ABSTRACT
It is necessary to develop new methods for the isolation of unknown actinomycetes from soils. To evaluate the effects of oligotrophic medium on the isolation of soil actinomycetes and develop a new isolation method, the Gause's synthetic medium was diluted to one tenth the recommended concentration in the present study. Soil dilution plate technique was used to isolate actinomycetes from the soil samples. Oligotrophy decreased actinomycete and streptomycete counts, as well as the number of antagonistic actinomycete species. Oligotrophy also decreased the number of actinomycete species in five samples. Some actinomycete species were cultured only on the oligotrophic medium, whereas other species could not be cultured. Oligotrophy decreased actinomycete counts more significantly for soils with organic matter content >40 g/kg. We used 16S rRNA sequence analysis to identify 22 actinomycete species that were only cultured on the oligotrophic medium. Oligotrophic medium was helpful for the isolation of Streptomyces spp., Micromonospora spp. and Streptosporangium spp. Slightly more than 80 % of the identified actinomycete species were biologically active. Therefore, we could draw a conclusion that oligotrophic medium could be helpful for the discovery of new antibiotic producers and the exploitation and utilization of new, biologically active compounds.
ABSTRACT
OBJECTIVE: To identify antithrombin III (AT-III) gene mutation and polymorphisms in pregnant women and parturients with cerebral venous thrombosis (CVT) using denaturing high-performance liquid chromatography (DHPLC). METHODS: The genomic DNA was extracted from the blood samples of 50 pregnant women and parturients with CVT and 52 matched healthy women for molecular analysis using a PCR/DHPLC assay followed by DNA sequence analysis. Ten primer pairs were designed for amplifying the AT- III promoter region and exons 1-6 including the exon/intron boundaries. A rapid screening assay based on DHPLC was established to screen the mutation and polymorphisms of AT- III gene. RESULTS: Six abnormal peaks were detected in 40 of the patients by DHPLC. Direct DNA sequencing was performed on representative samples detected by DHPLC profiling. One pathogenic heterozygous G13328A missense mutation in exon 6, and a novel silent mutation in exon 4+243 G>A were identified. Six single nucleotide polymorphism (SNP) sites were found, including 4 previously reported ones in the SNP library and two were novel SNP sites. An abnormal peak was detected in the control group by DHPLC. CONCLUSION: DHPLC allows automated and rapid high-throughput detection of AT- III gene mutation and polymorphisms in the clinical setting and prenatal diagnosis. Our findings suggested that AT- III gene mutation, as well as its polymorphisms, contributes to the occurrence of CVT in pregnant women and parturients.
