• Molecules and Cells
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• 제21권1호
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• pp.161-165
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• 2006

Dichlorodihydrofluorescein ($DCFH_2$) is a widely used probe for intracellular $H_2O_2$. However, $H_2O_2$ can oxidize $DCFH_2$ only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), $Cu^{2+}$, and $Fe^{3+}$ under various conditions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of $DCFH_2$ oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by $Cu^{2+}$ and $Fe^{3+}$. Oxidation of $DCFH_2$ by $H_2O_2$ in the presence of a cell lysate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of $H_2O_2$-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the $H_2O_2$-dependent oxidation of $DCFH_2$.

• KSBB Journal
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• 제14권1호
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• pp.82-90
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• 1999

병원성 미생물을 측정할 수 있는 분석시스템을 구성하기 위해 면역학적 성분들을 합성하였고, 이를 이용하여 모델 시스템을 구성함으로써 균 세포 분석원리가 연구되었다. 구성성분을 준비하기 위해 Salmonella thompson에 대한 복합 클론항체를 면역 친화 크로마토그래피를 이용하여 정제하였고, 이렇게 정제된 항체를 Streptavidin과 horseradush peroxdase에 화학결합시켰다. 항체와 Streptavidinfdm은 각각 SMCC와 SPDP에 의해 활성화 되있고 두 물질을 반응시킴으로써 중합체가 합성되었다. 중합체는diaminobiotion 젤과 sephades G-100젤을 이중 층으로 쌓은 칼럼을 이용하여 정제되었다. 항체- HRP 중합체의 합성을 위해, HRP를 $NAIO_4$ 처리에 의해 안정화된 중합체는 Biohel A5M을 이용한size exchusion크로토그래피로 정제되었다. 이렇게 준비된 중합체들과 dot-bloner 그리고 biotim이 고정화된 nitrocellulose membrane($12\mum$ pore size)을 이용하여 모델시스템을 구성하였다. 분석물질(S.Thormpson cells)을 먼적 액상에서 두 중합체와 반응되었고 반응먹을 membrane이 정착된 blotter에 옮긴 후 하부에 진공을 걸어 면역복합체를 biotin-streptavidin 반응에 의해 membrane 표면에 포획하였다.최적조건 하에서 시스템의 균 세포 분석원리를 확인하였으며 측정하한농도는 약 $1{\mu}g/m{\ell}(10^5 {\cdot} 10^6\;cells/m{\ell}$인 것으로 나타났다. 이러한 측정성능의 주요조절인자는 두항체 종합체 농도의 증가는 항원-항체 응집반응을 초래하는 것으로 나타났다.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.231-235
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• 1998

A sensitive enzyme immunoassay for serum TSH has been developed utilizing the tight binding between biotin and avidin, and three layered protein polystyrene beads as solid phase. To increase binding capacity of TSH and sensitivity of the assay, the polystyrene beads were coated sequentially with mouse immunoglobulin as first layer, rabbit antimouse immunoglobulin as second layer and monoclonal anti-TSH as third layer. A serum sample was incubated simultaneously with a monoclonal anti-TSH immobilized polystyrene beads and a second monoclonal anti-TSH covalently attached to biotin. After washing, the antibody bound serum TSH-anti-TSH-biotin complex is reacted with horseradish peroxidase (HRP)-labeled avidin. Following second wash, the bound HRP activity was measured calorimetrically. Reproducible results were obtained within 4 hours for serum TSH in the range between $0{\mu}\textrm{IU}$ml and ${50}{\mu}\textrm{IU}$ml with detection limit of $0.1{\mu}\textrm{IU}$ per test.

• Bulletin of the Korean Chemical Society
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• 제18권1호
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• pp.44-50
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• 1997

A method of dual-signal generation from two different enzymes was developed and utilized to simultaneously perform dual immunoassays in a single microwell. Two enzymes selected as tracers were horseradish peroxidase (HRP) and β-galactosidase (GAL). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) and chlorophenolred-β-galactopyranoside (CPRG) as chromogenic substrates for the respective enzyme were used. Although the two enzymes showed their maximum activities at distinct pH conditions (pH 5.1 for HRP and 7.5 for GAL), the enzyme reactions were able to be concurrently carried out at pH 5.75 in a dual-substrate solution without signal loss. This performance was achieved by increasing TMB concentration two-fold, introducing potassium salt as activator of GAL reaction, and extending total reaction time 50%. The signal generation method was then used for dual-enzyme immunoassays to detect antibodies with co-immobilized Hepatitis C virus antigens (core and NS5) and a Hepatitis B virus antigen (PreS(2)) in a microwell. Dose-response curves of the assays revealed cooperativity between different antigen-antibody complex formation, which suggested that dual immunoassays can only be used for qualitative screening tests unless the antigens immobilized were spatially separated.

• Nuclear Engineering and Technology
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• 제53권1호
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• pp.142-147
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• 2021

Hydrogen peroxide is a radiolysis product of water formed under gamma-irradiation; therefore, its reliable detection is crucial in the nuclear industry for spent fuel management and coolant chemistry. This study proposes an electrochemical sensor for hydrogen peroxide detection. Cysteamine (CYST), gold nanoparticles (GNPs), and horseradish peroxidase (HRP) were used in the modification of a gold electrode for fabricating Au/CYST/GNP/HRP sensor. Each modification step of the electrode was investigated through electrochemical and physical methods. The sensor exhibited strong sensitivity and stability for the detection and measurement of hydrogen peroxide with a linear range of 1-9 mM. In addition, the Michaelis-Menten kinetic equation was applied to predict the reaction curve, and a quantitative method to define the dynamic range is suggested. The sensor is highly sensitive to H₂O₂ and can be applied as an electrochemical H₂O₂-sensor in the nuclear industry.

• 한국동물학회지
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• 제39권1호
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• pp.26-35
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• 1996

쥐의 외측 망상핵으로부터 소뇌충부 제6엽 내의 각 소엽으로 신경 경로를 WGA-HRP를 이용한 역행수송법을 써서 조사하였다. 표지된 신경세포는 양측의 외측 망상핵에 모두 존재하였으나, 동축의 경우에 편중되어 있었다. 동측 또는 대측의 외측 망상핵의 큰 세포구획(magnocellular division)에서 국소순적으로 배열이 관찰되었는데, a소엽에서 c소엽으로의 투사가 동측의 큰 세포구획에서 등쪽에서 배쪽으로 분포양상을 보였으며, 대측의 큰세포구획에서는 다소 머리측에서 꼬리측 절편으로의 분포 양상을 보였다. 그 외 동측 또는 대측의 작은세포구획(parvocellular division) 및 삼차밑구획(subtrigeminal division)에서의 표지된 신경세포의 수는 극히 적었다. 한편 외측 망상핵으로부터 소뇌층부의 제6엽 a소엽/b소엽으로서의 투사에 관한 컴퓨터를 이용한 삼차원 재구성은 각 경우에 있어서 상당량의 투사의 중첩이 존재함을 보여주고 있다.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.773-777
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• 2003

The color of Congo red hinders the spectrometric measurements of a concentration of hydrogen peroxide and enzyme activity (Horseradish peroxidase; HRP) during enzymatic decoloring of Congo red. In this study, a method was developed to measure peroxidase activity and hydrogen peroxide concentration in the presence of Congo red. The oxidation product of HRP/hydrogen peroxide and ABTS(2,2'-azino-bis-(3-ethylbenzotriazoline-6-sulfonic acid)) formed a dark green color. The spectrum of this product showed absorption bands at 420 nm and 734 nm. When compared with the Congo red spectrum, the absorption at 734 nm of this product did not overlap with Congo red, thus making the hydrogen peroxide measurement possible even in the presence of Congo red. Kinetic study of decoloring of Congo red performed by this method showed that the decoloring reaction followed the Michaelis-Menten kinetics. Pulse feeding of hydrogen peroxide, upon depletion, significantly increased the decoloring of Congo red. This result shows that this newly developed technique can monitor, predict, and improve the enzymatic decoloring process.

• Journal of the Korean Wood Science and Technology
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• 제38권3호
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• pp.223-229
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• 2010

본 연구에서는 천연리그닌의 전구물질인 3종의 모노리그놀[p-coumaryl alcohol (PCA), coniferyl alcohol(CA), sinapyl alcohol (SA)]을 이용하여 horseradish peroxidase (HRP, EC. 1.11.1.7)/$H_2O_2$ 조건하에서 dehydrogenative polymers (DHPs)를 제조하였다. 합성한 DHPs와 천연리그닌의 구조적 특성을 비교하기 위해 소나무와 포플라 milled wood lignin (MWL)과 함께 Gel permeation chromatography (GPC)에 의한 분자량 측정과 Derivatization followed by reductive cleavage (DFRC) 분석에 의한 ${\beta}$-O-4 결합 빈도를 측정하였다. DHP 합성 수율은 CA만을 단독으로 주입한 G-DHP가 71%로 가장 높았고 PCA에 의한 H-DHP 수율은 42%로 나타났다. 그러나 horseradish peroxidase/$H_2O_2$ 조건하에서 SA 단독으로는 S-DHP는 전혀 합성되지 않았다. 합성한 DHPs의 분자량(Mw)은 3,000~4,700 범위로 측정되었는데, 이는 침엽수 리그닌인 소나무 MWL (G-type lignin: Mw 7340)의 절반 정도였고 활엽수인 포플라 MWL (GS-type lignin: Mw 13,250)의 1/3 수준으로 측정되었다. DHP 합성 과정에서 형성된 ${\beta}$-O-4 결합 빈도는 GS-DHP가 502 ${\mu}mol$/g으로 가장 높았지만 포플라 MWL (1107 ${\mu}mol$/g)의 1/2 수준에 머물렀고, G-DHP의 경우도 약 286 ${\mu}mol$/g으로 H-DHP (127 ${\mu}mol$/g)보다 약 2.5배 이상 많은 ${\beta}$-O-4 결합을 형성하였지만 소나무 MWL (651 ${\mu}mol$/g)과 비교하여 절반 정도로 측정되었다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.686-692
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• 2004

To develop the enzyme-linked immunosorbent assay (ELISA) for the analysis of N-acetylehitooligosaccharides (NACOS) and chitooligosaccharides (COS), specific antibodies (Abs) were produced, and their properties were compared. N-acetylehitohexaose (NACOS6), chitohexaose (COS6), and COS mixture (COSM) conjugated to bovine serum albumin (BSA) were used to immunize rabbits. By the use of specific Abs and NACOS6-horseradish peroxidase (HRP), COS6-HRP, and COSM-HRP conjugates, competitive direct ELISA (cdELISA) was developed. The detection limits of NACOS6 by the anti-NACOS6 Ab and COS6 by the anti-COS6 and the anti-COSM Abs in the cdELISAs were about 0.2, 2, ana 2 ng/ml (ppb), respectively. In the cdELISA, the anti-NACOS6 Ab was found to recognize NACOS3-NACOS6, but not N-acetyl-D-glucosamine (GlcNAc), NACOS2, and COSs; the anti-COS6 Ab recognized COS2-COS6 and COSM, but not glucosamine (GlcN) and NACOSs. The recognition pattern of the anti-COSM Ab was almost the same as that of the anti- COS6 Ab, except that the former recognized COS2 and COS3 slightly better than the latter.

• 약학회지
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• 제35권6호
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• pp.486-496
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• 1991

Spinal parasympathetic outflows originate in the sacral parasympathetic nuclei. The sacral parasympathetic nuclei receive inputs from the brainstem. Many areas in the medulla appear to influence sympathetic outflow of the spinal cord. Whether neurons in these areas of the medulla may project to the lumbosacral cord to affect the parasympathetic outflow has not been studied clearly. Thus, this study was intended to investigate origins of cells projecting from the medulla to the sacral parasympathetic nuclei of the spinal cord. In 3 cats, horseradish peroxidase (HRP) was injected into the lower lumbar spinal cord. HRP labeled neurons were found mainly in the following areas: nucleus retroambiguus, nucleus tractus solitarius, raphe complex and ventrolateral area of the rostral medulla. Most of these areas are known to be involved in regulation of sympathetic activity, and, thus, these results indicate that these areas are likely to affect the sacral parasympathetic outflow as they do for the sympathetic nerves.