• BMB Reports
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• 제46권5호
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• pp.264-269
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• 2013

Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-${\beta}$-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-${\beta}GRP$). Ap-${\beta}GRP$ was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-${\beta}GRP$ specifically bind 1,3-${\beta}$-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-${\beta}$-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-${\beta}GRP$ antibody could be recovered by addition of purified Ap-${\beta}GRP$. These results demonstrate that Ap-${\beta}GRP$ acts as a biosensor of 1,3-${\beta}$-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-${\beta}$-D-glucan or Ap-${\beta}GRP$ alone was unable to trigger the proPO system, but they both did. Ap-${\beta}GRP$ was specifically degraded following the activation of proPO with 1,3-${\beta}$-Dglucan. These results indicate the variation in the amount of Ap-${\beta}GRP$ after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response.

• 분석과학
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• 제29권1호
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• pp.35-42
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• 2016

A new nano-composite carbon ink for the development of disposable dopamine (DA) biosensors based on screen-printed carbon electrodes (SPCEs) is introduced. The method developed uses SPCEs coupled with a tyrosinase modified nano-composite carbon ink. The ink was prepared by an “in-house” procedure with reduced graphene oxide (rGO), Pt nanoparticles (PtNP), and carbon materials such as carbon black and graphite. The rGO-PtNP carbon composite ink was used to print the working electrodes of the SPCEs and the reference counter electrodes were printed by using a commercial Ag/AgCl ink. After the construction of nano-composite SPCEs, tyrosinase was immobilized onto the working electrodes by using a biocompatible matrix, chitosan. The composite of nano-materials was characterized by X-ray photoelectron spectroscopy (XPS) and the performance characteristics of the sensors were evaluated by using voltammetric and amperometric techniques. The cyclic voltammetry results indicated that the sensors prepared with the rGO-PtNP-carbon composite ink revealed a significant improvement in electro-catalytic activity to DA compared with the results obtained from bare or only PtNP embedded carbon inks. Optimum experimental parameters such as pH and operating potential were evaluated and calibration curves for dopamine were constructed with the results obtained from a series of amperometric detections at −0.1 V vs. Ag/AgCl. The limit of detection was found to be 14 nM in a linear range of 10 nM to 100 µM of DA, and the sensor’s sensitivity was calculated to be 0.4 µAµM⁻¹cm⁻².

• 대한의생명과학회지
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• 제15권2호
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• pp.161-166
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• 2009

Human erythropoietin(EPO) is a glycoprotein that enhances red blood cell production by stimulating proliferation and differentiation of erythroid progenitor cells in the bone marrow. Patients with chronic kidney disease(CKD) suffer from anemia caused by reduced production of EPO in the kidney. Recombinant human EPO protein has been used successfully for the treatment of anemia associated with CKD. Recently, attention has been paid to the development of side effect of EPO, pure red cell aplasia(PRCA), in some patients with CKD. PRCA is a rare disorder of erythropoiesis that leads to a severe anemia due to an almost complete cessation of red blood cell production. EPO-related PRCA is caused by the production of EPO-neutralizing antibodies(Abs) that eliminate the biological activity of EPO as well as endogenous EPO in patients undergoing therapy. Since 1988, almost 200 cases worldwide have been reported with Ab-positive PRCA after receiving EPO therapeutics. The underlying mechanisms of the breaking of immune tolerance to self-EPO have been investigated. Modification of formulation, organic compounds of container closures, and route of administration has been suggested for the possible mechanism of increased immunogenicity of EPO. A number of assays have been used to detect Abs specific to EPO. These assays are generally grouped into two major categories: binding Ab assays and neutralizing Ab assays(bioassays). There are several types of binding Ab assays, including radioimmunoprecipitation assay, enzyme-linked immunosorbent assay, and the BIAcore biosensor assay. In vitro cell-based bioassays have been utilized for the detection of neutralizing Abs. Finally, the recent experience with anti-EPO Abs may have considerable implications for the future development and approval of EPO preparations. Also, considering that millions of patients are being treated with EPO, clinicians need to be aware of signs and consequences of this rare but severe clinical case.

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.659-665
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• 2016

The oligosaccharides in human milk constitute a major innate immunological mechanism by which breastfed infants gain protection against infectious diarrhea. Clostridium difficile is the most important cause of nosocomial diarrhea, and the C-terminus of toxin A with its carbohydrate binding site, TcdA-f2, demonstrates specific abolishment of cytotoxicity and receptor binding activity upon diethylpyrocarbonate modification of the histidine residues in TcdA. TcdA-f2 was cloned and expressed in E. coli BL21 (DE3). A human milk oligosaccharide (HMO) mixture displayed binding with TcdA-f2 at 38.2 respond units (RU) at the concentration of 20 μg/ml, whereas the eight purified HMOs showed binding with the carbohydrate binding site of TcdA-f2 at 3.3 to 14 RU depending on their structures via a surface plasma resonance biosensor. Among them, Lacto-N-fucopentaose V (LNFPV) and Lacto-N-neohexaose (LNnH) demonstrated tight binding to TcdA-f2 with docking energy of −9.48 kcal/mol and −12.81 kcal/mol, respectively. It displayed numerous hydrogen bonding and hydrophobic interactions with amino acid residues of TcdA-f2.

• 한국축산식품학회지
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• 제37권1호
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• pp.134-138
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• 2017

Salmonella enterica infects a broad range of host animals, and zoonostic infection threatens both public health and the livestock and meat processing industries. Many antimicrobials have been developed to target Salmonella envelope that performs essential bacterial functions; however, there are very few analytical methods that can be used to validate the efficacy of these antimicrobials. In this study, to develop a potential biosensor for Salmonella envelope stress, we examined the transcription of the S. enterica serovar typhimurium spy gene, the ortholog of which in Escherichia coli encodes Spy (${\underline{s}}pheroplast$ ${\underline{p}}rotein$ ${\underline{y}}$). Spy is a chaperone protein expressed and localized in the periplasm of E. coli during spheroplast formation, or by exposure to protein denaturing conditions. spy expression in S. typhimurium was examined by constructing a spy-gfp transcriptional fusion. S. typhimurium spy transcription was strongly induced during spheroplast formation, and also when exposed to membrane-disrupting agents, including ethanol and the antimicrobial peptide polymyxin B. Moreover, spy induction required the activity of regulator proteins BaeR and CpxR, which are part of the major envelope stress response systems BaeS/BaeR and CpxA/CpxR, respectively. Results suggest that monitoring spy transcription may be useful to determine whether a molecule particularly cause envelope stress in Salmonella.

• Bulletin of the Korean Chemical Society
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• 제25권8호
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• pp.1195-1201
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• 2004

Tyrosinase was covalently immobilized on platinum electrode according to the method we developed for laccase (Bull. Korean Chem. Soc. 2002, 23(7), 385) and p-chlorophenol, p-cresol, and phenol could be detected with sensitivities of 334, 139 and 122 nA/ ${\mu}M$ and the detection limits of 1.0, 2.0, and 2.5 ${\mu}M$, respectively. The response time ($t_{90\%}$) is 3 seconds for p-chlorophenol, and 5 seconds for p-cresol and phenol. The optimal pHs of the sensor are in the range of 5.0- 6.0. This sensor can tolerate at least 500 times repeated injections of p-chlorophenol with retaining 80% of initial activity. In case of tyrosinase and laccase co immobilized platinum electrode, the sensitivities are 560 nA/ ${\mu}M$ for p-phenylenediamine (PPD) and 195 nA/ ${\mu}M$ for p-chlorophenol, respectively. The sensitivity of the bi-enzyme sensor for PPD increases 70% compared to that of only laccase immobilized one, but the sensitivity for p-chlorophenol decreases 40% compared to that of only tyrosinase immobilized one. The sensitivity increase for the bi-enzyme sensor for PPD can be ascribed to the additional catalytic function of the co-immobilized tyrosinase. The sensitivity decrease for p-chlorophenol can be explained by the “blocking effect” of the co-immobilized laccase, which hinders the mass transport through the immobilized layer. If PPD was detected with the electrode that had been used for p-chlorophenol, the sensitivity decreased 20% compared to that of the electrode that had been used only for PPD. Similarly, if p-chlorophenol was detected with PPD detected electrode, the sensitivity also decreased 20%. The substrate-induced conformation changes of the enzymes in a confined layer may be responsible for the phenomena.

• 전기화학회지
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• 제7권2호
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• pp.83-88
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• 2004

[ $DeniLite^{TM}$ ] laccase immobilized Pt electrode was used for amperometric detection of some catechol derivatives and o-aminophenol (OAP) derivative by means of substrate recycling. In case of catechol derivatives, the obtained sensitivities are 85, 79 and $57 nA/{\mu}M$ with linear ranges of $0.6\~30,\;0.6\~30\;and\; 1\~25 {\mu}M$ and detection limits (S/N=3) of 0.2, 0.2 and $0.3{\mu}M$ for 3,4-dihydroxycinnaminic acid (3,4-DHCA), 3,4-dihydroxybenzoic acid (3,4-DHBA) and 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), respectively. In case of OAP derivative, the obtained sensitivity is $237 nA/{\mu}M$ with linear range of $0.2\~15{\mu}M$ and detection limit of 70 nM for 2-amino-4-chlorophenol (2-A-4-CP). The response time $(t_{90\%})$ is about 2 seconds for each substrate and the long-term stability is around 40-50days for catechol derivatives and 30 days for 2-A-4-CP with retaining $80\%$ of initial activity. The optimal pHs of the sensor for these substrates are in the range of 4.5-5.0, which indicates that stability of the enzymatically oxidized product plays a very important role in substrate recycling. The different sensitivity of the sensor for each substrate can be explained by the electronic effect of the sugstituent on the enzymatically oxidized form.

• KSBB Journal
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• 제10권4호
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• pp.468-474
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• 1995

Glucose biosensor용 glucose oxidase 고정화 막의 단일화 벚 일회용의 간편화를 위한 연구로서, glucose oxidase의 고정화 대상막으로는 CTA와 PCL의 촌합비율이 80/20인 혼합막을 사용한 고저화 방법 중에서는 glutaraldehyde one-steD 방법으로 효소고정화층의 건조 전 두께가 $10{\mu}m$ 인 효소고정화 막이 효과적이었다. 이 고정화 막을 천자현미쇠 으로 관찰해 본 결과, 고밀도의 CTA/PCL 막층 위에 GOD-glutaraldehyde 층이 건조 후 $3{\mu}m$ 정도로 덮여 있는 것을 알 수 있었다. Dissolved oxygen전극을 사용하여 glucose농도의 증가에 따른 전류세기의 차이 값을 측정해 본 결과, glucose 7mM의 안도범위 내에서 선형성을 나타냈으므로 이 고정화원이 glucose sensor용 효소고정화 방법으로 가장 적합하였다. 한편, tyrosmase는 CTA와PCL의 혼 합비율이 80/20인 혼합막에 direct CDI 방법으로 고정화한 것이 가장 효과적이었으며, 8일 후에도고 정화된 tyrosmase의 활성이 35% 이상 유지되었다.

• 대한의용생체공학회:의공학회지
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• 제40권1호
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• pp.1-6
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• 2019

Human mesenchymal stem cells (hMSC) are multipotent stromal cells that have great potential to differentiate into a variety of cell types such as osteocytes, chondrocytes, and myocytes. Although there have been many studies on their clinical availability, little is known about how intracellular signals can be modulated by topographic features of the extracellular matrix (ECM). In this study, we investigated whether and how microwavy-patterned extracellular matrix (ECM) could affect the signaling activity of focal adhesion kinase (FAK), a key cellular adhesion protein. The fluorescence resonance energy transfer (FRET)-based FAK biosensor-transfected cells are incubated on microwavy-patterned surfaces and then platelet derived growth factor (PDGF) are treated to trigger FAK signals, followed by monitoring through live-cell FRET imaging in real time. As a result, we report that PDGF-induced FAK was highly activated in cells cultured on microwavy-patterned surface with L or M type, while inhibited by H type-patterned surface. In further studies, PDGF-induced FAK signals are regulated by functional support of actin filaments, microtubules, myosin-related proteins, suggesting that PDGF-induced FAK signals in hMSC upon microwavy surfaces are dependent on cytoskeleton (CSK)-actomyosin networks. Thus, our findings not only provide new insight on molecular mechanisms on how FAK signals can be regulated by distinct topographical cues of the ECM, but also may offer advantages in potential applications for regenerative medicine and tissue engineering.

• KSBB Journal
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• 제21권2호
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• pp.133-139
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• 2006

혈액 내 순환시 안정성 향상과 면역원성의 감소를 위해, rhIFN-${\alpha}$-2a은 N-terminus의 ${\alpha}$-아민기에 mPEG aldehyde를 solid-phase PEGylation 시킨다. CM-Sepharose와 같은 양이온 교환수지가 고체 지지체로 사용되었다. Mono-PEGylate는 양이온 교환 수지에서 unmodified 단백질과 분리되어 용출된다. Site-srecific PEGylation과 mono-PEGylate의 분리가 한 단계의 공정으로 얻어진다는 점은 solid-phase PEGylation의 이점을 뒷받침해준다. 위치 특이성은 peptide digest의 질량 분석과 Edman degradation을 이용한 N-terminal sequencing에 의해 확인하였다. Mono-PEGylate는 항바이러스 활성과 면역원성의 감소를 나타내고, 감소 정도는 결합되는 mPEG의 분자량에 비례한다. Trypsin 저항성과 온도 안정성은 mono-PEGylation에 의해 두드러지게 개선되었다. Solid-phase PEGylation을 통해 종래의 액상 반응에서 나타날 수 있는 재현성 낮은 반응, 부 반응물 생성, 부 반응물 제거 공정 등의 단점을 극복할 수 있었다. 그러나 solid-phase PEGylation의 문제점인 액상 반응에 비교하여 많은 양의 PEG를 사용하여야 한다는 점은 개선되어야 한다.