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박두상,박호용 한국곤충학회 2002 Entomological Research Vol.32 No.3
From Bombyx mori larvae, RT--PCR and cDNA library screening isolated masquerade--like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined.The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio’s 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disul.de bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis.
곤충 장내미생물로부터 lipase 생산능력이 우수한 Burkholderia sp. HY-10 균주의 분리 및 특성
박두상,오현우,배경숙,김향미,허선연,김남정,설광열,박호용,Park, Doo-Sang,Oh, Hyun-Woo,Bae, Kyung-Sook,Kim, Hyang-Mi,Heo, Sun-Yeon,Kim, Nam-Jung,Seol, Kwang-Youl,Park, Ho-Yong 한국응용곤충학회 2007 한국응용곤충학회지 Vol.46 No.1
곤충으로부터 유용 효소생산 미생물의 탐색 과정에서 우수한 lipase 생산균주 9종을 분리하고 lipase 생산능을 조사하였다 16S rDNA 분석 결과 분리된 균주는 주로 Serratia 속, Pseudomonas 속, Burkholderia 속에 속하는 그람음성균들로 분석되었다. 그 중 lipase 생산능이 가장 우수한 균주를 선별하고 16S rDNA 서열분석 및 생리 생화학적 분석 결과를 바탕으로 Burkholderia sp. HY-10으로 동정하였으며 균주의 lipase생산특성을 조사하였다. 이 균주는 톱하늘소의 장으로부터 분리되었으며 olive oil을 탄소원으로 포함하는 배지에서 배양하였을 때 세포밀도에 의존하여 lipase의 생산이 유도되는 특성을 나타내었고 0.5%의 yeast extract와 0.5%의 olive oil이 포함된 M9배지에서 $30^{\circ}C$, 36-42시간의 배양에 의해 lipase의 생산이 최대치에 도달하였다. From the course of screening of useful enzyme producing microorganism from insect guts, we isolated 9 lipase producing strains and their lipase producing activities were tested. 16S rDNA sequence analysis showed that they were Gram negative bacteria grouped on Serratia sp., Pseudomonas sp., and Burkholderia sp.. Among them, an excellent lipase producing strain, Burkholderia sp. HY-10 identified by 16S rDNA analysis and biochemical methods, was further studied its lipase producing characteristics. It was isolated from a longcorm beetle, Prionus insularis and showed cell density dependent lipase producing activity in the culture media that contained olive oil as a carbon source. Maximum lipase production was achieved in the M9 media containing 0.5% yeast extract and 0.5% olive oil when cultured at $30^{\circ}C$ for 36-42 hrs.
박두상,박호용 한국곤충학회 2004 Entomological Research Vol.34 No.4
Two types of serine proteases and a serine protease homologue cDNAs wereisolated from Hyphantria cunealarvae induced immune response due to an injection of amicroorganism through RT-PCR and cDNA library screening, and their characteristics wereexamined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, whichencoded 388, 390, 580 amino acid residues, and were designated as HcPE-1, HcPE-2 and HcPE-3, respectively. They were revealed as serine proteases or a serine protease homologue with theclip domain through a database search. The deduced amino acid sequence comparison showedhigh homology of 72-78% among them. Six Cys residues of the N-terminal clip domain formingthe disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter-bridgebetween clip domain and catalytic domain were also well preserved. Three amino acid residues,His, Asp, and Ser, within the active site were perfectly conserved in HcPE-2 and HcPE-3,however, His was replaced with Gln178 in HcPE-1. The Arg residues (HcPE-1, Arg132; HcPE-2,Arg134; HcPE-3, Arg325) known as the activation sites by proteolytic cleavage were preserved wellin all three types of protein. In case of HcPE-3, three continuous clip-like domains existed in theN terminal. As the result of phylogenetic analysis, three clip domain family of protein from H.cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showedall three genes were induced through an injection of Escherichia coli, but expression patterns were varied.
박두상,오현우,허선연,정우진,신동하,배경숙,박호용 한국미생물학회 2007 The journal of microbiology Vol.45 No.5
Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60°C. A broad range of lipase substrates, from C4 to C18 ρ-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was ρ-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.
박두상,윤미애,서명철,유하나,김주령,정태숙,박호용 한국생약학회 2004 생약학회지 Vol.35 No.3
- Chronical intake of alcohol can cause alcoholic fatty liver. Fatty liver is caused by fat infiltration: the state of high rate of fat in liver cells and by losing the balance between the synthesis and the secretion of fatty acid. It could be developed into liver necrosis and cirrhosis. Ka-Mi-Chung-Gan-Tang (KMCGT) is a decoction used for fatty liver as oriental medicines in China. The prescription is composed of Ginseng Radix, Bupleuri Radix, Scutellariae Radix, Pinelliae Tuber, Artemisiae capillaris Herba, Gardeniae Fructus, Zingiberis Rhizoma, Zizyphi Fructus and Glycyrrhizae Radix etc. We have induced alcoholic fatty liver by ethanol administration (6 g/kg, single dose/day, for a week) on rats and observed changes of triglyceride, cholesterol and lipid peroxidation in liver tissues of them. Also we checked the activities of GOT and GPT in blood of rats. KMCGT inhibited significantly the increase of triglyceride, cholesterol, lipid peroxidation level and effectively the increase of malondialdehyde (MDA).
복숭아심식나방 유충의 전사체 분석에 의한 종 동정 marker 탐색
박두상,김석원,권용국,최경희,오현우 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.10
복숭아심식나방의 신속한 동정을 위한 간이진단킷트용 marker 개발을 위하여 유충의 전사체를 454 NGS를 통하여 분석하였다. 그 결과 총 237,000개의 서열을 확보하였으며, 약 160,000개의 서열이 복숭아심식나방 유충의 전사체 정보를 포함하였다. 이 전사체 정보는 약 3,000개의 contigs를 구성하였으며 singleton의 수는 17,000개로서, 이들을 genome 서열이 밝혀진 B. mori의 유전자와 BlastX로서 상동성유전자를 탐색하였을 때 68%, 44%의 서열들이 각각 B. mori, D. melanogaster의 유전자와 상동성을 나타내었다. 그 중 기능이 알려진 유전자의 수는 총 4500여개로서 중복을 고려하였을 때 약 2000개의 유전자에 대한 annotation이 가능하였다. 서열이 밝혀진 유전자의 64%가 B. mori의 유전자와 70% 이상의 상동성을 나타내었으며 60% 이하의 상동성을 나타내는 서열은 약 20%를 차지하였다. 이들 서열로부터 유충의 진단에 사용 가능할 것으로 예상되는 다수의 후보 단백질을 선정하였으며 이들의 서열 상동성을 비교하였다.