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1.
J Nanobiotechnology ; 18(1): 15, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952530

RESUMO

BACKGROUND: The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Here we describe a novel platform of gene delivery by elastin-like polypeptide (ELP) based targeting biopolymers. RESULTS: For better tumor targeting and membrane penetrating characteristics, we designed various chimeric ELP-based carriers containing a cell penetrating peptide (Tat), single or multiple copies of AP1 an IL-4 receptor targeting peptide along with coding sequence of ELP and referred as Tat-A1E28 or Tat-A4V48. These targeted polypeptides were further analyzed for its ability to deliver siRNA (Luciferase gene) in tumor cells in comparison with non-targeted controls (Tat-E28 or E28). The positively charged amino acids of these polypeptides enabled them to readily complex with negatively charged nucleic acids. The complexation of nucleic acid with respective polypeptides facilitated its transfection efficiency as well as stability. The targeted polypeptides (Tat-A1E28 or Tat-A4V48) selectively delivered siRNA into tumor cells in a receptor-specific fashion, achieved endosomal and lysosomal escape, and released gene into cytosol. The target specific delivery of siRNA by Tat-A1E28 or Tat-A4V48 was further validated in murine breast carcinoma 4T1 allograft mice model. CONCLUSION: The designed delivery systems efficiently delivered siRNA to the target site of action thereby inducing significant gene silencing activity. The study shows Tat and AP1 functionalized ELPs constitute a novel gene delivery system with potential therapeutic applications.


Assuntos
Peptídeos Penetradores de Células/química , Elastina/química , Peptídeos/química , RNA Interferente Pequeno/química , Animais , Biopolímeros , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Luciferases/genética , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Imagem Óptica , RNA Interferente Pequeno/administração & dosagem , Receptores de Interleucina-4/metabolismo , Transfecção
2.
Chem Commun (Camb) ; 56(2): 281-284, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31807738

RESUMO

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.


Assuntos
Luciferases/química , Serina-Treonina Quinases TOR/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Animais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Linhagem Celular Tumoral , Humanos , Ligantes , Limite de Detecção , Luciferases/genética , Proteínas Luminescentes/química , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Ligação Proteica , Sirolimo/química , Sirolimo/metabolismo
3.
Zhonghua Zhong Liu Za Zhi ; 41(11): 820-825, 2019 Nov 23.
Artigo em Chinês | MEDLINE | ID: mdl-31770848

RESUMO

Objective: To establish a nude mouse model of subcutaneous lung cancer using dual fluorescence reporting genes of luciferase (Luc) and near-infrared fluorescent protein (iRFP). Methods: The Luc and iRFP expressed lentiviral vector was constructed by Gateway method. After verified by sequencing, the lentivirus particle was prepared and infected into lung cancer A549 cells. Successfully infected A549 (mA549) cells were selected by puromycin and amplified. The expression of Luc and iRFP were observed under fluorescence microscope, and the expression of c-Met protein on the cell surface was detected by immunofluorescence. Twelve female nude mice were randomly divided into 2 groups, 6 in each group. A549 and mA549 cells were inoculated subcutaneously into the right forelimb of nude mice. The growth and fluorescence expression of the tumor were observed by in vivo imaging. The tumor formation was evaluated by hematoxylin-eosin (HE) staining and immunohistochemistry. Results: The Luc and iRFP stably expressed mA549 cell line was successfully constructed. The expressions of iRFP and Luc in mA549 cells were observed under fluorescence microscope. The results of immunofluorescence showed that c-Met protein expressed in both A549 cells and mA549 cells. The growth period of mA549 xenograft in nude mice was moderate and the tumorigenesis rate was 100%. The growth trend of mA549 cells in vivo was not significantly different from that of A549 cells (P>0.05). HE staining and immunohistochemistry results showed that the tumor issues displayed typical histopathological features of tumor. Immunohistochemistry results showed that both A549 and mA549 tumors expressed c-Met protein. Conclusion: A stable, real-time monitoring model of iRFP-Luc-A549 lung cancer cell xenograft in nude mice was successfully constructed.


Assuntos
Neoplasias Pulmonares/patologia , Transplante de Neoplasias/métodos , Células A549 , Animais , Linhagem Celular Tumoral , Feminino , Fluorescência , Genes Reporter , Humanos , Imuno-Histoquímica , Luciferases/genética , Proteínas Luminescentes/genética , Pulmão , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/genética , Distribuição Aleatória
4.
Chem Commun (Camb) ; 55(77): 11619-11622, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31501844

RESUMO

Mesoporous organosilica nanoparticles (PHT-PMO) have been prepared from an octa-triethoxysilylated Zn phthalocyanine precursor. These PHT-PMO nanoparticles had no dark toxicity but high phototoxicity when irradiated at 650 nm, and remarkable near-infrared phototoxicity when excited at 760 and 810 nm. The PHT-PMO were then aminated to promote electrostatic complexation with siRNA. Transfection experiments were performed upon NIR irradiation and photochemical internalization was very efficient, leading to 65% luciferase extinction in MCF-7 cancer cells expressing stable luciferase.


Assuntos
Indóis/química , Nanopartículas/química , Compostos Organometálicos/química , Fotoquimioterapia/métodos , RNA Interferente Pequeno/química , Silanos/química , Sobrevivência Celular , Cetrimônio/química , Humanos , Raios Infravermelhos , Luciferases/genética , Células MCF-7 , Processos Fotoquímicos , Porosidade , RNA Interferente Pequeno/metabolismo , Eletricidade Estática , Propriedades de Superfície
5.
BMC Res Notes ; 12(1): 476, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370865

RESUMO

OBJECTIVE: A previous study undertaken at our centre to identify common genetic variants associated with sporadic breast cancer in Sri Lankan women showed that the T allele of rs3218550, located in the 3'untranslated region of X-ray repair cross-complementing gene-2 (XRCC2), increased breast cancer risk by 1.5-fold. Dual luciferase reporter assays performed in MCF-7 breast cancer cells showed a putative transcriptional repressor effect exerted mainly by the T allele. Electrophoretic mobility shift assays were conducted to further investigate the interaction of this variant with DNA-binding protein, using nuclear protein extracts derived from MCF-7 cells. RESULTS: An allele-specific differential binding was observed. The T allele resulted in differential DNA-protein complex binding as evidenced by the presence of multiple bands of increased intensity compared to the wild-type C allele. This implies possible alteration in binding of regulatory proteins by the variant allele. These results implicate XRCC2:rs3218550C>T as a potential low-penetrant susceptibility allele for sporadic breast cancer. XRCC2 is known to play an essential role in homologous recombination repair of DNA double-strand breaks. It is plausible that this variant may be exerting regulatory effects on XRCC2 gene expression leading to altered DNA repair capacity. Further functional studies are warranted to validate this finding.


Assuntos
Neoplasias da Mama/genética , Reparo do DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Regiões 3' não Traduzidas , Alelos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Quebras de DNA de Cadeia Dupla , DNA de Neoplasias/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Expressão Gênica , Frequência do Gene , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Células MCF-7 , Penetrância , Ligação Proteica
6.
DNA Cell Biol ; 38(10): 1155-1165, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31433201

RESUMO

Our previous genome-wide association study has identified a suggestive association at rs11264799 within FCRL3 (Fc receptor-like 3) locus on 1q23.1 for IgA nephropathy (IgAN) in a Chinese Han population. This study aims to investigate the association of FCRL3 variants with the susceptibility, clinicopathological phenotypes and prognosis of IgAN. Eleven FCRL3 single-nucleotide polymorphisms (SNPs) were selected and analyzed in this two-stage case/control study with a total of 1750 IgAN cases and 2500 healthy controls in a Chinese Han population. Unconditional logistic regression models were used to estimate odds ratios and 95% confidence intervals (CIs) as implemented in the PLINK software. Luciferase assays were applied to detect the allelic effect of rs11264794 on gene expression regulation. We found that four SNPs (rs11264794, rs7865684, rs11264799, and rs6691569) were significantly associated with IgAN susceptibility after Bonferroni correction in the combined samples. Genotype/phenotype association analysis observed that two SNPs (rs11264794 and rs11264793) were associated with less disease severity. After adjusting for confounders, rs11264794 was independently correlated with renal outcome in IgAN patients (hazard ratio = 0.64, 95% CI = 0.43-0.97, p = 0.033). In addition, the protective allele A of rs11264794 was significantly associated with higher FCRL3 gene expression. Furthermore, luciferase reporter gene assays demonstrated that the minor allele of rs11264794 obviously reduced the specific binding between miR-183-5p.1 and FCRL3 3'-untranslated region. Our results indicate that FCRL3 gene polymorphisms are associated with the development and progression of IgAN, and the rs11264794-A allele showed a protective role for IgAN.


Assuntos
Predisposição Genética para Doença , Glomerulonefrite por IGA/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Receptores Imunológicos/genética , Regiões 3' não Traduzidas , Alelos , Grupo com Ancestrais do Continente Asiático , Estudos de Casos e Controles , Feminino , Expressão Gênica , Genes Reporter , Estudos de Associação Genética , Glomerulonefrite por IGA/diagnóstico , Glomerulonefrite por IGA/etnologia , Glomerulonefrite por IGA/patologia , Humanos , Modelos Logísticos , Luciferases/genética , Luciferases/metabolismo , Masculino , MicroRNAs/metabolismo , Razão de Chances , Fenótipo , Prognóstico , Receptores Imunológicos/metabolismo , Índice de Gravidade de Doença
7.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366048

RESUMO

As aberrant activity of protein kinases is observed in many disease states, these enzymes are common targets for therapeutics and detection of activity levels. The development of non-natural protein kinase substrates offers an approach to protein substrate competitive inhibitors, a class of kinase inhibitors with promise for improved specificity. Also, kinase activity detection approaches would benefit from substrates with improved activity and specificity. Here, we apply a substrate-mediated selection to a peptidomimetic DNA-encoded chemical library for enrichment of molecules that can be phosphorylated by the protein tyrosine kinase, c-Src. Several substrates were identified and characterized for activity. A lead compound (SrcDEL10) showed both the ability to serve as a substrate and to promote ATP hydrolysis by the kinase. In inhibition assays, compounds displayed IC50's ranging from of 8-100 µM. NMR analysis of SrcDEL10 bound to the c-Src:ATP complex was conducted to characterize the binding mode. An ester derivative of the lead compound demonstrated cellular activity with inhibition of Src-dependent signaling in cell culture. Together, the results show the potential for substrate-mediated selections of DNA-encoded libraries to discover molecules with functions other than simple protein binding and offer a new discovery method for development of synthetic tyrosine kinase substrates.


Assuntos
Técnicas de Química Combinatória , DNA/química , Peptidomiméticos/síntese química , Bibliotecas de Moléculas Pequenas/química , Quinases da Família src/química , Trifosfato de Adenosina/química , Anticorpos Monoclonais/química , DNA/metabolismo , Genes Reporter , Humanos , Hidrólise , Cinética , Luciferases/genética , Luciferases/metabolismo , Peptidomiméticos/metabolismo , Fosforilação , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Quinases da Família src/metabolismo
8.
Int J Pharm ; 569: 118570, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31352048

RESUMO

Small interfering RNA (siRNA) represents a new class of therapeutic agents. Its successful intracellular delivery is a major challenge. Lipo-oligomeric carriers can complex siRNA into lipopolyplexes and thus mediate its cellular uptake. In this study, siRNA against the kinesin related mRNA EG5 gene (siEG5) and the microtubule inhibitor pretubulysin (PT) were co-formulated into polyplexes using azide-containing lipo-oligomer 1198. Nanoparticles were further modified by click reaction using shielding agent DBCO-PEG or EGFR targeting peptide GE11 (DBCO-PEG-GE11). Polyplexes displayed efficient payload incorporation and homogenous particle sizes of 200 nm. The biological effects of the unmodified and surface-functionalized polyplexes were investigated. The successful GE11-mediated intracellular delivery of siRNA into the EGFR overexpressing KB and Huh7 cell lines facilitated potent silencing of an EGFP-luciferase reporter gene by GFP siRNA. Specific downregulation of EG5 mRNA by siEG5 resulted in the expected antitumoral activity. The combination formulation 1198 siEG5 + PT provided superior antitumoral activity over free PT and 1198 siEG5.


Assuntos
Cinesina/genética , Oligopeptídeos/administração & dosagem , Peptídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Moduladores de Tubulina/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/genética , Proteínas de Fluorescência Verde/genética , Humanos , Luciferases/genética , Polietilenoglicóis/administração & dosagem
9.
Immunology ; 158(2): 121-135, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31322727

RESUMO

The Autoimmune Regulator (Aire) protein coordinates the negative selection of developing thymocytes by inducing the expression of hundreds of tissue-specific antigens within the thymic medulla, which is also a primary site of the expression of the immune checkpoint HLA-G molecule. Considering the immunomodulatory properties of Aire and HLA-G, and considering that the role of the constitutive thymus expression of HLA-G has not been elucidated, we studied the effect of AIRE cDNA transfection on HLA-G expression in 4D6 thymic cells and in the HLA-G-positive JEG-3 choriocarcinoma cells. Aire promoted the transactivation of HLA-G gene by increasing the overall transcription, inducing the transcription of at least G1 and G2/G4 isoforms, and incrementing the occurrence and distribution of intracellular HLA-G protein solely in 4D6 thymic cells. Luciferase-based assays and chromatin immunoprecipitation experiments performed in 4D6 cells revealed that Aire targeted at least two regions within the 5'-untranslated regulatory region (5'-URR) extending 1·4 kb from the first ATG initiation codon. The interaction occurs independently of three putative Aire-binding sites. These results indicate that the Aire-induced upregulation of HLA-G in thymic cells is likely to act through the interaction of Aire with specific HLA-G 5'-URR DNA-binding factors. Such a multimeric transcriptional complex might operate in the thymus during the process of promiscuous gene expression.


Assuntos
Células Epiteliais/imunologia , Antígenos HLA-G/genética , Fatores de Transcrição/genética , Ativação Transcricional/imunologia , Regiões 5' não Traduzidas , Autoimunidade/genética , Sítios de Ligação , Linhagem Celular Tumoral , Células Epiteliais/citologia , Genes Reporter , Antígenos HLA-G/imunologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Transdução de Sinais , Timo/citologia , Timo/imunologia , Fatores de Transcrição/imunologia , Transfecção
10.
Anal Sci ; 35(8): 835-838, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31281129

RESUMO

Glucose transporter 4 (GLUT4) is an insulin-regulated glucose transporter, which is vital for blood glucose homeostasis. To clarify the physiological roles of GLUT4, quantitative measurement of GLUT4 exocytosis is indispensable. Herein, we show a rapid detection system for GLUT4 on the cell surface using spontaneous split-luciferase reconstitution. Upon insulin-induced GLUT4 exocytosis, GLUT4 was exposed outside, where luciferase is reconstituted and emitted luminescence. Pretreatment with inhibitors reduced the insulin-induced signal elevation. The results indicate that the developed method is applicable to high-throughput analysis on GLUT4 trafficking, which will greatly accelerate comprehensive research on the physiological roles of GLUT4.


Assuntos
Exocitose , Teste de Complementação Genética , Transportador de Glucose Tipo 4/análise , Luciferases/metabolismo , Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Luciferases/genética
11.
Int J Pharm ; 567: 118500, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31288056

RESUMO

The development of new antibacterial molecules is essential in view of the emergence of pathogenic strains resistant to multiple antibiotics. Among the infectious pathologies, pulmonary infections are particularly difficult to treat due to the complexity of lung anatomy and the presence of natural barriers such as mucus. At present, the aerosol delivery of antibacterial compounds is still poorly employed. Furthermore, the presence of bacteria in lungs negatively affects aerosolized Cystic Fibrosis gene therapy efficiency. A multi-functional formulation (antibacterial and transfection activities) could increase the therapeutic effect. This work reports the synthesis of new N-heterocyclic carbene silver complexes (Ag-NHC) featuring a lipid chain and the evaluation of their antibacterial potency, especially when delivered following aerosolization. When formulated alone in water, these Ag-NHC displayed remarkable antibacterial activities against some Staphyloccocus aureus strains and Pseudomonas aeruginosa clinical strains. Moreover, combined with cationic lipid and DNA (ternary combination), they could be used to deliver therapeutic genes via aerosolization in infected lungs. Altogether, the data reported herein support n-alkyl chain Ag-NHC as a possible alternative to conventional antibiotics for treating respiratory infections and to combat the emergence of multi-resistant bacteria.


Assuntos
Antibacterianos/administração & dosagem , DNA/administração & dosagem , Metano/análogos & derivados , Prata/administração & dosagem , Transfecção/métodos , Aerossóis , Brônquios/citologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Humanos , Luciferases/genética , Metano/administração & dosagem , Plasmídeos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
12.
Acta Pharm ; 69(1): 49-61, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31259716

RESUMO

Use of exogenous small interfering RNA (siRNA) has shown potential in gene silencing. The need for target-specific siRNA delivery vehicles is crucial to successful gene silencing. This study is aimed at developing and evaluating the safety and efficiency of siRNA delivery using unmodified and folic acid (FA) modified poly(amidoamine) generation 5 (PAMAM G5D) functionalized gold nanoparticles (Au:G5D/Au:G5D:FA) in vitro. All formulations were physico--chemically characterized and nanocomplexes were evaluated using the band shift, dye displacement, nuclease protection, MTT cell viability, and luciferase reporter gene assays. Nanocomplexes bound and protected siRNA against degrading RNases, and were well tolerated by the cells. The Au:G5D:FA nanocomplexes elicited excellent gene silencing in folate receptor expressing HeLa-Tat-Luc cells, decreasing significantly in the presence of excess FA ligand, indicating nanocomplex uptake by the mechanism of receptor mediation. These results highlight the synergistic role played by Au and the dendrimer in enhancement of transgene silencing.


Assuntos
Dendrímeros/química , Ácido Fólico/química , Inativação Gênica/efeitos dos fármacos , Ouro/química , Luciferases/genética , Nanopartículas Metálicas/química , Células CACO-2 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dendrímeros/administração & dosagem , Ácido Fólico/administração & dosagem , Ouro/administração & dosagem , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Nanopartículas Metálicas/administração & dosagem , RNA Interferente Pequeno/genética
13.
Environ Sci Process Impacts ; 21(11): 1908-1914, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31332417

RESUMO

Previously, perfluoroalkyl substances (PFASs) have been found to be associated with many adverse effects mediated by the peroxisome proliferator-activated receptor α (PPARα) and PPARγ. Here, we found another subtype of the peroxisome proliferator-activated receptors (PPARs); the PPARß/δ mediated pathway might also be a potential adverse outcome pathway for PFASs. We investigated the direct binding and transcriptional activity of PFASs toward human PPARß/δ, and further revealed the structure-binding and structure-activity relationship between PFASs and PPARß/δ. The receptor binding experiment showed that their binding potency was dependent on the carbon chain length and the terminal functional group. For twelve perfluoroalkyl carboxylic acids (PFCAs), an inverted U-shaped relationship existed between the PPARß/δ binding potency and the carbon chain length, with perfluorododecanoc acid (C12) showing the highest binding potency. The three perfluoroalkane sulfonic acids (PFSAs) exhibited a stronger binding potency than their PFCA counterparts. The two fluorotelomer alcohols (FTOHs) showed no binding potency. In receptor transcriptional activity assays, they enhanced the PPARß/δ transcriptional activity. Their transcriptional activity was also related to the carbon chain length and the terminal functional group. Molecular docking analysis showed the PFASs fitted into the ligand binding pocket of PPARß/δ with a binding geometry similar to a fatty acid.


Assuntos
Ácidos Carboxílicos/química , Fluorcarbonetos/química , PPAR delta/química , PPAR beta/química , Animais , Ligação Competitiva , Genes Reporter , Células HEK293 , Humanos , Ligantes , Luciferases/genética , Simulação de Acoplamento Molecular , PPAR delta/genética , PPAR delta/metabolismo , PPAR beta/genética , PPAR beta/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Transfecção
14.
Mol Med Rep ; 20(2): 967-976, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173204

RESUMO

Ischemia reperfusion injury (IRI) is one of the primary causes of acute renal injury and even acute renal failure. An increasing body of evidence has indicated that the aberrant expression of microRNAs (miRNA/miR) is closely associated with the progression of IRI. In the process of renal IRI, inflammatory reactions, cell adhesion and the death of renal tubular epithelial cells serve important roles. The present study investigated the expression of renal miRNAs following renal IRI in an attempt to identify the miRNAs that exert pivotal functions in renal IRI. The present study revealed that miR­27a, which was downregulated in IRI, and the 3'­-untranslated region (UTR) of Toll­like receptor 4 (TLR4) have associated binding sites. The levels of TLR4, miR­27a and specific associated proteins in the renal tissues of gestational rats were determined by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis, immunohistochemistry and western blotting. The associations between miR­27a and TLR4 were analyzed, and the regulatory effect of miR­27a on TLR4 was detected via a luciferase reporter gene assay, western blotting and RT­qPCR in vivo and in vitro. In addition, the present study demonstrated that miR­27a suppressed the expression of TLR4 by binding to the 3'UTR of TLR4. The overexpression of miR­27a downregulated the expression of TLR4, which in turn inhibited the inflammation, cell adhesion and cell death in IRI. Therefore, miR­27a inhibited inflammation in IRI by decreasing the expression of TLR4.


Assuntos
Lesão Renal Aguda/genética , Células Epiteliais/metabolismo , MicroRNAs/genética , Traumatismo por Reperfusão/genética , Receptor 4 Toll-Like/genética , Regiões 3' não Traduzidas , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Animais , Apoptose/genética , Sequência de Bases , Sítios de Ligação , Adesão Celular , Linhagem Celular , Células Epiteliais/patologia , Regulação da Expressão Gênica , Genes Reporter , Rim/metabolismo , Rim/patologia , Luciferases/genética , Luciferases/metabolismo , Masculino , MicroRNAs/metabolismo , Ratos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
15.
Ecotoxicol Environ Saf ; 181: 164-171, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185430

RESUMO

Short-chain chlorinated paraffins (SCCPs) are frequently detected in environmental matrices and human tissues. It was hypothesized that SCCPs might interact with the peroxisome proliferator-activated receptor α (PPARα). In the present study, an in vitro, dual-luciferase reporter gene assay and in silico molecular docking analysis were employed together to study the interactions between SCCPs congeners and PPARα. Expressions of genes downstream in pathways activated by PPARα in liver of rats exposed to 1, 10, or 100 mg/kg bm/d of C10-13-CPs (56.5% Cl) for 28 days were examined to confirm activation potencies of SCCPs toward PPARα signaling. Effects of exposure to C10-13-CPs (56.5% Cl) on fatty acid metabolism in rat liver were also explored via a pseudo-targeted metabolomics strategy. Our results showed that C10-13-CPs (56.5% Cl) caused a dose-dependent greater expression of luciferase activity of rat PPARα. Molecular docking modeling revealed that SCCPs had a strong capacity to bind with PPARα only through hydrophobic interactions and the binding affinity was dependent on the degree of chlorination in SCCPs congeners. In livers of male rats, exposure to 100 mg/kg bm/d of C10-13-CPs (56.5% Cl) resulted in up-regulated expressions of 11 genes that are downstream in the PPARα-activated pathway and regulate catabolism of fatty acid. Consistently, accelerated fatty acid oxidation was observed mainly characterized by lesser concentrations of ∑fatty acids in livers of rats. Overall, these results demonstrated, for the first time, that SCCPs could activate rat PPARα signaling and thereby disrupt metabolism of fatty acid in livers of male rats.


Assuntos
Ácidos Graxos/metabolismo , Fígado/efeitos dos fármacos , PPAR alfa/metabolismo , Parafina/toxicidade , Animais , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Halogenação , Fígado/metabolismo , Luciferases/genética , Masculino , Simulação de Acoplamento Molecular , PPAR alfa/química , Parafina/química , Ratos , Transdução de Sinais , Regulação para Cima
16.
Dokl Biochem Biophys ; 485(1): 157-161, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201640

RESUMO

The results of a comparative study of the luciferin-luciferase systems of seven species of bioluminescing oligochaetes-Henlea petushkovi, Henlea rodionovae, Fridericia heliota (Enchytraeidae), Microscolex phosphoreus (Acanthodrilidae), Pontodrilus litoralis (Megascolecidae), Eisenia lucens, and Avelona ligra (Lumbricidae)-are presented.


Assuntos
Luciferases , Oligoquetos , Animais , Luciferases/genética , Luciferases/metabolismo , Oligoquetos/enzimologia , Oligoquetos/metabolismo
17.
BMC Biotechnol ; 19(1): 34, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31200673

RESUMO

BACKGROUND: In vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC). RESULTS: Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated. CONCLUSIONS: XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.


Assuntos
Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/citologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Luciferases/genética , Medições Luminescentes/métodos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Transplante Heterólogo
18.
Parasit Vectors ; 12(1): 321, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238993

RESUMO

BACKGROUND: Ticks are important vectors of disease-causing pathogens. With the rise of resistance to chemical acaricides, alternative methods in tick control are warranted. Gene manipulation has been successful in controlling mosquitoes and mosquito-borne diseases and is now looked upon as a candidate method to control ticks and tick-borne pathogens. Our previous study has identified the actin and ferritin promoter regions in the Haemaphysalis longicornis tick. RESULTS: Here, the ferritin-derived promoter from the H. longicornis tick was characterized in silico, and the core promoter sequences and some of its important components were identified. Several truncations of the promoter region were created and inserted to a reporter plasmid to determine the important components for its activity. The activities of the truncated promoters on the Ixodes scapularis tick cell line (ISE6) were measured via a dual luciferase assay using experimental and control reporter genes. To induce the promoter's activity, transfected ISE6 cells were exposed to ferrous sulfate. The 639 nucleotides truncated promoter showed the highest activity on ISE6 cells when exposed to 1 mM ferrous sulfate. CONCLUSION: In this study, we characterized an iron-inducible tick promoter that could be a valuable tool in the development of a gene-manipulation system to control ticks and tick-borne pathogens.


Assuntos
Ferritinas/genética , Ferro/metabolismo , Ixodes/citologia , Ixodes/genética , Regiões Promotoras Genéticas , Animais , Linhagem Celular , Compostos Ferrosos/farmacologia , Técnicas Genéticas , Luciferases/genética , Transfecção
19.
Int J Pharm ; 566: 141-148, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31125716

RESUMO

Small interfering RNAs (siRNAs) can down-regulate the expression of a target mRNA molecule in a sequence-specific manner, making them an attractive new class of drugs with broad potential for the treatment of diverse human diseases. Here, we report the synthesis of a series of cationic amphiphiles which were obtained by the coupling of amino acids and dipeptides onto a lipidic double chain. The new amphiphiles presenting a peptidic motif on a short hydrophilic spacer group were evaluated for selective gene silencing through RNA interference. Our results show that tryptophan residues boost siRNA delivery in an unexpected manner. The silencing experiments performed with very low concentrations of siRNA showed that the best formulations could induce significant death of tumor cells after silencing of polo-like kinase 1 which is implicated in cell cycle progression. In addition, these Trp containing peptide amphiphiles were highly efficient siRNA delivery vectors even in presence of competing serum proteins.


Assuntos
Peptídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Inativação Gênica , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Luciferases/genética , Triptofano , Tirosina
20.
Cell Biol Int ; 43(7): 789-798, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31042002

RESUMO

As a cationic non-viral gene delivery vector, poly(agmatine/ N, N'-cystamine-bis-acrylamide) (AGM-CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM-CBA/pDNA polyplexes was found to have a non-linear relationship with AGM-CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM-CBA), we used pGL3-control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM-CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate-limiting step for pLUC transfection expression. Further optimization of the non-viral gene delivery system can be focused on the improvement of gene intracellular availability.


Assuntos
Núcleo Celular/metabolismo , Genes Reporter/genética , Luciferases/genética , Luciferases/metabolismo , Plasmídeos/genética , Transfecção/métodos , Acrilamidas/química , Agmatina/química , Animais , Camundongos , Células NIH 3T3 , Polietilenoimina/química
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