• Clinical and Experimental Reproductive Medicine
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• 제22권2호
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• pp.191-201
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• 1995

Transcriptional activation of the embryonic genome initiates at 2-cell stage in mouse embryo and is characterized by the synthesis of TRC which is restricted to 2-cell stage. To investigate the roles of various protein kinases on the embryonic gene activation, the effects of protein kinase inhibitors on in vitro development and protein synthetic profiles of the early mouse embryos were examinded. None of ${\alpna}-amanitin$ which is a mRNA synthetic inhibitor, H8 which is a PKA inhibitor, and H7 which is a PKC inhibitor, affected on first cleavage of mouse 1-cell embryos in vitro. However, all of these drugs inhibited the second cleavage. When the drugs were removed following treatment for 6 hours, H8 or H7 treatment showed little inhibition on subsequent development of 1-cell embryos to 2-cell stage or further. In contrast, ${\alpna}-amanitin$ irreversibly inhibited the development of 1-cell embryos to 2-cell stage following removal of the drug. Genistein, a TPK inhibitor, inhibited both the first cleavage of 1-cell embryos and the second cleavage of 2-cell embryos, suggesting that TPK activity may be important during the early cleavages. All of the above four drugs inhibited TRC synthesis as shown by the fluorographic analysis of $[^{35}S]-Met$ labeled protein profiles. When late 1-cell embryos were treated with H7 and analyzed synthetic patterns of $[^{35}S]-Met$ labeled protein, the quantitative differences of protein synthesis on SDS-PAGE appeared on 77 kD and 33 kD region at $32{\sim}38$ hours post hCG. From these studies, transcriptional activation of embryonic genome is not essenting to the mouse 1-cell embryos to develop to 2-cell stage. Hawever, TPK activity is reguisite for both the first cleavage and second cleavage. Similarly, both PKC and PKA activities are required for the second cleavage of mouse embryos, but not for the first cleavage.

• Parasites, Hosts and Diseases
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• 제30권2호
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• pp.133-140
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• 1992

T. sergenti merozoite 수용성 항원을 T. sergenti 감염적혈구로부터 분리하고자 저삼투압액으로 용혈, 조직 분쇄기로 분쇄한 후에 고속 원심분리하여 수용성 항원을 얻었으며, SDS-PAGE와 Western blot의 방법으로 29, 34, 35 그리고 105 kD가 함유된 항원을 본 예방접종실험의 항원성 polypeptide로 정하였다. 본 수용성 항원(0.5 mg/ml)을 준비, Freund's adjunant를 이용하여 한우(5개월령)에 경피 접종하였으며, 다시 4주 후에 추가접종하였다. 추가접종 9주 후에 예방 접종군과 대조군에 동종의 냉동충주(5.6$\times$106RBC/dose, 40% 기생률)을 접종시킨 후에 적혈구용적비, 총적혈구수, 기생률, western biot에 의한 특이항체 그리고 간접형광항체(IFA) 등을 관찰하였던 바, 예방접종 후 18주(충 접종 6주 후)에 있어서 예방접종군의 IFA는 10,240이었으나, 대조군은 1,280이었다. 예방접종군에 있어서의 충접종 전후에 있어서의 총적혈구소와 적혈구웅적비는 유의적 차이 (p<0.05)를 나타내지 않았지만, 대조군에 있어서는 적혈구용적비와 총적혈구수에서 있어서 빈혈 소견을 관찰하였다 (p<0.05). 예방접종군의 충전종 후에 있어서의 western blot 반응에서는 29, 34, 35 그리고 105 kD polypeptide의 물질이 면역반응을 잘 나타내고 있어, 이들 polypeptide는 앞으로 vaccine 제조에 활용 가능성이 충분함을 예견할 수 있었다.

• 한국축산식품학회지
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• 제33권4호
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• pp.542-548
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• 2013

살균한 원유에 지방분해효소와 단백질 분해효소활성이 가장 높게 나타났던 균주를 접종하고 저장하면서 내냉성미생물이 생성하는 효소가 원유의 품질에 미치는 영향을 조사하였다. 지방분해효소활성이 높은 Acinetobacter genomospecies 10은 냉장저장기간 중 총 고형분과 유지방의 함량에 영향을 미치지 않았고, 지방의 분해는 냉장보관 14일째에 대조구와 비교하여 2.6배 이상의 유리지방산을 생성하였다. 생성된 지방산의 조성은 short chain free fatty acid(SCFFA), middle chain free fatty acid(MCFFA) 및 long chain free fatty acid(LCFFA)를 모두 생성하였으며, 특히 SCFFA와 MCFFA의 증가율이 높았다. 단백질 분해효소활성이 높은 Serratia liquefaciens은 원유의 총 고형분과 유단백질 함량에 영향을 미치지 않았고, 단백질 조성의 변화는 냉장저장기간 동안 케이신의 함량이 서서히 감소하다가 10일째부터 $\kappa$-케이신의 함량이 현저히 감소하였다. 유리아미노산은 대조구와 비교하여 냉장저장 14일째 2.8배 이상 생성되었으며 특히 소수성 아미노산으로 쓴맛을 내는 leucin, valine 등의 함량이 급격히 증가하였다.

• Plant Resources
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• 제5권1호
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• pp.29-44
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• 2002

In this study, plant regeneration through in vitro culture from plantlet stems of Yooja (C. junos Sieb.) and trifoliate orange (P. trifoliata Rafin.) was attempted to make mass-production system of virus-free plants having the same genotype with mother plant. In order to investigate physiological change depending on the developmental stage of plant regeneration, the changes of total protein, peroxidase and esterase activity and their isozyme patterns as well were examined in 1/2 MS medium. The results are as follows : 1. The MS medium for the optimal callus induction and shoot formation was utilized. The medium was supplemented either with 2,4-D and Kinetin or with BA and NAA. The optimal concentrations were the combination of 1.0mg/ 2,4-D +0.3mg/ Kinetin and 1.0mg BA +0.3mg NAA in callus induction and shoot formation, respectively. 2. For the plant regeneration from somatic embryos, 1/2 MS medium was used with supplements of growth regulators (free, 1.0mg/ IBA +1.0mg/ BA ,0.5mg/ IBA +0.5mg/ BA). Shooting and rooting were the best in the treatment of 0.5mg/ IBA and 0.5mg/ BA combination. 3. The total protein content has a tendency of increase with the developmental stage of embryo, but it was decreased at the plantlet. Also it was the highest at 8 and 6 weeks stage in C. junos Sieb. and P. trioliata Rafin, respectively. In the SDS-PAGE pattern of protein, C. junos Sieb. showed bands of 29.0 and 40kDa at 10 weeks. The 45,66 and 97.4 kDa bands at 10 weeks of culture were shown in P. trifoliata Rafin. 4. The highest esterase activity was shown at the 6 and 8 weeks of culture in C.junos Sieb. and P. trifoliata Rafin.., respectively. 5. Esterase isozyme patterns were shown difference according to the developmental stage. In C. junos Sieb. a new band was observed at pl 7.7 following 4 weeks culture. On the other hand, new bands in P. trifoliata Rafin. were observed at pl 7.5~6.5 following 4 and 6 weeks culture, respectively.

• 대한바이러스학회지
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• 제27권2호
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• pp.239-255
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• 1997

전염성 췌장괴저바이러스 (Infectious pancreatic necrosis virus) DRT 주의 VP1유전자를 대 장균 발현운반체와 Baculovirus에 삽입하여 대장균과 진핵세로에서 VP1단백질의 발현을 연구하였다. 재조합체 pMal-pol 클론 [7]에서 2.7 Kb 단편인 VP1 유전자를 제한효소 XbaI으로 절단하여 Baculovirus 운반체인 pBacPAK9에 클로닝하여 pBacVP1이라 명명하였다. 이 pBacVP1에 클로닝된 VP1유전자를 제한효소 SacI과 PstI으로 절단하여 대장균 발현 운반체인 pQE-30에 클로닝하여 pQEVP1이라 명명하였다. 또한 VP1 단백질의 C-말단에 6개의 히스티딘 $6{\times}His$이 붙어 있는 단백질을 만들기 위하여, pQEVP1 클론의 His부위를 EcoRI으로 절단하고, 또한 pBacVP1을 EcoRI으로 절단하여 생긴 부위에His-EcoRI DNA 단편을 교체시켜 재클로닝하여 pBacHis-VP1을 만들었다. pBacHis-VP1 DNA와 Bsu36I로 처리된 LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV)를 함께 lipofectin을 이용하여 곤충세포 (Spodoptera frugiperda cell)에 동시 감염을 시켜서 재조합 바이러스를 선발하여, VP1-HcNPV-1이라 명명하였다. pQEVP1 클론은 6개의 히스티딘 단편이 부착된 VP1단백질을 Ni-NTA resin 크로마토그래피법으로 정제하여 SDS-PAGE와 Western blot으로 확인하였고, 단백질의 활성과 구조에 영향을 주지 않는 6개의 히스티딘 단편 ($6\;{\times}\;His$)이 부착된 94 kDa의 VP1단백질을 정제할 수 있었다. 또한 재조합 바이러스에 감염된 곤충세포에서 VP1 단백질이 발현된 것을 전기영동과 Western blot으로 검색을 한 결과 95 kDa VP1 단백질이 발현이 되었음을 확인하였다.

• 한국잡초학회지
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• 제9권3호
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• pp.245-249
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• 1989

식물생장(植物生長) 억제제(抑制劑)인 butachlor의 작용기구(作用機構)를 구명(究明)하기 위하여 본(本) 제초제(除草劑)가 생장기본요소(生長基本要素)인 세포분열(細胞分裂) 및 단백질(蛋白質) 합성(合成) 억제(抑制)에 미치는 영향(影響)은 다음과 같다. 1. Butachlor의 세포분열(細胞分裂) 억제(抑制) 효과(效果)는 18시간(時間) 처리후(處理後) $10^{-6}M$은 9.3%, $10^{-5}M$은 28.2%의 억제(抑制)를 보였으며, 특히 $10^{-3}M$ 농도(濃度)에서는 81.6%의 현저한 억제(抑制)를 보임으로서 본(本) 제초제(除草劑)는 농도(濃度)가 높아질수록 세포분열(細胞分裂) 억제효과(抑制效果)도 증가(增加)하였다. 2. 단백질(蛋白質) 억제(抑制) 효과(效果)는 18시간(時間) 처리후(處理後) $10^{-4}M$ 농도(濃度)에서 22.9%, $10^{-3}M$은 34.1%의 억제효과(抑制效果)를 보였다. 또한 24시간(時間) 처리후(處理時) $10^{-4}M$은 34.3%로 로서 18시간(時間) 처리후(處理後)의 $10^{-3}M$과 거의 같은 억제효과(抑制效果)를 보이고 있다. 이와같이 butachlor 는 농도(濃度)와 처리시간(處理時間)이 길어짐에 따라서 단백질(蛋白質) 농도(濃度)의 증가(增加)를 보였다. 3. Butachlor 처리(處理)는 전체적인 단백질(蛋白質) 합성(合成)을 억제(抑制)하였고 특히 분자량 16, 18, 30, 43 그리고 43.5KD의 단백질(蛋白質)을 억제(抑制)하는 것으로 나타났으며 귀리(Avena sativa L.)의 root tip은 100,000 이하의 polypeptide subunit의 단백질로 구성(構成)되었다.

• Journal of Microbiology and Biotechnology
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• 제26권11호
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• 생명과학회지
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• 제28권1호
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• pp.99-104
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• 2018

Streptococcus pneumoniae는 pneumolysin과 같은 병원성 인자를 가지고 있으며, 지역사회에서 전파되는 심각한 병원성균에 포함된다. Pneumolysin (PLY)은 콜레스테롤 의존적으로 세포막에 구멍을 형성하는 세포독소로서 백신의 주요한 표적 항원이다. PLY의 연구를 위하여 Streptococcus pneumoniae D39 균주에서 추출한 genomic DNA를 주형으로 하여 PCR을 시행하였다. 합성된 PLY 유전자 DNA를 pQE-30 vector에 삽입하고, E. coli M15에 형질전환 시킨 후 LB 배지에 IPTG를 첨가하여 PLY 단백질을 생산하였다. 재조합 단백질은 $Ni^{2+}$-agarose column을 사용하여 정제하였다. 또한, EGFP를 PLY C-말단에 부착한 융합단백질도 동일한 방법으로 클로닝하여 재조합 단백질을 생산하였다. 500 ng/ml 농도의 재조합 PLY는 1.0% 적혈구 현탁액을 100% 용혈시켰으며, 240 ng/ml 농도는 50% 용혈을 나타내었다. 그러나 재조합 PLY-EGFP는 용혈 활성이 전혀 나타나지 않았으나 형광현미경으로 관찰하였을 때 적혈구 막에 결합되어 있었다. 즉, EGFP의 PLY C-말단 부착은 PLY의 세포막 결합능은 유지시켰으나 용혈기능은 방해하였다. PLY C-말단은 용혈기능에 아주 중요한 영역이며, 세포막 결합은 PLY의 다른 영역이 보다 중요하게 작용할 것으로 추측된다. 따라서, 육안으로 관찰이 가능한 결합능은 가졌으나 용혈 기능이 결여된 PLY-EGFP를 대조군으로 활용함으로써 두 재조합 단백질은 폐렴 유발에 있어서 PLY 작용 연구에 사용될 수 있을 것으로 기대된다.

• 한국환경생물학회:학술대회논문집
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• 한국환경생물학회 2002년도 학술대회
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• pp.141-144
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• 2002

Environmental estrogens are natural or synthetic substances present in the aquatic environment, especially in effluent from sewage treatment. However, the adverse effects of these estrogenic substances on fish reproduction are unknown. Di-2-ethylhexyl phthalate (DEHP) is the most common phthalate, which Ps used as a plasticizer in polyvinylchloride (PVC), and it is widespread in the environment and has been found in aquatic organisms and sediments. Therefore, juvenile common carps (Cyprinus carpio) were exposed to nominal concentrations of 17$\beta$-estradiol (E2) (0.5, 5, 50 $\mu\textrm{g}$/L) and DEHP (10, 100, 500 $\mu\textrm{g}$/L) for 21 days, to determine the adverse reproductive effects of these compounds on plasma vitellogenin (VTG) induction, sex steroid level, and gonad weight. Electrophoresis (SDS-PAGE) revealed that much of VTG was induced in fish exposed to 5 and 50 E$_2$ $\mu\textrm{g}$/L, but none of DEHP exposure showed induction. Enzyme-linked immunosorbent assay (ELISA) revealed that VTG was significantly induced in fish exposed to 5 and 50 E$_2$ $\mu\textrm{g}$/L, and combination of 50 E$_2$ $\mu\textrm{g}$/L with 10 and 500 DEHP $\mu\textrm{g}$/L, but none of DEHP exposure showed induction. Analysis of sex steroid levels in some fish revealed that testosterone (T) was detected in both male and female fish of the control and DEHP exposures, but none of fish exposed to 22 concentrations had detectable testosterone level. On the other hand, E$_2$ exposure induced 17$\beta$-estradiol in plasma of male fish, but there was no induction of 17$\beta$-estradiol in plasma of male fish exposed to DEHP. Comparison of gonadosomatic index (GSI) revealed that maximal E$_2$ exposure inhibited ovarian growth, but maximal DEHP exposure stimulated testicular growth. The results indicated that those comparisons can be a useful bio-indicator for determining adverse reproductive effect of endocrine disrupting chemicals (EDCs).

• Journal of Microbiology and Biotechnology
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• 제22권3호
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• pp.292-300
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• 2012

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters $K_m$, $V_{max}$, and $k_{cat}$ for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at $50^{\circ}C$, but the optimal temperature of activity was $37^{\circ}C$. It also showed maximal and optimal activities at pH 9.0. The values of $K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$ were $8.9{\pm}0.967{\times}10^{-3}$ M, $128{\pm}2.8$ U/mg protein, $106{\pm}2s^{-1}$, and $1.2{\pm}0.105{\times}10^4M^{-1}s^{-1}$, respectively. The L-asparaginase activity was reduced in the presence of $Mn^{2+}$, $Zn^{2+}$, $Ca^{2+}$, and $Mg^{2+}$ metal ions for about 52% to 31%. In addition, we found that $NH_4{^+}$, L-Asp, D-Asn, and ${\beta}$-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobial-type asparaginase II family.