RESUMEN
The complete natamycin (NTM) biosynthetic gene cluster of Streptomyces chattanoogensis was cloned and confirmed by the disruption of pathway-specific activator genes. Comparative cluster analysis with its counterpart in Streptomyces natalensis revealed different cluster architecture between these two clusters. Compared with the highly conserved coding sequences, sequence variations appear to occur frequently in the intergenic regions. The evolutionary change of nucleotide sequence in the intergenic regions has given rise to different transcriptional organizations in the two clusters and resulted in altered gene regulation. These results provide insight into the evolution of antibiotic biosynthetic gene clusters. In addition, we cloned a pleitropic regulator gene, adpA(ch), in S. chattanoogensis. Using the genetic system that we developed for this strain, adpA(ch) was deleted from the genome of S. chattanoogensis. The ΔadpA(ch) mutant showed a conditionally sparse aerial mycelium formation phenotype and defects in sporulation; it also lost the ability to produce NTM and a diffusible yellow pigment normally produced by S. chattanoogensis. RT-PCR analysis revealed that transcription of adpA(ch) was constitutive in YEME liquid medium. By using rapid amplification of 5' complementary DNA ends, two transcription start sites were identified upstream of the adpA(ch) coding region. Quantitative transcriptional analysis showed that the expression level of the NTM regulatory gene scnRI decreased 20-fold in the ΔadpA(ch) mutant strain, while the transcription of the other activator gene scnRII was not significantly affected. Electrophoretic mobility shift assay (EMSA) showed that AdpA(ch) binds to its own promoter but fails to bind to the promoter region of scnRI, indicating that the control of scnRI by AdpA(ch) is exerted in an indirect way. This work not only provides a platform and a new potential target for increasing the titre of NTM by genetic manipulation, but also advances the understanding of the regulation of NTM biosynthesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Natamicina/biosíntesis , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismo , Transactivadores/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Datos de Secuencia Molecular , Streptomyces/genética , Transactivadores/genéticaRESUMEN
A novel Streptomyces strain, L10, which is capable of producing natamycin, was isolated from a soil sample collected from Zhejiang province, China. On the basis of phylogenetic analysis of rpoB gene and 16S rDNA sequences, as well as phenotypic comparison, strain L10 (CGMCC 2644) is proposed to be a previously uncharacterized strain of S. chattanoogensis. By screening a cosmid library of strain L10 and primer walking, a partial sequence of scnRI and the entire sequence of scnRII were obtained, which are orthologues to the pathway-specific positive regulator genes of natamycin biosynthesis in S. natalensis. The engineered S. chattanoogensis Dl, generated by inserting an additional copy of scnRII into the chromosome of strain L10, increased its natamycin production by 3.3 fold in YSG medium and 4.6 fold in YEME medium without sucrose.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Genes Reguladores , Natamicina/biosíntesis , Streptomyces/aislamiento & purificación , Streptomyces/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , China , Clonación Molecular , ADN Bacteriano/genética , ADN Ribosómico/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Alineación de Secuencia , Microbiología del Suelo , Streptomyces/clasificación , Streptomyces/genéticaRESUMEN
Severe side effects and complications such as gastrointestinal and hematological toxicities because of current anticancer drugs are major problems in the clinical management of gastric cancer, which highlights the urgent need for novel effective and less toxic therapeutic approaches. Hispolon, an active polyphenol compound, is known to possess potent antineoplastic and antiviral properties. In this study, we investigated the efficacy of hispolon in human gastric cancer cells and explored the cell death mechanism. Hispolon induced ROS-mediated apoptosis in gastric cancer cells and was more toxic toward gastric cancer cells than toward normal gastric cells, suggesting greater susceptibility of the malignant cells. The mechanism of hispolon-induced apoptosis was that hispolon abrogated the glutathione antioxidant system and caused massive ROS accumulation in gastric cancer cells. Excessive ROS caused oxidative damage to the mitochondrial membranes and impaired the membrane integrity, leading to cytochrome c release, caspase activation, and apoptosis. Furthermore, hispolon potentiated the cytotoxicity of chemotherapeutic agents used in the clinical management of gastric cancer. These results suggest that hispolon could be useful for the treatment of gastric cancer either as a single agent or in combination with other anticancer agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Catecoles/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Antioxidantes/metabolismo , Catecoles/química , Línea Celular Tumoral , Glutatión/metabolismo , Humanos , Estructura MolecularRESUMEN
A medicinal mushroom Fomes fomentarius, was isolated from the fruiting body of a wild F. fomentarius and identified by ITS-5.8S rDNA sequencing analysis. Then, the optimization of submerged culture conditions and nutritional requirements of mycelial biomass and exopolysaccharide (EPS) production from F. fomentarius was studied using orthogonal matrix method. Under the optimal culture condition, the maximum EPS concentration reached 3.64 g l(-1), which is about four times higher than that at the basal medium. Furthermore, the EPS from F. fomentarius has a direct antiproliferative effect in vitro on SGC-7901 huaman gastric cancer cells in a dose- and time-dependent manner. Moreover, it was about three times that EPS at noncytocxity concentration of 0.25 mg ml(-1) could sensitize doxorubicin(Dox)-induced growth inhibition of SGC-7901 cells after 24h treatment.
Asunto(s)
Antineoplásicos/farmacología , Reactores Biológicos , Polyporales/fisiología , Polisacáridos/biosíntesis , Biomasa , División Celular/efectos de los fármacos , ADN de Hongos/genética , ADN Ribosómico/genética , Humanos , Polyporales/citología , Polisacáridos/farmacología , ARN de Hongos/genética , ARN Ribosómico 5.8S/genética , Neoplasias Gástricas/patologíaRESUMEN
OBJECTIVE: To study eleven organophosphorus insecticides residuals in four kinds of Chinese crude drugs. METHOD: The organophosphorus insecticides were extracted with dichloromethane and cleaned-up with a mixture of Celite 545-activated carbon. The extracts were analyzed by gas chromatography equipped with a flame photometric detector (FPD). RESULT: Analysis of fortified Chinese crude drug showed that the average recoveries ranged from 77.5% -112.3% at three different levels, the RSDs were below 10% (n = 4). Trace organophosphorous pesticide residues were found in samples of Rhizoma Atractylodis Macrocephalae and Flos Chrysanthemi. CONCLUSION: A method was established for determination multi-residues in Rhizma Atractylodis Macrocephalae, Radix Curcumae, Bulbus Fritillariae Thunbergii and Flos Chrysanthemi. It provides a method for the risk assessment of organophosphorous pesticide in Chinese crude drugs.
Asunto(s)
Medicamentos Herbarios Chinos/química , Compuestos Organofosforados/análisis , Residuos de Plaguicidas/análisis , Plantas Medicinales/química , Atractylodes/química , Cromatografía de Gases , Chrysanthemum/química , Curcuma/química , Contaminación de Medicamentos , Fritillaria/químicaRESUMEN
Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor. DNA microarray analysis revealed that ecrA1/A2, which mapped at distant sites from red locus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway. ecrA1 and ecrA2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover. Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain. However, the change of the actinorhodin (Act) production level was not significant compared with wild type. Thus, these experiment results confirmed that the two-component system ecrA1/A2 was positive regulatory element for red gene cluster.