• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.7-13
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• 2011

In general, expression of membrane protein in Escherichia coli is very toxic to the host organism, but the mechanism for the toxicity is not clear yet. Expression of human purinergic receptor $P2X_4$ was found to be extremely toxic to the host E. coli. We examined this toxicity by isolation and analysis of less toxic mutant proteins. We could isolate 30 less toxic mutants of $P2X_4$ after hydroxylamine mutagenesis. Western blot showed that all of them produced proteins smaller than the wild type $P2X_4$. DNA sequencing of two largest mutant proteins showed that they were lost its second transmembrane domain. Localization analysis of these mutant proteins showed that they are not in cytoplasmic membrane, but in inclusion bodies. These data showed that inactive truncated $P2X_4$ is not toxic to E. coli and membrane integration and functionality of $P2X_4$ may be needed to show host toxicity.

• Parasites, Hosts and Diseases
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• v.33 no.3
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• pp.155-164
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• 1995

The location of actin and myosin of the several stages of Cwptosporinium parvum was observed. The tissue antigen of C. pcruum was prepared through immunosuppression of IgG mice with Depomedrol . The thin sectioned specimens, which were incubated with the IgG fraction of the rabbit polyclonal antibodies raised against chicken back muscle actin and bovine uterus myosin, were treated with 10 nm gold-conjugated goat anti-rabbit IgG, Electrodense particles were located mainly on the pellicles of all observed developmental stages of the parasites. The number of actin gold particles in the cytoplasm increased when the parasite was dividing actively as in case of meronts. Especially in macrogametocytes, a lot of actin and myosin particles were synthesized and storaged as amilopectin-like bodies. There were many actin gold particles along the microspikes of cytoplasmic membranes in various developmental stages. The actin and myosin observed in this study may play important roles to control the shape of the parasites and movement of cytoplasmic membranes as cvtoskeletal proteins.

• Korean Journal of Microbiology
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• v.27 no.2
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• pp.117-123
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• 1989

Radioactive N-$\alpha$-p-nitrobenzoxycarbonyl (NBZ)-L-[2,$3-^{3}$H] arginyl diazomethane was used as an affinity label for the vacuolar arginine transporter in Neurospora crassa. Vacuolar matrix proteins were removed by fracturing the membranes with freeze-thaw method in dry ice/ethanol bath. Vacuolar membrane proteins were then wasged with 500mM NaCl to remove ionically bound derivatives and peripheral membrane proteins from vacuolar membranes. After dissolved in 1% Titon X-100, dissolved vacuolar memvrane proteins were separated with molecular sieve column chromatography, anion and cation exchange chromatographies. The arginine transporter was purified giving the purification factor of 1136.

• Proceedings of the KAIS Fall Conference
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• 2000.10a
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• pp.243-244
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• 2000

Serum albumin은 혈청 단백질 중 50-60%를 차지한다. 알부민은 순환계 내에서 삼투압을 유지시켜 세포내외의 체액을 유지 및 지용성 물질치 이동에 관여한다. 신사구체와 기저막의 투과성이 증가하거나 세뇨관의 흡수감소 등으로 단백질의 요배설량이 하루에 150mmg 이상되는 단백뇨는 신장내의 병변이 있음을 나타내는 중요한 지표이다. 이러한 단백뇨는 크게 사구체성 단백뇨, 세뇨관성 단백뇨, 혼합성 단백뇨로 나눌수 있는데 이중 사구체 기저막의 손상으로 발생되는 사구체성 단백뇨는 알부민과 같은 고분자량의 단백질이 소변 내로 배설되는 현상이다. 일반적으로 알부민이 정상치 이상으로 소변을 통하여 배설되는 것을 'microalbuminuria'라고 하며 당뇨병으로 인한 신장병변을 예견하는데 중요한 지표로 알려져 있다. Microalbuminuria는 방사 면역확산법, 방사성 면역측정법, 면역혼탁측정법, 비탁측정법, 효소결합면역측정법(ELISA) 등으로 검출할 수 있으나 최근에는 안전하면서도 민감도가 높고 측정이 용이한 방법인 ELISA를 이용한 면역분석방법이 많이 사용되고 있다. 면역특이성이 떨어지는 다세포군 항체를 사용한 방법의 문제점을 극복하기 위해서 세포융합기술을 이용한 단세포군항체를 생산하는 세포주를 개발하였고 그 특성을 규명하였다. 따라서 본 연구에서 세포융합기술을 도입하여 개발된 인간 혈청 알부민에 대한 단세포군항체는 glycohemoglobin에 대한 단세포군항체와 더불어 당뇨병의 진행 정도를 진단할 수 있는 키트의 원료로 사용될 수 있다.

• Applied Chemistry for Engineering
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• v.9 no.7
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• pp.1090-1097
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• 1998

Lidocaine compounds have widely been used as local anesthetics. Regarding the molecular mechanism for anesthesia by lidocaine, it is proposed that lidocaine molecules penetrate to the hydrophobic region of cell membrane and expand the membrane volume, producing a change in protein conformation that blocks sodium permeability or lidocaine molecules directly adsorb into lidocaine receptor in the protein channel without expanding the cell membrane. But these proposals have never been proven experimentally. In this study, the expansion of cell membrane by lidocaine compounds was investigated by employing lipid monolayer at the air/water interface as the mimetic system of cell membrane. It was found that oil-soluble lidocaine contracted the area/molecule of lipid in the monolayer of phosphatidyl choline, sphingomyelin, DS-PL95E and lipoid, but expanded the monolayer of phosphatidyl ethanolamine only in a certain range of mixing ratios. On the contrary, water-soluble lidocaine-HCl salt expanded the monolayers of all lipids used in this study.

• Journal of the Korea Society of Computer and Information
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• v.14 no.1
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• pp.73-80
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• 2009

G protein-coupled receptors(GPCRs) family is a cell membrane protein, and plays an important role in a signaling mechanism which transmits external signals through cell membranes into cells. But, GPCRs each are known to have various complex control mechanisms and very unique signaling mechanisms. Structural features, and family and subfamily of GPCRs are well known by function. and accordingly, the most fundamental work in studies identifying the previous GPCRs is to classify the GPCRs with given protein sequences. Studies for classifying previously identified GPCRs more easily with mathematical models have been mainly going on. In this paper Considering that functions of proteins are determined by their stereoscopic structures, the present paper proposes a method to compare secondary structures of two GPCRs having different amino acid sequences, and then detect an unknown GPCRs assumed to have a same function in databases of previously identified GPCRs.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.276-276
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• 1994

지구 환경문제의 하나인 오존충의 파괴는 지구상에 분포되어있는 자외선량을 증가시키는 외에 지금까지 지상에 도달하지 않았던 단파장역의 자외선량의 증가를 초래하여, 이것에 피부암, 백내장등의 발병율 증가등의 건강 피해가 염려된다. 이들 발병기전은 아직 확실치 않으나 에너지가 큰 단파영역의 자외선에 폭로되면 세포내의 물분자의 이온화에 기인되어 발생하는 활성산소종이 막지질, 핵산. 단백질등에 산화적 손상을 가져와 이것에 돌연변이, 세포사를 초래하는 것이 그 원인의 하나라고 생각된다. 본 연구는 자외선 중 UVB의 조사로 인한 장해와 유도단백질을 찾아내어, 자외선의 유해성을 밝히는데 목적을 두었다. UVB를 농도별로 1회 hairless mouse에게 조사하여, 경시적으로 피부를 채취하여, UVB 조사로 인해 유도되는 단백질을 2차 전기영동법을 이용해 관찰하고. 유도단백질이 HSP인지를 면역염색을 통해 밝히고, 유도된 단백질을 protein sequence를 하여, 어떤 단백질인지 밝혔다.

• Development and Reproduction
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• v.13 no.4
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• pp.297-303
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• 2009

Implantation of blastocyst into the uterine endometrium is established by the existence of histologically and functionally prepared uterine endometrium. Doc-1, an oral cancer suppressor gene, is expressed under the control of steroid hormones and has been suggested as a proliferation regulator of endometrial cells. However, the role is not much clear and in this study we examined the expression modulation of Doc-1 in decidualizing cells in vitro. In vitro decidualization was performed in endometrial stroma cells using progesterone and estrogen. Until 24 hr after decidual induction the proliferation of stroma cell was significantly increased but decreased after then. On the other hand, most of the cells differentiated into decidual cell after 48 hr of induction. The Doc-1 protein was co-localized in a specific deciudal cells and colocalization rate was increased in a parallel manner with the induction time. Based on these results, it is suggested that Doc-1 expression is under the control of both steroid hormones and decidual signals, and Doc-1 protein is involved in suppression of the proliferation of decidualizing cells.

• Proceedings of the KOR-BRONCHOESO Conference
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• 1991.06a
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• pp.16-16
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• 1991

렉틴(Lectins)은 세포막의 당단백질의 과당류 말단기에 특이적으로 결합하는 비면역성의 당단백질 혹은 단백질을 말하는데 정상세포와 변형된 세포에서 렉틴반응의 차이가 남으로써 요즘에는 렉틴을 병리학적 진단에 이용 가능하게 되었다. “Loss of contact inhibition”은 세포 악성화의 중요한 특징으로 세포의 악성화는 세포막의 구조의 변화와 관계가 있을 것으로 알려져 왔으며 1989년 후두암에서 PNA의 반응이 양성 반응을 나타낸다고 보고되어 저자들은 7가지의 렉틴을 이용하여 후두암전구증의 하나인 후두각화증에서 이형성(Atypia)의 존재유무에 따른 렉틴 반응의 변화를 알아보고자 본 연구를 시행하여 다음과 같은 결과를 얻었다. 1. 이형성이 없는 단순 후두각화증에서는 Con A와 WGA만이 기저세포에 반응이 있었으며, 후두각화증의 가장 중요한 부위인 극세포층에서는 WGA, RCA-I, PNA, SBA, UEA-I, DBA가 세포질에는 반응이 없이 세포간교에만 염색이 되는것을 관찰할 수 있었고 Con A는 반대로 극세포층의 세포질에 반응을 하였으나 세포간교에는 반응이 없었다. 2. 이형성을 동반한 후두각화증에서, 기저세포에서는 반응의 차이가 없었고 극세포층에서는 UEA-I, RCA-I. WGA, PNA, SBA는 세포간교에서 반응이 나타나지 않았고 세포질에서 양성 반응을 관찰 할 수 있었으며 DBA, Con-A에서는 반응의 차이를 관찰 할 수 없었다. 따라서 후두각화증의 극세포층 세포막에서 UEA-I, RCA-I, WGA, PNA, SBA 반응의 소실은 후두암으로의 진행과 밀접한 관계가 있는 이형성의 존재를 암시한다하겠다.