• Journal of Life Science
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• v.16 no.5
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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.

• Journal of Life Science
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• v.32 no.8
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• pp.611-621
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• 2022

Dimethyl sulfoxide (DMSO) is commonly used as control or vehicle solvent in preclinical research of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) due to its ability to dissolve lipophilic compounds and cross the blood brain barrier. However, the biochemical effects of DMSO on the outcomes of preclinical research are often overlooked. In the present study, we investigated whether the long-term oral administration of 5% DMSO affects the neurological, functional, and histological disease phenotype of the copper/zinc superoxide dismutase glycine 93 to alanine mutation (SOD1-G93A) mouse model of amyotrophic lateral sclerosis. SOD1-G93A transgenic mice showed shortened survival time and reduced motor function. We found that administration with DMSO led to increased mean survival time, reduced neurological scores, and improved motor performance tested using the rotarod and grip strength tests. On the other hand, DMSO treatment did not attenuate motor neuron loss in the spinal cord and denervation of neuromuscular junctions in the skeletal muscle. These results suggest that DMSO administration could improve the quality of life of the SOD1-G93A mouse model of ALS without affecting motor neuron denervation. In conclusion, the use of DMSO as control or vehicle solvent in preclinical research may affect the behavioral outcomes in the SOD1-G93A mouse model. The effect of the vehicle should be thoroughly considered when interpreting therapeutic efficacy of candidate drugs in preclinical research.

• Annals of Clinical Neurophysiology
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• v.15 no.2
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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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• v.31 no.3 s.62
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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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• v.25 no.1
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.4
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• pp.389-394
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• 2007

The study was carried out to optimize the electron donating ability (EDA), superoxide dismutase (SOD)-like activity, nitrite scavenging ability, and the inhibitory activities of xanthine oxidase and tyrosinase of Ailanthus altissima ethanol extracts from roots, stems and leaves. The EDA of the roots and stem extracts were 64.04% and 63.27% at $1,000{\mu}g/mL$, respectively. The SOD-like activity of the leaves extract was the highest (50.00%) at $1,000{\mu}g/mL$. The nitrite scavenging ability of the leaves extracts was over 98% at pH 1.2 and 3.0. The xanthine oxidase inhibitory rates of the extracts were $93.62%{\sim}95.40%$ and the tyrosinase inhibitory rate of the roots was the highest (62.01%) at the concentration of $1,000{\mu}g/mL$. These results indicated that the roots extract showed the highest EDA and tyrosinase inhibition, while the leaves extract had tile highest SOD-like activity and nitrate scavenging ability.

• Annals of Clinical Neurophysiology
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• v.8 no.1
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.7
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• pp.972-979
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• 2014

This study was conducted to investigate the physiological characteristics of water and ethanol extracts from leaves and stems of Achyranthes japonica. The highest contents of total polyphenol and flavonoid compounds were 58.27 and 42.22 mg/g in water extract from leaves, respectively. The protein content was the highest at 16.42 mg/g in water extract from leaves. Ethanol extract from stems showed the highest content of reducing sugars at 11.35 mg/g. In the measurement of electron donating ability (EDA), ethanol extract from stems showed the highest EDA at 93.41% at a concentration of 1.0 mg/mL. Superoxide dismutase-like activity of ethanol extract from leaves was the highest at 8.13% at a concentration of 1.0 mg/mL. In the analysis of nitrate scavenging activity, water extract from leaves showed the highest activity at 94.90% at pH 1.2, and the activity increased as concentration increased and pH decreased. In the measurement of xanthine oxidase inhibition, ethanol extract from stems showed the highest inhibitory activity at 66.67% at a concentration of 1.0 mg/mL. Especially, nitrate scavenging activities of water extract from leaves were the highest under all pH conditions. These results verify that extracts from leaves of A. japonica have strong antioxidant activity and can be used as an effective antioxidant source for nutraceutical foods, medicines, and cosmetic stuffs.

• Journal of Ginseng Research
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• v.45 no.3
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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.

• Journal of Life Science
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• v.22 no.9
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• pp.1207-1213
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• 2012

Onion peels is a natural source of high-value functional ingredients produced in the onion industry without suitable processing. The objective of this study was to evaluate characteristics of onion peel extract (OPE), including its biological activities, obtained from solvent extraction in 3 times pilot scales (Lot A, B, and C). Mineral analysis showed that K was present in the largest amount (13,767.56-15,506.78 ppm), followed by Na and Ca at 8,602.44-9,796.00 ppm and 4,255.78-4,903.33 ppm, respectively. The amounts of total phenol, total flavonoid, and quercetin in the OPE were in the ranges of 598.57~626.73, 211.73~233.64, and 93.78~107.29 mg/g, respectively. The biological activities such as antioxidant and effects of fibrinolysis increased in parallel with the concentration of OPE. The $IC_{50}$ value of DPPH radical scavenging activity was in the range of 517.58~557.32 ppm in the OPE. The $IC_{50}$ value for superoxide dismutase (SOD)-like activity was in the range of 11,900.91~12,690.35 ppm. A clear zone of OPE (20,000 ppm) in fibrinolysis test was three times higher than the plasmin as a reference. In conclusion, OPE could be used as a good source of antioxidants and fibrinolytic activities.