• Journal of Pharmaceutical Investigation
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• 제25권2호
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• pp.145-152
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• 1995

The superoxide dismutase (SOD) mimetic activity of copper complex of 3,5-disopropylsalicylic acid (Cu(II)-DIPS) was tested and compared to those of human recombinant SOD (hrSOD) and its conjugate form with polyethyleneglycol (PEG) using fer- ricytochrome c reduction assay. Stability constant of Cu(II)-DIPS was measured po- tentiometrically using SCOGS2 program. In the presence of 10 g/L albumin, Cu(II)-DIPS lost most of its SOD mimetic activity. HrSOD was modified with polyethylene glycol (PEG) of M.W. 5000. These conjugates have markedly prolonged plasma half-lives of enzymatic activity (15.5 hr) compared to native hrSOD (5 min). In summary, efficient SOD mimetics should be stable enough not to dissociate in blood by serum protein. HrSOD could have longer half-life by conjugation with inert PEG for sustained SOD effect.

• 한국가금학회지
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• 제35권3호
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• pp.241-245
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• 2008

형질 전환 닭 생산 방법들 가운데 목적 유전자 운반에 탁월한 능력이 있는 것으로 알려진 렌티바이러스는 배반엽 단계 수정란을 이용한 형질 전환 닭 생산 연구에 활발하게 이용되고 있다. 본 연구는 재조합 렌티바이러스를 이용하여 사람의 SOD-3 단백질이 닭의 ovalbumin 프로모터에 의해서 유도되는 형질 전환 닭을 생산하고자 하였다. 사람의 SOD-3 단백질은 호흡 과정에서 체내에서 생성되는 활성산소를 중화시키는 탁월한 기능이 있는 것으로 알려져 있다. 후보 병아리의 생산은 앞서 언급한 유전자를 가지는 $1{\times}10^6$ cfu/mL 재조합 렌티바이러스를 배반엽 단계 수정란의 미세 주입하고 대리난각 배양법을 이용하여 배양기에서 21일 동안 배양하는 방법으로 생산하였다. 유전자를 미세주입한 341개의 수정란에서 78수의 후보 형질 전환 병아리를 생산하였으며, 생산된 후보 형질 전환 병아리들의 유전 분석은 PCR 방법을 이용하여 검증하였다. 유전 분석 결과는 성 성숙에 이른 47수의 수컷들 가운데 2수의 정액에서 사람의 SOD-3 유전자가 존재함을 보였다. 이상의 연구 결과는 완전한 형태의 형질전환 닭 생산의 가능성을 보여주고 있다.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2004년도 International Meeting of the Microbiological Society of Korea
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• pp.47-50
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• 2004

The defenses against free radical damage include specialized repair enzymes that correct oxidative damages in DNA, and detoxification systems such as superoxide dismutases. These defenses may be coordinated genetically as global responses. We hypothesized that the expression of the SOD and the DNA repair genes would inhibit DNA damage under oxidative stress. Therefore, the protection of E. coli mutants deficient in SOD and DNA repair genes $(sod^-\;xth^-\;and\;nfo^-)$ was demonstrated by transforming the mutant strain with a plasmid pYK9 which encoded Photobacterium leiognathi CuZnSOD and human AP endonuclease. The results show that survival rates were increased in $sod^+\;xth^-\;nfo^+$ cells compared to $sod^-\;xth^-\;ap^+,\;sod^-\;xth^-\;ap^-,\;and\;sod^+\;xth^-\;ap^-$ cells under oxidative stress generated from 0.1 mM Paraquat or 3 mM $H_2O_2$. The data suggested that, at least, SOD and DNA repair enzymes may have collaborate protection and repair of the damaged DNA. Additionally, both enzymes are required for protection against free radicals.

• BMB Reports
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• 제48권2호
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• pp.91-96
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• 2015

Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of over-expressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

• 미생물학회지
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• 제28권2호
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• pp.91-97
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• 1990

생체내의 유해산소를 제거하는 superoxide dismutase (superoxide : superoxide oxidoreductase E.C.1.15.1.1) 중 세포질내에서 그 활성을 지니는 인체의 세포질 superoxide dismuta~ie (SODl) 유전자를 사람의 간 cDNA library로부터 동위원소로 표지된 oligonucleotide probe를 이용, in situ plaque hybridization 방법으로 선별 분리하여 내장균 벡터로 클로닝하였다. 이 클론은 SOD1 유전자의 5"L"TR과 3’UTR을 포함한 1.6 kb 정도의 cDNA였다 SOD1 구조유전자만을 선택적으로 분리하기 위해서 ATG를 포함하는 sense strand primer와 3’UTR 부위의 antisense strand primer를 이용하여 중합효소연쇄반응(Polymerase Chain Reaction) 방법을 써서 SOD1 구조유전자 부위만을 선택적으로 증폭시켰다. Taq DNA polymerase에 의해 증폭된 DNA를 벡터 pUCl9의 multiple cloning site (MCS) 내의 Hinc II 위치에 넣였으며 이 insert DNA를 M13 mp19으로 옮겨 dideoxy chain termination 방법으로 sequenase를 사용하여 염기서열을 결정하였다. 클론닝된 cDNA는 153개의 아미노산을 포함하고 있는 하나의 open reading frame (ORF)을 가셨다. 중합효소연쇄반응에 의해 이때 증폭된 SOD1 구조유전자를 $\lambda P_{L}$ 프로모터를 포함하고 있는 발현 벡터 pUPL에 옮긴 후 대장균에서 대량으로 발현시켰다. 이때 발현된 단백질 SOD1은 고유의 효소활성을 가지고 있었다.

• 한국산업보건학회지
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• 제19권3호
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• pp.261-269
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• 2009

To evaluate the effect of antioxidant on the cytotoxicity induced by oxidative stress of reactive oxygen species (ROS) in cultured human skin melanocytes, colorimeric assay of XTT and tyrosinase activity assay were adopted after human skin melanocytes were preincubated for 2 hours in the media containing various concentrations of superoxide dismutase (SOD) before the treatment of hydrogen peroxide. Light microscopic study was carried out in same cultures. The results of this study were as follows 1. Cell viability of human skin melanocytes was significantly decreased by 30 and $40{\mu}M$ of hydrogen peroxide($H_2O_2$), respectively. 2. XTT50 was determined at $30{\mu}M$ after human skin melanocytes were treated with $10{\sim}40{\mu}M$ of hydrogen peroxide for 6 hours. 3. The cell viability of cultured human skin melanocytes pretreated with SOD was increased than that of cultured human skin melanocytes treated with $H_2O_2$ dose-dependently. 4. In tyrosinase activity of human skin melanocytes, the cell treated with SOD showed brown stain compared with $H_2O_2$ treated cells, dark stain. 5. In light microscopy, cultured human skin melanocytes exposed to $H_2O_2$ showed morphological changes such as the decreased cell number and cytoplasmic processes, compared with control. 6. In light microscopy, cultured human skin melanocytes pretreated with SOD showed the increase of cell number and cytoplasmic processes compared with $H_2O_2-treated$ group. From these results, it is suggested that oxidative stress of ROS such as $H_2O_2$ has cytotoxicity by showing the decreased cell viability, the increased tyrosinase activity and mophological changes of the decreased cell number and cytoplasmic processes. While, antioxidant like SOD was effective in the prevention of oxidative stress-mediated cytotoxicity by the increased cell viability, decreased tyrosinase activity and the protection of degenerative morphological changes in cultured human skin melanocytes.

• Journal of Pharmaceutical Investigation
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• 제25권4호
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• pp.313-322
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• 1995

The covalent conjugation of human recombinant superoxide dismutase (hrSOD) with trichloros-triazine activated polyethylene glycol (PEG) 5000 formed soluble conjugates with molecular weight of 92KD, which retained $90{\sim}98%$ of original activity with a markedly prolonged plasma half-life of enzyme activity. The effect of hrSOD-PEG conjugates on acetaminophen (ACP)-induced hepatotoxicity was tested in male rats which were pretreated with 3-methylcholanthrene. HrSOD-PEG conjugates inhibited the hepatotoxicity produced by ACP, on the other hand, native hrSOD had no protective effect. The above results indicated that oxygen radicals might participate in the mechanism of the ACP-induced hepatotoxicity and that polymer conjugated-protein drugs with prolonged half-lives could be employed as an effective therapeutic agent.

• BMB Reports
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• 제40권5호
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• pp.684-689
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• 2007

The endogenous neurotoxin, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), has been considered a potential causative factor for the pathogenesis of Parkinson's disease (PD). In the present study, we examined the pattern of human Cu,Zn-superoxide dismutase (SOD) modification elicited by salsolinol. When Cu,Zn-SOD was incubated with salsolinol, some protein fragmentation and some higher molecular weight aggregates were occurred. Salsolinol led to inactivation of Cu,Zn-SOD in a concentration-dependent manner. Free radical scavengers and catalase inhibited the salsolinol-mediated Cu,Zn-SOD modificaiton. Exposure of Cu,Zn-SOD to salsolinol led also to the generation of protein carbonyl compounds. The deoxyribose assay showed that hydroxyl radicals were generated during the oxidation of salsolinol in the presence of Cu,Zn-SOD. Therefore, the results indicate that free radical may play a role in the modification and inactivation of Cu,Zn-SOD by salsolinol.

• BMB Reports
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• 제50권2호
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• pp.85-90
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• 2017

Recently, we demonstrated that superoxide dismutase 3 (SOD3) is a strong candidate for biomedicine. Anti-oxidant function of SOD3 was accomplished without cell penetration, and it inhibited the inflammatory responses via non-enzymatic functions. SOD3 has the heparin binding domain associating cell surface. Interestingly, we found that $Zn^{2+}$ promotes transduction effects of recombinant human SOD3 (rhSOD3) by increasing uptake via the heparin binding domain (HBD). We demonstrated an uptake of rhSOD3 from media to cell lysate via HBD, resulting in an accumulation of rhSOD3 in the nucleus, which was promoted by the presence of $Zn^{2+}$. This resulted in increased inhibitory effects of rhSOD3 on NF-{\kappa}B and STAT3 signals in the presence of $Zn^{2+}$, which shows elevated association of rhSOD3 into the cells. These results suggest that an optimized procedure can help to enhance the inflammatory efficacy of rhSOD3, as a novel biomedicine.

• Molecules and Cells
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• 제39권3호
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• pp.242-249
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• 2016

A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.