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1.
Mar Biotechnol (NY) ; 23(2): 294-307, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33570690

RESUMO

In mammals, mature miR-122 is 22 nucleotides long and can be involved in regulating a variety of physiological and biological pathways. In this study, the expression profile and effects of grouper Epinephelus coioides miR-122 response to Singapore grouper iridovirus (SGIV) infection were investigated. The sequences of mature microRNAs (miRNAs) from different organisms are highly conserved, and miR-122 from E. coioides exhibits high similarity to that from mammals and other fish. The expression of miR-122 was up-regulated during SGIV infection. Up-regulation of miR-122 could significantly enhance the cytopathic effects (CPE) induced by SGIV, the transcription levels of viral genes (MCP, VP19, LITAF and ICP18), and viral replication; reduce the expression of inflammatory factors (TNF-a, IL-6, and IL-8), and the activity of AP-1 and NF-κB, and miR-122 can bind the target gene p38α MAPK to regulate the SGIV-induced cell apoptosis and the protease activity of caspase-3. The results indicated that SGIV infection can up-regulate the expression of E. coioides miR-122, and up-regulation of miR-122 can affect the activation of inflammatory factors, the activity of AP-1 and NF-κB, and cell apoptosis to regulate viral replication and proliferation.

2.
Dev Comp Immunol ; 119: 104013, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33465381

RESUMO

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

3.
Dev Comp Immunol ; 119: 104020, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33476669

RESUMO

Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.

4.
Phytomedicine ; 80: 153400, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33157413

RESUMO

BACKGROUND: Vascular Endothelial Growth Factors (VEGFs) are a group of growth factor in regulating development and maintenance of blood capillary. The VEGF family members include VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. VEGF receptor activation leads to multiple complex signaling pathways, particularly in inducing angiogenesis. Besides, VEGF is produced by macrophages and T cells, which is playing roles in inflammation. In macrophages, VEGF receptor-3 (VEGFR-3) and its ligand VEGF-C are known to attenuate the release of pro-inflammatory cytokines. METHODS: Immunoprecipitation and molecular docking assays showed the binding interaction of kaempferol-3-O-rutinoside and VEGF-C. Western blotting and qRT-PCR methods were applied to explore the potentiating effect of kaempferol-3-O-rutinoside in VEGF-C-mediated expressions of proteins and genes in endothelial cells and LPS-induced macrophages. Enzyme-linked immunosorbent assay (ELISA) was employed to reveal the release of proinflammatory cytokines in LPS-induced macrophages. Immunofluorescence assay was performed to determine the effect of kaempferol-3-O-rutinoside in regulating nuclear translocation of NF-κB p65 subunit in the VEGF-C-treated cultures. In addition, Transwell® motility assay was applied to detect the ability of cell migration after drug treatment in LPS-induced macrophages. RESULTS: We identified kaempferol-3-O-rutinoside, a flavonoid commonly found in vegetable and fruit, was able to act on cultured macrophages in inhibiting inflammatory response, and the inhibition was mediated by its specific binding to VEGF-C. The kaempferol-3-O-rutinoside-bound VEGF-C showed high potency to trigger the receptor activation. In LPS-treated cultured macrophages, applied kaempferol-3-O-rutinoside potentiated inhibitory effects of exogenous applied VEGF-C on the secretions of pro-inflammatory cytokines, i.e. IL-6 and TNF-α, as well as expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). This inhibition was in parallel to transcription and translocation of NF-κB. Moreover, the binding of kaempferol-3-O-rutinoside with VEGF-C suppressed the LPS-induced migration of macrophage. CONCLUSION: Taken together, our results suggested the pharmacological roles of kaempferol-3-O-rutinoside in VEGF-C-mediated anti-inflammation.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Quempferóis/metabolismo , Quempferóis/farmacologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Quempferóis/química , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7
5.
Molecules ; 25(17)2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32824997

RESUMO

Piceatannol is also named as trans-3,4,3',5'-tetrahydroxy-stilbene, which is a natural analog of resveratrol and a polyphenol existing in red wine, grape and sugar cane. Piceatannol has been proved to possess activities of immunomodulatory, anti-inflammatory, antiproliferative and anticancer. However, the effect of piceatannol on VEGF-mediated angiogenesis is not known. Here, the inhibitory effects of piceatannol on VEGF-induced angiogenesis were tested both in vitro and in vivo models of angiogenesis. In human umbilical vein endothelial cells (HUVECs), piceatannol markedly reduced the VEGF-induced cell proliferation, migration, invasion, as well as tube formation without affecting cell viability. Furthermore, piceatannol significantly inhibited the formation of subintestinal vessel in zebrafish embryos in vivo. In addition, we identified the underlying mechanism of piceatannol in triggering the anti-angiogenic functions. Piceatannol was proposed to bind with VEGF, thus attenuating VEGF in activating VEGF receptor and blocking VEGF-mediated downstream signaling, including expressions of phosphorylated eNOS, Erk and Akt. Furthermore, piceatannol visibly suppressed ROS formation, as triggered by VEGF. Moreover, we further determined the outcome of piceatannol binding to VEGF in cancer cells: piceatannol significantly suppressed VEGF-induced colon cancer proliferation and migration. Thus, these lines of evidence supported the conclusion that piceatannol could down regulate the VEGF-mediated angiogenic functions with no cytotoxicity via decreasing the amount of VEGF binding to its receptors, thus affecting the related downstream signaling. Piceatannol may be developed into therapeutic agents or health products to reduce the high incidence of angiogenesis-related diseases.

6.
Fish Shellfish Immunol ; 106: 71-78, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32738512

RESUMO

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1ß, IL-6, as well as the signaling of NF-κB. The application of CoCl2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

7.
Front Pharmacol ; 11: 526, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32410995

RESUMO

Kaempferol is a major flavonoid in Ginkgo Folium and other edible plants, which is being proposed here to have roles in angiogenesis. Angiogenesis is important in both physiological and pathological development. Here, kaempferol was shown to bind with vascular endothelial growth factor (VEGF), probably in the heparin binding domain of VEGF: this binding potentiated the angiogenic functions of VEGF in various culture models. Kaempferol potentiated the VEGF-induced cell motility in human umbilical vein endothelial cells (HUVECs), as well as the sub-intestinal vessel sprouting in zebrafish embryos and formation of microvascular in rat aortic ring. In cultured HUVECs, application of kaempferol strongly potentiated the VEGF-induced phosphorylations of VEGFR2, endothelial nitric oxide synthase (eNOS) and extracellular signal-regulated kinase (Erk) in time-dependent and concentration-dependent manners, and in parallel the VEGF-mediated expressions of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were significantly enhanced. In addition, the potentiation effect of kaempferol was revealed in VEGF-induced migration of skin cell and monocyte. Taken together, our results suggested the pharmacological roles of kaempferol in potentiating VEGF-mediated functions should be considered.

8.
Fish Shellfish Immunol ; 97: 125-134, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31809835

RESUMO

Heat shock protein 22 (Hsp22) is an important regulatory factor response to various stresses in mammals. In this study, the full length cDNA of Epinephelus coioides Hsp22, which was 1680bp in length, with a 289 bp 5' UTR, a 725 bp 3'UTR, and a 666 bp open reading frame encoding 221 amino acids, was obtained. E. coioides Hsp22 contains a highly conserved α-crystallin domain. E. coioides Hsp22 mRNA was detected in all tissues examined by quantitative real-time PCR, with the highest expression in blood, followed by the spleen, skin, gill, head kidney, muscle, heart, liver, trunk kidney, stomach, pyloric caeca, intestine, brain and thymus. The expression patterns of E. coioides Hsp22 response to infection with Singapore grouper iridovirus (SGIV) and Vribro alginolyticus, the important pathogens of E. coioides, were studied. The expression levels of the gene were up-regulated in the tissues examined. Subcellular localization analysis demonstrated that E. coioides Hsp22 was distributed in both the cytoplasm and nucleus. In addition, E. coioides Hsp22 significantly inhibited the SGIV-induced cell apoptosis. In summary, the E. coioides Hsp22 might play a critical role in pathogenic stimulation.


Assuntos
Bass/imunologia , Proteínas de Peixes/genética , Proteínas de Choque Térmico/genética , Vibrioses/veterinária , Viroses/veterinária , Animais , Bass/microbiologia , Bass/virologia , Clonagem Molecular , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Expressão Gênica , Proteínas de Choque Térmico/imunologia , Iridovirus , Vibrioses/imunologia , Vibrio alginolyticus , Viroses/imunologia
9.
Phytomedicine ; 74: 152815, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30833146

RESUMO

BACKGROUND: According to traditional Chinese medicine (TCM) theory, the herbal property is the most important guiding principle of ancient medication in China. The classification of warm- and cold-stimulating TCM is defined mainly based on the effects of herbs in regulating body temperature; however, the underlying mechanism of such distinction has not been fully identified. METHODS: Here, four commonly used spleen-meridian herbs, Ginseng Radix and Astragali Radix as typical warm-stimulating herbs, and Nelumbinis Semen and Coicis Semen as typical cold-stimulating herbs, were selected to test their effects in regulating body temperature, as well as its triggered thermo-regulatory factors and energy related metabolites, in yeast-induced fever rats. RESULTS: The intake of Astragali Radix increased body temperature in yeast-induced fever rats; while Coicis Semen showed cooling effects in such rats. In parallel, the levels of cAMP, PGE2 and thermo-related metabolites, including choline, creatine, alanine, lactate and leucine, in the blood of yeast-induced rats were increased significantly by the intake of Astragali Radix. Oppositely, the cold-stimulating herbs, Nelumbinis Semen and Coicis Semen, showed cooling effects by increasing certain metabolites, e.g. histidine, tyrosine, lipid, myo-inositol, as well as AVP level. CONCLUSION: Here, we compared different effects of warm and cooling spleen-meridian herbs in the regulation of body temperature. By providing an intuitive comparison of thermo-regulatory factors and related metabolites after intake of selected herbs, the mechanism behind the warm and cooling effects of specific herbs were revealed.


Assuntos
Regulação da Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Animais , Coix/química , Medicamentos de Ervas Chinesas/química , Febre/tratamento farmacológico , Febre/etiologia , Masculino , Medicina Tradicional Chinesa/métodos , Meridianos , Panax/química , Plantas Medicinais/química , Ratos Endogâmicos , Baço , Leveduras/patogenicidade
10.
Fish Shellfish Immunol ; 92: 500-507, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31247318

RESUMO

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/imunologia , Sequência de Aminoácidos , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , MAP Quinase Quinase 6/química , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio alginolyticus/fisiologia
11.
Fish Shellfish Immunol ; 86: 1081-1087, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30593900

RESUMO

Antimicrobial peptides (AMPs) are small proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens (bacteria, fungi and viruses). In this study, the effects of AMPs from Bacillus subtilis on Epinephelus coioides were examined. E. coioides were fed with diets containing AMPs (0, 100, 200, 400 or 800 mg/kg) for four weeks. Results showed that the levels of total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose (GLU) and lipopolysaccharide (LPS) in the serum of E. coioides changed than those of the control group; compared to the control group, the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and lysozyme (LZM) levels in E. coioides fed with different dosages AMP diets were also different; in addition, the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin-1-beta (IL-1ß), and heat shock protein 90 (Hsp90) in the tissues of E. coioides were measured, the three genes in the tissues examined were significantly upregulated. The results demonstrated that diets containing AMPs can enhance the antioxidant capacity and innate immune ability of E. coioides, indicating that AMPs might be a potential alternative to antibiotics in E. coioides.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antioxidantes/metabolismo , Bass/imunologia , Imunidade Inata , Ração Animal/análise , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Bacillus subtilis/química , Bass/metabolismo , Análise Química do Sangue/veterinária , Dieta/veterinária
12.
J Agric Food Chem ; 67(4): 1127-1137, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30525561

RESUMO

Resveratrol is a polyphenol commonly found in plants and food health products, such as grape and red wine, and was identified for its binding to vascular endothelial growth factor (VEGF) by using HerboChips screening. The binding, therefore, resulted in alterations of VEGF binding to its receptor and revealed the roles of VEGF in angiogenesis. Several lines of evidence gave support to the inhibitory activities of resveratrol in VEGF-triggered angiogenesis. In human umbilical vein endothelial cells (HUVECs), compared with a VEGF-induced group, resveratrol, at a high concentration, suppressed VEGF-mediated endothelial cell proliferation, cell migration, cell invasion, and tube formation by 80 ± 9.01%, 140 ± 3.78%, 110 ± 7.51%, and 120 ± 10.26%, respectively. Moreover, resveratrol inhibited the subintestinal vessel formation in zebrafish embryo. In signaling cascades, application of resveratrol in HUVECs reduced the VEGF-triggered VEGF receptor 2 phosphorylation and c-Jun N-terminal kinase phosphorylation. Moreover, the VEGF-mediated phosphorylations of endothelial nitric oxide synthase, protein kinase B, and extracellular signal-regulated kinase were obviously decreased by (3 ± 0.37)-, (2 ± 0.27)- and (6 ± 0.23)-fold, respectively, in the presence of resveratrol at high concentration. Parallelly, the VEGF-induced reactive oxygen species formation was significantly decreased by 50 ± 7.88% to 120 ± 14.82% under resveratrol treatment. Thus, our results provided support to the antiangiogenic roles of resveratrol, as well as its related signaling mechanisms, in attenuating the VEGF-mediated responses. The present results supported possible development of resveratrol, which should be considered as a therapeutic agent in terms of prevention and clinical treatment of diseases related to angiogenesis.


Assuntos
Inibidores da Angiogênese/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Resveratrol/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Resveratrol/química , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/química , Peixe-Zebra
13.
Fish Physiol Biochem ; 44(1): 87-93, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29214431

RESUMO

A novel cell line, Epinephelus moara kidney cell line (EMK), was established from kidneys of kelp grouper E. moara. Cells were cultured at 24 °C in Leibovitz's L-15 medium (L15) supplemented with antibiotics, basic fibroblast growth factor (bFGF), foetal bovine serum (FBS) and 2-mercaptoethanol (2-ME). EMK cells, fibroblastic in morphology, proliferated to 100% confluency in 3-4 days and were subcultured for over 50 passages. The cells could grow from 18 to 30 °C, with optimal growth at 24 °C. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 42. Green fluorescent signals could be observed in EMK cells when the cells were transfected with pEGFP-N3 plasmid. Moreover, a significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or nervous necrosis virus (NNV), and viral replication was confirmed by quantitative real-time PCR (qPCR). These results suggested the potential of the EMK cell line for studies of transgene and pathogenesis of SGIV and NNV.


Assuntos
Gadiformes/fisiologia , Rim/citologia , Vírus/classificação , Animais , Técnicas de Cultura de Células/veterinária , Linhagem Celular , Iridovirus/fisiologia , Nodaviridae/fisiologia , Temperatura , Replicação Viral/fisiologia
14.
Fish Shellfish Immunol ; 57: 17-24, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27531577

RESUMO

Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/genética , Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Receptores Toll-Like/genética , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Astacoidea/metabolismo , Astacoidea/microbiologia , Análise de Sequência de DNA , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Vibrio/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
15.
Dev Comp Immunol ; 43(1): 87-95, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24239708

RESUMO

The IFN-γ gene was identified in a turtle, the Chinese soft-shelled turtle, Pelodiscus sinensis, with its genome consisting of 4 exons and 3 introns. The deduced amino acid sequence of this gene contains a signal peptide, an IFN-γ family signature motif (130)IQRKAVNELFPT, an NLS motif (155)KRKR and three potential N-glycosylation sites. As revealed by real-time quantitative PCR, the gene was constitutively expressed in all tested organs/tissues, with higher level observed in blood, intestine and thymus. An induced expression of IFN-γ at mRNA level was observed in peripheral blood leucocytes (PBLs) in response to in vitro stimulation of LPS and PolyI:C. The overexpression of IFN-γ in the Chinese soft-shelled turtle artery (STA) cell line resulted in the increase in the expression of transcriptional regulators, such as IRF1, IRF7 and STAT1, and antiviral genes, such as Mx, PKR, implying possibly the existence of a conserved signalling network and role for IFN-γ in the turtle. Furthermore, the infection of soft-shelled turtle iridovirus (STIV) in the cell line transfected with IFN-γ may cause the cell death as demonstrated with the elevated lactate dehydrogenase (LDH) level and cell mortality. However, the mechanism involved in the antiviral activity may require further investigation.


Assuntos
Antivirais/metabolismo , Infecções por Vírus de DNA/imunologia , Interferon gama/metabolismo , Iridovirus/fisiologia , Leucócitos Mononucleares/imunologia , Tartarugas/imunologia , Sequência de Aminoácidos , Animais , Antivirais/isolamento & purificação , Linhagem Celular , Sequência Conservada/genética , Interferon gama/genética , Interferon gama/isolamento & purificação , Mucosa Intestinal/metabolismo , Leucócitos Mononucleares/virologia , Lipopolissacarídeos/imunologia , Dados de Sequência Molecular , Poli I-C/imunologia , Transdução de Sinais , Ativação Transcricional/genética , Transcriptoma , Transgenes/genética , Regulação para Cima , Replicação Viral/imunologia
16.
Yi Chuan ; 34(6): 765-72, 2012 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-22698749

RESUMO

A pan-genome describes the full complement of genes in species. It is a superset of all the genes in all the individuals of a species, which is composed of a 'core genome' containing genes present in all individuals, and a 'dispensable genome' containing genes present only in some individuals and individual-specific genes. From pan-genome sight, 30 finished genomes from Escherichia coli were employed to analyze their gene and genome compositions and evaluation in this study. The results indicated that the core genes accounted for about 50% of the total number of genes, while about 146 strain-specific genes existed in the each strain tested. The data suggests that the E. coli pan-genome is vast, and unique genes will continue to be identified when more E. coli genomes are sequenced. After analyzing relationships of the gene conservation, GC content and selection pressure in different strains tested, we found that more conserved genes had a nar-row range of GC content, and they also bear more selection pressure. These results will be helpful for better understanding of the evolution profile of E. coli genome, and the dynamic changes of its gene compositions. The E. coli pan-genome pro-vides useful information for prevention and control of the diseases caused by pathogenic E. coli, and also provides a para-digm for the large-scale analysis of pathogenic bacteria genomes.


Assuntos
Escherichia coli/genética , Evolução Molecular , Genoma Bacteriano , Genômica/métodos
17.
Fish Shellfish Immunol ; 30(2): 559-68, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21145974

RESUMO

Orange-spotted grouper, Epinephelus coioides is one of the most important economic species of marine-cultured fish in China and Southeast Asia countries. However, very little information of the innate immune mechanisms against microbial pathogens is available in grouper, Epinephelus sp. Hepcidin, as an antimicrobial peptide (AMP), is a very important component in the innate immune system and widespread in fish. In this study, two novel types of hepcidin gene (designated EC-hepcidin1 and EC-hepcidin2) were cloned from E. coioides. They consist of open reading frames (ORFs) of 267 bp and 263 bp encoding the putative peptides of 88 and 87 amino acids, respectively. The homologous identity of deduced amino acid sequences between EC-hepcidin1 and EC-hepcidin2 is up to 79%, and predicted mature regions of both them have four cysteines residues. Genomic DNAs of both EC-hepcidin1 and EC-hepcidin2 consist of three exons and two introns. RT-PCR results showed that EC-hepcidin1 transcript was most abundant in liver and less in stomach. However, the transcript of EC-hepcidin2 was only detected in liver. The expressions of both EC-hepcidins were up-regulated by microbial and viral challenges, and iron overload, respectively, and EC-hepcidin1 was more responsive. The growth of Gram-negative bacterium of Vibrio vulnificus and Gram-positive bacterium of Staphylococcus aureus was inhibited by synthetic EC-hepcidins, and EC-hepcidin1 displayed stronger antimicrobial activity. The replication of Singapore grouper iridovirus (SGIV) was inhibited in the EC-hepcidin1 and EC-hepcidin2 over-expressed stable transfected fish cell lines (GS/pcDNA-Hep1, GS/pcDNA-Hep2) indicative of the antiviral activity of EC-hepcidins. These data should offer important information on the antimicrobial and antiviral roles of EC-hepcidins, and will be help to the better understanding of molecular mechanisms of grouper innate immunity.


Assuntos
Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Bass/genética , Bass/imunologia , Regulação da Expressão Gênica , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/veterinária , Sequência de Bases , Clonagem Molecular , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Ordem dos Genes , Hepcidinas , Iridovirus/imunologia , Ferro/farmacologia , Dados de Sequência Molecular , RNA Mensageiro/imunologia , Saccharomyces cerevisiae/imunologia , Alinhamento de Sequência
18.
Arch Virol ; 155(7): 1069-76, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20461535

RESUMO

The dUTPase is a ubiquitous and crucial enzyme responsible for regulating cellular levels of dUTP. In the present study, the expression pattern and translocation of a dUTPase homolog encoded by Singapore grouper iridovirus (SGIV) were elucidated. The SGIV ORF049R encodes a dUTPase homolog, which is a peptide of 155 amino acids that contains five conserved motifs. The temporal expression pattern during infection in vitro revealed that the SGIV dUTPase was an early transcript. A leucine-rich nuclear export signal (NES) at the C-terminus was predicted using CBS Online Servers. Subcellular location analysis showed that SGIV dUTPase is a cytoplasmic protein. Site-direct mutagenesis by overlap extension-PCR indicated that the NES is crucial for the translocation of SGIV dUTPase from the nucleus to the cytoplasm. We have discovered for the first time that the NES-dependent translocation of dUTPase is different for SGIV than for members of other species, which depend on a nuclear localization signal. These results provide new insights into the pathogenesis of fish iridoviruses.


Assuntos
Iridovirus/metabolismo , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Peixes , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica/fisiologia , Iridovirus/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
19.
Arch Virol ; 155(3): 351-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20130938

RESUMO

Singapore grouper iridovirus (SGIV) is an important pathogen isolated from grouper, Epinephelus tauvina, and characterized as a novel ranavirus. To better understand the function of viral structural genes involved in SGIV infection and virus-host interactions, a candidate gene, VP38 (ORF038L), was investigated in this study. SGIV VP38 was found to encode a 170-aa peptide containing an RGD motif, and it showed significant identity only to members of the genus Iridovirus, family Iridoviridae, except megalocytivirus. The VP38 gene was identified by temporal expression pattern analysis and drug inhibition assay as a late (L) gene. Immunofluorescence localization revealed that P38 was distributed predominately in the cytoplasm and that association of VP38 with viral factories increased at the late stage of SGIV infection. Consistent results from immunoelectron microscopy (IEM) and western blot analysis revealed that SGIV VP38 is a viral capsid protein. Furthermore, antibodies specific for SGIV VP38 exhibited substantial SGIV-neutralizing activity in vitro, suggesting that VP38 might play an important role in SGIV infectivity.


Assuntos
Proteínas do Capsídeo/genética , Ranavirus/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Bass/virologia , Western Blotting , Citoplasma/química , Perfilação da Expressão Gênica , Iridovirus/genética , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Testes de Neutralização , Fases de Leitura Aberta , Filogenia , Proteoma/análise , Ranavirus/isolamento & purificação , Homologia de Sequência de Aminoácidos , Vírion/química , Vírion/ultraestrutura
20.
Virology ; 377(1): 39-48, 2008 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-18555886

RESUMO

A DNA microarray containing all Singapore grouper iridovirus (SGIV) open reading frames (ORFs) was constructed to map the viral gene transcriptional profiles in virus-infected grouper spleen (GS) cells and in spleen tissues of virus-infected grouper. The results showed that viral genes started to be transcribed as early as 1 h postinfection (p.i.), and followed by a rapid increasing gene expression along with virus infection in cell cultures. The three temporal kinetic classes (15 immediate-early, 89 early and 53 late transcripts) were classified during an in vitro infection by their dependence on de novo protein synthesis and viral DNA replication inhibitors. In SGIV-infected grouper, Epinephulus coioides, most of the viral genes were expressed between 1 and 4 d p.i., and the number and expression levels started to decrease after 5 d p.i. These data were confirmed by real-time RT-PCR. This study provides an experimental basis for investigation of virus-host interactions and the development of control strategies against SGIV infection.


Assuntos
Bass/virologia , Iridovirus/genética , Iridovirus/patogenicidade , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA/genética , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , DNA Viral/genética , Doenças dos Peixes/virologia , Perfilação da Expressão Gênica , Genoma Viral , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
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