• 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.

• Tuberculosis and Respiratory Diseases
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• 제58권2호
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• pp.142-152
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• 2005

배 경 : 최근 결핵에 대한 새로운 백신 개발은 초회 면역 방법 및 추가 면역 방법을 이용하는 방향으로 연구되고 있다. 본 실험은 새로운 백신 후보 물질로서의 가능성을 알아보기 위하여 결핵균 adenylate kinase (AK), nucleoside diphosphate (NdK) 및 heat shock protein 70(Hsp70)의 결핵균에 대한 방어면역효능을 측정하였다. 방 법 : 재조합 단백질들을 정제하기 위하여 중합효소 연쇄반응으로 증폭한 결핵균 유전자 단편들을 E.coli expression vector, pQE30에 클로닝한 후, Ni-NTA resin을 이용하여 정제하였다. DDA와 재조합 단백질들을 마우스에 면역주사하고 면역반응 생성 유무를 확인하기 위하여 항체와 $IFN-{\gamma}$ 생성능을 측정하였다. 면역주사 한 마우스에 결핵균을 공기 감염시킨 후, 폐와 비장을 분리하여 결핵균 생균수 실험을 하였다. 결 과 : 재조합 단백질 AK, NdK 와 Hsp70을 면역보강제인 DDA를 이용하여 면역주사 한 결과에서, 생리식염수 혹은 DDA를 면역주사 한 마우스에 비교하여 재조합 단백질을 면역주사 한 마우스에서는 각 항원에 대해 항체와 $IFN-{\gamma}$ 생성능이 높게 나타났으나 결핵균에 대한 효과적인 방어면역효능은 나타나지 않았다. 결 론 : 마우스를 모델로 한 결핵균에 대한 방어면역효능 실험에서, 면역보강제 DDA를 이용한 재조합 단백질 AK, NdK 및 Hsp70을 면역주사 한 경우에는 결핵균의 성장을 효과적으로 조절하지 못하였다. 혼합 단백질 혹은 다른 T세포 면역보강제의 사용에 의한 추시가 필요하다.

• 한국발생생물학회지:발생과생식
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• 제19권3호
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• pp.135-144
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• 2015

Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii.

• 한국해양생명과학회지
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• 제3권2호
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• pp.59-66
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• 2018

갯벌의 건강 수준에 대한 평가는 갯벌에 서식하는 생물의 건강에 의해 평가될 수 있다. 생체지표유전자의 발현 분석을 통하여 갯벌에 서식하는 생물의 건강 수준을 평가할 수 있다. 본 연구의 목적은 heat shock protein 70 (Hsp70), heat shock protein 90 (Hsp90), glutathione S-transferases (GST) 및 thioredoxin (TRX)과 같은 생체지표유전자를 이용하여 서해안 갯벌의 건강을 평가하는 것이다. 이들 유전자는 스트레스, 면역 및 항산화 관련한 유전자들로서 이들 유전자의 발현을 통해 생물의 건강 정도를 관찰하는 데 사용할 수 있다. 본 연구에서는 서해안의 8개 정점에서 바지락(Ruditapes philippinarum)을 수집했다. 유전자의 발현은 RT-qPCR 방법으로 분석하였다. 연구 결과 Hsp70, Hsp90, GST 및 TRX 유전자들의 발현이 8개 정점에서 차별적으로 발현되는 것으로 나타났다. 특히, Hsp90 및 GST의 발현 또는 Hsp70 및 TRX의 발현은 유사하였다. 이것은 각 유전자에 특이적으로 반응하는 물질이 존재하는 것으로 생각된다. 따라서 이화학적 분석결과에 근거하여 분석에 적합한 유전자를 선택할 수 있다고 생각한다. 이 결과는 Hsp70, Hsp90, GST 및 TRX 유전자는 갯벌의 건강을 평가하기 위한 생체지표유전자로서의 역할을 수행함을 시사한다.

• Animal Bioscience
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• 제36권12호
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• pp.1796-1805
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• 2023

Objective: This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. Methods: The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. Results: Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. Conclusion: HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

• IMMUNE NETWORK
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• 제4권1호
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• pp.7-15
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• 2004

Background: The heat shock proteins (HSPs) play an important role in cellular protection mechanisms against physical or chemical stresses. In this study scFv antibodies specific for human HSP70.1 were isolated from a semi-synthetic human scFv library with the ultimate goal of developing anti-HSP70.1 intracellular antibody (intrabody) that may offer an attractive alternative to gene targeting to study the function of the protein in cells. Methods: A semi-synthetic human scFv display library ($5{\times}10^{8}$ size) was constructed using pCANTAB-5E vector and the selection of the library against bacterially expressed recombinant human HSP70.1 was attempted by panning. Results: Three positive clones specific for recombinant HSP70.1 were identified. All three clones used $V_{H}$ subgroup III. On the other hand, $V_{L}$ of two clones belonged to the kappa light chain subgroup I, but the other utilized $V_{k}$ subgroup IV Interestingly, these scFv molecules specifically reacted to the recombinant HSP70.1, yet failed to recognize native HSP70 induced in U937 human monocytic cells by heat treatment. Conclusion: Our results indicated that affinity selection of an scFv phage display library using recombinant antigens produced in E. coli might not guarantee the isolation of scFv antibody molecules specific for a native form of the antigen. Therefore, the source of target antigens needs to be chosen carefully in order to isolate biofunctional antibody molecules.

• 한국발생생물학회지:발생과생식
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• 제14권3호
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• pp.179-184
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• 2010

Water temperature influences on various key biological events in fish, but the internal pathway of the temperature effects are not well understood. Heat shock proteins (HSPs), known to respond in the level of cells to many environmental factors including temperature, could improve our understanding on the pathway. Some biological processes such as gonadal development and sex differentiation in the Nile tilapia Oreochromis niloticus is particularly sensitive to water temperature. In this study, we have investigated the expressions of HSP70 and HSP90 genes in young tilapia at an ordinary temperature ($28^{\circ}C$) and elevated water temperature ($36^{\circ}C$). The distribution of the expressions of HSP70 and HSP90 mRNA in this species were found to be almost ubiquitous, being detected in all tissues studied here (brain, gonad, liver and muscle), suggesting the house keeping functions of these genes. Heat shock by elevating temperature from $28^{\circ}C$ to $36^{\circ}C$ significantly increased the expression of HSP70 mRNA in the gonad, liver and muscle for several hours (P<0.05) (brain tissue was not examined for this). The increased level of HSP70 gene expression recovered to the level at control temperature ($28^{\circ}C$) when fish were kept continuously at high temperature ($36^{\circ}C$) for 24 hours. Contrary to this, expression of HSP90 mRNA did not show significant increase in the gonad and muscle by the same heat shock (P>0.05), except in the liver where the expression of HSP90 mRNA increased continuously for 24 hours at $36^{\circ}C$. The results obtained in this study suggest that response to temperature change in different tissue or organ may utilize different heat shock proteins, and that HSP70 may have some importance in temperature-sensitive gonadal event in the Nile tilapia.

• 한국가금학회지
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• 제43권4호
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• pp.213-218
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• 2016

본 연구는 고온 스트레스 하에 타우린 첨가가 육계의 간에서 heat shock protein 70(hsp 70) 및 cell free system 상태에서 단백질 합성에 미치는 영향을 조사하였다. 공시동물은 1일령 육계 120수를 2주간 사육 후, 대조(CO, $24^{\circ}C$)와 고온스트레스 처리($34^{\circ}C$)에서 타우린을 공급하지 않은 처리구(HO)와 타우린 0.1%를 공급한 처리구(HT)로 나누어 3처리 4반복, 반복 당 10수씩 배치하였다. 고온 스트레스는 3, 6, 9, 12일간 진행하였다. 최종 체중과 간 무게는 HO와 HT가 CO보다 유의적으로 낮았다(P<0.05). 그러나 타우린을 첨가한 HT은 HO보다 유의적으로 높았다(P<0.05). 육계 간에서 hsp70 발현은 HO가 CO와 HT보다 유의적으로 높았으나(P<0.05), CO와 HT는 유의적인 차이가 없었다. In vitro 실험에서 고온 및 타우린은 21일령 육계 간의 총 단백질 합성률에 영향을 미치지 않았다. 따라서 타우린 섭취가 in vivo 육계의 성장에서 고온 스트레스를 완화시키지만, 생체 내의 단백질 합성에 직접적인 영향을 미치지 않는 것을 보이며, 이러한 결과는 타우린이 다양한 생리학적 기전을 통해 단백질 turnover 대사에 간접적으로 영향을 미칠 수 있다는 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제25권8호
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• pp.1096-1101
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• 2012

Intrauterine growth retardation (IUGR) leads to the dysfunction in digestive system, as well as the alteration in the expression of some functional proteins. Heat shock protein 70 (Hsp70) could be induced by various stress factors, but whether Hsp70 expression is changed in neonatal IUGR infants has not been demonstrated. This study was conducted to explore the expression of Hsp70 in the liver by using the IUGR piglet model. Liver and plasma samples were obtained from IUGR and normal birth weight (NBW) piglets at birth. The neonatal IUGR piglets had significantly lower liver weight than their counterparts. The activities of aspartate aminotransferase and alanine aminotransferase in serum were enhanced significantly in IUGR indicating liver dysfunction. The activities of superoxide dismutase (p<0.01), glutathione peroxidase (p<0.01) and catalase (p>0.05) were lower and the level of malondialdehybe was higher (p<0.05) in IUGR liver compared with in NBW. According to the results of histological tests, fatty hepatic infiltrates and cytoplasmic vacuolization were present in the liver of IUGR piglets, but not in NBW liver. The expression of Hsp70 protein was significantly higher (p<0.05) in IUGR piglet liver than in NBW. Similar to where the hepatic injuries were observed, location of Hsp70 was mostly in the midzonal hepatic lobule indicating that oxidative stress might be responsible for the increased expression of Hsp70.

• KSBB Journal
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• 제30권1호
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• pp.21-26
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• 2015

Plasma is an ionized gas mixture, consisting of neutral particles, positive ions, negative electrons, electronically excited atoms and molecules, radicals, UV photons, and various reactive species. Also, plasma has unique physical properties distinct from gases, liquids, and solids. Until now, non-thermal plasmas have been widely utilized in bio-medical applications (called bio-plasma) and have been developed for the plasma-related devices that are used in the medical field. Although numerous bio-plasma studies have been performed in biomedicine, there is no confirmation of the nonthermal effect induced by bio-plasma. Standardization of the biological application of plasma has not been evaluated at the molecular level in living cells. In this context, we investigated the biological effect of bio-plasma on living cells. Hence, we treated the fibroblasts with Dielectric Bauvier Discharge bio-plasma (DBD), and assessed the characteristic change at the molecular level, one of the typical cellular responses. Heat shock protein 70 (HSP70) regulates its own protein level in response to stimuli. HSP70 responds to heat shock by increasing its own expression at the molecular level in cells. Hence, we confirmed the level of HSP70 after treatment of mouse embryonic fibroblasts (MEFs) with DBD. Interestingly, DBD-plasma induced cell death, but there was no difference in the level of HSP70, which is induced by heat shock stimuli, in DBD-treated MEFs. Our data provide the basic information on the interaction between MEFs and DBD, and can help to design a molecular approach in this field.