• BMB Reports
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• 제42권3호
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• pp.160-165
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• 2009

The temperate Staphylococcus aureus phage ${\Phi}11$ harbors cI and cro repressor genes similar to those of lambdoid phages. Using extremely pure ${\Phi}11$ Cro (the product of the ${\Phi}11$ cro gene) we demonstrated that this protein possesses a single domain structure, forms dimers in solution at micromolar concentrations and maintains a largely $\alpha$-helical structure even at $45^{\circ}C$. ${\Phi}11$ Cro was sensitive to thermolysin at temperatures ranging from $55-75^{\circ}C$ and began to aggregate at ${\sim}63^{\circ}C$, suggesting that the protein is moderately thermostable. Of the three homologous 15-bp operators (O1, O2, and O3) in the ${\Phi}11$ cI-cro intergenic region, ${\Phi}11$ Cro only binds efficiently to O3, which is located upstream of the cI gene. Our comparative analyses indicate that the DNA binding capacity, secondary structure and dimerization efficiency of thermostable ${\Phi}11$ Cro are distinct from those of P22 Cro and $\lambda$ Cro, the best characterized representatives of the two structurally different Cro families.

• 한국산학기술학회논문지
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• 제10권3호
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• pp.648-653
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• 2009

p62는 산화스트레스가 주요 원인 중 하나인 퇴행성 뇌질환과 연관된 엉긴 물질(aggregate)의 주요 구성성분이다. 퇴행성 뇌질환과 관련된 것으로 알려진 hydroxydopamin이나 $C_2-ceramide$를 신경모세포종 세포주인 SH-SY5Y에 처리하였을 때 p62의 발현이 유도되며 시간이 지날수록 그 양이 증가되었다. 또한 p62를 과발현시키면 ceramide에 의한 SH-SY5Y 세포의 사멸이 지연되었다. 이 과정에서 P62는 시간이 경과됨에 따라 침전화되며 절단되었다. 이 결과로 p62의 신경보호효과와 여러 종류의 퇴행성 뇌질환에서 p62가 엉긴 물질에서 발견되는 이유를 추측할 수 있다.

• 대한약리학회지
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• 제32권3호
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• pp.433-444
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• 1996

본 연구에서는 열충격에 의한 세포내 단백질 변성을 정량하는 방법을 소개하고 있다. Thiol compound인 diamide [azodicarboxylic acid bis (dimethylamide)]는 단백질변성시 노출된 sulfyhydryl기를 cross-link 시킨다. 정상 상태에서는 노출되지 않는 sulfyhydryl group이 변성된 단백질에서는 노출되기 때문에 diamide에 의한 cross-linking이 선택적으로 일어날 것이다. 그러므로 diamide는 변성된 단백질을 "trap"하는 작용을 할 수 있다. 본 연구진은 세포내 열충격후 고분자 단백질 응집물 (high molecular weight protein aggregate, HAA)이 나타남을 비환원 (non reducing) SDS-PAGE에서 관찰하였고 이를 gas flow counter로 scanning하여 정량하였다. 실험 결과 세포에 열충격을 가한후 diamide를 처리하면 HMA가 열충격 용량의존적으로 증가함을 관찰하였다. 이는 HMA의 양을 측정함으로써 열충격에 의하여 변성된 단백질을 정량할 수 있음을 반증한다. 열내성이 유도된 세포와 그렇지 않은 세포를 비교하였을 때 열내성이 유도된 세포에서는 열충격에 의한 HMA의 형성이 억제됨을 관찰하였다. 열충격후 정상온도에서 회복기를 주면서 시간대별로 diamide를 첨가하고 이때 형성된 HMA양을 측정하여, 단백질 원형복구의 역동성을 실험하였다. 그 결과, HMA는 열내성의 유도 여부와 상관없이 빠르게 없어짐을 알 수 있었다. 그러나 열내성이 유도된 세포에서 HSP70 단항체를 electroporation에 의하여 투여하였을 때 HMA가 현저히 증가하였고, 이는 열내성이 유도된 세포에서는 HSP70의 증가에 의하여 HMA생성이 억제되었음을 나타낸다. HSP70 항체를 이용하여 면역침전을 시행한 결과 변성된 세포내 단백질이 HSP70과 같이 침전됨이 관찰되었다. 이 결과는 HSP70 단백질이 변성된 단백질과 일시적으로 결합하여 정상 상태로 돌아가거나 복구될 수 있도록 도와줄 수 있음을 시사한다.

• 미생물학회지
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• 제46권4호
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• pp.313-318
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• 2010

D. radiodurans RecA 단백질은 DNA에 결합한 DNA-단백질 복합체만이 ATPase 활성을 나타내며. 보통의 낮은 염 농도조건에서는 DNA가 존재하지 않으면 RecA 단백질에 의한 ATP 가수분해는 거의 일어나지 않았으나 이러한 ATP 가수분해현상은 높은 농도의 염을 첨가하게 되면 1,000배 활성화 되었으며 1.6 M KCl이 존재할 때 ATP 혹은 dATP를 가수분해 하였다. DNA가 존재하지 않을 때 염에 의해 촉진되는 활성은 RecA 단백질 농도에 비례하였고, 더 높은 염농도에서 더 높은 ATP 가수분해 활성이 나타났다. 이러한 활성화 현상을 다양한 종류의 이온 형태에서 분석하였을 때 1.6 M Cl 음이온이 존재할 때 양이온의 형태에 따른 활성화 정도는 $K^+{\geq}Na^+$> $NH_4^+$의 경향을 보였으며, 1.6 M의 K 양이온 존재할 때 음이온의 형태에 따른 활성화는 glutamate > $Cl^-$ > acetate > $PO_4^-$의 순서로 높게 나타났다. 고농도의 염이 존재하는 조건에서 DNA 비의존성 ATPase의 활성은 비교적 넓은 범위 최적 조건인 pH7과 pH 8 사이에서 최대 활성을 보였고, 기질에 대한 친화도면에서도 외가닥 DNA 의존성 활성보다는 이중가닥 DNA 의존성 활성형태를 보였다. 고농도의 염이 첨가되고 DNA가 존재하지 않을 때 RecA 단백질에 의한 ATP 가수분해를 위한 RecA 단백질의 활성 종 형태는 최소 3개의 RecA 단백질이 결합되어 있는 과량체로 작용하는 것으로 나타났다.

• BMB Reports
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• 제36권5호
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• pp.488-492
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• 2003

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for neuronal survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of copper and peroxide in the modification of NF-L. When disassembled NF-L was incubated with copper ion and hydrogen peroxide, then the aggregation of protein was proportional to copper and hydrogen peroxide concentrations. Dityrosine crosslink formation was obtained in copper-mediated NF-L aggregates. The copper-mediated modification of NF-L was significantly inhibited by thiol antioxidants, N-acetylcysteine, glutathione, and thiourea. A thioflavin-T binding assay was performed to determine whether the copper/$H_2O_2$ system-induced in vitro aggregation of NF-L displays amyloid-like characteristics. The aggregate of NF-L displayed thioflavin T reactivity, which was reminiscent of amyloid. This study suggests that copper-mediated NF-L modification might be closely related to oxidative reactions which may play a critical role in neurodegenerative diseases.

• 한국가축번식학회지
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• 제26권4호
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• pp.311-320
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• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.

• 한국동물생명공학회지
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• 제37권4호
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• pp.246-254
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• 2022

Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and anti-inflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient's synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RA-hMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• 미생물학회지
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• 제37권4호
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• pp.245-252
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• 2001

MLS (macrolide-lincosamide-streptogramin B) 항생제 내성인자 단백질인 Erm 단백질들은 아미노산 서열 중 그 동일성과 유사성이 높아 구조적으로도 동등한 단백질의 한 집단을 형성한다. 최근 X-ray crystallography에 의해 구조가 결정된 ErmC\` 및 ErmAM 단백질의 구조에 근거하여 ErmSF 단백질도 catalytic domain과 substrate binding domain으로 구분하였고 N-terminal end에 존재하는 catalytic domain의 대량생산을 다양한 pET 발현 vector를 사용하여 시도하였다. 그리고 catalytic domain을 coding하는 DNA 절편은 세 종류를 사용하였다: DNA 절편 1은 Met 1부터 Glu 186까지를 coding하고 DNA 절편 2는 Arg 60부터 Glu 186까지의 정보를 가진 DNA이고 DNA 절편 3은 Arg 60부터 Arg 240까지를 encoding하는 DNA이다. 사용된 다양한 발현 vector중에서 pET19b는 DNA 절편 3, pET23b는 DNA 절편 1과 2를 성공적으로 대량생산하였다. 그러나 대량생산된 catalytic domain들은 불용성 단백질 집합체인 inclusion body를 형성하였다. ErmSF catalytic domain들의 용해성 단백질의 생산을 위하여 chaperone GroESL과 Thioredoxin의 동시 발현 및 배양온도를 $22^{\circ}C$로 낮추어 시도했으나 대량 발현된 단백질의 용해에는 도움을 얻지 못하였다.

• Journal of Nutrition and Health
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• 제29권1호
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• pp.9-21
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• 1996

This study was carried out to evaluate the fatty acid intakes of employees in employee feeding operations in Seoul and to provide prudent dietary guidelines with special concern on dietary fat. Four establishments were selected in large scale group and other four were selected as small scale group according to feeding numbers and food cost. Food intake was measured by substracting the leftover from the averaged portion amount. The leftover was measured by the modified aggregate selection plate waste measurement technique. The results were as follows : Employees from the large scale institution consumed more energy, protein, carbohydrate and niacin compared to those from the small scale institution(p<0.05). The mean calorie compositions of carbohydrate, protein and fat of all subjects were 66.7, 16.4 and 16.9%. The mean fat intake was 12.1g/lunch. Linoleic acid(C18:2 $\omega$6, 3.67g) was the most abundant fatty acid contained in the diet, followed by oleic acid (C18:1 $\omega$9, 3.53g) and palmitic acid(C16:0, 1.83g). The subjects consumed 5.2g polyunsaturated fatty acids(PUFA), 4.6g monounsaturated fatty acid(MUFA), 3.2g saturated fatty acid(SFA) per lunch per person. The average ratios of P/M/S and $\omega$6/$\omega$3 fatty acids were 1.6/1.5/1.0 and 8.5/1/0., respectively. the dietary $\omega$3 fatty acid status can be improved, even though the ratios found belong to the desirable range, by including $\omega$3 fatty acid rich-foods such as bean products and seafoods more frequently in the diet. Caution is needed for higher unsaturated nature of $\omega$3 series fatty acids to be prevented from peroxidation.