PURPOSE: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. MATERIALS AND METHODS: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. RESULTS: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. CONCLUSION: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.
Acetonitriles ; Alkaline Phosphatase ; Blotting, Western ; Bone Morphogenetic Protein 2 ; Chromatography, Affinity ; Chromatography, High Pressure Liquid ; Durapatite ; Electrophoresis, Polyacrylamide Gel ; Genetic Engineering ; Heparin ; Humans ; Molecular Weight ; Osteoblasts ; Recombinant Proteins ; Sprains and Strains ; Transforming Growth Factor beta

Objectives: This study aimed to test the effect of photodynamic therapy (PDT) on the inhibition and removal of biofilms containing multidrug-resistant Acinetobacter baumannii. Methods: Using multidrug-resistant A. baumannii strains, an antibiotic susceptibility test was performed using the Gram-negative identification card of the Vitek 2 system (bioMérieux Inc., France), as well as an analysis of resistance genes, the effects of treatment with a light-emitting diode (LED) array using Radachlorin (RADA-PHARMA Co., Ltd., Russia), and transmission and scanning electron microscopy to confirm the biofilm-inhibitory effect of PDT. Results: The antibiotic susceptibility test revealed multiple resistance to the antibiotics imipenem and meropenem in the carbapenem class. A class-D–type β-lactamase was found, and OXA-23 and OXA-51 were found in 100% of 15 A. baumannii strains. After PDT using Radachlorin, morphological observations revealed an abnormal structure due to the loss of the cell membrane and extensive morphological changes, including low intracellular visibility and small vacuoles attached to the cell membrane. Conclusion PDT involving a combination of LED and Radachlorin significantly eliminated the biofilm of multidrug-resistant A. baumannii. Observations made using electron microscopy showed that PDT combining LED and Radachlorin was effective. Additional studies on the effective elimination of biofilms containing multidrug-resistant bacteria are necessary, and we hope that a treatment method superior to sterilization with antibiotics will be developed in the future.

In order to investigate the occurrence of norovirus in rivers and beaches, a total of 81 samples were tested at seven sites of Oncheon stream, Suyeong river and Gwanganri beach in Busan from January to November, 2017. To improve the detection of norovirus from sea water, we applied the inorganic cation-coated filter method which showed 48.8% ± 12.2% (n=3) and 27.4% ± 6.0% (n=3) recovery yields from river water and sea water inoculated with Norovirus, respectively. Norovirus was detected in a total of four samples (4.9%), which all were GII genotype. Norovirus GII was detected in three samples at two waste water treatment plants (WWTP) outlet and one sample at about 500 meter downstream from WWTP in both the winter and spring seasons. We also monitored fecal indicator organisms, Escherichia coli (E. coli), Enterococcus and coliphages [somatic coliphages (SC), male-specific coliphages (MSC)] to analyze the potential transmission of enteritis causative agent in dry and wet days. Bacterial influences were found at the site of the WWTP effluents in the dry days and spread further to the costal beach in the wet days. But no viral influences were found in the river downstream in both dry and wet days.
Busan* ; Coliphages ; Enteritis ; Enterococcus ; Escherichia coli ; Genotype ; Korea* ; Methods ; Norovirus* ; Rivers* ; Seasons ; Seawater ; Waste Water ; Water