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87V Scrapie Agent 에 감염된 IM 마우스 뇌조직에서 Sulfated Glycoprotein - 2 ( SGP - 2 ) 의 발현 증가
양형모,민본홍,최은경,김용선 대한바이러스학회 1995 Journal of Bacteriology and Virology Vol.25 No.2
Scrapie-induced amyloid plaque formation in mouse is very useful animal model in research of human Alzheimer's disease. It has been suggested that sulfated glycoprotein-2 (SGP-2) is involved in neuronal cell death. In this study, we have investigated the role of SGP-2 in formation of amyloid plaques in brains of IM rnouse infected with 87V scrapie agent. Both control and scrapie-injected mice were sacrificed after 300 days incubation period and total cellular RNAs were extracted from cerebral cortex, brain stem, cerebellum and entire brain. Northern blot analysis showed that SGP-2 mRNA label was significantly increased in brains of scrapie-infected mice than in control mice. In dissected tissue samples, the level of SGP-2 mRNA was elevated in cerebellum and brain stem but not in cerebral cortex. Since amyloid plaques were mainly found in cerebral cortex of scrapie-injected mice, the observed changes of SGP-2 mRNA level were not exactly correlated with the site of amyloid plaque formation. It has previously shown that SGP-2 mRNA levels were also increased in several nonneural tissues under going programmed cell death and in some tissues of neuronal degeneration. We speculate that the overall increase of SGP-2 mRNA levels in brains of scrapie-infected mice may be a compensatory response to a neurodegenerative cascade control.
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내재형 Plasmid pBL1이 제거된 Brevibacterium lactofermentum 개발과 형질전환
이규남,민본홍,윤기홍 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.2
코리네형 아미노산 생산균을 위한 plasmid vectors의 개발에 이용되어 온 내재형 cryptic alsmid pBL1을 Brevibacterium lactofermentum으로부터 제거 하였다. B. lactofermentum에서는 복제할 수 없으나 ㎞^r 유전자를 지니고 있는 plasmid pEM1으로 제한 효소 Sau3AI에 의해 절단된 pBL1의 DNA fragments를 도입시킨 pEM1 유도체를 제조한 후 이를 Escherichia coli로부터 B. lactofermentum으로 conjugal transfer 시킴으로써 ㎞에 대해 내성을 갖는 B. lacto-fermentum transconjugant를 얻었다. ㎞^r transconjugant에는 pBL1과 pEM1 유도체간에 상동성 재조합으로 생겨난 plasmid pEB14가 존재하였으며 이것은 pEM1으로부터 유래된 ㎞^r 유전자를 지니고 있는 것으로 확인되었다. B. lactofermentum transconjugant의 ㎞^r 표현형질을 선별인자로 이용하여 ㎞에 대한 내성과 함께 pEB14이 동시에 상실된 균주를 선별함으로써 최종적으로 pBL1-free B. lactofermentum 균주가 제조되었다. plasmid pBL1이 존재하는 균과 상실된 균을 숙주균으로 사용하여 plasmid DNA에 의한 형질전환 효율과 형질전환체에서 plasmid 안정성을 비교한 결과 pBL1-free strain이 원래의 균주보다 유전자조작을 위한 숙주균으로서 우수하였다. 또한 pBL1이 존재하지 않으므로 pBL1-free strain에서 외래 plasmid DNAs가 간편하게 분리 및 분석될 수 있다. An endogenous cryptic plasmid, pBL1, which has been used to construct plasmid vectors for coryneform bacteria producing amino acids, was eliminated from Brevibacterium lactofermentum. The pBL1 was partially digested with Sau3AI and the resulting DNA fragments were subcloned into a suicide vector pEM1 which contains a kanamycin-resistant (㎞^r) gene. Km^r B. lactofermentum transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. Transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. A ㎞^r transconjugant was analyzed to contain a plasmid pEB14, which occurred in vivo by homologous recombination between pBL1 and the conjugal-transferred plasmid. The pEB14 including the pEM1-derived ㎞^r gene was found to be lost concomitantly with ㎞^r phenotype, resulting in the construction of a pBL1-free strain of B. lactofermentum. Based on transformation efficiencies and palsmid stability, the resultant pBL1-free strain is more useful than wild strain as a host cell for genetic manipulation. It could be concluded that foreign plasmid DNAs are efficiently isolated and analyzed from the pBL1-free strain because of the absence of endogenous pBL1 plasmid.
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이자섬 세포의 발달과 베타세포의 분화에서 clusterin의 역할
홍석우,박인선,민본홍,신용재,Ranjan KC,이송 대한당뇨병학회 2007 Diabetes and Metabolism Journal Vol.31 No.1
-Clusterin is a highly glycosylated heterodimeric glycoprotein that plays diverse biological roles in various organs. The secreted clusterin has been established as a major form of the protein that exerts diverse tissue effects. For instance, clusterin is known to act in cell protection through the actions of extra-cellular molecular chaperones. In the extracellular milieu, clusterin participates in specific interactions with a diverse array of native biological molecules including LRP-2 (Lipoprotein receptor-related protein 2, also known as gp330 or megalin), which is involved in ligand endocytosis at the surfaces of certain epithelia.Clusterin is expressed transiently in developing and differentiating endocrine pancreatic cells and might be involved in pancreas development. This transient expression of clusterin at specific time points of pancreas development and cell differentiation during pancreas regeneration implies that the protein is a regulatory factor for cytodifferentiation as well as for replication. A specific action of the clusterin in the reconstruction and remodeling of the endocrine pancreas has been demonstrated. It also strongly stimulates duct cell differentiation into insulin-secreting cells under in vitro culture conditions. Clusterin appears thus as a potent regulator of insulin cell morphogenesis. (J Kor Diabetes Assoc 31:1~8, 2007)
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BC₃H1 혈관평활근세포의 증식 및 분화과정에 있어서 Polyamine 대사의 역할에 관한 연구
최상현,문창택,민본홍,전보권,천연숙 고려대학교 의과대학 1992 고려대 의대 잡지 Vol.29 No.3
The influences of spermine (SM) and DFMO. an irreversible ODC-inhibitor, on the polyamine metabolism, CK-activity, [^(3)H]-thymidine DNA synthesis, and isoactin mRNA expression of BC_(3)H1 in culture were studied, with the references of phosphodiesterase(PDE)-inhibitors including isobutyl-methylxanthine (IBMX) and KR30075. BC_(3)Hl cells cultured in 10% fetal bovine serum (FBS)-DMEM for 24 hrs (the first : pre-drug phase) were cultivated in 10% FBS-DMEM added with one of the test drugs for further 96 hrs(the second proliferation phase), and then continue to grow in 2% FBS-DMEM for 48 hrs (the third : differentiation phase). The putrescine content of BC_(3)Hl harvested after the first phase was relatively higher in comparison with those of the other phases and was gradually decreased through the full phases of culture, and the spermine content was little changed. The spermidine (SD) content was. unlike other polyamines, increased by 26.5% at the end of the second phase, but that SD increase was moderately attenuated by PDE-inhibitors, particularly KR30075, and furthermore. the SD content of BC_(3)Hl cultured in the presence of DFMO, unlike other polyamines. showed the marked and gradual decrease throughout the full phases of culture. The [^(3)H]-thymidine DNA synthesis during the third : differentiation phase was not affected by IBMX and slightly inhibited by KR30075, but was significantly inhibited by SM plus DFMO or each of them. And the creatine kinase (CK) activity gradually increased in advancing with culture duration was not changed by PDE-inhibitors. but both SM and DFMO significantly enhanced the increase of CK activity. The synthesis of BC_(3)Hl α-actin and β-/ γ-actin mRNAs was significantly enhaced by DFMO, and the DFMO-induced enhancement was dramatically inhibited by SM. And PDE-inhibitors and SM little affected the synthesis of α-actin mRNA, but PDE-inhibitors attenuated the synthesis of β-/ γ-mRNAs, especially in the second phase. These results suggest that polyamines have a pivotal role in the proliferation and differentiation of BC_(3)Hl cells, that the enhancement induced by DFMO of expression of VSM isoactin gene seems to be an attractive subject to be studied in future, and that PDE-inhibitors may inhibit the synthesis of VSM β-/ γ-actin mRNAs.
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내열성 Cellulase-free xylanase를 생산하는 고온성 Bacillus sp.의 분리 및 효소 특성
김대준,신한재,민본홍,윤기홍 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.3
토양으로부터 cellulase-free xylanase를 세포외로 분비 생산하는 고온성 Bacillus sp. KK-1을 분리하였다. Bacillus sp.의 최적 배양조건은 50℃와 배지의 초기 pH 7.0이었으며 탄소원을 따로 첨가하지 않은 배지에서 최대 효소 생산량은 2.2 units/㎖이었다. Strain KK-1을 배양할 때 배지에 trehalose나 sucrose가 존재하면 효소 생성이 저해되었고 xylan과 xylose에 의해서는 효소의 생성이 증가되었다. Bacillus sp.로부터 생산된 부분정제된 xylanase는 70℃, pH, 7.0에서 최대 활성을 보였고 65℃에서는 10시간 동안 방치한 경우에 75% 이상의 활성을 유지하였다. 그리고 pH 5.0∼9.0에서 효소 반응 활성이 최적조건인 pH 7.0에서의 효소 활성의 90% 이상을 보이고 pH 10.0에서도 60% 이상의 활성을 지니는 내알칼리적 특성을 나타내었다. 또한 pH 5.0∼9.0에서 8시간 이상 효소 활성이 안정하게 유지되었다. Xylanase 활성이 금속이온에 의해 감소되는 경우가 많았는데 특히 Hg^(2+)와 Fe^(2+)에 의해서는 효소 활성이 완전히 상실되었으나 EDTA, phenylmethylsulfonyl fluoride, β-mercaptoethanol과 SDS에 의해서는 영향을 받지 않는 것으로 밝혀졌다. 또한 효소 기질 특이성을 조사한 결과 carboxymethylcellulose, laminarin, soluble starch과 galactomannan 등을 전혀 분해하지 못했다. A thermophilic bacterium producing the extracellular cellulase-free xylanase was isolated from soil and has been identified as Bacillus sp. The optimal growth temperature was 50℃ and the optimal pH, 7.0. Under the optimal growth condition, maximal xylanase production was 2.2 units/㎖ in the flask culture. The enzyme production was induced by xylan and xylose, but was repressed by sucrose or trehalose. The partially purified xylanase was most active at 70℃. It was found that the enzyme was stable at 65℃ for 10 hours with over 75% of the activity. The enzyme was most active at pH 7.0 and retained 90% of its maximum activity between pH 5.0 and pH 9.0 though Bacillus sp. was not grown on alkaline conditions (>pH 8.0). In addition, the activity of xylanase was over 60% at pH 10.0. At the ambient temperature, the enzyme was stable over a pH range of 5.0 to 9.0 for 10 h, indicating that the enzyme is thermostable and alkalotolerant. The activity of xylanase was completely inhibited by metal ions including Hg^(2+) and Fe^(2+), while EDTA, phenylmethylsulfonyl fluoride (PMSF), β-mercaptoethanol and SDS didn’t affect its activity. The enzyme was also identified to exert no activity on carboxymethylcellulose, laminarin, galactomannan, and soluble starch.
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