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1.
Fish Shellfish Immunol ; 154: 109906, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39278379

RESUMO

Interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms in the innate immune system. The activated PKR performs its antiviral function by inhibiting protein translation and inducing apoptosis. In our previous study, we identified grass carp TARBP2 as an inhibitor of PKR activity, thereby suppressing cell apoptosis. This study aimed to explore the effects of grass carp TARBP2 on PKR activity and cell apoptosis. Grass carp TARBP2 comprises two N-terminal dsRBDs and a C-terminal C4 domain. Subcellular localization analysis conducted in CIK cells revealed that TARBP2-FL (full-length TARBP2), TARBP2-Δ1 (lack of the first dsRBD), and TARBP2-Δ2 (lack of the second dsRBD) are predominantly located in the cytoplasm, while TARBP2-Δ3 (lack of the two dsRBDs) is distributed both in the nucleus and cytoplasm. Colocalization and immunoprecipitation assays confirmed the interaction of TARBP2-FL, TARBP2-Δ1, and TARBP2-Δ2 with PKR, while TARBP2-Δ3 showed no binding. Furthermore, our findings suggested that the inhibitory effect of TARBP2-Δ1 or TARBP2-Δ2 on the PKR-eIF2α pathway is depressed compared to TARBP2-FL. In cell apoptosis assays, it was observed that TARBP2-FL inhibits PKR-mediated cell apoptosis. TARBP2-Δ1 or TARBP2-Δ2 exhibits decreased inhibition to PKR-mediated cell apoptosis, whereas TARBP2-Δ3 nearly completely loses this inhibitory effect. These findings highlight the critical importance of two dsRBDs of TARBP2 in interaction with PKR, as well as in the inhibition of PKR activity, resulting in the suppression of cell apoptosis triggered by prolonged PKR activation.

2.
Gene ; 927: 148735, 2024 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-38944166

RESUMO

BACKGROUND: OCIAD2(Ovarian carcinoma immunoreactive antigen-like protein 2) is a protein reported in various cancers. However, the role of OCIAD2 has not been explored in pan-cancer datasets. The purpose of this research lies in analyzing the expression level and prognostic-related value of OCIAD2 in different human cancers, as well as revealing the underlying mechanism in specific cancer type (pancreatic adenocarcinoma, PAAD). METHODS: The correlation between OCIAD2 expression level and clinical relevance in different human cancers was investigated from bioinformatical perspective (GTEx and TCGA). The OCIAD2 expression level and clinical significance in PAAD were explored in GEO datasets and tissue microarray. Functional experiments were used to determine the OCIAD2 cell functions in vitro and in vivo. GSEA, western blot and immunohistochemistry were used to uncover the potential mechanism. RESULTS: OCIAD2 expression level was closely correlated with clinical relevance in many cancer types through pan-cancer analysis, and we found OCIAD2 was highly expressed in PAAD and associated with poorer prognosis. OCIAD2 acted as the promotor of Warburg effect and influenced PAAD cells proliferation, migration and apoptosis. Mechanistically, OCIAD2 upregulation may boost glycolysis in PAAD via activating the AKT signaling pathway in PAAD. CONCLUSIONS: In PAAD, OCIAD2 promotes Warburg effect via AKT signaling pathway and targeting cancer cells metabolic reprogramming could be a potential treatment.


Assuntos
Proteínas de Neoplasias , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , Feminino , Humanos , Masculino , Camundongos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/metabolismo , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
3.
Math Biosci Eng ; 17(1): 105-121, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31731342

RESUMO

Public water supply facilities are vulnerable to intentional intrusion. In particular, Water Distribution Network (WDN) has become one of the most important public facilities that are prone to be attacked because of its wide coverage and constant open operation. In recent years, water contamination incidents happen frequently, causing serious losses and impacts to the society. Various measures have been taken to tackle this issue. Pollution or contamination isolation by localizing the contamination via sensors and scheduling certain valves have been regarded as one of the most promising solutions. The main challenge is how to schedule water valves to effectively isolate contamination and reduce the residual concentration of contaminants in WDN. In this paper, we are motivated to propose a reinforcement learning based method for valve real time scheduling by treating the sensing data from the sensors as state, and the valve scheduling as action, thus we can learn scheduling policy from uncertain contamination events without precise characterization of contamination source. Simulation results show that our proposed algorithm can effectively isolate the contamination and reduce the risk exclosure to the customers.


Assuntos
Aprendizado Profundo , Monitoramento Ambiental/métodos , Medição de Risco/métodos , Poluição da Água/análise , Abastecimento de Água , Algoritmos , Difusão , Cadeias de Markov , Poluentes Químicos da Água/análise , Qualidade da Água
4.
Fish Shellfish Immunol ; 35(6): 1874-81, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24084043

RESUMO

The interferon-induced, dsRNA-activated protein kinase (PKR) is considered as an important component of innate immune system and as a representative effector protein of interferon system. In the present study, PKR gene (CiPKR, JX511974) from grass carp (Ctenopharyngodon idellus) was isolated and identified using homology-based PCR. CiPKR shares high sequence identity with the counterparts of goldfish (Crucian carp) and zebrafish (Danio rerio). The full-length cDNA of CiPKR was found to be 2436 bp, with an ORF of 2067 bp that encodes a polypeptide of 688 amino acids. The deduced polypeptide CiPKR contains three tandem dsRNA-binding motifs (dsRBMs) at the N-terminus and a conserved Ser/Thr kinase domain at the C-terminus. CiPKR was expressed ubiquitously at a low-level under normal conditions, but it could be up-regulated after intraperitoneal (ip) injection with grass carp haemorrhagic virus (GCHV). CiPKR was dramatically up-regulated at 6 h post-injection and then recovered rapidly to normal levels within 24 h; however, it was obviously up-regulated once again at 48 h or 72 h post-injection. It seemed that CiPKR could respond to GCHV infection in an IFN-independent as well as an IFN-dependent pathway. To further investigate its mechanism of biological actions, we constructed a series of recombinant plasmids including pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C (deletion of dsRBD sequence) and pcDNA3.1/PKR-C-K430R, and then each recombinant plasmid was transfected into CIK cells. In comparison with those of controls, a 79% and a 64% decrease of luciferase activities were detected in the tested cells transfected with CiPKR and CiPKR-C, respectively; however, luciferase activities were increased in those cells transfected with PKR-K430R and PKR-C-K430R, with a 160% and 115% up-regulation, respectively. Similarly, MTT colorimetric assay indicated that cell viabilities of CIK cells transfected with pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C and pcDNA3.1/PKR-C-K430R were 49%, 90%, 54% and 100%, respectively. Our observations suggested that the expression of CiPKR could be up-regulated following viral infection, and then resulted in the inhibition of protein synthesis and the induction of potential apoptosis.


Assuntos
Carpas/genética , Carpas/imunologia , Proteínas de Peixes/genética , eIF-2 Quinase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/virologia , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reoviridae , eIF-2 Quinase/química , eIF-2 Quinase/metabolismo
5.
Fish Shellfish Immunol ; 33(1): 42-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22510210

RESUMO

With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 °C or cold stress at 4 °C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 °C to 42 °C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4°C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct ΔN-CiHsp90 (256-798aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and ΔN-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature.


Assuntos
Carpas/genética , Carpas/metabolismo , Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Temperatura Alta , Estresse Fisiológico , Animais , Carpas/classificação , Linhagem Celular Tumoral , Células Cultivadas , Citrato (si)-Sintase/genética , Temperatura Baixa , Escherichia coli/genética , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/química , Camundongos , Dados de Sequência Molecular , Mutação , Filogenia , Homologia de Sequência de Aminoácidos , Estresse Fisiológico/genética
6.
Fish Shellfish Immunol ; 31(6): 1173-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22008285

RESUMO

The new teleost fish PKZ (PKR-like) full-length cDNA (GU299765) had been cloned and identified from grass carp (Ctenopharyngodon idellus). The cDNA of grass carp PKZ (CiPKZ) has 2185 bp in length with a largest open reading frame (ORF) encoding 513aa. CiPKZ possesses a conserved C-terminal catalytic domain of eIF2α kinase family. Within its N-terminal there are two binding domain (Zα) named Zα1 (1-67aa) and Zα2 (81-152aa). BLAST homologous search reveals that CiPKZ has a high-level homology with other fish PKZs and PKRs. Like other fish PKZs and PKRs, CiPKZ is a ubiquitous tissue expression gene that had a very low level of constitutive expression but up-regulated in response to Poly I:C or hot stress (34 °C). For the purpose of searching for the potential function of CiPKZ, we obtained CiPKZ polypeptide via Escherichia coli Rosetta prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. CiPKZ polypeptide was used for the test of phosphorylating eIF2αin vitro. The results demonstrated that CiPKZ could be activated by Z-DNA but not by Poly I:C, and with subsequent could phosphorylate eIF2α. Meanwhile, four pcDNA3.1/PKZ recombinant plasmids, including pcDNA3.1/PKZ-wet, pcDNA3.1/PKZ-wet-K198R, pcDNA3.1/PKZ-wet-C, pcDNA3.1/PKZ-wet-C-K198R had been constructed, respectively. Mouse Myeloma cells (Sp2/0) and Human Umbilical Vein Endothelial Cells (HUVEC) were transiently cotransfected with pcDNA3.1/PKZ recombinant plasmid and PGL-3-promoter plasmid. The results revealed that CiPKZ could greatly decrease luciferase level in these cells. Zα and the K198 amino acid residue may play a key role in its function.


Assuntos
Carpas/genética , DNA Forma Z/metabolismo , Regulação da Expressão Gênica/imunologia , eIF-2 Quinase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cromatografia de Afinidade , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Componentes do Gene , Regulação da Expressão Gênica/efeitos dos fármacos , Temperatura Alta , Humanos , Luciferases , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Fosforilação , Plasmídeos/genética , Poli I-C/imunologia , Poli I-C/farmacologia , Análise de Sequência de DNA , Transfecção , eIF-2 Quinase/metabolismo
7.
Fish Shellfish Immunol ; 28(5-6): 783-8, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20139004

RESUMO

PKZ was the most recently discovered member of eIF2alpha kinase family in fish. CaPKZ, the first identified fish PKZ, possessed a conserved eIF2alpha kinase catalytic domain in C-terminal and two Z-DNA binding domains (Zalpha) in N-terminal. The Zalpha of CaPKZ closely resembled that of other Z-DNA binding proteins: ADAR1, DLM-1, and E3L. In order to understand more about the function of CaPKZ, we expressed and purified three constructed peptides of CaPKZ (P(Zalpha)): P(Zalpha1Zalpha2), P(Zalpha1Zalpha1) and P(Zalpha2)(Zalpha2). Moreover, most of the plasmids containing d(GC)(n) inserts were maintained in the Z-conformation, as confirmed by using inhibition of methylation experiments and anti-Z-DNA antibody. Gel mobility shift assays were then used to examine the affinity of these P(Zalpha) to the recombinant plasmids. Meanwhile, a competition experiment using P(Zalpha1Zalpha2) and anti-Z-DNA antibody was performed. The results revealed that P(Zalpha1Zalpha2) and P(Zalpha1Zalpha1) were able to bind to the recombinant plasmids with high affinity, whereas P(Zalpha2)(Zalpha2) could not bind to it. In addition, dimerization of P(Zalpha1Zalpha2) indicated the function unit of Zalpha of CaPKZ would be a dimer.


Assuntos
DNA Forma Z/metabolismo , Carpa Dourada/genética , Carpa Dourada/metabolismo , eIF-2 Quinase/metabolismo , Animais , DNA Forma Z/química , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Enzimológica da Expressão Gênica , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , eIF-2 Quinase/química , eIF-2 Quinase/isolamento & purificação
8.
Yi Chuan ; 29(12): 1519-24, 2007 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-18065389

RESUMO

Branch-Site Model is a statistical method for detecting molecular adaptation at individual along specific lineages. Not only the model could indicate whether genes in phylogeny under the positive selection or not, but also could forecast positive selection sites in these genes. The sites promote divergence and polymorphism of genes. Chemokines are chemotactic cytokines that can induce immune cells migration, conducting their functions via chemokine receptors. In the test, the molecular adaptation of chemokines and chemokines receptors genes had been analyzed by Branch-Site model. The results showed that only a few of genes, such as RANTES and CCR5, are in the course of positive selection. Several CCR5 positive selection sites are located in a region that involved in binding to receptor and its chemokine.


Assuntos
Adaptação Biológica/genética , Quimiocinas/genética , Evolução Molecular , Receptores de Quimiocinas/genética , Sequência de Aminoácidos , Animais , Quimiocina CCL5/química , Quimiocina CCL5/genética , Quimiocinas/química , Humanos , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Receptores CCR5/química , Receptores CCR5/genética , Receptores de Quimiocinas/química , Seleção Genética , Biologia de Sistemas
9.
Fish Shellfish Immunol ; 17(4): 353-66, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15312662

RESUMO

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zalpha). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs.


Assuntos
Carpas/genética , Filogenia , Reoviridae , eIF-2 Quinase/biossíntese , eIF-2 Quinase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Blástula/citologia , Blástula/metabolismo , Células Cultivadas , Clonagem Molecular , Análise por Conglomerados , Cicloeximida , Primers do DNA , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Interferons/metabolismo , Interferons/farmacologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Raios Ultravioleta , Inativação de Vírus
10.
Fish Shellfish Immunol ; 15(5): 453-66, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14550671

RESUMO

Interferon (IFN) can induce an antiviral state via interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). Here we describe a fish IRF, termed CaIRF7, cloned from a subtractive cDNA library which is constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. CaIRF7 cDNA was found to be 1816 bp in length, with a 42 bp 5'UTR and a 508 bp 3'UTR. The open reading frame translates into 421 amino acids in which a DNA-binding domain (DBD) containing the repeated tryptophan motif and IRFs association domain have been identified. Like chicken GgIRF3, CaIRF7 was most similar to mammalian IRF7 with 27 to 30% identity overall and some 37% identity in their DBDs. A single transcript of 1.9 kb was detected in virally induced CAB cells by virtual Northern blotting. RT-PCR analysis revealed a wide tissue distribution of CaIRF7 constitutive expression, with detectable transcript in non-infected CAB cells and various tissues of healthy crucian carp. In addition, CaIRF7 expression was differentially increased by stimulation of the CAB cells with active GCHV, UV-inactivated GCHV or CAB IFN, indicating that the activation of CaIRF7 was directly regulated by IFN.


Assuntos
Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Carpa Dourada/imunologia , Animais , Sequência de Bases , Blástula/imunologia , Northern Blotting , Análise por Conglomerados , Primers do DNA , DNA Complementar/genética , Perfilação da Expressão Gênica , Carpa Dourada/genética , Fator Regulador 7 de Interferon , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência
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