Eock-Kee Hong

Study of Macrophage Activation and Structural Characteristics of Purified Polysaccharides from the Fruiting Body of Hericium erinaceus

Most, if not all, Basidiomycetes mushrooms have biologically active polysaccharides showing potent antitumor activity with immunomodulating properties. These polysaccharides have various chemical compositions and belong primarily to the beta-glucan group. In this study, the crude water-soluble polysaccharide HEF-P, which was obtained from the fruiting body of Hericium erinaceus by hot water extraction and ethanol precipitation, was fractionated by DEAE-cellulose and Sepharose CL-6B column chromatographies. This process resulted in four polysaccharide fractions, named HEF-NP Fr I, HEF-NP Fr II, HEF-AP Fr I, and HEF-AP Fr II. Of these fractions, HEF-AP Fr II was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide and expression of cytokines (IL-1beta and TNF-beta). The molecular mass of HEF-AP Fr II was estimated by gel filtration to be 13 kDa. Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicate that HEF-AP Fr II is a low-molecular-mass polysaccharide with a laminarin-like triple helix conformation of a beta-1,3-branched-beta-1,6-glucan.

Characterization of the nar promoter to use as an inducible promoter

SummaryThe nar promoter of Escherichia coli was characterized, which is maximally induced under anaerobic conditions in the presence of nitrate. The following results were obtained; Expression of β-galactosidase was optimal at 1 % of nitrate and was not affected much by molybdate; the amount of β -galactosidase per unit volume was maximal when the nar promoter was induced at OD600 = 1.7, and when anaerobic condition was made by supplying nitrogen gas. At the optimal condition, the ratio of β-galactosidase between before and after induction was approximately 250 and Miller units were approximately 7,500. The results showed that the nar promoter can be used as an inducible promoter.

Gene expression usingnar promoter under anaerobic condition with recombinantE. coli

Thenar promoter as an inducible promoter was characterized for the process development for the gene expression and the protein production under anaerobic condition. The LB medium was selected as a main culture medium showing the enzyme activity of 18,000 units/min/g cell in the flask cultivation. The optimum concentration of nitrate was 1%. Under anaerobic conditions, the gene expression was fully induced in the presence of nitrate.

Fabrication of Cytochrome c Multi-Layers by Schaefer Technique

Abstract To spread cytochrome c, the water soluble protein, on subphase homogeneously, cytochrome c buffer solution was mixed with ethanol diluted with a deionized distilled water (DDW). After spreading it, cytochrome c multi-layers were deposited onto ITO coated glass by Schaefer technique (the horizontal lifting technique), and Langmuir-Blodgett (LB) technique (vertical lifting technique). Cytochrome c multi-layers deposited onto the substrate by Schaefer technique and LB technique were analyzed by UV-visible spectroscopy, and multi-layers of cytochrome c fabricated using the Schaefer technique was analyzed by Atomic Force Microscopy (AFM).