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Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae, and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin-induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Botrytis , Resistência à Doença , Doenças das Plantas , Pseudomonas syringae , Tiazóis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Botrytis/fisiologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Indóis/metabolismo , Fosforilação , Fitoalexinas , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Pseudomonas syringae/patogenicidade , Pseudomonas syringae/fisiologia , Tiazóis/metabolismoRESUMO
To effectively detect faults in transmission lines, monitoring the operating status of these lines is imperative. However, providing power to monitoring devices for transmission line status presents a significant challenge. In this research, a hybrid energy harvesting approach based on micro thermoelectric generator (MTEG) and triboelectric nanogenerator (TENG) is proposed, and a theoretical model for MTEG-TENG hybrid energy harvesting is established. This study develops an integrated energy harvesting prototype, which incorporates oscillating-TENG (O-TENGs), MTEGs, and a power management control unit. This prototype not only harvests energy from the vibrations of transmission lines but also converts the lines thermal energy into electricity. The Experiment results show that the maximum open-circuit voltages of O-TENG and MTEG reach 80.3 V and 1.094 V, respectively. Compared to a single MTEG energy harvesting device, the prototype of the MTEG-TENG hybrid energy harvesting device demonstrates a 5.36% improvement in energy harvesting and battery charging performance. Consequently, this approach achieves self-powered monitoring with excellent stability and lower manufacturing costs. It provides an efficient and durable power approach for transmission line status monitoring devices.
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Four new norlignans, noralashinols D-F (1a/b-3), and two known analogues (4 and 5) were isolated from the peeled stems of Syringa pinnatifolia Hemsl. The structures were elucidated by analysis of spectroscopic data, such as IR, HR-ESI-MS, 1D and 2D NMR, and ECD. All compounds were evaluated for anti-inflammatory activities against NO production induced by LPS in BV2 microglia cells. Compounds 1b and 2 exhibited moderate activities with IC50 values of 32.39 ± 9.1 and 47.83 ± 10.44 µM, respectively, compared with positive control indomethacin (IC50 = 21.62 µM). It is worth to note that 1, 3, and 4 have a distinctive woody fragrance.
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In the era of climate change and carbon neutrality, China is actively coping with its carbon lock-in dilemma. In this context, the development of the digital economy is considered a possible path to carbon unlocking. This study contributes to the literature by providing a comprehensive analysis of how the digital economy can be leveraged to address carbon lock-in, highlighting the importance of formal environmental regulation and informal environmental regulation in enhancing this effect. Accordingly, following findings are highlighted. (1) Our primary findings provide strong evidence on the negative effect of the digital economy on carbon lock-in; by implication, improving the digital economy is an efficient measure for eradicating carbon lock-in. (2) The digital economy shows the greatest marginal impact on industrial lock-in, while its inhibiting effect on behavior lock-in is the least pronounced. Moreover, the digital economy plays a prominent role in curbing carbon lock-in in provinces with a higher level of physical, human, and social capital. And the asymmetric impacts of the digital economy on carbon lock-in are significant at most quantiles. (3) Environmental regulation is a significant moderator. Put differently, the synergy of formal environmental regulation and the digital economy, as well as informal environmental regulation and the digital economy, are important means to break carbon lock-in. (4) The carbon lock-in mitigation effect of the digital economy is caused mainly by increasing technical market turnover and the efficiency of energy consumption.
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Carbono , Mudança Climática , China , Humanos , Conservação dos Recursos NaturaisRESUMO
Three quinone-terpenoid alkaloids, alashanines A-C (1-3), possessing an unprecedented 6/6/6 tricyclic conjugated backbone and quinone-quinoline-fused characteristic, were isolated from the peeled stems of Syringa pinnatifolia. Their structures were elucidated by analysis of extensive spectroscopic data and quantum chemical calculations. A hypothesis of biosynthesis pathways for 1-3 was proposed on the basis of the potential precursor iridoid and benzoquinone. Compound 1 exhibited antibacterial activities against Bacillus subtilis and cytotoxicity against HepG2 and MCF-7 human cancer cell lines. The results of the cytotoxic mechanism revealed that compound 1 induced apoptosis of HepG2 cells through activation of ERK.
Assuntos
Alcaloides , Antineoplásicos , Syringa , Humanos , Syringa/química , Terpenos , Estrutura Molecular , Extratos Vegetais , Alcaloides/farmacologia , Benzoquinonas , QuinonasRESUMO
Conventional double differential phase-shift keying modulation amplifies the phase noise and performs poorly under the time-varying direct-sequence spread-spectrum (DSSS) communication system. Therefore, the authors propose an iterative reception for DSSS communication in time-varying underwater acoustic channels. First, bit-interleaved coded modulation with iterative decoding integrated with multi-symbol differential detection is used. Second, this paper uses cross correlation method to estimate and track the Doppler shift of each symbol. Based on Doppler estimates, a dynamic linear prediction model is proposed to estimate and track the channel phase variation. Third, an algorithm for adaptive selection of reference signals is utilized to recover the magnitude attenuation of correlation peaks. Numerical simulation results demonstrate that the proposed reception achieves around 9 dB gain compared to conventional differential decision reception under constant acceleration of 0.14 m/s2. During the acoustic communication experiment in Songhua Lake, the proposed reception was tested by using a moving source at a speed of 1-6 knots at 2-m depth and the farthest distance between the transceivers is 2.8 km. The proposed reception achieves only one frame error from a total of 205 frames collected in the lake experiment, and it also achieves error-free communications over 96 frames during a 10 km depth deep-sea experiment.
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Underwater vehicles can operate independently in the exploitation of marine resources. However, water flow disturbance is one of the challenges underwater vehicles must face. The underwater flow direction sensing method is a feasible way to overcome the challenges but faces difficulties such as integrating the existing sensors with underwater vehicles and high-cost maintenance fees. In this research, an underwater flow direction sensing method based on the thermal tactility of the micro thermoelectric generator (MTEG) is proposed, with the theoretical model established. To verify the model, a flow direction sensing prototype is fabricated to carry out experiments under three typical working conditions. The three typical flow direction conditions are: condition No. 1, in which the flow direction is parallel to the x-axis; condition No. 2, in which the flow direction is at an angle of 45° to the x-axis; and condition No. 3, which is a variable flow direction condition based on condition No. 1 and condition No. 2. According to the experimental data, the variations and orders of the prototype output voltages under three conditions fit the theoretical model, which means the prototype can identify the flow direction of three conditions. Besides, experimental data show that in the flow velocity range of 0~5 m/s and the flow direction variation range of 0~90°, the prototype can accurately identify the flow direction in 0~2 s. The first time utilizing MTEG on underwater flow direction perception, the underwater flow direction sensing method proposed in this research is cheaper and easier to be applied on the underwater vehicles than traditional underwater flow direction sensing methods, which means it has great application prospects in underwater vehicles. Besides, the MTEG can utilize the waste heat of the underwater vehicle battery as the energy source to achieve self-powered work, which greatly enhances its practical value.
Assuntos
Fontes de Energia Elétrica , Temperatura Alta , PercepçãoRESUMO
Corydalis hendersonii(CH) is a Tibetan folk medicine with the functions of clearing heat, detoxifying, cooling blood, checking diarrhea, and lowering blood pressure. It is often used to treat high altitude polycythemia, vasculitis, peptic ulcer, and diarrhea. Nine compounds were separated from the ethanol extract of CH by silica gel, ODS, Sephadex LH-20 chromatography and semi-preparative HPLC. Their structures were identified as hendersine H(1),hendersine I(2), dehydrocheilanthifoline(3), protopine(4), izmirine(5), 6,7-methylenedioxy-1(2H)-isoquinolinone(6), icariside D_2(7), ethyl 4-(ß-D-glucopyranosyloxy)-3-methoxybenzoate(8), 3-hydroxy-4-methoxybenzoic acid(9), respectively, by the spectroscopic data analysis and comparison with those in the literature. Among them, compounds 1 and 2 are new isoquinoline alkaloids, and compounds 7-9 are reported the first time for Corydalis. The hypoglycemic model of H9c2 cardiomyocytes and the inflammatory model of H9c2 cardiomyocytes induced by conditional supernatant were employed to determine the activities of the above compounds. The results showed that 20 µmol·L~(-1) compound 1 had a protective effect on H9c2 cardiomyocytes and 10 µmol·L~(-1) compounds 4 and 5 inhibited H9c2 cardiomyocyte inflammation induced by conditional supernatant.
Assuntos
Alcaloides , Corydalis , Humanos , Corydalis/química , Alcaloides/farmacologia , Alcaloides/química , Inflamação , Análise Espectral , Isoquinolinas/farmacologiaRESUMO
The peeled stems of Syringa pinnatifolia(SP) is a representative Mongolian folk medicine with the effects of anti-depression, heat clearance, pain relief, and respiration improvement. It has been clinically used for the treatment of coronary heart disease, insomnia, asthma, and other cardiopulmonary diseases. As part of the systematic study on pharmacological substances of SP, 11 new sesquiterpenoids were isolated from the terpene-containing fractions of the ethanol extract of SP by liquid chromatography-mass spectrometry(LC-MS) and proton nuclear magnetic resonance(~1H-NMR) guided isolation methods. The planar structures of the sesquiterpenoids were identified by MS, 1D NMR, and 2D NMR data analysis, and were named pinnatanoids C and D(1 and 2), and alashanoids T-ZI(3-11), respectively. The structure types of the sesquiterpenoids included pinnatane, humulane, seco-humulane, guaiane, carryophyllane, seco-erimolphane, isodaucane, and other types. However, limited to the low content of compounds, the existence of multiple chiral centers, the flexibility of the structure, or lack of ultraviolet absorption, the stereoscopic configuration remained unresolved. The discovery of various sesquiterpenoids enriches the understanding of the chemical composition of the genus and species and provides references for further analysis of pharmacological substances of SP.
Assuntos
Asma , Sesquiterpenos , Syringa , Terpenos , Cromatografia LíquidaRESUMO
A phytochemical investigation guided by 1H NMR and LC-MS data on the ethanol extract of Syringa pinnatifolia stems led to the isolation of 11 new dimeric eremophilane sesquiterpenoids, namely, syringenes A-K (1-11) and one known analog (12, syringene L). These structures were elucidated by extensive analysis of spectroscopic data, single-crystal X-ray diffraction, and computational methods. Biological assays revealed that 1-12 exhibited different degrees of anti-inflammatory effects, and 5 and 6 showed significant cytotoxicity against human hepatoma HepG2 cells with IC50 values of 12.3 and 12.9 µM, respectively. Furthermore, flow cytometry assays and western blot analysis revealed that 5 and 6 promoted the apoptosis of HepG2 cells by activating ERK. Finally, the molecular docking analysis implied that the carbonyl and hydroxy groups at the C-11/C-6' of 5 and 6 had a good binding affinity with ERK.
Assuntos
Sesquiterpenos , Syringa , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Sesquiterpenos Policíclicos , Sesquiterpenos/química , Syringa/químicaRESUMO
Five new sesquiterpenoids, alashanoids O-S (1-5), along with three known analogs (6-8) were isolated from the peeled stems of Syringa pinnatifolia. Their structures were elucidated by analysis of extensive spectroscopic data including ESI-MS, 1D, 2D NMR. The absolute configurations were determined by comparing its experimental and calculated electronic circular dichroism, calculated OR, calculated NMR, and single crystal X-ray diffraction data analysis. Compounds 1 and 2 belong to the seco-humulane type and possess a rare 13-membered oxygen heterocycle framework, and 3-5 belong to eremophilane-type. Compounds 1, 2, and 5 showed inhibitory effects against NO production in LPS-induced RAW264.7 macrophage cells with its IC50 values of 11.86±2.34, 72.08±7.72, and 69.22±15.29â µM, respectively, compared with the positive control indomethacin (IC50 =31.52â µM).
Assuntos
Sesquiterpenos , Syringa , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Sesquiterpenos Policíclicos , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Syringa/químicaRESUMO
Three pairs of enantiomeric sesquiterpenoids, (∓)-syringanoid A (1a and 1b) and (±)-pinnatanoids A (2a and 2b) and B (3a and 3b), that represent an unprecedented 5/4/6 tricyclic backbone and a rare 6/7 bicyclic backbone, respectively, were isolated from the peeled stems of Syringa pinnatifolia. The structures were elucidated by extensive spectroscopic analysis, single-crystal X-ray diffraction, a modified Mosher's method, and quantum chemical calculations. A plausible biotransformation pathway for 1-3 was proposed, and their cardiomyocyte-protective and anti-inflammatory activities were evaluated.
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Sesquiterpenos , Syringa , Cristalografia por Raios X , Estrutura Molecular , EstereoisomerismoRESUMO
Two enantiomeric humulane sesquiterpenes, namely (+)-alashanoid N (1a) and (-)-alashanoid N (1b), along with two known analogs ((2R,3R,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (2) and (2R,3S,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (3)), were described from the peeled stems of a folk Mongolian herbal medicine Syringa pinnatifolia. Their structures were characterized based on UV, IR, NMR, and HR-ESI-MS data analyses, and the absolute configurations were determined by data analysis of X-ray diffraction and quantum chemical calculations. (+/-)-Alashanoid N showed inhibition against NO production in RAW 264.7 macrophage cells with IC50 values of 90.1â µM and 71.7â µM, and protective effect against oxygen-glucose deprivation injury to H9c2 cells at a concentration of 20â µM and 5â µM, respectively.
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Substâncias Protetoras/farmacologia , Syringa/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glucose/antagonistas & inibidores , Glucose/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Conformação Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Oxigênio/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Células RAW 264.7 , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In this study, the compound search was completed through SciFinder and CNKI databases, and the drug-like properties were screened in FAFdrugs4 and SEA Search Server databases. In addition, based on the target sets related to acute myocardial ischemia(AMI) searched in disease target databases such as OMIM database, GeneCards database and DrugBank, a network diagram of chemical component-target-pathway-disease was established via Cytoscape to predict the potential active components of Corydalis Herba, a traditional Tibetan herbal medicine which derived from the aerial parts of Corydalis hendersonii and C. mucronifera against AMI. A protein-protein interaction(PPI) network was constructed through the STRING database and the core targets in the network were predicted. And the enrichment analyses of core targets were completed by DAVID database and R software. Furthermore, a molecular docking method was used to verify the binding of the components with core targets using softwares such as Autodock Vina. The present results showed that there were 60 compounds related to AMI in Corydalis Herba, involving 73 potential targets. The GO functional enrichment analysis obtained 282 biological processes(BP), 49 cell components(CC) and 78 molecular functions(MF). KEGG was enriched into 85 pathways, including alcoholism pathway, endocrine resistance pathway, calcium signaling pathway, cAMP signaling pathway, vascular endothelial growth factor signaling pathway and adrenergic signaling transduction pathway of myocardial cells. The results of network topology analysis showed that the key components of anti-AMI of Corydalis Herba might be tetrahydropalmatine, etrahydrocolumbamine, N-trans-feruloyloctopamine, N-cis-p-coumaroyloctopamine, N-trans-p-coumaroylnoradrenline and N-trans-p-coumaroyloctopamine, and their core targets might be CDH23, SCN4 B and NFASC. The results of molecular docking showed that the key components of Corydalis Herba had stable binding activity with the core targets. This study provides reference for further elucidation of the pharmacological effects of Corydalis Herba against AMI, subsequent clinical application, and development.
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Corydalis , Medicamentos de Ervas Chinesas , Isquemia Miocárdica , Medicamentos de Ervas Chinesas/farmacologia , Medicina Tradicional Tibetana , Simulação de Acoplamento Molecular , Isquemia Miocárdica/tratamento farmacológico , Fator A de Crescimento do Endotélio VascularRESUMO
Polyamines are small amines that accumulate during stress and contribute to disease resistance through as yet unknown signaling pathways. Using a comprehensive RNA-sequencing analysis, we show that early transcriptional responses triggered by each of the most abundant polyamines (putrescine, spermidine, spermine, thermospermine and cadaverine) exhibit specific quantitative differences, suggesting that polyamines (rather than downstream metabolites) elicit defense responses. Signaling by putrescine, which accumulates in response to bacteria that trigger effector triggered immunity (ETI) and systemic acquired resistance (SAR), is largely dependent on the accumulation of hydrogen peroxide, and is partly dependent on salicylic acid (SA), the expression of ENHANCED DISEASE SUSCEPTIBILITY (EDS1) and NONEXPRESSOR of PR GENES1 (NPR1). Putrescine elicits local SA accumulation as well as local and systemic transcriptional reprogramming that overlaps with SAR. Loss-of-function mutations in arginine decarboxylase 2 (ADC2), which is required for putrescine synthesis and copper amine oxidase (CuAO), which is involved in putrescine oxidation, compromise basal defenses, as well as putrescine and pathogen-triggered systemic resistance. These findings confirm that putrescine elicits ROS-dependent SA pathways in the activation of plant defenses.
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Arabidopsis/efeitos dos fármacos , Reguladores de Crescimento de Plantas/metabolismo , Putrescina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Arabidopsis/metabolismo , Cadaverina/farmacologia , Perfilação da Expressão Gênica , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Espermidina/farmacologia , Espermina/análogos & derivados , Espermina/farmacologiaRESUMO
Saussurea lappa originates in India, and now mainly grow in Yunnan, Sichuan and other places in China. It is one of the commonly used traditional herbal medicines in Tibet and other minority regions, with effects in regulating qi to relieve pain and invigo-rating spleen to promote food. It has been used in clinic for gastrointestinal diseases, such as Qi stagnation syndrome of spleen and stomach, diarrhea and tenesmus. More than 200 compounds have been identified from S. lappa. Among them, sesquiterpenoids attracted much attention. In terms of the number of compounds, eudesmanetype is dominant, guaiane and germacranetypes have also been reported frequently. Pharmacological studies have involved extracts, volatile oils and monomeric components represented by dehydrocostus lactone. Anti-tumor, anti-inflammatory and anti-bacterial effects on digestive system have attracted great attention. However, due to the complex sources of S. lappa and widely used in clinical practice, there is few research progress on relevant chemical constituents and pharmacological activities. This paper systematically summarizes terpenes and the pharmacological effects of S. lappa, in order to provide basis for further studies and clinical applications.
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Saussurea , Sesquiterpenos , China , Extratos Vegetais , Terpenos , TibetRESUMO
Genetic divergence between populations can lead to reproductive isolation. Hybrid incompatibilities (HI) represent intermediate points along a continuum toward speciation. In plants, genetic variation in disease resistance (R) genes underlies several cases of HI. The progeny of a cross between Arabidopsis (Arabidopsis thaliana) accessions Landsberg erecta (Ler, Poland) and Kashmir2 (Kas2, central Asia) exhibits immune-related HI. This incompatibility is due to a genetic interaction between a cluster of eight TNL (TOLL/INTERLEUKIN1 RECEPTOR-NUCLEOTIDE BINDING-LEU RICH REPEAT) RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like genes (R1-R8) from Ler and central Asian alleles of a Strubbelig-family receptor-like kinase (SRF3) from Kas2. In characterizing mutants altered in Ler/Kas2 HI, we mapped multiple mutations to the RPP1-like Ler locus. Analysis of these suppressor of Ler/Kas2 incompatibility (sulki) mutants reveals complex, additive and epistatic interactions underlying RPP1-like Ler locus activity. The effects of these mutations were measured on basal defense, global gene expression, primary metabolism, and disease resistance to a local Hyaloperonospora arabidopsidis isolate (Hpa Gw) collected from Gorzów (Gw), where the Landsberg accession originated. Gene expression sectors and metabolic hallmarks identified for HI are both dependent and independent of RPP1-like Ler members. We establish that mutations suppressing immune-related Ler/Kas2 HI do not compromise resistance to Hpa Gw. QTL mapping analysis of Hpa Gw resistance point to RPP7 as the causal locus. This work provides insight into the complex genetic architecture of the RPP1-like Ler locus and immune-related HI in Arabidopsis and into the contributions of RPP1-like genes to HI and defense.
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Proteínas de Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Resistência à Doença/genética , Mutação , Doenças das Plantas/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Sistemas CRISPR-Cas , Quimera , Resistência à Doença/imunologia , Epistasia Genética , Regulação da Expressão Gênica de Plantas , Proteínas NLR/genética , Oomicetos/patogenicidade , Plantas Geneticamente Modificadas , Polônia , Proteínas Proto-Oncogênicas c-myb/genética , Locos de Características Quantitativas , Autoincompatibilidade em Angiospermas/genética , Autoincompatibilidade em Angiospermas/imunologia , NicotianaRESUMO
SAMHD1 is an innate immunity restriction factor that inhibits virus infection through IRF3-mediated antiviral and apoptotic responses. Fish SAMHD1 shares some similar properties with those in mammals. In this study, a SAMHD1 orthologue from grass carp (Ctenopharyngodon idellus) was cloned and characterized. The full-length cDNA of CiSAMHD1 is 2792 bp with an ORF of 1884 bp encoding a polypeptide of 627 amino acids. Multiple alignments showed that SAMHD1 is highly conserved among different species. Phylogenetic tree analysis revealed that CiSAMHD1 shared a high degree of homology with Sinocyclocheilus rhinocerous SAMHD1. Expression analysis indicated that CiSAMHD1 was widely expressed in all tissues tested including the brain, eyes, spleen, gill, intestine, liver, heart and kidney. It was significantly up-regulated in spleen, liver and intestines after treatment with poly I:C. Also, CiSAMHD1 can be induced following stimulation with recombinant IFN in CIK cells. The promoter sequence of CiSAMHD1 was identified to explore the mechanism underlying the transcriptional regulation of CiSAMHD1. The promoter sequence of CiSAMHD1 (1370 bp) consists of IRF1, IRF3, IRF9 and p65 binding elements. Gel mobility shift assay also showed that IRF1, IRF3, IRF9 and p65 prokaryotic proteins can separately interact with CiSAMHD1 promoter. Dual luciferase assay and q-PCR suggested that the promoter of CiSAMHD1 can be activated by the overexpression of CiIRF3 and CiIRF9, but cannot be triggered by CiIRF1 and Cip65. In contrast, knockdown of CiIRF3 or CiIRF9 inhibits the transcription of CiSAMHD1. Intriguingly, CCK assay suggested that CiSAMHD1 decreased cell viability. TUNEL apoptosis assay and Hoechst 33258 staining assay indicated that apoptosis is induced by the overexpression of CiSAMHD1. Crystal violet staining, detection of two GCRV genes (vp3 and vp5) and viral titration showed that CiSAMHD1 can suppress the proliferation of grass carp reovirus (GCRV) in CIK cells.
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Apoptose , Carpas/genética , Proteínas de Peixes/genética , Orthoreovirus/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Replicação Viral , Animais , Proliferação de Células , Clonagem Molecular , DNA Complementar/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Regiões Promotoras Genéticas , Infecções por Reoviridae , Proteína 1 com Domínio SAM e Domínio HD/metabolismoRESUMO
In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an absolutely vital role in response to cytokines and growth factors. In mammals, IL-6/JAK/STAT3 pathway is closely linked to immune response and promotes cell proliferation, survival and metastasis. Some recent studies have already demonstrated that STAT3 regulates autophagy. As a downstream target gene of STAT3, Bcl-2 (B-cell lymphoma 2) not only participates in regulating apoptosis, but also responds to autophagy. STAT3 regulates autophagy through Bcl-2. In general, the generation of autophagy is always accompanied by the change of apoptosis, and the occurrence of apoptosis is often accompanied by the decreased of cell viability. In grass carp (Ctenopharyngodon idella), LPS-induced autophagy is involved in the release of pro-inflammatory cytokines. However, only the relationship between autophagy and cytokines was illustrated, in which the signaling pathways were not discussed. In the present study, we found that the autophagy inducer, Tunicamycin (Tm), can induce C.Idella Kidney cells (CIK) autophagy. When the cells were incubated with the recombinant human IL-6 (rIL-6) for a short period of times, the mRNA expression level of C.Idella IL-6R and STAT3 were increased. At the same time, the number of GFP-LC3 puncta and the ratio of LC3-II/LC3-I were both decreased obviously in cells. It indicated that the rIL-6 can significantly alleviate autophagy induced by Tm. We speculated that CiSTAT3 may play a key role in the process. To confirm this hypothesis, we performed a rIL-6 activating CiSTAT3 assay. The result demonstrated that rIL-6 can induce CiSTAT3 to form homologous dimmer. The activated CiSTAT3 regulated the transcription activity of CiBcl-2, finally led to a decrease of autophagy. In addition, when cells were in the state of autophagy, apoptosis was increased and cell viability was decreased. When CiSTAT3 was activated, cell apoptosis weakened and cell viability was increased. The results suggest that CiSTAT3 plays an important role in maintaining the normal physiological process of cells.
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Autofagia/genética , Carpas/fisiologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição STAT3/genética , Regulação para Cima/imunologia , Animais , Carpas/imunologia , Dimerização , Proteínas de Peixes/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
In mammals, signal transducer and activator of transcription 6 (STAT6) is a broad-spectrum transcriptional regulator involved in cellular immune responses and apoptosis by regulating the immune-related genes and various functional genes. The structure, expression and tyrosine-based phosphorylation of STAT6 are conserved from fish to mammal. However, except the sporadic reports from zebra fish, the function of fish STAT6 has not been well reported. Here, we cloned and characterized the full length cDNA sequence of grass carp (Ctenopharyngodon idella) STAT6 (CiSTAT6). Meanwhile, the activation mechanism and the potential function of CiSTAT6 were studied. The full length cDNA of CiSTAT6 is 2747 bp with an ORF of 2313 bp encoding a polypeptide of 770â¯amino acids. Phylogenetic tree analysis revealed that CiSTAT6 shares the maximum homology with Cyprinus carpio STAT6. CiSTAT6 was significantly up-regulated and interacted with each other to form the homodimer after treatment with poly I:C. The transfected CiSTAT6 in fish cell lines can activate the promoter activities of CCL20 and Bcl-xl and increase their mRNA levels. In addition, we also found that CiSTAT6 can increase cell viability and inhibit cell apoptosis. Taken together, grass carp STAT6 plays an important part in innate immunity and anti-apoptosis.