RESUMEN
Based on genome mining, a new antibacterial peptide named actinokineosin was isolated from a rare actinomycete Actinokineospora spheciospongiae. The amino acid sequence of the C-terminus of actinokineosin was established by TOF-MS/MS experiments. The amino acid sequence in the macrolactam ring was determined by TOF-MS/MS analyses after cleavage with BNPS-skatole and successive trypsin treatment. As a result of an antibacterial assay using a paper disk, actinokineosin showed antibacterial activity against Micrococcus luteus at a dosage of 50 µg per disk. From the genome sequence data of A. spheciospongiae, the biosynthetic gene cluster of actinokineosin was found and was indicated to consist of 10 genes. Among the genes, the gene aknA encoded the precursor of actinokineosin and the genes including aknC, aknB1 and aknB2 were proposed as modification enzymes to give mature actinokineosin. SIGNIFICANCE AND IMPACT OF THE STUDY: Genome mining is a powerful tool to find new bioactive compounds from the genome database. In this report, we succeeded in isolation and structure determination of a new antibacterial peptide named actinokineosin based on genome mining.
Asunto(s)
Actinobacteria/química , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Micrococcus luteus/efectos de los fármacos , Actinobacteria/genética , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Secuencia de Bases , Genoma Bacteriano , Pruebas de Sensibilidad Microbiana , Familia de Multigenes , Escatol/análogos & derivados , Escatol/química , Espectrometría de Masas en Tándem , Tripsina/químicaAsunto(s)
Células Epiteliales/patología , Células Epiteliales/virología , Herpesvirus Humano 4/fisiología , Neoplasias Gástricas/patología , Neoplasias Gástricas/virología , Animales , División Celular , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Ratones , Ratones SCID , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Neoplasias Gástricas/genética , Activación Viral , Latencia del Virus/genéticaRESUMEN
An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.
Asunto(s)
ADN Viral/genética , ADN Viral/aislamiento & purificación , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Neoplasias Gástricas/virología , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN/genética , Enzimas de Restricción del ADN , Desoxirribonucleasa BamHI , Desoxirribonucleasa EcoRI , Infecciones por Virus de Epstein-Barr/complicaciones , Estudios de Evaluación como Asunto , Globinas/genética , Herpesvirus Humano 4/patogenicidad , Humanos , Albúmina Sérica Bovina , Neoplasias Gástricas/etiología , Virología/métodosRESUMEN
We characterized the expression of Epstein-Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF 1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA 1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or n-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cells lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with a Xhol probe in both cell lines. These epithelial cells lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.
