Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 2 de 2
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
APMIS ; 118(1): 45-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20041870

RESUMO

Chlamydia pneumoniae is an intracellular gram-negative bacterium, which replicates only in eukaryotic cells. Quantification of C. pneumoniae in cell culture is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally, this has been performed by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary, and multiple inclusions are also seen. Therefore, methods are needed to quantify exact amounts of C. pneumoniae in cells. Here, we describe a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes in cultures. In human epithelial (HL) cell cultures, the C. pneumoniae inclusion numbers and the ratio of C. pneumonia genomes/human genome (Cpn/Hum) correlated significantly (r = 0.978, p < 0.001); thus with HL cells, both methods are usable. However, in macrophage cultures, the correlation was weaker (r = 0.133, p = 0.036) and we recommend PCR quantification for exact measurements.


Assuntos
Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/isolamento & purificação , Macrófagos/microbiologia , Infecções por Chlamydophila/diagnóstico , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/imunologia , DNA Bacteriano/química , DNA Bacteriano/genética , Ensaio de Imunoadsorção Enzimática , Células Epiteliais , Genoma Bacteriano , Humanos , Lipopolissacarídeos/análise , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Estatísticas não Paramétricas
2.
Biochemistry ; 41(42): 12729-38, 2002 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-12379115

RESUMO

Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.


Assuntos
Epitopos/química , Epitopos/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/fisiologia , Proteínas/química , Proteínas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos/genética , Linhagem Celular , Epitopos/genética , Humanos , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Biblioteca de Peptídeos , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/imunologia , Proteínas Proto-Oncogênicas c-crk , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Ratos , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de src/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...