RESUMO
Thioredoxins (TRXs) are central to redox regulation, modulating enzyme activities to adapt metabolism to environmental changes. Previous research emphasized mitochondrial and microsomal TRX o1 and h2 influence on mitochondrial metabolism, including photorespiration and the tricarboxylic acid (TCA) cycle. Our study aimed to compare TRX-based regulation circuits towards environmental cues mainly affecting photorespiration. Metabolite snapshots, phenotypes and CO2 assimilation were compared among single and multiple TRX mutants in the wild-type and the glycine decarboxylase T-protein knockdown (gldt1) background. Our analyses provided evidence for additive negative effects of combined TRX o1 and h2 deficiency on growth and photosynthesis. Especially metabolite accumulation patterns suggest a shared regulation mechanism mainly on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1)-dependent pathways. Quantification of pyridine nucleotides, in conjunction with 13C-labelling approaches, and biochemical analysis of recombinant mtLPD1 supported this. It also revealed mtLPD1 inhibition by NADH, pointing at an additional measure to fine-tune it's activity. Collectively, we propose that lack of TRX o1 and h2 perturbs the mitochondrial redox state, which impacts on other pathways through shifts in the NADH/NAD+ ratio via mtLPD1. This regulation module might represent a node for simultaneous adjustments of photorespiration, the TCA cycle and branched chain amino acid degradation under fluctuating environmental conditions.
Assuntos
Di-Hidrolipoamida Desidrogenase , Mitocôndrias , Tiorredoxina h , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Dióxido de Carbono/metabolismo , Di-Hidrolipoamida Desidrogenase/metabolismo , Di-Hidrolipoamida Desidrogenase/genética , Meio Ambiente , Mitocôndrias/metabolismo , Mutação , NAD/metabolismo , Oxirredução , Fotossíntese , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Tiorredoxinas/metabolismo , Tiorredoxina h/genética , Tiorredoxina h/metabolismoRESUMO
Thiol-based redox regulation is a crucial posttranslational mechanism to acclimate plants to changing light availability. Here, we conducted a biotin switch-based redox proteomics study in Arabidopsis (Arabidopsis thaliana) to systematically investigate dynamics of thiol-redox networks in response to temporal changes in light availability and across genotypes lacking parts of the thioredoxin (Trx) or NADPH-Trx-reductase C (NTRC) systems in the chloroplast. Time-resolved dynamics revealed light led to marked decreases in the oxidation states of many chloroplast proteins with photosynthetic functions during the first 10â min, followed by their partial reoxidation after 2 to 6â h into the photoperiod. This involved f, m, and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys peroxiredoxins, and Trx y2 showed an opposing pattern, being more oxidized in light than dark. In Arabidopsis trxf1f2, trxm1m2, or ntrc mutants, most proteins showed increased oxidation states in the light compared to wild type, suggesting their light-dependent dynamics were related to NTRC/Trx networks. While NTRC deficiency had a strong influence in all light conditions, deficiencies in f- or m-type Trxs showed differential impacts on the thiol-redox proteome depending on the light environment, being higher in constant or fluctuating light, respectively. The results indicate plant redox proteomes are subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. The importance of the individual elements of the NTRC/Trx networks mediating these responses depend on the extent of light variability, with NTRC playing a crucial role to balance protein-redox states in rapidly fluctuating light.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Luz , Oxirredução , Proteoma , Compostos de Sulfidrila , Tiorredoxinas , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteoma/metabolismo , Compostos de Sulfidrila/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Dissulfetos/metabolismo , Fotossíntese/efeitos da radiação , Proteômica/métodos , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Cloroplastos/metabolismoRESUMO
Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , NADP/metabolismo , Dióxido de Carbono/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Arabidopsis/metabolismo , Fotossíntese/fisiologia , Cloroplastos/metabolismo , Oxirredução , Folhas de Planta/metabolismo , Tiorredoxinas/metabolismo , AclimataçãoRESUMO
BACKGROUND: We have limited knowledge on the impact of hydrometeorological conditions on dengue incidence in China and its associated disease burden in a future with a changed climate. This study projects the excess risk of dengue caused by climate change-induced hydrometeorological conditions across mainland China. METHODS: In this modelling study, the historical association between the Palmer drought severity index (PDSI) and dengue was estimated with a spatiotemporal Bayesian hierarchical model from 70 cities. The association combined with the dengue-transmission biological model was used to project the annual excess risk of dengue related to PDSI by 2100 across mainland China, under three representative concentration pathways ([RCP] 2·6, RCP 4·5, and RCP 8·5). FINDINGS: 93â101 dengue cases were reported between 2013 and 2019 in mainland China. Dry and wet conditions within 3 months lag were associated with increased risk of dengue. Locations with potential dengue risk in China will expand in the future. The hydrometeorological changes are projected to substantially affect the risk of dengue in regions with mid-to-low latitudes, especially the coastal areas under high emission scenarios. By 2100, the annual average increased excess risk is expected to range from 12·56% (95% empirical CI 9·54-22·24) in northwest China to 173·62% (153·15-254·82) in south China under the highest emission scenario. INTERPRETATION: Hydrometeorological conditions are predicted to increase the risk of dengue in the future in the south, east, and central areas of mainland China in disproportionate patterns. Our findings have implications for the preparation of public health interventions to minimise the health hazards of non-optimal hydrometeorological conditions in a context of climate change. FUNDING: National Natural Science Foundation of China.
Assuntos
Mudança Climática , Dengue , Humanos , Teorema de Bayes , Cidades , China/epidemiologia , Dengue/epidemiologiaRESUMO
BACKGROUND: Global warming has led to methods of planting late-maturing maize varieties in northeast China that have hindered the development of physiological maturity (PM) at harvest and the use of mechanical grain harvesting (MGH). Under these conditions it is difficult to balance the drying characteristics of maize varieties and to make full use of accumulated temperature resources in such a way as to reduce grain moisture content (GMC) at harvest. RESULTS: The effective accumulated temperature (AcT) and the drying rates of different varieties vary. In northeast China, with a GMC of 25%, the growth periods of a fast-drying variety (FDV) and a slow-drying variety (SDV) were 114-192 days and 110-188 days respectively. After PM, the FDV needed 47 days and the SDV needed 51 days to reduce the GMC to be ready for MGH. Harvested with a GMC of 20%, the growth period for the FDV was 97-175 days and for the SDV it was 90-171 days. After PM, the FDV required 64 days and the SDV needed 70 days to reduce the GMC to be ready for MGH. CONCLUSION: Matching cultivars with AcT can help farmers to choose suitable varieties. Promoting MGH may boost maize production, thus ensuring China's food security. © 2023 Society of Chemical Industry.
Assuntos
Grão Comestível , Zea mays , Temperatura , Grão Comestível/química , Aquecimento Global , ChinaRESUMO
Protein purification is a basic technology in both biological research and industrial production, and efficient, convenient, economical, and environmentally friendly purification methods have always been pursued. In this study, it was found that alkaline earth metal cations (Mg2+, Ca2+) and alkali metal cations (Li+, Na+, K+) and even nonmetal cations (e.g., NH4+, imidazole, guanidine, arginine, lysine) can precipitate multi-histidine-tagged proteins (at least two tags in a whole protein) at low salts concentrations that are 1-3 orders of magnitude lower than salting-out, and precipitated proteins could be dissolved at moderate concentration of corresponding cation. Based on this finding, a novel cation affinity purification method was developed, which requires only three centrifugal separations to obtain highly purified protein with purification fold similar to that of immobilized metal affinity chromatography. The study also provides a possible explanation for unexpected protein precipitation and reminds researchers to consider the influence of cations on the experimental results. The interaction between histidine-tagged proteins and cations may also have broad application prospects. KEY POINTS: ⢠Histidine-tagged proteins can be precipitated by low-concentrations common cations ⢠A novel nonchromatographic protein purification method was developed ⢠Purified protein can be obtained in pellet form by only three centrifugations.
Assuntos
Histidina , Histidina/química , Histidina/metabolismo , Indicadores e Reagentes , Cátions , Cromatografia de Afinidade/métodos , Proteínas RecombinantesRESUMO
Importance: Dengue fever is a climate-sensitive infectious disease. However, its association with local hydrological conditions and the role of city development remain unclear. Objective: To quantify the association between hydrological conditions and dengue fever incidence in China and to explore the modification role of city development in this association. Design, Setting, and Participants: This cross-sectional study collected data between January 1, 2013, and December 31, 2019, from 54 cities in 4 coastal provinces in southeast China. The Standardized Precipitation Evapotranspiration Index (SPEI) was calculated from ambient temperature and precipitation, with SPEI thresholds of 2 for extreme wet conditions and -2 for extreme dry conditions. The SPEI-dengue fever incidence association was examined over a 6-month lag, and the modification roles of 5 city development dimensions were assessed. Data were analyzed in May 2022. Exposures: City-level monthly temperature, precipitation, SPEI, and annual city development indicators from 2013 to 2019. Main Outcomes and Measures: The primary outcome was city-level monthly dengue fever incidence. Spatiotemporal bayesian hierarchal models were used to examine the SPEI-dengue fever incidence association over a 6-month lag period. An interaction term between SPEI and each city development indicator was added into the model to assess the modification role of city development. Results: Included in the analysis were 70 006 dengue fever cases reported in 54 cities in 4 provinces in China from 2013 to 2019. Overall, a U-shaped cumulative curve was observed, with wet and dry conditions both associated with increased dengue fever risk. The relative risk [RR] peaked at a 1-month lag for extreme wet conditions (1.27; 95% credible interval [CrI], 1.05-1.53) and at a 6-month lag for extreme dry conditions (1.63; 95% CrI, 1.29-2.05). The RRs of extreme wet and dry conditions were greater in areas with limited economic development, health care resources, and income per capita. Extreme dry conditions were higher and prolonged in areas with more green space per capita (RR, 1.84; 95% CrI, 1.37-2.46). Highly urbanized areas had a higher risk of dengue fever after extreme wet conditions (RR, 1.80; 95% CrI, 1.26-2.56), while less urbanized areas had the highest risk of dengue fever in extreme dry conditions (RR, 1.70; 95% CrI, 1.11-2.60). Conclusions and Relevance: Results of this study showed that extreme hydrological conditions were associated with increased dengue fever incidence within a 6-month lag period, with different dimensions of city development playing various modification roles in this association. These findings may help in developing climate change adaptation strategies and public health interventions against dengue fever.
Assuntos
Dengue , Humanos , Incidência , Dengue/epidemiologia , Teorema de Bayes , Estudos Transversais , China/epidemiologiaRESUMO
SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis.
Assuntos
Escherichia coli , Corpos de Inclusão , Reagentes de Ligações Cruzadas/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Glutaral/metabolismo , Agregados Proteicos , Proteínas/metabolismoRESUMO
Daphne retusa Hemsl. (Thymelaeaceae) is an evergreen shrub plant. First, we characterized the complete nucleotide sequence of chloroplast (cp) genome of D. retusa. The total length of cp genome was found to be 170,553 bp, including a large single copy (LSC) region of 84,886 bp, a small single copy (SSC) region of 2,437 bp, and a pair of inverted repeats (IRs) of 41,617 bp. The cp genome of Daphne retusa Hemsl. contains 134 genes, including 90 protein-coding genes (75 PCG species), 37 transfer RNA genes (29 tRNA species), and 6 rRNA genes (3 RNA species). A total of 13 genes (trnK-UUU, trnS-CGA, atpF, rpoC1, trnL-UAA, trnC-ACA, petD, rpl16, rpl2, ndhB, trnE-UUC, ndhA, and trnA-UGC) contain a single intron, and one gene (ycf3) contains two introns. The GC content in whole cp genome, LSC region, SSC region, and IR region was 36.75%, 34.83%, 28.19%, and 38.96% respectively, like other Thymelaeaceae plants. Phylogenetic analysis suggested that D. retusa has a close relationship with congeneric Daphne tangutica.
RESUMO
Arabidopsis contains eight different h-type thioredoxins (Trx) being distributed in different cell organelles. Although Trx h2 is deemed to be confined to mitochondria, its subcellular localization and function are discussed controversially. Here, cell fractionation studies were used to clarify this question, showing Trx h2 protein to be exclusively localized in microsomes rather than mitochondria. Furthermore, Arabidopsis trxo1, trxh2 and trxo1h2 mutants were analyzed to compare the role of Trx h2 with mitochondrial Trx o1. Under medium light, trxo1 and trxo1h2 showed impaired growth, while trxh2 was similar to wild type. In line with this, trxo1 and trxo1h2 clustered differently from wild type with respect to nocturnal metabolite profiles, revealing a decrease in ascorbate and glutathione redox states. Under fluctuating light, these genotypic differences were attenuated. Instead, the trxo1h2 double mutant showed an improved NADPH redox balance, compared to wild type, accompanied by increased photosynthetic efficiency, specifically in the high-light phases. Conclusively, Trx h2 and Trx o1 are differentially localized in microsomes and mitochondria, respectively, which is associated with different redox-active functions and effects on plant growth in constant light, while there is a joint role of both Trxs in regulating NADPH redox balance and photosynthetic performance in fluctuating light.
RESUMO
Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O2 conditions. However, an increase in the stoichiometry of photorespiratory CO2 release was found during O2 -dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD+ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Tiorredoxina h/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dióxido de Carbono/metabolismo , Respiração Celular/fisiologia , Clorofila A , Regulação da Expressão Gênica de Plantas , Glicina Desidrogenase (Descarboxilante)/metabolismo , Glicina Hidroximetiltransferase , Oxirredução , Fotossíntese/fisiologia , Folhas de Planta/metabolismo , Tiorredoxina h/genética , TranscriptomaRESUMO
NADPH-thioredoxin reductase C (NTRC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NTRC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NTRC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NTRC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NTRC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NTRC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NTRC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NTRC regulates the osmotic balance of developing fruits. These results indicate that NTRC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.
Assuntos
Frutas/enzimologia , Solanum lycopersicum/enzimologia , Amido/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Metabolismo dos Carboidratos , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/fisiologia , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/fisiologia , Metabolômica , Oxirredução , Fotossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , Tiorredoxina Dissulfeto Redutase/genéticaRESUMO
Taxonomically distinct Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) are two of the most prevalent viruses worldwide; when co-infecting orchids, they cause synergistic symptoms. Because of the huge economic loss in quality and quantity in the orchid industry with virus-infected orchids, virus-resistant orchids are urgently needed. To date, no transgenic resistant lines against these two viruses have been reported. In this study, we generated transgenic Nicotiana benthamiana expressing various constructs of partial CymMV and ORSV genomes. Several transgenic lines grew normally and remained symptomless after mixed inoculation with CymMV and ORSV. The replication of CymMV and ORSV was approximately 70-90% lower in protoplasts of transgenic lines than wild-type (WT) plants. Of note, we detected extremely low or no viral RNA or capsid protein of CymMV and ORSV in systemic leaves of transgenic lines after co-infection. Grafting experiments further revealed that CymMV and ORSV trafficked extremely inefficiently from co-infected WT stocks to transgenic scions, presumably due to RNA-mediated interference. This study reports the first successful creation of dual resistant transgenic lines against CymMV and ORSV. Our studies shed light on the commercial development of transgenic orchid production to combat the global viral threat.
Assuntos
Nicotiana/genética , Potexvirus/genética , Tobamovirus/genética , Proteínas do Capsídeo/genética , Primers do DNA/genética , Engenharia Genética/métodos , Orchidaceae/genética , Orchidaceae/virologia , Plantas Geneticamente Modificadas/genética , Potexvirus/patogenicidade , Protoplastos , Interferência de RNA , RNA Viral/genética , Tobamovirus/patogenicidade , Replicação Viral/genéticaRESUMO
Bisphenol A (BPA) is a widely studied typical endocrine-disrupting chemical. The present study aimed to verify whether BPA could induce proliferation of cardiac fibroblasts and collagen production leading to cardiac interstitial fibrosis. After exposure to BPA for 30 consecutive days, decreased cardiac function was observed in rats using echocardiography, and the deposition of collagen was detected by Masson's trichrome staining and electron microscope. BPA remarkably stimulated proliferation and migration of cultured cardiac fibroblasts and collagen production in a concentration-dependent manner, as revealed by MTT, wound healing assay and collagen assay. Meanwhile, BPA treatment also enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In contrast, pretreatment with estrogen receptor inhibitor ICI182780 or ERK inhibitor PD98059 prevented the enhanced phosphorylation of ERK1/2, and subsequently inhibited the up-regulation of transforming growth factor-ß1 (TGF-ß1) expression induced by BPA. As a consequence, these inhibitors also decreased proliferation and collagen production, as well as the fibrosis-related genes expression. Taken together, our results indicated that BPA may act as a promoting factor in proliferative process and collagen production of cardiac fibroblasts via activating ERK1/2.
Assuntos
Compostos Benzidrílicos/toxicidade , Cardiomiopatias/induzido quimicamente , Disruptores Endócrinos/toxicidade , Poluentes Ambientais/toxicidade , Fibroblastos/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/enzimologia , Fenóis/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Cardiomiopatias/enzimologia , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Antagonistas de Estrogênios/farmacologia , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Fibrose , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Miocárdio/ultraestrutura , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Non-small cell lung cancer (NSCLC), one of the most common causes of cancer-related death, is a worldwide public health problem. MicroRNAs (miRNAs) have recently been identified as a novel class of regulators of carcinogenesis and tumor progression, including miRNAs associated with NSCLC. This study aimed to explore the role of miR-522 in NSCLC and the mechanisms underlying this role. We report here that miR-522 expression was significantly increased in both human NSCLC tissues and cell lines. Furthermore, an MTT assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay kit and flow cytometry confirmed that the inhibition of miR-522 suppressed NSCLC cells proliferation and induced apoptosis. Compared with miR-522 overexpression, miR-522 inhibitor markedly reduced cells migration and invasion, as indicated by wound-healing and transwell assays. In addition, a luciferase assay identified DENN/MADD domain containing 2D (DENND2D) as a direct target of miR-522. qRT-PCR and western blot analyses indicated the reciprocal expression of miR-522 and DENND2D in NSCLC tissue samples. DENND2D was involved in miR-522 induced proliferation and metastasis of NSCLC cells by a miRNA-masking antisense oligonucleotides (miR-mask) technology. These data highlight a novel molecular interaction between miR-522 and DENND2D, which indicates that targeting miR-522 may constitute a potential therapy for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Interferência de RNA , Proteínas Supressoras de Tumor/genética , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Proteínas Supressoras de Tumor/metabolismoRESUMO
Oxidative stress is a causal factor and key promoter of a variety of cardiovascular diseases associated with apoptotic cell death by causing deregulation of related genes. Though carvedilol, a ß-adrenergic blocker, has been shown to produce cytoprotective effects against cardiomyocyte apoptosis, the mechanisms are not fully understood. The present study was designed to investigate whether the beneficial effects of carvedilol are related to microRNAs which have emerged as critical players in cardiovascular pathophysiology via post-transcriptional regulation of protein-coding genes. In vivo, we demonstrated that carvedilol ameliorated impaired cardiac function of infarct rats and restored miR-133 expression. In vitro, carvedilol protected cardiomyocytes from H2O2 induced apoptosis detected by TUNEL staining and MTT assays, and increased miR-133 expression in cardiomyocytes. Overexpression of miR-133, a recognized anti-apoptotic miRNA, produced similar effects to carvedilol: reduction of reactive oxygen species (ROS) and malondialdehyde (MDA) content and increment of superoxide dismutase (SOD) activity and glutathione peroxidase (GPx) level, so as to protect cardiomyocytes from apoptosis by downregulating caspase-9 and caspase-3 expression in the presence of H2O2. Transfection with AMO-133 (antisense inhibitor oligodeoxyribonucleotides) alone abolished the beneficial effects of carvedilol. Caspase-9-specific inhibitor z-LEHD-fmk, caspase-3-specific inhibitor z-DEVD-fmk, caspase-9 siRNA and caspase-3 siRNA were used to establish caspase-3 as a downstream target of miR-133. In conclusion, our data indicated that carvedilol protected cardiomyocytes by increasing miR-133 expression and suppressing caspase-9 and subsequent apoptotic pathways.
Assuntos
Apoptose/efeitos dos fármacos , Carbazóis/farmacologia , Citoproteção/efeitos dos fármacos , MicroRNAs/genética , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Propanolaminas/farmacologia , Regulação para Cima/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Animais , Apoptose/genética , Carbazóis/uso terapêutico , Cardiotônicos/farmacologia , Carvedilol , Caspase 9/metabolismo , Técnicas de Silenciamento de Genes , Testes de Função Cardíaca , Peróxido de Hidrogênio/toxicidade , Masculino , MicroRNAs/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Propanolaminas/uso terapêutico , Ratos WistarRESUMO
Global warming has seriously decreased world crop yield. High temperatures affect development, growth and, particularly, reproductive tissues in plants. A gene encoding ß-ureidopropionase (SlUPB1, EC 3.5.1.6) was isolated from the stamens of a heat-tolerant tomato (CL5915) using suppression subtractive hybridization. SlUPB1 catalyzes the production of ß-alanine, the only ß-form amino acid in nature. In the anthesis stage, SlUPB1 expression in CL5915 stamens, growing at 35/30°C (day/night), was 2.16 and 2.93 times greater than that in a heat-sensitive tomato (L4783) cultivated at 30/25°C or 25/20°C, respectively. Transgenic tomatoes, upregulating SlUPB1 in L4783 and downregulating SlUPB1 in CL5915, were constructed, and the amount of ß-alanine measured by liquid chromatography-electrospray ionization-mass spectrometry in the transgenic overexpression of SlUPB1 was higher than that of L4783. However, the ß-alanine in the transgenics downregulating SlUPB1 was significantly lower than the ß-alanine of CL5915. Pollen germination rates of these transgenics were analyzed under different developmental and germinating temperatures. The results indicated that germination rates of transgenics overexpressing SlUPB1 were higher than germination rates of the background tomato L4783. Germination rates of transgenics downregulating SlUPB1 were significantly lower than germination rates of background tomato CL5915, indicating the necessity of functional SlUPB1 for pollen germination. Pollen germinating in the buffer with the addition of ß-alanine further indicated that ß-alanine effectively enhanced pollen germination in tomatoes with low SlUPB1 expression. Together, these results showed that the expression of SlUPB1 is important for pollen germination, and ß-alanine may play a role in pollen germination under both optimal and high temperatures.
Assuntos
Amidoidrolases/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Pólen/genética , Solanum lycopersicum/genética , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Regulação para Baixo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/métodos , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Regulação para Cima , beta-Alanina/metabolismoRESUMO
Few studies have focused on biosorption by microorganisms under culture conditions. To explore the biosorption of uranium by Saccharomyces cerevisiae under culture conditions, the S. cerevisiae growth curve, biosorption capacity and surface interaction under batch culture conditions were investigated in this study. The growth curve showed that uranium (<300mgL(-1)) did not markedly inhibit the growth of S. cerevisiae under short culture time. The maximum scavenging efficiency reached 92.4% under 6-10h culture conditions, and the adsorption quantity of S. cerevisiae increased with initial uranium concentration. Centrifuging and drying after biosorption caused the volume reduction ratio to reach 99%. Scanning electron microscope results demonstrated that uranium interacted with yeast cell surfaces, as well as culture medium, and produced uranium precipitate on cell surfaces. Fourier transformed infrared spectra revealed that cell walls were the major sorption sites, and -O--H, -C==O and -PO(2-) contributed to the major binding groups.