RESUMO
We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10(3) CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.
Assuntos
Bactérias/genética , Colorimetria , Análise de Sequência com Séries de Oligonucleotídeos , RNA Ribossômico 16S/análise , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Animais , Bactérias/isolamento & purificação , Primers do DNA/metabolismo , Sondas de DNA/metabolismo , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Genoma Bacteriano , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Estreptavidina/química , Sais de Tetrazólio/químicaRESUMO
BACKGROUND: Many cancerous cells accumulate beta-catenin in the nucleus. We examined the role of epidermal growth factor receptor (EGFR) signaling in the accumulation of beta-catenin in the nuclei of oral cancer cells. RESULTS: We used two strains of cultured oral cancer cells, one with reduced EGFR expression (OECM1 cells) and one with elevated EGFR expression (SAS cells), and measured downstream effects, such as phosphorylation of beta-catenin and GSK-3beta, association of beta-catenin with E-cadherin, and target gene regulation. We also studied the expression of EGFR, beta-catenin, and cyclin D1 in 112 samples of oral cancer by immunostaining. Activation of EGFR signaling increased the amount of beta-catenin in the nucleus and decreased the amount in the membranes. EGF treatment increased phosphorylation of beta-catenin (tyrosine) and GSK-3beta(Ser-(9), resulting in a loss of beta-catenin association with E-cadherin. TOP-FLASH and FOP-FLASH reporter assays demonstrated that the EGFR signal regulates beta-catenin transcriptional activity and mediates cyclin D1 expression. Chromatin immunoprecipitation experiments indicated that the EGFR signal affects chromatin architecture at the regulatory element of cyclin D1, and that the CBP, HDAC1, and Suv39h1 histone/chromatin remodeling complex is involved in this process. Immunostaining showed a significant association between EGFR expression and aberrant accumulation of beta-catenin in oral cancer. CONCLUSIONS: EGFR signaling regulates beta-catenin localization and stability, target gene expression, and tumor progression in oral cancer. Moreover, our data suggest that aberrant accumulation of beta-catenin under EGFR activation is a malignancy marker of oral cancer.
Assuntos
Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/enzimologia , Neoplasias Bucais/genética , Transcrição Gênica , beta Catenina/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Imunoprecipitação da Cromatina , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Bucais/patologia , Mutação/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Frações Subcelulares/metabolismo , beta Catenina/genéticaRESUMO
BACKGROUND: Cytoplasmic and nuclear accumulation of beta-catenin in mucoepidermoid carcinoma (MEC) is frequently noted, but the mechanism is unknown. METHODS: The methylation status of adenomatous polyposis coli (APC) and secreted frizzled-related proteins (SFRPs) was examined by methylation-specific polymerase chain reaction (MSP) assay. The association of SFRP1, beta-catenin, and cyclin D1 expression in MEC was evaluated by immunohistochemical staining. RESULTS: A high percentage of methylation in APC and the SFRP genes was found in MEC compared with adjacent normal tissues, in which SFRP1 (58.6%) was the most frequent methylated gene. Moreover, abundant expression of SFRP1 was noted in normal tissues, whereas reduced SFRP1 expression was detected in 71.7% (33/46) of MECs. There was significant association between methylation and reduced expression of SFRP1. Cytoplasmic/nuclear (C/N) beta-catenin and high cyclin D1 expression were found in 13/55 (23.6%) and 36/55 (65.5%) of cases, respectively. There was significant correlation between C/N beta-catenin expression and reduced SFRP1 expression (P = 0.009). In addition, SFRP1 and beta-catenin expression correlated with tumor malignancy index such as tumor grade and stage. Overall patient survival was significantly worse in patients with reduced SFRP1 and C/N beta-catenin expression (P = 0.009 and P = 0.002, respectively). CONCLUSIONS: Methylation of the SFRP1 gene was the major cause of reduced SFRP1 expression. Reduced SFRP1 led to C/N accumulation of beta-catenin and was associated with tumor malignancy. Therefore, examination of SFRP1 expression and beta-catenin location could be useful predictors of tumor progression and prognosis in patients with MEC.