Production and Characterization of Enzymes Involved in Chitin Catabolic Cascade from a Bacterial Strain Isolated from Soil

( Ji Hyun Lee ),( Jeong In Lee ),( So Yeon Park ),( Yoo Kyeong Jang ),( You Bin Lim ),( Hye Yoon Lee ),( Young Min Jung ),( Hae Chang Lee ),( Jong Hwa Lee ),( Shaheen Amna ),( Jae Kweon Park ) 한국키틴키토산학회 2019 한국키틴키토산학회지 Vol.24 No.1

The present study was aimed to isolate the bacteria producing chitinolytic enzymes including chitin deacetylase (CDA) from soil origin. About 24 bacteria were screened for their chitinolytic enzymes producing ability on the basis of colloidal chitin. Amongst one of the most potent isolates designated to JI-02 was further selected as the most prominent chitinolytic and CDAlike enzyme (CDA-X) producer based on the ability of deacetylation of an artificial substrate pNP-acetamilide. The maximum production of CDA-X in crude enzyme from JI-02 was observed in the presence of 1% colloidal chitin at 37°C and pH 6.4 after 120 h of incubation. Potent enzyme activity of CDA-X was determined toward pNP-acetamilide, demonstrating that optimal pH, temperature and ionic strength of the CDA-X were assessed to be 7.0, 52°C and 125 mM, respectively. Unexpectedly, Nacetylglucosamine (GlcNAc) was not fully converted by the action of CDA-X to glucosamine (GlcN) under the established conditions. Further study on enzyme activity toward chitin-oligosaccharides consisting of multi-N-acetylglucosamine (GlcNAc)n, n=2-5 may be necessary to elucidate the mode of action which requires the minimum size of (GlcNAc)n. However, our data suggest that CDA-X can convert chitin to chitosan with the maximum yield of approximately 0.08g/L during the fermentation of the strain in the presence of chitin. Furthermore, we found that crude enzyme showed the significant chitosanolytic activity. Taken together, our data suggest that the strain JI-02 may be a potential candidate as a particular strain for better understating of chitin catabolic pathway.

D-Amphetamine이 니코틴성 흥분작용에 의한 카테콜아민 분비작용에 미치는 영향

임건한,서유석,민선영,임지연,김용직,나광문,임동윤 朝鮮大學校 附設 醫學硏究所 2005 The Medical Journal of Chosun University Vol.30 No.1

본 연구의 목적은 d-arphetamine이 흰쥐의 적출부신 관류모델에서 니코틴 수용체 흥분에 의한 카테콜아민(CA) 유리작용에 미치는 영향을 검색하여 그 작용기전을 규명하고자 하였다. D-amphetamine은 흰쥐 부신정맥내로 60분간 관류시 d-amphetamine자체는 약한 CA 분비작용을 나타내었으나, d-amphetamine (30 μM)을 비롯한 강력한 neuronal nicotine 수용체 작용제인 cytisine (50 μM) 및 epibatidine (30 μM)에 의한 CA 유리작용을 처음 4-10분 동안만 유의하게 증강시켰다. 또한, d-amphetamine (30 μM)은 60분간 부신정맥 내로 관류한 상태에서 dihydropyridine L-형 칼슘통로 개방약물인 Bay-K-8644 (10 μM)과 세포질내 칼슘저장고에서 Ca^(2+) ATPase 억제제인 cyclopiazonic acid (10 μM)의 CA 유리작용을 처음 4분간만 유의하게 증강시켰다. 그러나, 고농도의 d-amphetamine (500 μM)은 상기한 모든 분비촉진제의 CA분비작용을 오히려 억제하였다. 이와 같은 연구결과로 보면, 흰쥐 관류 부신수질에서 d-amphetamine은 낮은 농도에서는 콜린성 니코틴 수용체 흥분에 의한 카테콜아민 분비반응을 증강시키지만, 고농도에서는 오히려 억제적으로 작용함을 시사한다. 따라서, d-amphetamine은 용량에 따라서 흰쥐 적출 관류부신수질의 니코틴 수용체의 작용제 및 길항제로 이중 작용(dual action)을 나타내는 것으로 생각된다. 이러한 d-amphetamine의 작용은 흰쥐 부신수질 크롬친화세포의 dihydropyridine계 L-형 칼슘통로의 활성화 및 세포 내 칼슘저장고로부터 칼슘유리작용과 관련성이 있는 것으로 사료된다. The purpose of the present study was to examine the effect of d-amphetamine on CA release evoked by nocotinic receptor stimulation from the isolated perfused model of the rat adrenal gland, and to establish its mechanism of action. D-amphetamine(30 μM), when perfused into an adrenal vein of the rat adrenal gland for 60 min, enhanced the CA secretory responses evoked by ACh (5.32 mM), nicotine (30 μM), cytisine (50 μM, a selective neuronal nicotinic Nn-receptor agonist) and epibatidine (30 nM, a selective neuronal nicotinic Nn receptor agonist) only for the first period (4~10 min), although it alone has weak effect on CA secretion. Moreover, d-amphetamine (30 μM) in to an adrenal vein for 60 min also augmented the CA release evoked by BAY-K-8644, an activator of the dihydropyridine L-type Ca^(2+) channels, and cyclopiazonic acid, an inhibitor of cytoplasmic Ca^(2+) ATPase only for the first peroid (4 min). However, in the presence rather inhibited the CA secretory responses evoked by the above all of secretagogues. Taken together, these experimental results suggest that d-amphetamine at a low concentration enhances the CA secretion from the rat adrenal medulla evoked by stimulation of cholinergic nicotininc receptors, but at a high concentration it rather inhibits them. It semms that d-amphetamine has dual action acting as both agonist and antagonist at nicotinic receptors of the isolated perfused rat adrenal medulla, which are might be dependent on the concentration. It is also thought that these actions of d-amphetamine are probably relevant to the activation of the dihydropyridine L-type Ca^(2+) channels located on the rat adrenomedullary chromaffin cell membrane and release of Ca^(2+) from the cytoplasmic store.

Chlorpropamide 2-hydroxylation is catalysed by CYP2C9 and CYP2C19 <i>in vitro</i>: chlorpropamide disposition is influenced by CYP2C9, but not by CYP2C19 genetic polymorphism

Shon, Ji-Hong,Yoon, Young-Ran,Kim, Min-Jung,Kim, Kyoung-Ah,Lim, Young-Chae,Liu, Kwang-Hyeon,Shin, Dong-Hoon,Lee, Chung Han,Cha, In-June,Shin, Jae-Gook Blackwell Science Ltd 2005 British journal of clinical pharmacology Vol.59 No.5

<P>Aims</P><P>We evaluated the involvement of cytochrome P450 (CYP) isoforms 2C9 and 2C19 in chlorpropamide 2-hydroxylation <I>in vitro</I> and in chlorpropamide disposition <I>in vivo</I>.</P><P>Methods</P><P>To identify CYP isoforms(s) that catalyse 2-hydroxylation of chlorpropamide, the incubation studies were conducted using human liver microsomes and recombinant CYP isoforms. To evaluate whether genetic polymorphisms of CYP2C9 and/or CYP2C19 influence the disposition of chlorpropamide, a single oral dose of 250 mg chlorpropamide was administered to 21 healthy subjects pregenotyped for CYP2C9 and CYP2C19.</P><P>Results</P><P>In human liver microsomal incubation studies, the formation of 2-hydroxychlorpropamide (2-OH-chlorpropamide), a major chlorpropamide metabolite in human, has been best described by a one-enzyme model with estimated <I>K</I><SUB><I>m</I></SUB> and <I>V</I><SUB>max</SUB> of 121.7 ± 19.9 µ<SMALL>M</SMALL> and 16.1 ± 5.0 pmol min<SUP>−1</SUP> mg<SUP>−1</SUP> protein, respectively. In incubation studies using human recombinant CYP isoforms, however, 2-OH-chlorpropamide was formed by both CYP2C9 and CYP2C19 with similar intrinsic clearances (CYP2C9 <I>vs.</I> CYP2C19: 0.26 <I>vs.</I> 0.22 µl min<SUP>−1</SUP> nmol<SUP>−1</SUP> protein). Formation of 2-OH-chlorpropamide in human liver microsomes was significantly inhibited by sulfaphenazole, but not by <I>S</I>-mephenytoin, ketoconazole, quinidine, or furafylline. In <I>in vivo</I> clinical trials, eight subjects with the <I>CYP2C9</I>*<I>1/</I>*<I>3</I> genotype exhibited significantly lower nonrenal clearance [*<I>1/</I>*<I>3 vs.</I>*<I>1/</I>*<I>1</I>: 1.8 ± 0.2 <I>vs.</I> 2.4 ± 0.1 ml h<SUP>−1</SUP> kg<SUP>−1</SUP>, <I>P</I> < 0.05; 95% confidence interval (CI) on the difference 0.2, 1.0] and higher metabolic ratios (of chlorpropamide/2-OH-chlorpropamide in urine: *<I>1/</I>*<I>3 vs.</I>*<I>1/</I>*<I>1</I>: 1.01 ± 0.19 <I>vs.</I> 0.56 ± 0.08, <I>P</I> < 0.05; 95% CI on the difference − 0.9, − 0.1) than did 13 subjects with <I>CYP2C9</I>*<I>1/</I>*<I>1</I> genotype. In contrast, no differences in chlorpropamide pharmacokinetics were observed for subjects with the <I>CYP2C19</I> extensive metabolizer <I>vs.</I> poor metabolizer genotypes.</P><P>Conclusions</P><P>These results suggest that chlorpropamide disposition is principally determined by CYP2C9 activity <I>in vivo</I>, although both CYP2C9 and CYP2C19 have a catalysing activity of chlorpropamide 2-hydroxylation pathway.</P>

DVB-S2 기반 고속 LDPC 복호를 위한 효율적인 CNU 계산방식에 관한 연구

임병수,김민혁,정지원,Lim, Byeong-Su,Kim, Min-Hyuk,Jung, Ji-Won 한국정보통신학회 2012 한국정보통신학회논문지 Vol.16 No.9

본 논문은 DVB-S2 기반 고속 LDPC 복호를 하기 위한 효율적인 CNU(Check Node Update) 계산방식에 대해 분석하였다. LDPC의 복호 속도는 CNU 계산 과정에 의존한다. 기존의 CNU 계산방식에서 LUT를 고려한 SP(Sum-Product)방식은 속도가 늦어지는 단점이 있다. 이에 본 논문에서는 SC-NMS(Self-Corrected Normalized Min-Sum) 방식을 제안한다. LUT 연산을 제거한 MS(Min-Sum) 방식에 정규화 계수 '${\alpha}$'를 곱하는 Normalized Min-Sum(NMS) 방식은 MP 방식보다 성능은 약간 감소하지만 critical path를 없애고 클럭 주기를 줄일 수 있어 구현에 있어서 고속화를 위한 효율적인 CNU 계산방식이다. 또한, 복호과정에서 반복 시 이전 반복에서의 엣지 값과 현재 반복에서의 엣지 값을 비교하여 부호가 바뀌면 신뢰성이 없음을 간주하여 현재 엣지에 "0"을 할당하는 SC(Self-Corrected) 방식을 연구하였다. SC-NMS 방식을 적용하여 시뮬레이션 한 결과, SC-NMS 방식은 SP 방식에 비해 0.1dB의 성능열화를 보였지만, 계산의 복잡도와 복호 속도를 고려했을 때, SC-NMS 방식이 최적의 CNU 계산 방식이라는 것을 확인하였다. In this paper, efficient CNU(Check Node Update) algorithms are analyzed for high speed LDPC decoding in DVB-S2 standard. In aspect to CNU methods, there are some kinds of CNU methods. Among of them, MP (Min Product) method is quite often used in LDPC decoding. However MP needs LUT (Look Up Table) that is critical path in LDPC decoding speed. A new SC-NMS (Self-Corrected Normalized Min-Sum) method is proposed in the paper. NMS needs only normalized scaling factor instead of LUT and compensates the overestimation of MP approximation. In addition, SC method is proposed. It gives a faster convergence toward a decoded codeword. If a message change its sign between two iterations, it is not reliable and to avoid to propagate noisy information, its module is set to 0. The performance of SC-NMS has a little degrade compare to MP by 0.1 dB, however considering computational complexity and decoding speed, SC-NMS algorithm is optimal method for CNU algorithm.

세 개의 히드록실기를 가진 양이온 계면활성제 합성 및 계면 특성에 관한 연구

이병민 ( Byung Min Lee ),김지현 ( Ji Hyun Kim ),김승수 ( Sung Soo Kim ),임종주 ( Jong Choo Lim ) 한국공업화학회 2012 공업화학 Vol.23 No.5

본 연구에서는 도데실 글리시딜 에테르와 아미노에탄올을 반응시킨 후 디메틸 설페이트로 4급화시켜 분자 내에 2개의 라우릴기, 3개의 히드록실기를 가진 양이온 계면활성제 BHMAS (N, N-bis-(3`-n-dodecyloxy-2`-hydroxypropyl)-N-methyl- 2-hydroxyethylammonium methyl sulfate)를 합성하였고, 생성물의 분자구조는 1 H-NMR, FT-IR 등의 기기분석을 통하여 확인하였다. 합성한 계면활성제의 CMC (critical micelle concentration)는 9.12 × 10 -4 mol/L이며, CMC에서의 표면장력은 28.71mN/m이었다. Maximum bubble pressure tensiometer를 사용하여 동적 표면장력을 측정한 결과, 공기와 수용액의 계면이 계면활성제 단분자에 의하여 포화되는 데 비교적 오랜 시간이 소요되었다. 1 wt% 계면활성제 수용액과 n-decane 사이의 계면장력은 0.045mN/m이며, 평형에 도달하는 데 약 5 min의 시간이 소요되었다. 합성된 계면활성제의 흡착 특성이 매우 우수하였으며, 합성한 양이온 계면활성제가 섬유표면에 효과적으로 흡착되어 유연효과를 나타낼 수 있음을 확인하였다. In this study, a cationic surfactant BHMAS (N,N-bis-(3`-n-dodecyloxy-2`-hydroxypropyl)-N-methyl-2-hydroxyethylammonium methyl sulfate) having two lauryl and three hydroxyl groups was synthesized by the reaction of n-dodecyl glycidyl ether and 2-aminoethanol followed by the quarternization with dimethyl sulfate. The structure of the product was elucidated by 1 H-NMR and FT-IR. The CMC (critical micelle concentration) and surface tension of BHMAS at CMC condition were found to be 9.12 × 10 -4mol/L and 28.71mN/m respectively. Dynamic surface tension measurements using a maximum bubble pressure tensiometer indicated that a relatively long time was required to saturate the interface between air and aqueous surfactant solution. The interfacial tension measured between 1 wt% surfactant solution and n-decane reached an equilibrium value of 0.045mN/m in 5 min. The adsorption capacity of the synthesized surfactant was observed to be excellent, which suggests that the surfactant can be used as a softening agent during a laundry process.

플라즈마 및 직접 기상 불소화에 따른 활성탄소섬유의 초산가스 흡착 특성

이란은 ( Raneun Lee ),임채훈 ( Chaehun Lim ),김민지 ( Min-ji Kim ),이영석 ( Young-seak Lee ) 한국공업화학회 2021 공업화학 Vol.32 No.1

실내 환경 오염물질인 휘발성 유기화합물(volatile organic compounds)인 초산에 대한 핏치계 활성탄소섬유의 흡착 성능을 향상시키기 위해 불소화를 진행하였다. 불소화에는 플라즈마 불소화와 직접 기상 불소화법을 사용하였으며, 두 가지 방식으로 불소화한 활성탄소섬유의 초산가스 흡착 성능을 고찰하였다. 불소가 도입된 활성탄소섬유의 표면 특성을 알아보기 위하여 X선 광전자 분광법(XPS)을 분석하였고 그 기공특성은 77K 질소 흡착법을 통하여 분석하였다. 초산가스의 흡착 성능은 가스 크로마토그래피를 통하여 측정하였으며, 플라즈마 불소화 된 활성탄소섬유의 파과시간이 790 min으로 650 min인 미처리 활성탄소섬유에 비해 파과시간이 지연되었음을 확인할 수 있으나, 직접 기상 불소화 표면처리한 활성탄소섬유의 파과시간은 390 min으로 오히려 흡착성능이 저해되었다. 이는 플라즈마 불소화 된 활성탄소섬유는 그 비표면적의 변화 없이, 표면에 도입된 불소 관능기가 초산가스(CH₃COOH)와 정전기적 인력으로 흡착성능이 증가하였다. 반면, 직접 기상 불소화 된 활성탄소섬유는 그 비표면적이 55%까지 크게 감소하여 초산가스에 대한 물리적 흡착 효과가 현저히 감소하였다. Fluorination was carried out to improve the adsorption performance of pitch-based activated carbon fibers (ACFs) onto acetic acid. Both plasma and direct gas fluorination were used for fluorination, and the acetic acid gas adsorption performance of fluorinated ACFs was investigated. X-ray photoelectron spectroscopy (XPS) is analyzed to determine the surface characteristics of ACFs, and the pore characteristics were analyzed by 77 K nitrogen adsorption. An adsorption performance was measured through gas chromatography, and it was confirmed that the breakthrough time of plasma fluorinated sample was 790 min and that the breakthrough time was delayed compared to that of using untreated one of 650 min. However, the breakthrough time of direct gas fluorinated sample was 390 min, indicating that the adsorption performance was inhibited. The plasma fluorinated ACFs showed an increase in the adsorption performance due to an electrostatic attraction between the acetic acid gas (CH₃COOH) with the fluorine group introduced to the surface without changing its specific surface area. On the other hand, the specific surface area of the direct gas fluorinated ACFs decreased significantly up to 55%, and the physical adsorption effect on the acetic acid gas also reduced.

Korean physician prescription patterns for home oxygen therapy in chronic obstructive pulmonary disease patients

( Youlim Kim ),( Hwan Il Kim ),( Ji Young Park ),( Ji Young Hong ),( Joo-hee Kim ),( Kyung Hoon Min ),( Chin Kook Rhee ),( Sunghoon Park ),( Chang Youl Lee ),( Seong Yong Lim ),( Seung Hun Jang ),( Yo 대한내과학회 2022 The Korean Journal of Internal Medicine Vol.37 No.1

Background/Aims: Hypoxemia in chronic obstructive pulmonary disease (COPD) leads to reduced ability to exercise, decreased quality of life, and, eventually, increased mortality. Home oxygen therapy in patients with severe COPD reduces distress symptoms and mortality rates. However, there have been few studies on physicians’ prescription behavior toward home oxygen therapy. Therefore, we investigated the respiratory specialists’ perspective on home oxygen therapy. Methods: In this cross-sectional, study, a questionnaire was completed by 30 pulmonary specialists who worked in tertiary hospitals and prescribed home oxygen therapy. The questionnaire consisted of 28 items, including 15 items on oxygen prescription for outpatients, four for inpatients, and nine on service improvement. Results: All physicians were prescribing less than 2 L/min of oxygen for either 24 (n = 10, 33.3%) or 15 hours (n = 9, 30.3%). All (n = 30) used pulse oximetry, 26 (86.7%) analyzed arterial blood gas. Thirteen physicians had imposed restrictions and recommended oxygen use only during exercise or sleep. Sixteen (53.3%) physicians were educating their patients about home oxygen therapy. Furthermore, physicians prescribed home oxygen to patients that did not fit the typical criteria for long-term oxygen therapy, with 30 prescribing it for acute relief and 17 for patients with borderline hypoxemia. Conclusions: This study identified the prescription pattern of home oxygen therapy in Korea. Respiratory physicians prescribe home oxygen therapy to hypoxemic COPD patients for at least 15 hours/day, and at a rate of less than 2 L/ min. More research is needed to provide evidence for establishing policies on oxygen therapy in COPD patients.

헬스케어용 가다랑어(Katsuwonus pelamis) 통조림의 제조 및 특성

김현정 ( Hyeon Jeong Kim ),김민지 ( Min Ji Kim ),김기현 ( Ki Hyun Kim ),지성준 ( Seung Jun Ji ),임경훈 ( Kyung Hun Lim ),박권현 ( Kwon Hyun Park ),신준호 ( Joon Ho Shin ),허민수 ( Min Soo Heu ),김진수 ( Jin Soo Kim ) 한국수산과학회 2012 한국수산과학회지 Vol.45 No.3

This study was conducted to prepare canned skipjack tuna Katsuwonus pelamis in the mixture of isotonic beverage and tomato paste (ST-S) for use as a health food. An analysis of the time-temperature profile and viable cells showed that a reasonable F0 value for ST-S preparation was 4 min. The proximate composition of ST-S was 76.8% moisture, 20.2% crude protein, 0.8% crude lipid, 1.7% ash and 0.5% carbohydrate. The calorie content of ST-S was 94.8 kcal, which is 47.4% lower than that of commercial canned skipjack tuna in oil (ST-O) and 2.2% lower than that of commercial chicken breast in water (CB-B). The total amino acid content of ST-S was 18.54 g/100 g, which is 31.4% lower than that of ST-O and 7.9% lower than that of CB-B. The major amino acids in ST-S were aspartic acid and glutamic acid. An enrichment effect due to such minerals as phosphorus, potassium and iron would be expected on consuming 100 g of ST-S. The major fatty acids in ST-S were 16:0 (27.4%), 18:1n-9 (14.3%) and 22:6n-3 (27.8%), which are different from those in ST-O and CB-B. The major free amino acids in ST-S were glutamic acid (8.1%), histidine (38.6%) and its related dipeptide, such as anserine (15.7%). In an evaluation of taste, flavor and color, ST-S was found to be superior to ST-O and CB-B.

Impact of Irradiation Time on the Hydrolysis of Waste Activated Sludge by the Dielectric Heating of Microwave

Tae Joo Park,Im Gyu Byun,Jae Ho Lee,Ji Sung Lim,Jeong Min Lee 대한환경공학회 2014 Environmental Engineering Research Vol.19 No.1

The effects of initial solid concentration and microwave irradiation (MWI) time on the hydrolysis of waste activated sludge (WAS)were investigated. MWI time strongly influenced WAS hydrolysis for all initial solid concentrations of 8.20, 31.51, and 52.88 g VSS/L. For all WAS, the volatile suspended solids (VSS) solubilization degree ranged from 35.6% to 38.4% during a total MWI time of 10 min. Solublechemical oxygen demand (SCOD) concentration increased at a rate proportional to the decrease of VSS during the MWI. However, the clearly different VSS solubilization patterns that were observed during the MWI were explained by the 2-step hydrolysis of WAS, consisting of the initial disintegration of the easily degradable part of the sludge, followed by the subsequent disintegration of the hardlydegradable part of the sludge. WAS hydrolysis rates for 3 to 6 min of MWI were significantly lower than those for less than 3 min, or more than 6 min. From these results, 3 min MWI time and WAS of 31.51 g VSS/L (centrifugal thickener WAS) showed the most efficienthydrolysis of WAS at 36.0%. The profiles of total nitrogen (T-N) concentrations corresponded well to the SCOD increases in terms of the empirical formula of bacterial cell mass (C5H7O₂N). The negligible T-N increase and pH decrease during WAS hydrolysis by MWI will allowthe application of this process to subsequent biological processes, such as anaerobic digestion.

저산소성 허혈성 뇌손상을 유발시킨 어린 백서에서 Myeloperoxidase 측정 검사

양혜정(Hae Joung Yang),피대훈(Dae Hun Pee),임지혜(Ji Hye Lim),최병민(Byung Min Choi),이기형(Kee Hyoung Lee),은백린(Baik-Lin Eun) 대한소아신경학회 2002 대한소아신경학회지 Vol.10 No.2

목 적 : 예전에는 뇌경색 부위에서 발견되는 백혈구를 단지 뇌손상에 뒤따르는 생리적인 반응으로만 생각하였으나 최근 10년 사이 재관류 손상에서도 백혈구에 의한 염증 작용이 중요한 역할을 한다고 알려지면서 이들의 역할에 대한 연구가 새롭게 이루어지고 있다. PML의 존재는 세포질내 과립에서 분비되는 MPO를 생화학적으로 측정함으로서 증명할 수 있는데 저자들은 미성숙 뇌에서 저산소성 허혈증에 의한 뇌손상이 발생할 때 백혈구가 침윤되는 과정을 MPO 측정 검사를 통하여 관찰하였고, P와 L-selectin 억제제인 Fucoidin을 투여한 후 MPO의 변화를 확인하였다. 방 법 : 생후 7일된 어린 흰쥐를 사용하여 우측총 경동맥을 전기 응고시켜 자르고 8% 산소에 노출시켰다. 저산소 노출 후 회복 시간에 따라 동물을 희생시키고 뇌를 추출하여 뇌조직 1 g당 10mL의 20 mM potassium Phosphate buffer(pH 7.4)를 첨가하여 50초간 분쇄하였다. 각각의 균질회된 조직 샘플들은 4℃에서 20분 동안 원심 분리하여 상층액을 제거한 후, 침전물은 처음 조직량에 따라 조직 1 g당 10 mL의 0.5% cetylditrimethylethyl ammonium bromide(wt/vol)가 첨가된 50 mM potassium phosphate buffer(pH 6.0)를 첨가하였다. Vortex를 이용하여 buffer와 침전물을 잘 섞고 60℃수조에서 120분 동안 방치하였다. 그 후 각 조직 샘플들을 4 watts에서 음파처리하고 4℃에서 15분 동안 원심 분리를 시행하여 상층액을 새로운 tube에 옮겼다. MPO 측정검사를 위한 MPO reaction buffer는 50 mM potassium phosphate buffer(pH 6.0) 100 mL에 0.53 mM odianisidine edihydrochlorde와 0.0005% HO를 섞어 만들었다. MPO reaction solution 2.9 mL에 각각의 MPO sample 0.1 mL을 첨가하여 460 nm의 파장에서 5분 동안 MPO에 다른 흡광도 차이를 관찰하였다. 결 과 : 조직내 백혈구 침윤의 지표로 측정한 MPO 활성도는 대조군에서는 미미하였으나 총 경동백을 절단한 우측 대뇌군에서는 허혈 및 저산소증 유발 후 8시간째부터 현저히 증가되기 시작하여 24시간 경과 후 발현이 가장 높았으며, Fucoidin 50 mg/kg으로 전처치한 약물 실험군에서는 MPO 활성도가 현저히 감소하였다. 결 론 : 백혈구는 미성숙 뇌의 저산소성 허혈증에 의한 뇌손상에서 중요한 매개체 역할을 하며, 백혈구의 생화학적 활성도를 나타내는 MPO 활성도는 저산소성 허혈성 뇌손상의 정도를 측정하는 지표로 이용될 수 있다고 생각된다. Purpose : Neutrophils found around an infarcted area in the brain was once considered as only the physiologic response following the brain injury, but recent studies have shown that inflammatory responses by neutrophils play an important role in the reperfusion injury. The presence of ploymorphonuclear leukocytes(PML) is proven by biochemical assay of myeloperoxidase(MPO) Secreted in the cytoplasmic granules. We observed the process of PML infiltration on hypoxic-ischemic brain injury of immature rats by the assay of MPO activity and changes of the MPO activity after the administration of fucoidin, inhibitor of P- and L-selectin. Methods : We used a well characterized model of the brains of 7 day-old-rats, which had unilateral hypoxic and ischemic injuries(HI). Those injuries were induced by unilateral carotid artery ligation followed by timed exposure to hypoxic inspiratory gas mixture(8% O). MPO activity was measured in the brain tissue homogenates of HI rats(n=18) at 0, 2, 8, 24 and 48 hrs and in rats that received fucoidin immediately before and again after hypoxia(50 mg/kg, n=6) at 8 and 24 hrs. Controls(n=2) were rats with neither hypoxia nor ischemia. The brain samples were homogenized in 20 mM potassium phosphate buffer(pH 7.4) for 50 secs. The homogenate was centrifuged at 14,000 g at 4℃ for 15 mins and the supernatant was discarded. The tissue was pulverized, weighed, and suspended in 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) contatining 0.5% cetylditrimethylammonium bromide(wt/vol). The tissue was sonicated and centrifuged at 10,000 g for 15 mins. 200 µL of the supernatant was mixed with 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) containing 10 μL of 1.325 mM o-dianisidine hydrochloride and 170 µL of 3% hydrogen peroxide(vol/vol). Changes in absorbance at 460 nm were measured for 5 mins by using microplate reader. One unit of MPO activity was defined as that degrading 1 µmol peroxide/min at 25℃, and the result was expressed as units of MPO/100 gm tissue. Results : In HI rats, MPO activity increased at 2 hrs after HI and peaked at 24 hrs in the right hemisphere. In rats with fucoidin treatment immediately before and again after hypoxia, the MPO activity significantly decreased in both hemispheres compared with HI rats(P<0.05). MPO activity in the tissue of control rats was insignificant. Conclusion : The dynamic changes of the MPO activity suggest the important role of PMN on hypoxic-ischemic brain injuries in immature rats. MPO activity could be used as an index of the severity of injuries of hypoxic-ischemic brains.