RESUMEN
In mammals, nemo-like kinase 2 (NLK2) is a conservative protein kinase involved in Wnt/ß-catenin signaling pathway and immune response. However, the role of NLK2 in immune response in teleost remain unclear. In this study, we identified an ortholog of mammalian NLK from grass carp (Ctenopharyngodon idellus) named CiNLK2. CiNLK2 shares a high level of homology with the counterparts, especially with that of Cyprinus carpio. CiNLK2 was ubiquitously expressed in all tested tissues (liver, brain, spleen, gill, kidney and eye) and its expression was up-regulated under the treatment with poly I:C or GCRV. Overexpression of CiNLK2 suppressed the production of IFN I in CIK cells whether or not treated with poly I:C. However, knockdown of CiNLK2 increased the expression level of IFN I. The analysis of subcellular localization showed that CiNLK2 protein was scattered throughout the cytoplasm and nucleus. In terms of mechanism, CiNLK2 can directly interact with MAVS and inhibit MAVS-induced IFN I response. Moreover, CiNLK2 increased the phosphorylation level of MAVS, which led to the degradation of MAVS protein. On the other hand, CiNLK2 suppressed the phosphorylation and nuclear translocation of IRF3. In general, CiNLK2 served as an inhibitor for IFN I response by targeting MAVS-IRF3 signal axis.
Asunto(s)
Carpas , Enfermedades de los Peces , Interferón Tipo I , Reoviridae , Animales , Carpas/genética , Carpas/metabolismo , Interferón Tipo I/metabolismo , Poli I-C/farmacología , Fosforilación , Proteínas de Peces , Inmunidad Innata/genética , Reoviridae/fisiología , Mamíferos/metabolismoRESUMEN
Isoprocarb is a widely used carbamate insecticide in agriculture and aquaculture. Overuse of isoprocarb always leaves toxic residues in soil and water, however, the potential ecotoxicity of isoprocarb to organisms is still confusing. In this study, zebrafish embryo was used as a model to evaluate the toxicity of isoprocarb. Zebrafish embryos (96 hpf) were separately exposed at different concentrations of isoprocarb. The mortality rate, hatchability rate, average heart beat of the zebrafish embryo were separately calculated. Our results suggested that exposure to isoprocarb induced developmental toxicity in zebrafish embryos. HE staining showed that exposure to isoprocarb caused developmental defect in the hindbrain of zebrafish embryos. As expected, the behavioral analysis also showed that the motor ability of zebrafish embryos were significantly inhibited following exposure to isoprocarb. In terms of mechanism, The expressions of genes involved in neurodevelopment signaling pathways, such as foxo3a, gfap, syn2a, elavl3 and sox19b, were inhibited in zebrafish embryos after exposure to isoprocarb. The acetylcholinesterase (AChE) activity was also reduced in isoprocarb-treated zebrafish embryos. Moreover, oxidative stress was induced by increasing the reactive oxygen species (ROS) level and decreasing the activity of antioxidant enzyme (SOD) after exposure to isoprocarb. Expectedly, acridine orange (AO) staining and the detection of some apoptosis-related genes revealed that oxidative stress resulted in apoptosis. In short, the expressions of genes associated with the neurodevelopmental signaling pathway are inhibited, and oxidative stress is also induced in zebrafish embryos after exposure to isoprocarb, which may be the molecular basics of isoprocarb-induced neurotoxicity in zebrafish embryos.
Asunto(s)
Síndromes de Neurotoxicidad , Contaminantes Químicos del Agua , Acetilcolinesterasa/metabolismo , Animales , Apoptosis/genética , Carbamatos/metabolismo , Embrión no Mamífero/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Estrés Oxidativo , Factores de Transcripción SOX/metabolismo , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Diflubenzuron is an insecticide that serves as a chitin inhibitor to restrict the growth of many harmful larvae, including mosquito larvae, cotton bollworm and flies. The residue of diflubenzuron is often detected in aquaculture, but its potential toxicity to aquatic organisms is still obscure. In this study, zebrafish embryos (from 6 h to 96 h post-fertilization, hpf) were exposed to different concentrations of diflubenzuron (0, 0.5, 1.5, 2.5, 3.5 and 4.5 mg/L), and the morphologic changes, mortality rate, hatchability rate and average heart rate were calculated. Diflubenzuron exposure increased the distance between the venous sinus and bulbar artery (SV-BA), inhibited proliferation of myocardial cells and damaged vascular development. In addition, diflubenzuron exposure also induced contents of reactive oxygen species (ROS) and malondialdehyde (MDA) and inhibited the activity of antioxidants, including SOD (superoxide dismutase) and CAT (catalase). Moreover, acridine orange (AO) staining showed that diflubenzuron exposure increased the apoptotic cells in the heart. Q-PCR also indicated that diflubenzuron exposure promoted the expression of apoptosis-related genes (bax, bcl2, p53, caspase3 and caspase9). However, the expression of some heart-related genes were inhibited. The oxidative stress-induced apoptosis damaged the cardiac development of zebrafish embryos. Therefore, diflubenzuron exposure induced severe cardiotoxicity in zebrafish embryos. The results contribute to a more comprehensive understanding of the safety use of diflubenzuron.
Asunto(s)
Diflubenzurón , Insecticidas , Contaminantes Químicos del Agua , Naranja de Acridina , Animales , Antioxidantes/metabolismo , Cardiotoxicidad/metabolismo , Catalasa/metabolismo , Quitina/metabolismo , Embrión no Mamífero/metabolismo , Insecticidas/metabolismo , Malondialdehído/metabolismo , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.
Asunto(s)
Carpas/inmunología , Proteínas de Peces/inmunología , Glucógeno Sintasa Quinasa 3 beta/inmunología , Interferón Tipo I/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/inmunología , Secuencia de Aminoácidos , Animales , Carpas/genética , Línea Celular , Proteínas de Peces/genética , Fosforilación , Filogenia , Poli I-C/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genéticaRESUMEN
Ethoprophos is an effective and widely pesticide that used in controlling nemathelminth and soil insect. However, ethoprophos has been frequently detected in environment and freshwater. The potential toxicity to aquatic organisms is still not be explored. In this study, zebrafish embryo model was used to evaluated the toxicity of ethoprophos during cardiovascular developmental process of zebrafish. Zebrafish embryos were separately exposed to 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L of ethoprophos exposure at 96 h post-fertilization (hpf), which induced cardiac defects, such as low heart rate, pericardium edema and long SV-BA distance, but had no influence to vascular development. Mechanistically, the expression of cardiac-related genes were abnormal. Moreover, ethoprophos exposure significantly increased oxidative stress in zebrafish embryos by inhibiting the production of antioxidant enzyme (SOD) and activating reactive oxygen species. Expectedly, some apoptosis genes were induced and the apoptotic cardiomyocytes were detected by acridine orange staining. In addition, ethoprophos exposure also inhibited the expression of genes in wnt signaling pathway, such as ß-catenin, Axin2, GSK3ß and Sox9b. BML284, an activator of wnt signaling pathway, can rescue the cardiotoxic effect of embryos. These results indicated that oxidative stress and blocking wnt signaling pathway were molecular basis of ethoprophos-induced injure in zebrafish. Generally, our study showed that ethoprophos exposure led to severe cardiotoxicity to zebrafish embryo.
RESUMEN
As a member of DExD/H-box helicase family, DDX41 (DEAD box polypeptide 41) acts as an intracellular DNA sensor that induces type I IFN expression in mammals. Fish DDX41 shares some similar properties with the mammalian counterparts. In this study, a DDX41 orthologous gene from grass carp (Ctenopharyngodon idellus) (CiDDX41) was cloned and characterized. The ORF of CiDDX41 encodes a polypeptide of 614 amino acids. Multiple alignments showed that DDX41 is highly conserved among different species. Phylogenetic tree analysis revealed that CiDDX41 shares a high degree of homology with Sinocyclocheilus rhinocerous DDX41. CiDDX41 is highly expressed in kidney, intestines, liver and spleen. Their expressions are up-regulated more obviously after the treatment with GCRV. Over-expression of CiDDX41 in CIK cells increases the transcription level of grass carp IFN I and ISG15. On the contrary, knockdown of CiDDX41 inhibits the IFN I and ISG15 transcription. Moreover, a part of CiDDX41 translocates from the nuclear to cytoplasm to interact with grass carp STING (CiSTING). In CIK cells, overexpression of CiDDX41 and CiSTING can promote the phosphorylation and nuclear-cytoplasm translocation of grass carp IRF7 (CiIRF7) and then acutely up-regulate the IFN I and ISG15 expression. However, the knockdown of CiDDX41 inhibits the phosphorylation IRF7. Taken together, all these results above suggested that CiDDX41 performs as an activator for innate immune through STING-IRF7 mediated signaling pathway.
Asunto(s)
Carpas/genética , Carpas/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Secuencia de Aminoácidos , Animales , Citocinas/química , Citocinas/genética , Citocinas/inmunología , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , Proteínas de Peces/química , Perfilación de la Expresión Génica/veterinaria , Interferón Tipo I/química , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Filogenia , Alineación de Secuencia/veterinaria , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/inmunologíaRESUMEN
As a NAD+-dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity.
Asunto(s)
Apoptosis/genética , Carpas/inmunología , Proteínas de Peces/inmunología , Inmunidad Innata/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/inmunología , Sirtuina 1/inmunología , Proteína p53 Supresora de Tumor/inmunología , Animales , Carpas/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica/inmunología , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Sirtuina 1/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
AHTN (tonalide) is a polycyclic musk that is widely used as fragrance additive in numerous consumer products. AHTN is of great worldwide concern owing to its adverse effects on aquatic organisms and frequent detection in both domestic and foreign aquatic environments. Therefore, derivation of the aquatic life criteria for AHTN exposure is urgently needed. In this work, AHTN toxicity data for eight Chinese native freshwater organisms were used to derive a criterion maximum concentration of 59.39 µg/L and a criterion continuous concentration of 22.43 µg/L using United States Environmental Protection Agency guidelines. Toxicity tests showed that the annelid L. hoffmeisteri and the amphibian R. nigromaculata were the least and most sensitive species to AHTN, respectively. The sensitivity of the planktonic crustacean D. magna to AHTN obviously differed from that of the benthic crustacean M. nipponense. The AHTN and HHCB correlation analysis exhibited a strong positive linear correlation (R2 = 0.8622) in water. The ecological risk assessment showed that AHTN and HHCB posed a higher risk in foreign surface waters than Chinese waters, but a lower risk in foreign wastewater treatment plant effluent than in China. The ecological risks of AHTN and HHCB in most surveyed water bodies of various countries were at acceptable levels, with a few exceptions.
Asunto(s)
Tetrahidronaftalenos/toxicidad , Contaminantes Químicos del Agua/toxicidad , Organismos Acuáticos/efectos de los fármacos , Medición de RiesgoRESUMEN
Tonalide or acetyl hexamethyl tetralin (AHTN) is used as a fragrance additive in various household products. Recently, AHTN has drawn attention owing to its negative health effects on aquatic organisms. Data on AHTN toxicity toward aquatic species are limited. Therefore, this study tested the oxidative stress induced by AHTN exposure on the Rhodeinae sinensis Gunther and Macrobrachium nipponense. In this study, malonaldehyde (MDA) content and the activities of acetyl cholinesterase (AchE), superoxide dismutase (SOD), glutathione S-transferase (GST), and catalase (CAT) in R. sinensis Gunther were tested after 30 days of exposure to 30.093, 34.005, 38.426, 43.421, 49.067, 55.444, 62.652, 70.800, and 80.000 µg/L AHTN, respectively. The MDA, AchE, SOD, GST and CAT in M. nipponense were tested after 40 days of exposure to 60.000, 72.000, 86.400, 103.680, 124.416, 149.299, 179.159, 214.991, and 257.989 µg/L AHTN, respectively. In addition, an integrated biomarker response (IBR) index was utilised to evaluate the integrated toxic effects of AHTN on R. sinensis Gunther and M. nipponense. Finally, the predicted no-effect concentrations (PNECs) of AHTN, based on reproduction, biochemistry, survival, chronic toxicity, and acute toxicity endpoints were derived. The results indicated that low concentrations of AHTN can induce significant changes of oxidative stress biomarkers. The no observed effect concentrations (NOECs) of SOD, GST, AchE, CAT, and MDA were 103.680, 72.000, <60.000, 72.000, and <60.000 µg/L for R. sinensis Gunther and 38.426, 43.421, 30.093, 30.093, and 38.426 µg/L for M. nipponense, respectively. The IBR calculation results showed that 149.299 µg/L AHTN caused the highest toxic effect on R. sinensis Gunther after 30 days of exposure, whereas 70.797 µg/L AHTN caused the greatest damage to M. nipponense after 40 days of exposure. The PNECs of AHTN based on the non-traditional endpoints of biochemistry and reproduction were 0.00145 µg/L and 0.000395 µg/L, respectively, which were significantly lower than the PNEC of 2.636 µg/L for traditional endpoint survival. Therefore, the protection of aquatic organisms based on non-traditional toxicity endpoints should be considered in ecological risk assessment.
Asunto(s)
Antioxidantes/metabolismo , Organismos Acuáticos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Palaemonidae/efectos de los fármacos , Perfumes/toxicidad , Tetrahidronaftalenos/toxicidad , Contaminantes Químicos del Agua/toxicidad , Acetilcolinesterasa/metabolismo , Animales , Organismos Acuáticos/enzimología , Catalasa/metabolismo , Relación Dosis-Respuesta a Droga , Determinación de Punto Final , Glutatión Transferasa/metabolismo , Malondialdehído/metabolismo , Nivel sin Efectos Adversos Observados , Palaemonidae/enzimología , Valor Predictivo de las Pruebas , Superóxido Dismutasa/metabolismoRESUMEN
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.
Asunto(s)
Autofagia/genética , Carpas/fisiología , Proteínas de Peces/genética , Regulación de la Expresión Génica/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factor de Transcripción STAT3/genética , Regulación hacia Arriba/inmunología , Animales , Carpas/inmunología , Dimerización , Proteínas de Peces/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Recently, studies have shown that IκB kinase ß (IKKß), a critical kinase in the nucleus factor kappa-B (NF-κB) pathway, participates in inflammatory responses associated with unfolded protein response (UPR) and plays an important role in ER stress-induced cell death. The unfolded protein response (UPR), which is a regulatory system to restore cellular homeostasis in the endoplasmic reticulum (ER), such as oxidative stress, bacterial infection, and virus invasion. The UPR pathways have been reported to be involved in immune responses in mammals, including the classical NF-κB pathway. However, the molecular mechanism of their crosstalk remains to be elucidated. Previously, we demonstrated that IKKß also has some conserved functions between fish and human, as grass carp (Ctenopharyngodon idella) IKKß (CiIKKß) can activate NF-κB pathway. In this study, we found that CiIKKß level in nucleus was elevated under ER stress and CiIKKß can interact with grass carp X-box-binding protein 1 (CiXBP1S), a key transcription factor in UPR. Consistently, fluorescent histochemical analysis of grass carp kidney (CIK) cells indicated that CiIKKß and CiXBP1S colocalized under ER stress. Furthermore, overexpression of CiIKKß in CIK cells enhanced ER stress tolerance by regulating UPR signaling and resulted in the significant increase of cell viability.
Asunto(s)
Carpas/genética , Estrés del Retículo Endoplásmico , Proteínas de Peces/genética , Regulación de la Expresión Génica/inmunología , Quinasa I-kappa B/genética , Proteína 1 de Unión a la X-Box/genética , Animales , Carpas/inmunología , Núcleo Celular/genética , Supervivencia Celular , Proteínas de Peces/inmunología , Quinasa I-kappa B/inmunología , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-Box/inmunologíaRESUMEN
Cyclic GMP-AMP synthase (cGAS) is known as a DNA sensor for the initiation of innate immune responses in human and other mammals. However, the knowledge about fish cGAS is limited. In this study, we identified two paralogs of cGAS genes from grass carp (Ctenopharyngodon idellus), namely, CicGASa and CicGASb. Grass carp cGASa and cGASb share some conservative domains with mammalian cGASs; however, cGASb contains a unique transmembrane domain. Grass carp cGASa and cGASb responded to GCRV and poly (dA:dT) infection, but they played opposite roles in the regulation of type I IFN response, i.e. cGASa served as an activator for ISGs and NF-κB in a dose-dependent manner, while cGASb acted as an inhibitor. We found that cGASa and cGASb interacted with STING. Similarly, cGASa is an activator for IRF7, but cGASb inhibited IRF7 expression. Both cGASa and STING can protect cells from GCRV infection. Grass carp cGASb inhibited cGASa-induced type I IFN response by the competitive interaction with STING, suggesting that cGASb may be a negative regulator of cGASa-STING-IRF7 axis.
Asunto(s)
Carpas/inmunología , Proteínas de Peces/genética , Nucleotidiltransferasas/genética , Isoformas de Proteínas/genética , Infecciones por Reoviridae/inmunología , Animales , Proteínas de Peces/metabolismo , Humanos , Inmunidad Innata/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Nucleotidiltransferasas/metabolismo , Reoviridae/fisiologíaRESUMEN
Type I interferon and apoptosis elicit multifaceted effects on host defense and various diseases, such as viral infections and cancers. However, the gene/protein network regulating type I interferon and apoptosis has not been elucidated completely. In this study, we selected grass carp (Ctenopharyngodon idella) as an experimental model to investigate the modulation of RNASEK on the secretion of type I interferon and apoptosis. We first cloned two paralogs RNASEK-a and -b in grass carp, defined three exons in each gene, and found the length of both coding regions is 306 bp with 73.27% of protein homology. The protein sequences of the two paralogs are highly conserved across species. Two proteins were mainly localized in early and late endosomes and endoplasmic reticulum. Further, quantitative real-time PCR demonstrated that dsRNA poly I:C and grass carp reovirus upregulated RNASEK-a and -b in grass carp cells and tissues. Overexpression of RNASEK-a and -b individually induced type I interferon expression and the phosphorylation of IRF3/IRF7 shown by Western blot and immunofluorescent staining, increased Bax/Bcl-2 mRNA ratio, DNA fragmentations, TUNEL-positive cells, and the proportion of Annexin V-positive signals in flow cytometry, and activated eIF2α, opposite to that observed when RNASEK-a and -b were knocked down in multiple cell types. Taken together, we claim for the first time that fish paralog proteins RNASEK-a and -b enhance type I interferon secretion and promote apoptosis, which may be involved in the phosphorylation of IRF3/IRF7 and eIF2α, respectively. Our study reveals a previously unrecognized role of RNASEK as a new positive regulator of type I interferon and apoptosis.
Asunto(s)
Apoptosis , Proteínas de Peces/genética , Interferón Tipo I/metabolismo , Animales , Carpas , Línea Celular , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/genéticaRESUMEN
TNK1 (thirty-eight-negative kinase 1) belongs to the ACK (Activated Cdc42 Kinases) family of intracellular non-receptor tyrosine kinases that usually acts as an important regulator in cytokine receptor-mediated intracellular signal transduction pathways. JAK-STAT signal pathway acts as a key point in cellular proliferation, differentiation and immunomodulatory. Mammalian TNK1 is involved in antiviral immunity and activation of growth factors. However, TNK1 has rarely been studied in fish. To evaluate the role of fish TNK1 in JAK-STAT pathway, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TNK1 (CiTNK1). CiTNK1 protein consists of N-terminal Tyrkc (tyrosine kinase) domain, C-terminal SH3 (Src homology 3) domain and Pro-rich domain. Phylogenetic analysis showed that CiTNK1 has a closer relationship with Danio rerio TNK1. The expression and phosphorylation of CiTNK1 in grass carp tissues and cells was increased under poly(I:C) stimulation. Subcellular localization and co-immunoprecipitation indicated that CiTNK1 is targeted in the cytoplasm and interacts with grass carp STAT1 (CiSTAT1). Co-transfection of CiTNK1 and CiSTAT1 into cells facilitated the expression of IFN I. This is because that the presence of CiTNK1 enhanced the phosphorylation of CiSTAT1 and causes activation of CiSTAT1. Our results revealed that TNK1 can potentiate the phosphorylation of STAT1 and then regulates JAK-STAT pathway to trigger IFN I expression in fish.
Asunto(s)
Carpas/metabolismo , Quinasas Janus/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1/metabolismo , Secuencia de Aminoácidos , Animales , Citoplasma/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Expresión Génica , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Fosforilación , Filogenia , Poli I-C/metabolismo , Unión Proteica , Proteínas Tirosina Quinasas/genética , Alineación de Secuencia , Transducción de SeñalRESUMEN
Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.
Asunto(s)
Carpas/genética , Regulación hacia Abajo/genética , Proteínas de Peces/genética , Factor 2 Regulador del Interferón/genética , Factores Reguladores del Interferón/genética , Interferones/genética , Animales , Células Cultivadas , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Inmunidad Innata/genética , Poli I-C/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Regulación hacia Arriba/genéticaRESUMEN
In mammals, interferon regulatory factor 5 (IRF5) can be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). Upon activation, IRF5 translocates into the nucleus, where it binds to IFN promoter and up-regulates IFN expression. However, there are few reports on the molecular mechanism by which TRAF6 up-regulates IFN expression in fish. In this study, we explored how Grass carp (Ctenopharyngodon idellus) TRAF6 initiated innate immunity by activating IRF5. We found that CiTRAF6, CiIRF5 and CiIFN1 were all significantly up-regulated in LPS-stimulated CIK cells and the expression of CiTRAF6 was earlier than the expressions of CiIRF5 and CiIFN1. These findings suggested that CiIFN1 expression might be induced by CiTRAF6 in fish. CiIFN1 expression, CiIFN1 promoter activity and CO cells viability were all significantly up-regulated in the overexpression experiments, but they were significantly down-regulated in the gene silencing experiments. This indicated that CiTRAF6, along with CiIRF5, regulated CiIFN1 expression. The localization analysis found that both CiTRAF6 and CiIRF5 located in the cytoplasm. Following LPS stimulation, CiIRF5 was observed to translocate to the nucleus. GST-pull down and co-IP experiments revealed that CiTRAF6 interacted with CiIRF5. The colocalization analysis also showed that CiTRAF6 bound with CiIRF5 in the cytoplasm. Overexpression of CiTRAF6 increased the endogenous CiIRF5, promoted its ubiquitination and nuclear translocation. In conclusion, CiTRAF6 bound to CiIRF5 in the cytoplasm, and then activated CiIRF5, resulting in up-regulating the expression of CiIFN1.
Asunto(s)
Carpas/inmunología , Proteínas de Peces/metabolismo , Factores Reguladores del Interferón/metabolismo , Interferón Tipo I/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Núcleo Celular/metabolismo , Supervivencia Celular , Células Cultivadas , Proteínas de Peces/genética , Inmunidad Innata , Factores Reguladores del Interferón/genética , Interferón Tipo I/genética , Lipopolisacáridos/farmacología , Unión Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Factor 6 Asociado a Receptor de TNF/genética , Activación Transcripcional/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Radix plicatula is broadly distributed in China, as well as Russia. It is one of the intermediate hosts of Fasciola species which leads to the spread of fascioliasis. Here, we first described the complete mitochondrial genome of R. plicatula. The mitogenome is 13,751 bp in length, containing 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The contents of each base are 30.7% A, 39.6% T, 15.7% G, and 13.9% C. The sequence is AT rich (70.3%). Mitochondrial phylogenomic analysis showed that R. plicatula is close to R. auricularia.
RESUMEN
In this study, the complete mitochondrial genome (mitogenome) sequence of Rhodeus lighti (Cypriniformes: Cyprinidae) was determined by long PCR and primer walking methods. The complete mitochondrial genome is 16,677 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A (28.87%), T (27.22%), C (26.53%) and G (17.38%). The mitogenome of R. lighti displayed novel gene order arrangement compared with published Rhodeus sinensis to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Acheilognathinae.
Asunto(s)
Cyprinidae/genética , Genoma Mitocondrial , Mitocondrias/genética , Animales , Composición de Base , Orden Génico , Tamaño del Genoma , Filogenia , Análisis de Secuencia de ADN/métodosRESUMEN
In this paper, the complete mitochondrial genome (mitogenome) sequence of Gallus domesticus was determined by long PCR and primer walking methods. The complete mitochondrial genome is 16,783 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A(30.25%), T(23.79%), C(32.44%), G(13.52%), respectively. The mitogenome of G. domesticus displayed novel gene order arrangement compared with published Gallus gallus var. domesticus to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Gallus.
Asunto(s)
Pollos/genética , Galliformes/genética , Genoma Mitocondrial/genética , Animales , Composición de Base/genética , ADN Mitocondrial/genética , Orden Génico/genética , Genes Mitocondriales/genética , Filogenia , ARN Ribosómico/genética , ARN de Transferencia/genética , Análisis de Secuencia de ADN/métodosRESUMEN
In this study, the complete mitochondrial genome (mitogenome) sequence of Paracheilognathus imberbis (Cypriniformes: Cyprinidae: Acheilognathinae) was determined by long PCR and primer-walking methods. The complete mitochondrial genome is 16,819 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A(29.73%), T(27.25%), C(26.63%) and G(17.40%), respectively. The mitogenome of P. imberbis displayed novel gene order arrangement compared with published Rhodeus sinensis to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Acheilognathinae.