• Food Science and Biotechnology
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• 제15권5호
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• pp.694-697
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• 2006

The initiation of immunoglobulin E (IgE)-mediated allergic reactions requires binding of IgE antibody to its high-affinity receptor. Human basophilic KU812F cells express $Fc{\varepsilon}RI$ on the cell surface and act as effector cells in the allergic response. In this study, we investigated the effects of Scutellaria radix extract on the expression of the $Fc{\varepsilon}RI$ in human KU812F cells. Flow cytometric analysis showed that S. radix extract treatment caused a concentration-dependent decrease in $Fc{\varepsilon}RI$ expression on the cell surface. Furthermore, the level of $Fc{\varepsilon}RI$ ${\alpha}$, ${\beta}$, and ${\gamma}$ chain mRNA in KU812F cells was examined by RT-PCR. S. radix extract reduced total cellular $Fc{\varepsilon}RI$ $\alpha$ and ${\gamma}$ chain mRNA expression in a concentration-dependent manner. $Fc{\varepsilon}RI$-mediated histamine release was reduced from $21.75{\pm}1.34\;ng/10^6$ cells in non-treated cells to $16.46{\pm}1.98\;ng/10^6$ cells in S. radix extract treated cells. These results suggested that S. radix extract has the potential to down-regulate of FcRI expression and to inactivate basophils.

• Animal cells and systems
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• 제15권4호
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• pp.259-267
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• 2011

This study was undertaken to produce a $F_{ab}$ fragment of a human monoclonal antibody reactive to oxidized and carbamylated low-density lipoprotein (oxLDL and cLDL) using phage display technology. An analysis of DNA sequences of this $F_{ab}$, termed plaque 15,16-46 $F_{ab}$, revealed that the rearranged $V_H$ was highly mutated. Complementarity-determining regions of the $V_H$ showed a very high R/S ratio and contained many positively charged amino acids. In direct binding and competitive ELISA, the $F_{ab}$ reacted strongly with both MDA-LDL and Cu-oxLDL forms of oxLDL, and also showed high affinity for cLDL. Immunofluorescence and immunohistochemical analyses showed that this $F_{ab}$ positively stained atherosclerotic aortic plaques in $ApoE^{-/-}$ mice as well as those in patients with atherosclerosis. The $F_{ab}$ also showed positive staining in placental decidua from patients with preeclampsia. It is suggested that the plaque 15,16-46 $F_{ab}$ against oxLDL and cLDL might possibly be applicable for developing a diagnostic reagent for both human and rodent animal research to detect and characterize atherosclerotic disease progression in atherosclerotic lesions as well as exploring the pathogenesis of atherogenic diseases such as preeclampsia.

• Molecules and Cells
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• 제39권11호
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• pp.807-813
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• 2016

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of $1.3{\times}10^6CFU/ml$. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate ($5{\times}10^4CFU/ml$). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of $2{\times}10^4CFU/mL$ of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2016년도 제50회 동계 정기학술대회 초록집
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• pp.378.2-378.2
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• 2016

Biosensors currently suffer from severe non-specific adsorption of proteins, which causes false positive errors in detection through overestimation of the affinity value. Overcoming this technical issue motivates our research. Polyethylene glycol (PEG) is well known for its ability to reduce the adsorption of biomolecules; hence, it is widely used in various areas of medicine and other biological fields. Likewise, amine functionalized surfaces are widely used for biochemical analysis, drug delivery, medical diagnostics and high throughput screening such as biochips. As a result, many coating techniques have been introduced, one of which is plasma polymerization - a powerful coating method due to its uniformity, homogeneity, mechanical and chemical stability, and excellent adhesion to any substrate. In our previous works, we successfully fabricated plasmapolymerized PEG (PP-PEG) films [1] and amine functionalized films [2] using the plasma enhanced chemical vapor deposition (PECVD) technique. In this research, an amine functionalized PP-PEG film was fabricated by using the plasma co-polymerization technique with PEG 200 and ethylenediamine (EDA) as co-precursors. A biocompatible amine functionalized film was surface characterized by X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FT-IR). The density of the surface amine functional groups was carried out by quantitative analysis using UV-visible spectroscopy. We found through surface plasmon resonance (SPR) analysis that non-specific protein adsorption was drastically reduced on amine functionalized PP-PEG films. Our functionalized PP-PEG films show considerable potential for biotechnological applications such as biosensors.

• 접착 및 계면
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• 제17권4호
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• pp.149-154
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• 2016

일반적으로, 화학 물리적 변성이 수반되는 멸균방법을 적용하지 못하는 경우 필터여과에 의한 멸균 방법을 주로 사용한다. 하지만 바이러스의 경우 100 nm의 작은 크기로 인해 박테리아 제거에 사용되는 필터여과에 의한 멸균 방법을 적용하기가 어렵다. 본 논문에서는 화학 물리적 변성 없이 바이러스를 비활성화하는 물질 개발을 위하여, 카테콜기가 도입된 헤파린 고분자(Hep-C)를 합성하였다. Hep-C의 바이러스 비활성화 효과를 알아보기 위해서 표면이 노르에피네프린으로 코팅된 조직배양접시에 아데노연관바이러스(Adeno-associated virus; AAV)를 고정화하였으며, 그 위에 다시 Hep-C를 코팅하여 녹색형광단백질(GFP) 유전자를 전달할 수 있는 AAV의 비활성화 정도를 유세포분석기(FACS)를 이용하여 분석하였다. 그 결과 AAV에 Hep-C를 도포하면 99%에 달하는 비활성화 효과를 보였으며, 헤파린 고분자를 도포한 결과에 비해 강한 염 저항성을 확인할 수 있었다. 이러한 결과로 미루어 보아 제조된 Hep-C는 AAV 바이러스 제거의 효과적 방법으로 충분한 가능성을 가짐을 증명하였다.

• BMB Reports
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• 제43권5호
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• pp.362-368
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• 2010

Dermcidin is a human antibiotic peptide that is secreted by the sweat glands and has no homology to other known antimicrobial peptides. As an initial step toward understanding dermcidin's mode of action at bacterial membranes, we used homonuclear and heteronuclear NMR to determine the conformation of the peptide in 50% trifluoroethanol solution. We found that dermcidin adopts a flexible amphipathic $\alpha$-helical structure with a helix-hinge-helix motif, which is a common molecular fold among antimicrobial peptides. Spin-down assays of dermcidin and several related peptides revealed that the affinity with which dermcidin binds to bacterial-mimetic membranes is primarily dependent on its amphipathic $\alpha$-helical structure and its length (>30 residues); its negative net charge and acidic pI have little effect on binding. These findings suggest that the mode of action of dermcidin is similar to that of other membrane-targeting antimicrobial peptides, though the details of its antimicrobial action remain to be determined.

• BMB Reports
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• 제43권7호
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• pp.485-490
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• 2010

Neurotrophins regulate many aspects of neuronal function through activation of the high affinity Trk receptors. Shc family proteins are implicated in the coupling of RTK to the Ras/mitogen-activated protein kinase signaling cascade. Here we report that the fourth Shc family member, ShcD, associates with TrkB receptor and regulates BDNF-induced MAPK activation. Yeast two-hybrid assay and Co-IP experiments demonstrate ShcD interacts with TrkB in a kinase-activity-dependent manner. Confocal analysis shows ShcD cololizes well with TrkB in transfected 293T cells. Subsequent mapping experiments and mutational analysis indicate that both PTB and SH2 domains are capable of binding to TrkB and PTB domain binds to TrkB NPQY motif. Furthermore, ShcD is involved in BDNF-induced MAPK activation. In summary, we demonstrate that ShcD is a substrate of TrkB and mediates TrkB downstream signaling pathway.

• 한국임상수의학회지
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• 제8권1호
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• pp.1-10
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• 1991

The B-lymphocyte differentiation from committed B-cell progenitors to antibody-secreting cells was discussed. B-cell progenitors derived from hematopoietic stem cells undergo the rearrangement of immunoglobulin(Ig) gene. The earliest cells as B-cell precursors have cytoplasmic Is(${\mu}$ chain). The entire Is molecule is expressed on the surface after synthesis of L chain. The resting B cells(Go stage) stimulated by binding antigen via Ig-receptors are activated(G$_1$ stage) and followed by proliferation(S stage), coupled with further selection(affinity maturation. class switch). The production of antibody against a particular antigen depends on the activation of B cells with surface Is capable of reacting with that antigen. This process does not occur in isolation but is controlled by helper and suppressor T cells and antigen presenting cells(APC). The mechanism of T cell-dependent B-cell response for production of antibody is largely explained by the cell to cell cooperation and soluble helper factors of T cells. 1) The antigen specific B cells and helper T cells are linked by Is-receptors, leading to the delivery of helper signals to the B cells. 2) Helper T cells recognize the processed antigen-derived peptides with the MHC class II molecules(la antigen) and is stimulated to secrete B-cell proliferation and differentiation factors which activate B cells of different antigenic specificity. The two models are shown currently 1) At low antigen concentration, only the antigen-specific B cell binds antigen and presents antigen-derived peptides with la molecules to helper T cells, which are stimulated to secrete cytokines(IL-4, IL-5, etc.) and 2) At high antigen concentration, antigen-derived peptides are presented by specific B cells, by B cells that endocytose the antigens, as well as by APC Cytokines secreted from helper T cells also lead to the activation of B cells and even bystander B cells in the on- vironmment and differentiate them into antibody-secreting plasma cells.

• KSBB Journal
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• 제11권2호
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• pp.218-224
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• 1996

The adsorption of cellulase on celluloses with different crystallinities was carried out In nonionic surfactant(Tween 20) solution. Highly crystallized celluloses were prepared by enzymatic prehydrolyzation. From the experiments, the Langmuir isotherm parameters, maximum adsorption amount (Amax) and adsorption equilibrium constant(Kad) for the adsorption, were obtained in the presence and absence of nonionic surfactant. It was found that the Kad values were decreased by adding Tween 20. This indicates that the adsorption affinity is reduced by nonionic surfactant, and Amax decreased with increasing crystallinity under conditions accompanying in both the presence and absence of surfactant. The thermodynamic parameters such as $\Delta$Ha, $\Delta$Ga, and $\Delta$Sa for the adsorption were calculated by using the experimental data. From these results, it was found that the adsorption processes are exothermic reactions in both the presence and absence of surfactant. The heats of adsorption in surfactant solution(-4.68∼-3.62KJmol-1) are smaller than that of the adsorption in the absence of surfactant(-15.60∼-12.10KJmol-1). These results indicated that the tightness of adsorption was reduced by the addition of surfactant. The $\Delta$Sa values were estimated to be positive. This may suggest that the water and solute are released from cellulose on adsorption. The $\Delta$Sa values in surfactant solution are larger than that of the adsorption in the absence of surfactant. This may suggest that the binding of surfactant on hydrophobic region of cellulase cause dispersion of water and solute molecule orienting around the enzyme molecule. The surfactant played an important role in the desorption of enzyme from cellulose functional groups, and enhance the saccharification of the cellulose.

• BMB Reports
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• 제38권2호
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• pp.225-231
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• 2005

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus-as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.