• Toxicological Research
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• v.12 no.1
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• pp.21-27
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• 1996

Vitamin C has been well known to be a potential chemopreventive agent for several toxic compounds. It reduced the mutation frequencies of 6-sulfooxymethylbenzo[a]pyrene (SMBP) and 6hydroxymethylbenzo[a]pyrene (HMBP) in Salmonella typhimurium TA98 and TA100, indicating that corbic acid affects both frameshift and base-pair substitution mtltations. A similar type of dose-response relationship was shown in the V79 cells, although the inhibitory effect was less dramatic compared with that in S. typhimurium. However, SMBP or HMBP binding to calf thymus DNA was not affected by the presence of vitamin C, suggesting that SMBP seems to be much more reactive to calf thymus DNA than vitamin C. This was supported by migration pattern and fluorescence intensity of SMBP-modified plasmid on the gel. These restilts were not correlated with mutation tests in bacterial and mammalian cell systems. It has been already reported that vitamin C inactivates SMBP through the formation of covalently bound addact. It was found from HPLC analysis that the reaction between vitamin C and SMBP was accomplished within just 5 min and then produced the several products. These findings indicate that the beneficiary of vitamin C is not merely derived from the covalent adducts. On the other hand, the addition of DNA to incubation mixture reduced the amounts of vitamin C adducts while the magnitude of HMBP peak increased, suggesting that DNA accelerates the SMBP hydrolysis to intercept the interaction between SMBP and vitamin C or forms rapidly complex with SMBP.

• Mass Spectrometry Letters
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• v.12 no.4
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• pp.126-130
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• 2021

The mass spectrometry (MS) provides the mass-to-charge ratios of atoms, molecules, stable/metastable complexes, and their fragments. I have taken a long journey with MS to address outstanding issues and problems by experiments and theory and gain insights into underlying principles in chemistry. By looking through the mass-to-charge ratio, I have studied thermochemical problems in silicon chemistry, the infrared multiphoton dissociation spectroscopy of organometallic intermediates, unimolecular dissociations of halotoluene radical cations, and the kinetics of association/dissociation of alkali halide triple ions with Lewis bases. Various MS platforms have been used to characterize non-covalent interactions between porphyrins and fullerenes and those between the group IIB ions and trioctylchalcogenides, and to examine the binding of the group IA, IIA and porphyrin ions to G-quadruplex DNA. Recently, I have focused on mass-balanced H/D isotope dipeptide tags for MS-based quantitative proteomics, a simple chemical modification method for MS-based lipase assay, and the kinetics and dynamics of energy-variable collision-induced dissociation of chemically modified peptides. Now, I see an important role of MS in global issues in the post-COVID era, as the society demands high standards for indoor air quality to contain the airborne-pathogen transmission as well as in-situ monitoring and tracking of carbon emissions to reduce global warming.

• Proceedings of the Korean Institute of Building Construction Conference
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• 2022.11a
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• pp.73-74
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• 2022

The corrosion inhibitive effect of a new hydrazone-based heterocyclic compound for steel in simulated concrete pore solution with 3.5 wt.% sodium chloride was investigated by experimental and computational techniques. Electrochemical studies, up to 30 days of immersion, and surface analysis (X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and scanning electron microscope (SEM)) were performed to assess the corrosion protection abilities of investigated compound for steel rebar. Results showed that adding the organic compound to the chloride contaminated concrete pore solution decreased the corrosion rate of the steel rebar thanks to the effective adsorption of inhibitor molecules. After 30 days of immersion of steel rebar in inhibited chloride contaminated synthetic concrete pore solution, the inhibition efficiency exceeded 80% at low concentration of 1 mmol/L. Computational studies by Density Functional based Tight Binding (DFTB) method revealed the formation of covalent bonds between the hydrazone molecule and the iron surface.

• Proceedings of the Korean Institute of Building Construction Conference
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• 2023.05a
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• pp.255-256
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• 2023

The development of corrosion inhibitors with outstanding performance is a never-ending and complex process engaged in by researchers, engineers and practitioners. Computational assessment of organic corrosion inhibitors performance is a crucial step towards the design of new task-pecific materials. Herein, electronic features, adsorption characteristics and bonding mechanisms of two pyridine oximes, namely 2-pyridylaldoxime (2POH) and 3-pyridylaldoxime (3POH) with the iron surface were investigated using molecular dynamics (MD), and self-consistent-charge density-unctional tight-binding (SCC-DFTB) simulations. SCC-DFTB simulations revealed that 3POH molecule can form covalent bonds with iron atoms in its neutral and protonated states, while 2POH molecule can only bond with iron through its protonated form, resulting in interaction energies of -2.534, -2.007, -1.897, and -0.007 eV for 3POH, 3POH+, 2POH+, and 2POH, respectively. Projected density of states (PDOSs) analysis of pyridines-Fe(110) interactions indicated that pyridine molecules chemically adsorbed on the iron surface.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.10-17
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• 2016

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

• Journal of Life Science
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• v.25 no.6
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• pp.631-637
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• 2015

Streptavidin, a protein produced by Streptomyces avidinii, strongly binds up to four molecules of vitamin H, d-biotin exhibiting the dissociation constant of about 10⁻¹⁵ M. This strong binding affinity has been applied for detection and characterization of numerous biological molecules suggesting expression and purification of functional streptavidin should be very useful for the application of this streptavidin-biotin interaction. To express a soluble streptavidin in Escherichia coli, We synthesized streptavidin genes and cloned into pET-22b plasmid, which uses T7 RNA polymerase/T7 promoter expression systems containing pelB leader for secretion into periplasmic space and six polyhistidine tags at C-terminus for purification of expressed proteins. Although streptavidin is toxic to Escherichia coli due to strong biotin binding property, streptavidin was expressed very sufficiently in a range of 10-20 mg/ml. In SDS-PAGE, the size of purified protein was shown as 17 kDa in denatured condition (boiling) and 68 kDa in native condition (without boiling) suggesting tetramerization of monomeric subunit by non-covalent association. Further analysis by size-exclusion chromatography supported streptavidin’s tetrameric structure as well. In addition, soluble streptavidin detected biotinylated proteins in westernblot indicating its functional activity to biotin. Taken these results together, it concluded that our simple expression system was able to show high yield, homotetrameric formation and biotin binding activity analogous to natural streptavidin.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.368-376
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• 2003

Cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. JB-13 was immobilized on various carriers by several immobilization methods such as ionic binding, covalent linkage and ultrafiltration to improve the process performance. The ultrafiltration and covalent linkage with CNBr-activated sepharose 4B were found as the best method for immobilization of CGTase. The ability of CGTase immobilization onto CNBr-activated sepharose 4B was as high as 18,000 units/g resin when the conditions was as follows: contact time 9 hrs at $37^{\circ}C$, pH 6.0, 100 nm and enzyme loading 24,000 units/g resin. The optimum conditions for production of 2-O-$\alpha$-D-Glucopyranosyl L-Ascorbic acid by immobilized CGTase turned out to be: pH 5.0, temperature $37^{\circ}C$, 20% substrate solution containing 8% (w/v) of soluble starch and 12% (w/v) of L-ascorbic acid sodium salt, 100 rpm, far 25 hrs and with 800 units of immobilized CGTase/ml substrate solution. Moreover the CGTase activity could be stably maintained for 8 times of repetitive reactions after removing products by ultrafiltration through YM 10 membrane.

• Journal of Life Science
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• v.28 no.12
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• pp.1416-1423
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• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

• Journal of Pharmaceutical Investigation
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• v.40 no.3
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• pp.139-153
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• 2010

Nitrosative stress is defined as pathophysiological conditions that are related to covalent modifications of proteins by nitration/nitrosylation by forms of nitrogen oxide ($NO_x$), leading to DNA damage, ultimately, cell death. This type of stress condition appears to be associated with a number of disease states, including diabetes, inflammation and neurodegenerative diseases. Since these pathological conditions are frequently chronic in nature and, thus, require long-term treatment, changes in pharmacokinetics are likely to affect the therapy. Transporters are membrane proteins that facilitate the movement of substrates, including drugs, across plasma membranes of epithelial / endothelial cells. Since it is now increasingly evident that transporters are pharmacokinetically significant, functional alteration of transporters by this stress condition may have therapeutic relevance. In this review, experimental techniques that are used to study both in vivo and in vitro nitrosative stress are summarized and discussed, along with available literature information on the functional implication of transporters under conditions of nitrosative stress conditions. In the literature, both functional induction and impa irment were apparently present for both drug transporter families [i.e., ATP-binding cassette (ABC) and solute carrier families (SLC)]. Furthermore, a change in the function of a certain transporter appears to have temporal dependency by impairment in the early phase of nitrosative stress and induction thereafter, suggesting that the role of nitrosative stress is complex in terms of functional implications of the transporters. Although the underlying mechanisms for these alterations are not fully understood, protein nitration/nitrosylation appears to be involved in the functional impairment whereas transcript factor(s) activated by nitrosative stress may play a role, at least in part, in functional induction. Interestingly, functional induction under conditions of nitrosative stress has not been observed for SLC transporters while such impairment has been documented for both ABC and SLC transporters. Further investigations appear to be necessary to fully delineate the underlying reasons for these differences on the impact and importance of nitrosative stress conditions.

• Preventive Nutrition and Food Science
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• v.5 no.4
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• pp.200-204
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• 2000

Protein conjugation of curdlan belonging to $\beta$-1, 3-glucan was carried out to improve it surface functionalities. The glucan was mixed with phosvitin, {TEX}$$\alpha$_{s}${/TEX}-casein, lysozyme or ovalbumin, respectively. The mixture was freeze-dried, and he resulting powder was incubated at 6$0^{\circ}C$ and 79% relative humidity for 12 days in order to generate a controlled Maillard reaction between curdlan and proteins. conjugation with unfolding proteins, i.e., phosvitin and {TEX}$$\alpha$_{s}${/TEX}-casein, drastically increased the solubility of the glucan, whereas lysozyme and ovalbmin did not. The solubility in water of curdlan was 3.44% for the phosvitin conjugate and 1.09% for the {TEX}$$\alpha$_{s}${/TEX}-casein conjugate. SDS-slab polyacrylamide gel electrophoresis showed that curdlan was solubilized due to covalent binding with phosvitin. Emulsifying properties of curdlan were substantially improved by the conjugation with phosvitin and {TEX}$$\alpha$_{s}${/TEX}-casein. Emulsion stability of the curdlan-phosvitin conjugate was about 2.9 times greater than that of the curdlan-phosvitin mixture.