Enhanced expression of the human ferritin H- and L-chain genes (hfH and hfL) was achieved in Saccharomyces cerevisiae by modifying the N-terminal region of the structural genes. The yeast episomal vector YEp352 with the galactokinase1 (GAL1) promoter was used to construct expression plasmids. The expression of each gene was examined using SDS-PAGE and Western blot analysis. Iron uptake was examined and the cellular iron concentration was increased in S. cerevisiae expressing hfH. When cultured cells were incubated with 14.3 mM Fe(2+), the recombinant yeast expressing hfH had a cellular iron concentration 1.5 times greater than that of the control strain. The relationship between the iron taken up by the cells and the expressed proteins was examined. Iron-binding H-chain ferritin (H-ferritin) was seen in the recombinant S. cerevisiae incubated with iron, while small amounts of iron-binding L-chain ferritin (L-ferritin) were observed. Combined, these observations demonstrate that human H-ferritin has a function in iron storage in S. cerevisiae, while L-ferritin does not.
Recombinant human H-ferritins produced from Saccharomyces cerevisiae were purified and their molecular properties were characterized. Electrophoresis of the recombinant H-ferritins showed revealed bands with mobilities similar to those of the H-ferritins produced by Escherichia coli. The pI of H-ferritins from yeast was more basic than that of H-ferritins produced by E. coli. When the cells were treated with tunicamycin, the pI of H-ferritins was driven to a sharp band with the pI range similar to that of those produced by E. coli, implying that the H-ferritins from yeast are glycosylated. The iron uptake of H-ferritins was rapid in the initial stage, with a slight reduction when compared to ferritins from E. coli. Recombinant ferritins are commonly used during synthesis of inorganic nanoparticles in their cores for chemical and industrial purposes. Transmission electron microscopy revealed that the reconstitution yield and size distribution of the core minerals were affected depending on the protein shells. The Hferritins with higher reconstitution yields (64.4%) showed slightly larger sizes (mean 6.52 nm) with narrower size distribution.
In this study, horse spleen apoferritins were induced to form biominerals using up to 3000 Fe atoms per protein molecule. The morphology and crystallinity of the nanometer-sized biominerals formed in the ferritins were then analyzed using field emission-energy filtering-transmission electron microscopy (FE-TEM). The ferritins were found to have reconstitution yields of 60-70% in the experiments. The mean core size of the ferritins varied somewhat with protein concentrations, indicating that crystal growth in ferritins could be controlled via protein concentrations. The core mineral size increased with the amount of Fe used. Lattice fringes of the core, associated with good crystallinity, were found in all samples. The lattice fringe images of a single domain ferrihydrite mineral appeared frequently in the (011)
Cervi cornu extracts have been used in traditional medicine for the treatment of various disorders, including osteoporosis. However, since it is not easy to separate the active ingredients, limited research has been conducted on their functional properties. In this study, we extracted the low-molecular-weight (843 Da) collagen NP-2007 from cervi cornu by enzyme hydrolyzation to enhance absorption and evaluated the therapeutic effect in monosodium iodoacetate-induced rat osteoarthritis (OA) model. NP-2007 was orally administered at 50, 100, and 200 mg/kg for 21 days. We showed that the production of matrix metalloproteinase-2, -3, and -9, decreased after NP-2007 treatment. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and prostaglandin E2 were also reduced after treatment of NP-2007. Furthermore, the administration of NP-2007 resulted in effective preservation of both the synovial membrane and knee cartilage and significantly decreased the transformation of fibrous tissue. We verified that the treatment of NP-2007 significantly reduced the production of nitric oxide and pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 in lipopolysaccharides-stimulated RAW 264.7 cells by regulation of the NF-kB and MAPK signaling pathways. This study indicates that NP-2007 can alleviate symptoms of osteoarthritis and can be applied as a novel treatment for OA treatment.
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