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1.
Arch Virol ; 162(3): 677-686, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27858289

RESUMO

Microtubule (MT) and dynein motor proteins facilitate intracytoplasmic transport of cellular proteins. Various viruses utilize microtubules and dynein for their movement from the cell periphery to the nucleus. The aim of this study was to investigate the intracellular transport of porcine circovirus type 2 (PCV2) via 8 kDa dynein light chain (DYNLL1, LC8) subunit along the MTs. At 20 µM, vinblastine sulfate inhibited tubulin polymerization resulting in disorganized morphology. In PCV2-infected PK-15 cells, double immunofluorescent labeling showed that the viral particles appeared at the cell periphery and gradually moved to the microtubule organization center (MTOC) at 0-12 hour post inoculation (hpi) while at 20-24 hpi they accumulated in the nucleus. Co-localization between DYNLL1 and PCV2 particles was observed clearly at 8-12 hpi. At 20-24 hpi, most aggregated tubulin had a paracrystalline appearance at the MTOC around the nucleus in vinblastine-treated, PCV2-infected PK-15 cells. Between 12 and 24 hpi, PCV2 particles were still bound to DYNLL1 before they were translocated to the nucleus in both treatments, indicating that vinblastine sulfate had no effect on the protein-protein co-localization. The DYNLL1 binding motif, LRLQT, was found near the C-terminus of PCV2 capsid protein (Cap). Molecular docking analysis confirmed the specific interaction between these residues and the cargo binding site on DYNLL1. Our study clearly demonstrated that dynein, in particular DYNLL1, mediated PCV2 intracellular trafficking. The results could explain, at least in part, the viral transport mechanism by DYNLL1 via MT during PCV2 infection.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/metabolismo , Microtúbulos/virologia , Doenças dos Suínos/virologia , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Infecções por Circoviridae/genética , Infecções por Circoviridae/metabolismo , Infecções por Circoviridae/virologia , Circovirus/genética , Dineínas/genética , Dineínas/metabolismo , Interações Hospedeiro-Patógeno , Microtúbulos/metabolismo , Ligação Proteica , Transporte Proteico , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/metabolismo
2.
Biomol Detect Quantif ; 8: 15-28, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27335807

RESUMO

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

3.
Oncol Res ; 17(7): 301-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19408575

RESUMO

Upregulation of the PI3K pathway has often been reported in androgen-independent prostate cancer and is implicated in cancer cell survival and proliferation in the absence of androgen. Inhibition of PI3K by LY294002 suppressed cell invasion and motility of the highly metastatic androgen-independent Dunning rat prostate cancer MLL cell line with similar IC50 values and inhibition profile. Moreover, LY294002 attenuated expression of urokinase plasminogen activator (uPA) without any significant effect on that of matrix metalloproteinase 2. These results indicated that the role of PI3K in MLL cell invasion is by regulating cell motility and uPA expression.


Assuntos
Androgênios/farmacologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Morfolinas/farmacologia , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/secundário , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Próstata/tratamento farmacológico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Animais , Movimento Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
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