• KSBB Journal
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• v.21 no.2
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• pp.133-139
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• 2006

In 'solid-phase' PEGylation, the conjugation reaction occurs as the proteins are attached to a solid matrix, and thus it can have distinct advantages over the conventional, solution-phase process. We report a case study: rhIFN-${\alpha}$-2a was first adsorbed to cation exchange resin and then N-terminally PEGylated by aldehyde mPEG of 5, 10, and 20 kD through reductive alkylation. After the PEGylation, salt gradient elution efficiently recovered the mono-PEGylate in a purified form from the unwanted species such as unmodified IFN, unreacted PEG, and others. The mono-PEGylation and its purification were integrated in a single chromatographic step. Depending on the molecular weight of the mPEG aldehyde used, the mono-PEGylation yield ranged 50-64%. We could overcome the major problems of random, or uncontrollable, multi-PEGylation and the post-PEGylation purification difficulties associated with the solution-phase process. N-terminal sequencing and MALDI-TOF MS confirmed that a PEG molecule was conjugated only to the N-terminus. Compared with the unmodified IFN, the mono-PEGylate showed the reduced anti-viral activity as measured by the cell proliferation assay. The bioactivity was reduced more as the higher molecular weight PEG was conjugated. Immunoreactivity, evaluated indirectly by antibody binding activity using a surface plasmon resonance biosensor, also decreased. Nevertheless, trypsin resistance as well as thermal stability was considerably improved.

• Food Engineering Progress
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• v.14 no.1
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• pp.49-53
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• 2010

Surface plasmon resonance (SPR) which is utilized in thin film refractometry-based sensors has been concerned on measurement of physical, chemical and biological quantities because of its high sensitivity and label-free feature. In this paper, an application of SPR to detection of alcohol content in wine and liquor was investigated. The result showed that SPR sensor had high potential to evaluate alcohol content. Nevertheless, food industry may need SPR sensor with higher sensitivity. Herein, we introduced a nano-technique into fabrication of SPR chip to enhance SPR sensitivity. Using Langmuir-Blodgett (LB) method, gold film with nano-structured surface was devised. In order to make a new SPR chip, firstly, a single layer of nano-scaled silica particles adhered to plain surface of gold film. Thereafter, gold was deposited on the template by an e-beam evaporator. Finally, the nano-structured surface with basin-like shape was obtained after removing the silica particles by sonication. In this study, two types of silica particles, or 130 nm and 300 nm, were used as template beads and sensitivity of the new SPR chip was tested with ethanol solution, respectively. Applying the new developed SPR sensor to a model food of alcoholic beverage, the sensitivity showed improvement of 95% over the conventional one.

• Journal of Gastric Cancer
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• v.21 no.2
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• pp.191-202
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• 2021

Purpose: A near-infrared (NIR) fluorescence imaging is a promising tool for cancer-specific image guided surgery. Human epidermal receptor 2 (HER2) is one of the candidate markers for gastric cancer. In this study, we aimed to synthesize HER2-specific NIR fluorescence probes and evaluate their applicability in cancer-specific image-guided surgeries using an animal model. Materials and Methods: An NIR dye emitting light at 800 nm (IRDye800CW; Li-COR) was conjugated to trastuzumab and an HER2-specific affibody using a click mechanism. HER2 affinity was assessed using surface plasmon resonance. Gastric cancer cell lines (NCI-N87 and SNU-601) were subcutaneously implanted into female BALB/c nu (6-8 weeks old) mice. After intravenous injection of the probes, biodistribution and fluorescence signal intensity were measured using Lumina II (Perkin Elmer) and a laparoscopic NIR camera (InTheSmart). Results: Trastuzumab-IRDye800CW exhibited high affinity for HER2 (K_D=2.093(3) pM). Fluorescence signals in the liver and spleen were the highest at 24 hours post injection, while the signal in HER2-positive tumor cells increased until 72 hours, as assessed using the Lumina II system. The signal corresponding to the tumor was visually identified and clearly differentiated from the liver after 72 hours using a laparoscopic NIR camera. Affibody-IRDye800CW also exhibited high affinity for HER2 (K_D=4.71 nM); however, the signal was not identified in the tumor, probably owing to rapid renal clearance. Conclusions: Trastuzumab-IRDye800CW may be used as a potential NIR probe that can be injected 2-3 days before surgery to obtain high HER2-specific signal and contrast. Affibody-based NIR probes may require modifications to enhance mobilization to the tumor site.

• Journal of Ginseng Research
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• v.46 no.1
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• pp.156-166
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• 2022

Background: Panax ginseng Meyer (P. ginseng), a herb distributed in Korea, China and Japan, exerts benefits on diverse inflammatory conditions. However, the underlying mechanism and active ingredients remains largely unclear. Herein, we aimed to explore the active ingredients of P. ginseng against inflammation and elucidate underlying mechanisms. Methods: Inflammation model was constructed by lipopolysaccharide (LPS) in C57BL/6 mice and RAW264.7 macrophages. Molecular docking, molecular dynamics, surface plasmon resonance imaging (SPRi) and immunofluorescence were utilized to predict active component. Results: P. ginseng significantly inhibited LPS-induced lung injury and the expression of proinflammatory factors, including TNF-α, IL-6 and IL-1β. Additionally, P. ginseng blocked fluorescencelabeled LPS (LPS488) binding to the membranes of RAW264.7 macrophages, the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Furthermore, molecular docking demonstrated that ginsenoside Ro (GRo) docked into the LPS binding site of toll like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex. Molecular dynamic simulations showed that the MD2-GRo binding conformation was stable. SPRi demonstrated an excellent interaction between TLR4/ MD2 complex and GRo (K_D value of 1.16 × 10^-9 M). GRo significantly inhibited LPS488 binding to cell membranes. Further studies showed that GRo markedly suppressed LPS-triggered lung injury, the transcription and secretion levels of TNF-α, IL-6 and IL-1β. Moreover, the phosphorylation of NF-κB and MAPKs as well as the p65 subunit nuclear translocation were inhibited by GRo dose-dependently. Conclusion: Our results suggest that GRo exerts anti-inflammation actions by direct inhibition of TLR4 signaling pathway.

• Journal of Ginseng Research
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• v.47 no.6
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• pp.743-754
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• 2023

Background: Myocardial fibrosis post-myocardial infarction (MI) can induce maladaptive cardiac remodeling as well as heart failure. Although 20(S)-ginsenoside Rg3 (Rg3) has been applied to cardiovascular diseases, its efficacy and specific molecular mechanism in myocardial fibrosis are largely unknown. Herein, we aimed to explore whether TGFBR1 signaling was involved in Rg3's anti-fibrotic effect post-MI. Methods: Left anterior descending (LAD) coronary artery ligation-induced MI mice and TGF-β1-stimulated primary cardiac fibroblasts (CFs) were adopted. Echocardiography, hematoxlin-eosin and Masson staining, Western-blot and immunohistochemistry, CCK8 and Edu were used to study the effects of Rg3 on myocardial fibrosis and TGFBR1 signaling. The combination mechanism of Rg3 and TGFBR1 was explored by surface plasmon resonance imaging (SPRi). Moreover, myocardial Tgfbr1-deficient mice and TGFBR1 adenovirus were adopted to confirm the pharmacological mechanism of Rg3. Results: In vivo experiments, Rg3 ameliorated myocardial fibrosis and hypertrophy and enhanced cardiac function. Rg3-TGFBR1 had the 1.78×10^-7 M equilibrium dissociation constant based on SPRi analysis, and Rg3 inhibited the activation of TGFBR1/Smads signaling dose-dependently. Cardiac-specific Tgfbr1 knockdown abolished Rg3's protection against myocardial fibrosis post-MI. In addition, Rg3 downregulated the TGF-β1-mediated CFs growth together with collagen production in vitro through TGFBR1 signaling. Moreover, TGFBR1 adenovirus partially blocked the inhibitory effect of Rg3. Conclusion: Rg3 improves myocardial fibrosis and cardiac function through suppressing CFs proliferation along with collagen deposition by inactivation of TGFBR1 pathway.

• Journal of Life Science
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• v.28 no.11
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• pp.1354-1360
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• 2018

The aim of this study was to develop a simple, environmentally friendly synthesis of silver nanoparticles (SNPs) without the use of chemical reducing agents by exploiting the extracellular synthesis of SNPs in a culture supernatant of Bacillus thuringiensis CH3. Addition of 5 mM $AgNO_3$ to the culture supernatant at a ratio of 1:1 caused a change in the maximum absorbance at 418 nm corresponding to the surface plasmon resonance of the SNPs. Synthesis of SNPs occurred within 8 hr and reached a maximum at 40-48 hr. The structural characteristics of the synthesized SNPs were investigated by various instrumental analysis. FESEM observations showed the formation of well-dispersed spherical SNPs, and the presence of silver was confirmed by EDS analysis. The X-ray diffraction spectrum indicated that the SNPs had a face-centered cubic crystal lattice. The average SNP size, calculated using DLS, was about 51.3 nm and ranged from 19 to 110 nm. The synthesized SNPs exhibited a broad spectrum of antimicrobial activity against a variety of pathogenic Gram-positive and Gram-negative bacteria and yeasts. The highest antimicrobial activity was observed against C. albicans, a human pathogenic yeast. The FESEM observations determined that the antimicrobial activity of the SNPs was due to destruction of the cell surface, cytoplasmic leakage, and finally cell lysis. This study suggests that B. thuringiensis CH3 is a potential candidate for efficient synthesis of SNPs, and that these SNPs have potential uses in a variety of pharmaceutical applications.

• Journal of Acupuncture Research
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• v.22 no.3
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• pp.169-183
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• 2005

Objective : The purpose of this study was to investigate the anti-caner effect of cobrotoxin on the prostatic cancer cell line (PC-3).The goal of study is to ascertain whether cobrotoxin inhibits tile cell growth and cell cycle of PC-3, or the expression of relative genes and whether the regression of PC-3 cell growth is due to cell death or the expression of gene related to apoptosis. Methods : After the treatment of Pc-3 cells with cobrotoxin, we performed 형광현미경, MTT assay, Western blotting, Flow cytometry, PAGE electrophoresis and Surface plasmon resonance analysis to identify the cell viability, cell death, apoptosis, the changes of cell cycle and the related protein, Adk, MAP kinase. Results : 1. Compared with normal cell, the inhibition of cell growth reduced in proportion with the dose of cobrotoxin(0-16nM) in PC-3. 2. Cell viabilities of 0.1, 1, 4nM cobrotoxin treatment were decreased and those of 8, 16nM were decreased significantly. 3. S phase of cell cycle was decreased at the group of 1, 2, 4, 8, 16nM cobrotoxin, but M phase was increased at 0.1, 1, 2, 4, 8, 16nM cobrotoxin. 4. Cox-2 expression after cobrotoxin was peaked at 12hours and was decreased significantly after 6, 12, 24 hours. 5. The expression of Cdk4 was decreased dose-dependently at 1, 2, 4, 8nM cobrotoxin and was decreased siginificantly at 4, 8nM Cyclin D1 was decreased at 1, 2, 4, 8nM and Cycline E was not changed. Cycline B was decreased at 1, 2, 4, 8nM dose-dependently and was decreased siginificanlty at 2, 4, 8nM. 6. The expression of Akt was decreased at 1, 2, 4, 8nM dose-dependently and was decreased significantly at 2, 4, 8nM. 7. ERK was increased at 1, 2nM and decreased at 4, 8nM, p-ERK was increased at 1, 2, 4 nM, but decreased at 8nM. JNK and p-JNK were increased at 1, 4, 8 nM. p38 was increased at 2nM p-p38 was increased at lnM but decreased significantly at 2, 4, 8nM. 8. The nucli of normal cells were stained round and homogenous in DAPI staining, but those of PC-3 were stained condense and splitted. Apoptosis was increased dose-dependently at 2, 4, 8, 16nM and increased significantly at 2, 4, 8, 16nM. 9. Bax wasn`t changed at 1, 2, 4, 8nM and Bcl-2 was decreased significantly at 1, 2, 4, 8nM. Caspase 3 and 9 weren`t changed at 1, 2, 4nM but were decreased significantly at 8nM. Conclusions : These results indicate that cobrotoxin inhibits the growth of prostate Cancer cells, has anti-cancer effects by inducing apoptosis.

• Journal of Acupuncture Research
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• v.22 no.3
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• pp.155-167
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• 2005

Objectives : The purpose of this study was to investigate the anti-inflammatory effect of Cobrotoxin on binding affinity of cobrotoxin with P50, $IKK{\alpa}$ and $IKK{\beta}$, activities of NF-${\kappa}B$, Cell viability of astrocyte, expressions of protein molecules of NF-${\kappa}B$ such as P50, P-$1{kappa}B$, $1{\kappa}B$ and iflammation related genes such as Cox-2, iNOS, cPLA2 in the SNP or LPS induced Inflammatory pathway of Rats' astrocytes. Methods : In this study, The expression of cytosolic phospholipase A2, Nitric oxcide, Cyclooxygenase-2 and inducible nitrogen oxide synthase was determined by western blotting with corresponding antibodies, and the generation of NF-${\kappa}B$ was assayed by EMSA method in astrocytes of rats. The Cell viability of astrocytes was determined by MTT assay, and Binding affinity of Cobrotoxin with P50, $IKK{\alpha}$ and $IKK{\beta}$ was assayed by Surface plasmon resonance analysis, and NF-${\kappa}B$ dependent luciferase activity was determined by luciferase analysis, and Uptake of cobrotoxin in astrocytes was identified by Confocal laser scanning microscope Results : 1. Compared with control, LPS-induced NF-${\kappa}B$ DNA binding activity was decreased significantly by 0.1, $0.5{\mu}g/m{\ell}$ of Cobrotoxin in Astrocyte. 2. Compared with control, LPS-induced NF-kB dependent luciferase expression was decreased significantly by 0.1, 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin in Astrocyte. 3. Compared with control, SNP induced P50, $I{\kappa}B$ expressions in astrocyte were decreased significantly by 0.1, 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin and P-$1{\kappa}B$ expression was decreased significantly by 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin. 4. Compared with control, LPS induced P50, $1{\kappa}B$ expressions in astrocyte were decreased significantly by 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin. 5. Compared with control, SNP induced Cox-2, iNOS, CPLA2 expressions in astrocyte were decreased significantly by $1{\mu}g/m{\ell}$ of Cobrotoxin. 6. Compared with control, LPS induced Cox-2, cPLA2 expressions in astrocyte were decreased significantly by 0.1, 0.5, $1{\mu}g/m{\ell}$ of Cobrotoxin and iNOS expression was decreased significantly by 0.5, $1{\mu}g/m{\ell}$ of Cobrotoxin. 7. Compared with $0.5{\mu}g/m{\ell}$ of Cobrotoxin, SNP-induced NF-${\kappa}B$ DNA bindins activity in astrocyte was increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM. 8. Compared with $0.5{\mu}g/m{\ell}$ of Cobrotoxin, LPS-induced NF-${\kappa}B$ DNA binding activity in astrocyte was increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM, Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM, Cobrotoxin $0.5{\mu}g/m{\ell}$with GSH 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 5mM 9. Compared with $0.1{\mu}g/m{\ell}$ of cobrotoxin, SNP induced P50 expressions in astrocyte were increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM, Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 5mM. 10. The uptake of the labeled cobrotoxin into the cells was shown under a confocal laser scanning microscope. cobrotoxin was uptaken into the membrane and nucleus of astrocytes. Conclusions : In summary, the present results demonstrate that cobrotoxin directly binds to sulfhydryl group of p50 and IKKS resulting In the reduction of translocation of p50 and IkB release, thereby inhibits activation of NF-${\kappa}B$, and suggest that pico to nanomolar range of cobrotoxin could inhibit the expression of genes in the NF-${\kappa}B$ signal pathway.