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1.
Biomed Res Int ; 2021: 5521058, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337018

RESUMO

Background: Gastric cancer (GC) is the most common type of cancer. It is highly malignant and is characterized by rapid and uncontrolled growth. The antitumour activity of Baicalin was studied in multiple cancers. However, its mechanism of action has not been fully elucidated. We provided a systematic understanding of the mechanism of action of baicalin against GC using a transcriptome analysis of RNA-seq. Methods: Human GC cells (SGC-7901) were exposed to 200 µg/ml baicalin for 24 h. RNA-seq with a transcriptome, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify the antitumour effects of baicalin on SGC-7901 cells in vitro. A protein-protein interaction (PPI) network of differentially expressed genes (DEGs) was constructed. A competitive endogenous RNA (ceRNA) network was constructed and further analysed after validation using qRT-PCR. Results: A total of 68 lncRNAs, 20 miRNAs, and 1648 mRNAs were differentially expressed in baicalin-treated SGC-7901 GC cells. Three lncRNAs, 6 miRNAs, and 7 mRNAs were included in the ceRNA regulatory network. GO analysis revealed that the main DEGs were involved in the biological processes of the cell cycle and cell death. KEGG pathway analysis further suggested that the p53 signalling pathway was involved in the baicalin-induced antitumour effect on SGC-7901 cells. Further confirmation using qPCR indicated that baicalin induced an antitumour effect on SGC-7901 cells, which is consistent with the results of the sequencing data. Conclusions: In summary, the mechanism of baicalin against GC involves multiple targets and signalling pathways. These results provide new insight into the antitumour mechanism of baicalin and help the development of new strategies to cure GC.


Assuntos
Flavonoides/uso terapêutico , Perfilação da Expressão Gênica , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Mapas de Interação de Proteínas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
2.
Front Microbiol ; 11: 569105, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33101241

RESUMO

O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.

3.
Biosci Rep ; 38(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29899163

RESUMO

To investigate the effect of stimulation of human bronchial epithelial cells (HBECs) by arterial traffic ambient PM2.5 (TAPM2.5) and wood smoke PM2.5 (WSPM2.5) on the expression of long non-coding RNAs (lncRNAs) in order to find new therapeutic targets for treatment of chronic obstructive pulmonary disease (COPD). HBECs were exposed to TAPM2.5 and WSPM2.5 at a series of concentrations. The microarray analysis was used to detect the lncRNA and mRNA expression profiles. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene ontology (GO) enrichment were conducted to analyze the differentially expressed lncRNAs and mRNAs. Quantitative real-time PCR (qRT-PCR) was performed to confirm the differential expression of lncRNAs. Western blot was performed to study the expression of autophagy and apoptosis-associated proteins. Flow cytometry was used to detect the apoptotic cells. The results indicated that fine particulate matter (PM2.5)-induced cell damage of HBECs occurred in a dose-dependent manner. The microarray analysis indicated that treatment with TAPM2.5 and WSPM2.5 led to the alteration of lncRNA and mRNA expression profiles. LncRNA maternally expressed gene 3 (MEG3) was significantly up-regulated in HBECs after PM2.5 treatment. The results of Western blot showed that PM2.5 induced cell apoptosis and autophagy by up-regulating apoptosis-associated gene, caspase-3, and down-regulating autophagy-associated markers, Bcl-2 and LC3 expression. In addition, we demonstrated that TAPM2.5 and WSPM2.5 accelerated apoptosis of human bronchial (HBE) cells, silencing of MEG3 suppressed apoptosis and autophagy of HBE cells. These findings suggested that the lncRNA MEG3 mediates PM2.5-induced cell apoptosis and autophagy, and probably through regulating the expression of p53.


Assuntos
Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Material Particulado/toxicidade , RNA Longo não Codificante , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Brônquios/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Inativação Gênica , Humanos , Material Particulado/administração & dosagem , RNA Longo não Codificante/genética , RNA Mensageiro , Fumaça/efeitos adversos
4.
Am J Physiol Cell Physiol ; 309(3): C148-58, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25994789

RESUMO

The nuclear factor erythroid 2-related factor 2 (Nrf2) is required to combat increases in oxidative stress. The chemical compound tert-butylhydroquinone (tBHQ) can downregulate Kelch-like ECH-associated protein 1 (Keap1), a repressor of Nrf2, thus maintaining the stability of Nrf2. tBHQ can also increase intracellular "free" zinc in human bronchial epithelial (16HBE) cells. We aim to investigate whether the intracellular free zinc change plays a role in Nrf2 activation. tBHQ exposure dose-dependently increases intracellular free zinc concentrations within 30 min in 16HBE cells by mobilizing intracellular zinc pools. Active Nrf2 and the antioxidant enzyme heme oxygenase-1 (HO-1) increase at 3 h after tBHQ treatment. Chelating intracellular free zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) during tBHQ exposure partially abrogates the tBHQ-induced activation of Nrf2 and HO-1 expression, while Keap1 is further decreased. These results indicate that tBHQ-induced stability of Nrf2 is associated with the intracellular free zinc level. Because the activated Nrf2 is phosphorylated, the serine/threonine protein phosphatase activity, which is known to be inhibited by zinc, is assayed. The results showed that tBHQ treatment can suppress cellular protein phosphatase-2A (PP2A) and protein phosphatase-2C (PP2C) activity, which can be abrogated by adding TPEN. This finding is verified in a cell-free protein extract experiment by supplying zinc or by chelating zinc with TPEN. These results provide a novel mechanistic insight into Nrf2 activation in antioxidant enzyme induction involving zinc signaling. The increase of intracellular free zinc may be one mechanism for Nrf2 activation. The inhibition of PP2A and PP2C activity may be involved in Nrf2 phosphorylation modulation.


Assuntos
Hidroquinonas/metabolismo , Membranas Intracelulares/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2/metabolismo , Zinco/metabolismo , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Hidroquinonas/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2C
5.
Chem Eng J ; 253: 538-543, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32288623

RESUMO

This study investigates and compares the virucidal performances of photocatalytic (PC) and photoelectrocatalytic (PEC) treatments in the presence and absence of halides, such as Br- and Cl-, under comparable experimental conditions. The results confirm that the PC virucidal efficiency can be enhanced in the presence of low halide concentrations (e.g., X = Br- or Cl-) and further enhanced by applying potential bias onto the photoanode in a PEC system. The PEC treatment in the presence of 1.0 mM Br (PEC-Br) shows the highest virucidal efficiency, enabling complete inactivation of a ∼1000 TCID50 replication-deficient recombinant adenovirus (RDRADS) population within 31.7 s. The superior virucidal performances of PEC-X treatments can be attributed to the increased production of active oxygen species and additional viricides resulting from the PEC halide oxidation, as well as prolonged lifetime of photoholes (h+ ) for direct inactivation. The findings of this work confirm that new forms of active species generated in situ via a PC or PEC process are effective for viruses.

6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 23(9): 856-8, 2007 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-17825236

RESUMO

AIM: To induce the expression of F10 in E.coli and prepare the rabbit polyclonal antibody against it. METHODS: The gene of F10 was amplified by PCR, and cloned into expression vector pET-GST to construct recombinant expression plasmid pET-GST/F10. The recombinant plasmid was transformed into E.coli BL21 and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and analyzed by SDS-PAGE and Western blot. A rabbit was immunized with the purified F10 fusion protein to produce polyclonal antibody, and the production of antibody was confirmed by ELISA. RESULTS: Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained correct coding region of F10. SDS-PAGE demonstrated that the recombinant protein was expressed with the expected molecular weight at 61 kD. After purified, the purity of the fusion protein was above 90%. Western blot confirmed the recombinant protein was GST/F10 fusion protein. Rabbit polyclonal antibody was obtained, the titer of which was 1:20 000. CONCLUSION: F10 recombinant expression vector has been successfully constructed and F10 protein has been expressed. The obtained rabbit anti F10 antibody has a high titer and will facilitate the study of the biological function of F10.


Assuntos
Anticorpos/imunologia , Anticorpos/isolamento & purificação , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Anticorpos/análise , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Solubilidade
7.
Sheng Wu Gong Cheng Xue Bao ; 21(5): 748-53, 2005 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-16285516

RESUMO

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.


Assuntos
Proteínas de Fluorescência Verde/genética , Proteínas Mutantes/genética , RNA Catalítico/genética , Tetrahymena/enzimologia , Trans-Splicing , Animais , Sequência de Bases , Células HeLa , Humanos , Íntrons/genética , Dados de Sequência Molecular , Mutação , RNA Mensageiro/genética , Transcrição Genética
8.
Yi Chuan Xue Bao ; 32(10): 1011-7, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16252695

RESUMO

Female precocious puberty is caused by premature activation of the hypothalamic-pituitary-gonadal axis, exposure to exogenous sex steroid hormones, and the presence of endogenous sex steroids caused by various factors. Estrogen is the final key factor to start onset of puberty. However,in some cases of precocious puberty in girls estrogen elevation could not be detected. The raised sensitivity of estrogen receptor, which may caused by ESR1 mutation or polymorphism, has been frequently mentioned for interpreting the etiology of sporadic low estrogen type cases. But no case evidence has been found in clinical practice. For the purpose of screening possible mutations in estrogen receptor gene, leukocyte genomic DNA were collected from 16 girls with precocious puberty of sporadic low estrogen,and exons of ESR1 were amplified and analysized using PCR-SSCP/silver staining method. A single strand conformation change in exon 8 was found in one of the patients (No. 14). The suspected fragment were cloned to a T vector and sequenced for analysis. Sequencing of these clones revealed that this conformation change is caused by a C to T transition. This mutation results in the replacement of arginine by cystine at position 548 of ESR1 protein. The mutation created an extra Btsl digest site and made it can be readily identified by PCR-PFLP method. Further detection using this method, and sequencing of cloned exon8 colonies from patients proved that the patient No. 14 is Arg548/Cys548 heterozagous in genotype. This mutation increased hydrophobility of the area dramatically. The position and the conservative of this residue in vertebrates suggested Arg548 may play an important role in ESR1 function. For study the role of this mutation in the onset of precocious puberty, a firefly luciferase reporter plasmid pGL3-promoter-ERE was constructed,and a pCR3. 1-hermut pisimid expressing Cys548 ER was constructed based on wild type pCR3. 1her. Co-transfection of reporter and pCR3. 1 -hermut in CMF-7 cell strain proved that Cys548 mutant can significantly increase the transcription activity over the Arg548 wild type.


Assuntos
Receptor alfa de Estrogênio/genética , Mutação , Puberdade Precoce/genética , Substituição de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Pré-Escolar , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Lactente , Luciferases/genética , Luciferases/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
9.
Di Yi Jun Yi Da Xue Xue Bao ; 25(2): 171-3, 2005 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-15698997

RESUMO

OBJECTIVE: To study the expressions of the novel gene F10 associated with hydatidiform mole in different trophoblastic tumors and explore the relation of F10 expression with the invasiveness of malignant trophoblastic tumor. METHODS: In situ hybridization was used to study the expression of F10 in 12 cases of hydatidiform mole, 6 cases of invasive mole, and 8 cases of choriocarcinoma. RESULTS: F10 mRNA was positive in all cases of hydatidiform mole, invasive mole, and choriocarcinoma, and the expression intensity significantly increased in the order of hydatidiform mole, invasive mole and choriocarcinoma (P<0.001). CONCLUSION: The expression of F10 gene may relate to the occurrence and invasiveness of trophoblastic tumor, with possible involvement in the invasion or malignant changes of trophoblastic cells.


Assuntos
Genes Neoplásicos/genética , Mola Hidatiforme Invasiva/genética , Mola Hidatiforme/genética , Neoplasias Trofoblásticas/genética , Neoplasias Uterinas/genética , Adulto , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Gravidez , Neoplasias Trofoblásticas/patologia , Neoplasias Uterinas/patologia
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