRESUMEN
The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.