ABSTRACT
Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase beta-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.
Subject(s)
Bacterial Proteins/genetics , Bacterial Typing Techniques , DNA-Directed RNA Polymerases/genetics , Fusobacterium nucleatum/classification , Fusobacterium nucleatum/genetics , Metalloendopeptidases/genetics , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Fusobacterium Infections/microbiology , Genotype , Humans , Molecular Sequence Data , Phylogeny , Polymorphism, Genetic , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
We carried out in vitro and in vivo assays to investigate the immunomodulatory and immunochemotherapeutic action mechanism of BRD-glucan, a high molecular weight ( approximately 3,500 kDa) polysaccharide isolated from Aureobasidium sp, and assessed the efficacy of BRD-glucan/adriamycin co-treatment of animal cancer models. RT-PCR and suspension hemolytic, plaque forming, wounding, invasion and cell proliferation assays were utilized to investigate the in vitro immunochemotherapeutic effects of BRD-glucan. In vivo, the effects of BRD-glucan and BRD-glucan/adriamycin co-treatment were tested in a B16 melanoma initiation model and in C57BL/6 mice. In vitro, BRD-glucan did not affect the cellular wounding response or invasion activity; treatment with BRD-glucan led to increase proliferation of B cells, natural killer cells and macrophages, but not T cells. In addition, we found that the BRD-glucan activation of B cells and macrophages was dependent on Toll-like receptor2 (TLR2) and TLR4, which play important roles in innate and adaptive immunity. In vivo, BRD-glucan/adriamycin co-treatment effectively reduced the number and size of metastatic colonies. Based on the results of our in vitro and in vivo toxicity, safety and immunochemotherapy assays, we propose that BRD-glucan is a promising immunochemotherapeutic anti-tumor agent.
Subject(s)
Combined Modality Therapy/methods , Glucans/therapeutic use , Immunotherapy/methods , Polysaccharides/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Ascomycota/metabolism , B-Lymphocytes/drug effects , Cell Line, Tumor , Cell Proliferation , Chromatography, High Pressure Liquid , Cytokines/metabolism , DNA Primers/chemistry , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Humans , Lung/pathology , Macrophages/metabolism , Magnetic Resonance Spectroscopy , Melanoma, Experimental , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Nude , Neoplasm Metastasis , Receptors, Cell Surface/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/drug effects , Time Factors , Toll-Like Receptor 2 , Toll-Like Receptor 4 , Toll-Like Receptors , Wound HealingABSTRACT
Streptomyces sp. Y-110, isolated from soil, modified compactin to pravastatin, a therapeutic agent for hypercholesterolemia. In a batch culture, the highest production of pravastatin was 340 mg l(-1) from 750 mg compactin l(-1) in 24 h. By intermittent feeding of compactin into the culture medium, both the compactin concentration and its conversion increased to 2000 mg l(-1) and 1000 mg pravastatin l(-1), respectively, with the conversion rate of 10 mg l(-1) h(-1). Continuous feeding of compactin increased production of pravastatin to 15 mg l(-1) h(-1).