• 생명과학회지
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• 제16권5호
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.

• 생명과학회지
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• 제32권8호
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• pp.611-621
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• 2022

DMSO (dimethyl sulfoxide)는 친유성 화합물을 용해하는 성질과 뇌혈관장벽(Blood-brain barrier)을 투과하는 화학적 특성으로 인해 근위축성 측삭경화증(amyotrophic lateral sclerosis) 등의 퇴행성 뇌신경질환을 타겟으로 하는 전임상 연구에서 용매로 널리 활용되고 있다. 그러나 DMSO를 활용한 연구 결과에 대하여 본 물질에 대한 생화학적 효과는 간과되고 있다. 본 연구에서는 근위축성 측삭경화증의 질환동물 모델인 SOD1-G93A형질 전환 마우스에 5% DMSO를 장기간 경구 투여하여 질병 표현형에 미치는 영향을 생존기간을 포함하여 신경학적, 기능학적, 조직학적으로 분석하였다. DMSO를 투여한 SOD1-G93A 동물군에서 DMSO 비투여군 보다 생존 기간과, 로타로드와 악력 측정으로 평가한 근육 기능이 유의미하게 증가했고, neurological score 가 감소했다. 반면, DMSO 투여군에서 DMSO 비투여군 대비하여 척수 운동 신경 세포와 신경근접합부가 보존되지 않았다. DMSO 투여는 SOD1-G93A형질 전환 마우스의 운동 신경 세포의 조직학적 영향을 미치지 않았지만, 신경 증상 완화와 생존 기간 등 개선된 마우스의 quality of life을 확인하였다. 본 연구 결과, DMSO를 이용한 퇴행성 뇌 질환 전임상 연구 및 후보 약물 효능 평가 시 DMSO의 생화학적 특성에 대한 종합적인 고려가 필요한 것으로 보인다.

• Annals of Clinical Neurophysiology
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• 제15권2호
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• pp.53-58
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• 2013

Background: The autophagy is the major route for lysosomal degradation of misfolded protein aggregates and oxidative cell components. We hypothesized that rapamycin (autophagy enhancer) would prolong the survival of motor neuron and suppress the disease progression in amyotrophic lateral sclerosis (ALS). Methods: A total of 24 transgenic mice harboring the human G93A mutated SOD1 gene were used. The clinical status involving rotarod test and survival, and biochemical study of ALS mice model were evaluated. Results: The onset of symptoms was significantly delayed in the rapamycin administration group compared with the control group. However, after the clinical symptom developed, the rapamycin exacerbated the disease progression and shortened the survival of ALS mice model, and apoptosis signals were up-regulated compared with control group. Conclusions: Even though further detailed studies on the relevancy between autophagy and ALS will be needed, our results revealed that the rapamycin administration was not effective for being novel promising therapeutic strategy in ALS transgenic mice and exacerbated the apoptosis.

• Journal of Preventive Medicine and Public Health
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• 제31권3호
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• pp.449-459
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• 1998

This study was started to find out if plasma malondialdehyde(MDA), $\alpha-tocopherol$ and erythrocyte superoxide dismutase(SOD) activity could be markers of biological activity resulting from exposed to lead in workers. Blood samples were randomly obtained from lead-exposed workers(n=29), $CO_2$, welders(n=60) and office workers(n=60). We used whole blood to analyse blood lead with atomic absorption spectrophotometer. Superoxide dismutase activity in erythrocyte was measured with spetrophotometer. MDA and $\alpha-tocopherol$ in plasma were measured with high performance liquid chromatography. Lead-exposed workers was significantly high in blood lead concentration$(29.37{\mu}g/d\ell)$ compared with welders$(6.42{\mu}g/d\ell)$ and office workers$(5.01{\mu}g/d\ell)$. The level of plasma MDA was significantly higher in the lead-exposed workers($1.87{\mu}mol/g$ cholesterol) than the welders($1.41{\mu}mol/g$ cholesterol) and office workers($1.41{\mu}mol/g$ cholesterol). Erythrocyte SOD activity in lead-exposed workers(56.80 U/g Hb) was significantly increased than those of welders(37.63 U/g Hb) and office workers(20.47 U/g Hb). The plasma $\alpha-tocopherol$ level of lead-exposed workers($4.93{\mu}g/g$ cholesterol) was statistically different from welders($4.25{\mu}g/g$ cholesterol) and office workers$4.28{\mu}g/g$ cholesterol). Neither age nor smoking was related to SOD or MDA level. Blood lead was significantly correlated with erythrocyte SOD activity(r=0.405), plasma MDA(r=0.296) and $\alpha-tocopherol$ (r=0.207). Plasma MDA was also significantly correlated with SOD (r=0.217). In multiple regression analysis, the change of MDA and SOD activity level related to the blood lead concentration. These results suggested that the increase of plasma MDA and erythrocyte SOD activity in lead-exposed workers had a close relationship with the oxidative stress induced by lead.

• Molecules and Cells
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• 제25권1호
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• pp.55-63
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• 2008

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of motor neurons. Mutations in the SOD1 gene are responsible for a familial form of ALS (FALS). Although many studies suggest that mutant SOD1 proteins are cytotoxic, the mechanism is not fully understood. To investigate the role of mutant SOD1 in FALS, human SOD1 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce in-frame PEP-1-SOD fusion proteins (wild type and mutants). The expressed and purified PEP-1-SOD fusion proteins were efficiently transduced into neuronal cells. Neurones harboring the A4V, G93A, G85R, and D90A mutants of PEP-1-SOD were more vulnerable to oxidative stress induced by paraquat than those harboring wild-type proteins. Moreover, neurones harboring the mutant SOD proteins had lower heat shock protein (Hsp) expression levels than those harboring wild-type SOD. The effects of the transduced SOD1 fusion proteins may provide an explanation for the association of SOD1 with FALS, and Hsps could be candidate agents for the treatment of ALS.

• 한국식품영양과학회지
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• 제36권4호
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• pp.389-394
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• 2007

한방생약자원으로 사용되는 가죽나무(A. altissima)의 생리학적 활성을 조사하고자 뿌리와 줄기, 잎을 에탄올을 용매로 추출하여 농도에 따라 전자공여능, SOD유사활성, 아질산염 소거능을 측정하였으며, xanthine oxidase와 tyrosinase 저해 활성을 측정하였다. 전자공여능은 $1,000{\mu}g/mL$의 농도에서 뿌리와 줄기 추출물이 64.04%와 63.27%이었으며, 잎 추출물은 17.47%를 나타내었다. SOD 유사활성능은 $7.66%{\sim}50.00%$로 잎 추출물이 뿌리와 줄기에 비해 약 5배 이상 높았고, 아질산염 소거능에 있어서도 가죽나무 잎 추출물이 $1,000{\mu}g/mL$의 농도에서 pH 1.2와 3.0에서 98%이상의 높은 소거효과를 나타내었으며 $500{\mu}g/mL$의 농도에서도 90% 이상의 소거효과를 나타내었다. Xanthine oxidase저해 활성은 $1,000{\mu}g/mL$ 농도에서 뿌리와 줄기, 잎 추출물 모두에서 90% 이상의 저해율을 보였고, tyrosinase저해는 $2,000{\mu}g/mL$의 농도에서 뿌리 추출물이 62.01%로 가장 높은 저해효과를 나타내었으며, $100{\mu}g/mL$의 농도에서도 57.70%의 저해 활성을 나타내었다. 그러므로 가죽나무는 한방생약자원으로 이용되는 뿌리(저근백피) 이외에도 잎은 높은 SOD 유사활성능과 아질산염 소거능을 나타내며 줄기에서도 뿌리보다 높은 전자공여능과 아질산염 소거능 및 유사한 xanthine oxidase 저해 활성을 나타내므로 유용한 한방생약자원인 것으로 판단된다.

• Annals of Clinical Neurophysiology
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• 제8권1호
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• pp.63-70
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• 2006

Background: Testosterone is reported to have neuroprotective effect in various neurological diseases. Recently, the mechanism involved in nitric oxide (NO)-mediated motor neuron death is under extensive investigation. The Cu/Zn-superoxide dismutase (SOD1) mutations has been implicated in selective motor neuron death of amyotrophic lateral sclerosis (ALS) and it is said to play an important role in NO-mediated motor neuron death. However, neuroprotective effect of testosterone on motor neuron exposed to NO has rarely been studied. Methods: Motor neuron-neuroblastoma hybrid cells expressing wild-type or mutant (G93A or A4V) SOD gene were treated with $200{\mu}M$ S-nitrosoglutathione. After 24 hr, cell viability was measured by MTT assay. To see the neuroprotective effect of testosterone, pretreatment with 1 nM testosterone was done 1 hr before S-nitroglutathione treatment. To study the mechanism of protective effect, $20{\mu}M$ flutamide (androgen receptor antagonist) was also pretreated with testosterone 1 hr before S-nitroglutathione treatment. Results: S-nitrosoglutathione showed significant neurotoxic effect in all three cell lines. Percentage of cell death was somewhat different in each cell line. 1 nM testosterone showed neuroprotective effect in G93A and wild-type cell line. In A4V cell line, testosterone did not showed neuroprotective effect. The neuroprotective effect of testosterone was reversed by $20{\mu}M$ flutamide. Conclusions: These results indicate that testosterone induces neuroprotection in NO-mediated motor neuron death directly through the androgen receptor. This neuroprotective effect of testosterone varies according to the types of SOD1 gene mutation. These data suggest that testosterone may be of therapeutic value against ALS.

• 한국식품영양과학회지
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• 제43권7호
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• pp.972-979
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• 2014

한방에서 건조된 뿌리를 '우슬'이라는 약재로 이용되는 쇠무릎의 잎과 줄기를 물과 에탄올을 용매로 추출하여 유용성분과 항산화 활성을 측정하였다. 폴리페놀과 플라보노이드 화합물의 총 함량을 측정한 결과 쇠무릎 잎의 물 추출물인 LWE에서 각각 58.27 mg/g과 42.22 mg/g으로 가장 많은 함량을 나타내었고, 단백질 함량에서도 LWE에서 16.42 mg/g으로 가장 많이 함유된 것으로 분석되었다. 환원당 함량은 줄기의 에탄올 추출물인 SEE에서 11.35 mg/g으로 가장 많았으며 전자공여능은 1.0 mg/mL의 농도에서 SEE가 93.41%로 가장 우수한 전자공여 효과를 보였다. SOD 유사활성능은 1.0 mg/mL의 농도에서 모든 추출물이 10% 미만의 낮은 활성을 나타내었다. 아질산염 소거능은 pH 1.2의 조건의 1.0 mg/mL의 농도에서 LWE가 94.90%의 가장 높은 아질산염 소거능을 나타내었으며, pH 3.0에서도 LWE가 45.20%로 가장 높은 소거 활성을 나타내었다. LWE는 1.0 mg/mL 66.67%로 가장 높은 XO 저해 효과를 나타내었으며, LEE와 SWE도 각각 31.17%와 32.06%의 XO 저해율을 나타내었다. 이상의 결과 쇠무릎 잎은 다량의 폴리페놀 및 플라보노이드 화합물을 함유하였으며 전자공여능과 SOD 유사활성능, 아질산염 소거 효과도 줄기보다 우수하며 식용 및 약용식물로 이용되는 여러 식물보다 우수하여 쇠무릎 잎을 식용뿐만 아니라 기능성 식품의 첨가제나 천연 항산화제로서 활용될 수 있을 것이라 판단된다.

• Journal of Ginseng Research
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• 제45권3호
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• pp.390-400
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• 2021

Background: We recently showed that gintonin, an active ginseng ingredient, exhibits antibrain neurodegenerative disease effects including multiple target mechanisms such as antioxidative stress and antiinflammation via the lysophosphatidic acid (LPA) receptors. Amyotrophic lateral sclerosis (ALS) is a spinal disease characterized by neurodegenerative changes in motor neurons with subsequent skeletal muscle paralysis and death. However, pathophysiological mechanisms of ALS are still elusive, and therapeutic drugs have not yet been developed. We investigate the putative alleviating effects of gintonin in ALS. Methods: The G93A-SOD1 transgenic mouse ALS model was used. Gintonin (50 or 100 mg/kg/day, p.o.) administration started from week seven. We performed histological analyses, immunoblot assays, and behavioral tests. Results: Gintonin extended mouse survival and relieved motor dysfunctions. Histological analyses of spinal cords revealed that gintonin increased the survival of motor neurons, expression of brain-derived neurotrophic factors, choline acetyltransferase, NeuN, and Nissl bodies compared with the vehicle control. Gintonin attenuated elevated spinal NAD(P) quinone oxidoreductase 1 expression and decreased oxidative stress-related ferritin, ionized calcium-binding adapter molecule 1-immunoreactive microglia, S100β-immunoreactive astrocyte, and Olig2-immunoreactive oligodendrocytes compared with the control vehicle. Interestingly, we found that the spinal LPA1 receptor level was decreased, whereas gintonin treatment restored decreased LPA1 receptor expression levels in the G93A-SOD1 transgenic mouse, thereby attenuating neurological symptoms and histological deficits. Conclusion: Gintonin-mediated symptomatic improvements of ALS might be associated with the attenuations of neuronal loss and oxidative stress via the spinal LPA1 receptor regulations. The present results suggest that the spinal LPA1 receptor is engaged in ALS, and gintonin may be useful for relieving ALS symptoms.

• 생명과학회지
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• 제22권9호
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• pp.1207-1213
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• 2012

본 연구에서는 양파가공부산물인 양파껍질을 이용하여 기능성 식품소재로 개발하고자 양파껍질추출물을 제조하였으며 양파껍질추출물에 대한 생리적 기능성과 같은 품질 특성을 조사하였다. 양파껍질추출물에서는 K가 13,767.56~15,506.78 ppm으로 가장 많은 함량을 차지하였고 Na이 8,602.44~9,796.00 ppm, Ca은 4,255.78~4,903.33 ppm으로 세 번째로 많은 함량을 차지하였다. 그리고 Mg, P, Fe은 각각 1,433.11~1,561.22 ppm, 1,212.44~1,428.89 ppm, 760.67~858.89 ppm의 함량을 차지하였고 Zn은 34.11~66.89 ppm으로 미량 검출되었다. Total phenol 함량은 598.57~626.73 mg/g, total flavonoid 함량은 211.73~238.52 mg/g 범위로 quercetin 함량은 93.67~107.29 mg/g의 범위로 나타났다. 양파껍질추출물의 DPPH radical 소거활성은 100 ppm에서 16.45~17.92%, 200 ppm은 29.96~35.41%, 1,000 ppm은 81.05~84.60%의 소거활성을 보였고, SOD 유사활성은 10,000 ppm에서 31.92~39.29%, 20,000 ppm에서 85.85~91.58%의 활성을 나타내었다. 혈전용해능은 20,000 ppm 농도에서는 plasmin에 비해 약 3배 높은 활성을 가졌다. 따라서 양파껍질 추출물은 항산화력 및 항혈전효과능을 가진 기능성소재로서의 활용 가능성을 보였다.