• Archives of Plastic Surgery
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• 제40권5호
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• pp.517-521
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• 2013

Background Diabetes is characterized by chronic hyperglycemia, which can increase reactive oxygen species (ROS) production by the mitochondrial electron transport chain. The formation of ROS induces oxidative stress and activates oxidative damage-inducing genes in cells. No research has been published on oxidative damage-related extracellular superoxide dismutase (EC-SOD) protein levels in human diabetic skin. We investigated the expression of EC-SOD in diabetic skin compared with normal skin tissue in vivo. Methods The expression of EC-SOD protein was evaluated by western blotting in 6 diabetic skin tissue samples and 6 normal skin samples. Immunohistochemical staining was also carried out to confirm the EC-SOD expression level in the 6 diabetic skin tissue samples. Results The western blotting showed significantly lower EC-SOD protein expression in the diabetic skin tissue than in the normal tissue. Immunohistochemical examination of EC-SOD protein expression supported the western blotting analysis. Conclusions Diabetic skin tissues express a relatively small amount of EC-SOD protein and may not be protected against oxidative stress. We believe that EC-SOD is related to the altered metabolic state in diabetic skin, which elevates ROS production.

• 한국환경농학회지
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• 제29권1호
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• pp.77-85
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• 2010

Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese superoxide dismutase (MnSOD) gene isolated from wheat. Although all QC 871 transformants grown at $37^{\circ}C$ expressed mRNA of MnSOD variants, only MnSOD2 transformant had functional SOD activity. MnSOD3 expressed active protein when grown at $22^{\circ}C$, however, MnSOD1 did not express functional protein at any growing and induction conditions. The sequence comparison of the wheat MnSOD variants revealed that the only amino acid difference between the sequence MnSOD2 and sequences MnSOD1 and 3 is phenylalanine/serine at position 58 amino acid. We made MnSOD2S58F gene, which was made by altering the phenylalaine to serine at position 58 in MnSOD2. The expressed MnSOD2S58F protein had functional SOD activity, even at higher levels than the original MnSOD2 at all observed temperatures. These data suggest that amino acid variation can result in highly active forms of MnSOD and the MnSOD2S58F gene can be an ideal target used for transforming crops to increase tolerance to environmental stresses.

• 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.

• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.70-79
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• 2014

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including $P_{tac}$, $P_{sod}$, $P_{sod}$ with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, $P_{ilvC}$, $P_{ilvC}$ with a conserved SD-1 ($P_{ilvC-M1}$), and $P_{ilvC}$ with a conserved SD-2 ($P_{ilvC-M2}$), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, $P_{sod}$ and $P_{sod-M}$ were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and $P_{sod-M}$ displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of $P_{ilvC}$ > $P_{ilvC-M1}$ > $P_{sod}$ > $P_{ilvC-M2}$ > $P_{sod-M}$, whereas all plasmids except pCSP30 with $P_{sod}$ displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with $P_{sod-M}$, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.

• 대한한의학방제학회지
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• 제26권2호
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• pp.103-111
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• 2018

Objectives : The purpose of this study was to examine the antioxidant effects of the extract of Rubi Fructus on TM4 Sertoli cells. Methods : The extract was studied for diphenyl-picryl-hydrazyl (DPPH) radical scavenging activity and cell viability assays on Sertoli cells. In addition, hydrogen peroxide-induced oxidative stress on Sertoli cells were examined by MTT assay. The antioxidant enzyme of Cu/Zn SOD, Mn SOD, catalase protein expression on Sertoli cells were also measured. Results : The results showed that the extract scavenged DPPH radical dose-dependent manner. The extract showed no cytotoxicity at concentration of 1, 5, 10, 50, $100{\mu}g/ml$. The hydrogen peroxide-induced cytotoxicity of Sertoli cells was protected to 88.3% by the extract at concentration of $100{\mu}g/ml$. Cu/Zn SOD and Mn SOD protein expression were significantly increased on Sertoli cells, but catalase protein expression was not significantly changed. Conclusions : In conclusion, the extract of Rubi Fructus has antioxidant effects on Sertoli cells and protect male reproductive system against oxidative stress.

• 운동영양학회지
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• 제13권2호
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• pp.161-167
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• 2009

The purpose of this study was to investigate whether two different exercises, acute exhaustive exercise and long-term endurance exercise training could affect to the expression of Mn-SOD, HSP70, and PPAR-γ protein in myocardium. The Wistar-Kyoto rats(n=24, 4 weeks) were used and randomly divided into 3 groups; endurance exercise training group (EET, n=8), acute exhaustive exercise group (AEE, n=8) or control group (CON, n=8). EET performed treadmill exercise for 12 weeks (5 days/week, 30~60 min/day). AEE exercised treadmill running (speed increased gradually to 14-26 m/min, 60 min ±10min) until exhausted when EET finished the program. Then, all the rats were sacrificed 48 hours rest at least after the last session of their own exercise program. Hearts were isolated and then the expression of Mn-SOD, HSP70, and PPAR-γ were analyzed by western blotting. One-way repeated ANOVA was used and p value under 0.05 was considered as statistical significance. The results were followed as; the expression of Mn-SOD of AEE was decreased compared with CON. However, the expression of Mn-SOD of EET was increased compared with CON. There was significant difference between AEE and EET in the expression of Mn-SOD. The expressions of HSP70 and PPAR-γ in the both AEE and EET were significantly increased compared with CON. In conclusion, acute exhaustive exercise might induce oxidative stress wheres endurance exercise training could ameliorate the oxidative conditions by increase of Mn-SOD, HSP70, and PPAR-γ. Therefore, we suggested that endurance exercise training could enhance the complementary antioxidant system and improve to prevent apoptosis. Further, a long-term moderat aerobic exercise program might play a important role in mitochondrial biogenesis in the heart.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.229-234
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• 2006

We have generated transgenic mice that expressed mouse extracellular superoxide dismutase (EC-SOD) in their skin. In particular, the expression plasmid DNA containing human keratin K14 promoter was used to direct the keratinocyte-specific transcription of the transgene. To compare intron-dependent and intron-independent gene expression, we constructed two vectors. The vector B, which contains the rabbit -globin intron 2, was not effective for mouse EC-SOD overexpression. The EC-SOD transcript was detected in the skin, as determined by Northern blot analysis. Furthermore, EC-SOD protein was detected in the skin tissue, as demonstrated by Western blot analysis. To evaluate the expression levels of EC-SOD in various tissues, we purified EC-SOD from the skin, lungs, brain, kidneys, livers, and spleen of transgenic mice and measured its activities. EC-SOD activities in the transgenic mice skin were approximately 7 fold higher than in wild-type mice. These results suggest that the mouse overexpressing vector not only induces keratinocyte-specific expression of EC-SOD, but also expresses successfully functional EC-SOD. Thus, these transgenic mice appeared to be useful for the expression of the EC-SOD gene and subsequent analysis of various skin changes, such as erythema, inflamation, photoaging, and skin tumors.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.700-706
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• 2000

Iron- and zinc-containing superoxide dismutase (FeZnSOD) and nickel-containing superoxide dismutase (NiSOD) are cytoplamic enzymes in Streptomyces griseus. The sodF gene coding for FeZnSOD was cloned from genomic Southern hybridization analysis with a 0.5-kb DNA probe, which was PCR-amplified with facing primers corresponding to the N-terminal amino acid of the purified FeZnSOD of S. griseus and a C-terminal region which is conserved among bacterial FeSODs and MnSODs. The sodF open reading frame (ORF) was comprised of 213 amino acid (22,430 Da), and the deduced sequence of the protein was highly homologous (86% identity) to that of FeZnSOD of Streptomyces coelicolor. The FeZnSOD expression of exponentially growing S. griseus cell was approximately doubled as the cell growth reached the early stationary phase. The growth-associated expression of FeZnSOD was mainly controlled at the transcriptional level, and the regulation was exerted through the 110 bp regulatory DNA upstream from the ATG initiation codon of the sodF gene.

• 생명과학회지
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• 제22권7호
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• pp.857-862
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• 2012

본 연구는 안정 시 고지방식이 흰쥐의 신장, 간장 그리고 골격근에서 지질과산화와 SOD 단백질 발현에 손바닥 선인장 보충이 미치는 효과에 대하여 연구하였다. SD계 수컷 흰쥐 16마리를 무작위로 대조군(CG, n=8)과 실험군(EG, n=8)으로 분류하였다. 8주 동안 대조군은 고지방식이를 부하하였으며, 실험군은 5% 손바닥선인장을 보충식 이하였다. 본 실험결과, 간장과 신장의 MDA 농도는 EG군이 CG군에 비해 유의하게 낮게 나타났다(p<0.01). 또한 골격근의 MDA 농도도 EG군이 CG군에 비해 높은 경향이 나타났으나, 통계적으로 유의한 차이는 나타나지 않았다. Cu,Zn-SOD 단백질 발현은 신장에서 EG군이 CG군에 비해 유의하게 증가하였다(p<0.01). Mn-SOD 단백질 발현은 골격근에서 EG군이 CG군에 비해 유의하게 증가하였다(p<0.01). 이상의 결과로부터 손바닥선인장 보충이 고지방식이 흰쥐의 지질과산화 억제작용과 SOD단백질 발현이 기관 특이적인 반응의 항산화 효과가 있는 것으로 나타났다.

• Clinical and Experimental Pediatrics
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• 제62권12호
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• pp.444-449
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• 2019

Background: Sixty percent of infants with severe neonatal hypoxic-ischemic encephalopathy die, while most survivors have permanent disabilities. Treatment for neonatal hypoxic-ischemic encephalopathy is limited to therapeutic hypothermia, but it does not offer complete protection. Here, we investigated whether hypoxia-inducible factor (HIF) promotes cell survival and suggested neuroprotective strategies. Purpose: HIF-1α deficient mice have increased brain injury after neonatal hypoxia-ischemia (HI), and the role of HIF-2α in HI is not well characterized. Copper-zinc superoxide dismutase (SOD)1 overexpression is not beneficial in neonatal HI. The expression of HIF-1α and HIF-2α was measured in SOD1 overexpressing mice and compared to wild-type littermates to see if alteration in expression explains this lack of benefit. Methods: On postnatal day 9, C57Bl/6 mice were subjected to HI, and protein expression was measured by western blotting in the ipsilateral cortex of wild-type and SOD1 overexpressing mice to quantify HIF-1α and HIF-2α. Spectrin expression was also measured to characterize the mechanism of cell death. Results: HIF-1α protein expression did not significantly change after HI injury in the SOD1 overexpressing or wild-type mouse cortex. However, HIF-2α protein expression increased 30 minutes after HI injury in the wild-type and SOD1 overexpressing mouse cortex and decreased to baseline value at 24 hours after HI injury. Spectrin 145/150 expression did not significantly change after HI injury in the SOD1 overexpressing or wild-type mouse cortex. However, spectrin 120 expression increased in both wild-type and SOD1 overexpressing mouse at 4 hours after HI, which decreased by 24 hours, indicating a greater role of apoptotic cell death. Conclusion: HIF-1α and HIF-2α may promote cell survival in neonatal HI in a cell-specific and regional fashion. Our findings suggest that early HIF-2α upregulation precedes apoptotic cell death and limits necrotic cell death. However, the influence of SOD was not clarified; it remains an intriguing factor in neonatal HI.