• BMB Reports
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• 제36권5호
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• pp.488-492
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• 2003

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for neuronal survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of copper and peroxide in the modification of NF-L. When disassembled NF-L was incubated with copper ion and hydrogen peroxide, then the aggregation of protein was proportional to copper and hydrogen peroxide concentrations. Dityrosine crosslink formation was obtained in copper-mediated NF-L aggregates. The copper-mediated modification of NF-L was significantly inhibited by thiol antioxidants, N-acetylcysteine, glutathione, and thiourea. A thioflavin-T binding assay was performed to determine whether the copper/$H_2O_2$ system-induced in vitro aggregation of NF-L displays amyloid-like characteristics. The aggregate of NF-L displayed thioflavin T reactivity, which was reminiscent of amyloid. This study suggests that copper-mediated NF-L modification might be closely related to oxidative reactions which may play a critical role in neurodegenerative diseases.

• BMB Reports
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• 제41권9호
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• pp.635-639
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• 2008

Acrolein is a highly reactive by product of lipid peroxidation and individuals with neurodegenerative disorders have been shown to contain elevated concentrations of this molecule in the brain. In the present study, we examined the pattern of neurofilament-L (NF-L) modification elicited by acrolein. When NF-L was incubated with acrolein, protein aggregation occurred in a acrolein concentration-dependent manner. Exposure of NF-L to acrolein also led to the generation of protein carbonyl compounds. Through the addition of free radical scavengers we observed a significant decrease in acrolein-mediated NF-L aggregation. These results indicate that free radicals may be involved in the modification of NF-L by acrolein. In addition, dityrosine crosslink formation was observed in acrolein-mediated NF-L aggregates and these aggregates displayed thioflavin T reactivity, reminiscent of amyloid. This study suggests that acrolein-mediated NF-L aggregation might be closely related to oxidative reactions, thus these reactions may play a critical role in neuro-degenerative diseases.

• 한국응용과학기술학회지
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• 제35권1호
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• pp.194-204
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• 2018

본 연구는 신경퇴행성질환과 밀접한 관련이 있는 neurofilament-L(NF-L)의 산화적 손상에 엄나무 발효물이 미치는 영향을 알아보고자 하였다. 용액 상에서 peroxyl radical을 생성하는 AAPH를 처리하여 NFL의 산화적 변형을 유도하고 엄나무 추출물(KP), 노루궁뎅이버섯 균사체 추출물(HE), 엄나무 발효물(KP-HE)을 각각 처리하여 어떤 영향을 미치는 지를 알아보고자 하였다. KP와 HE는 peroxyl radical에 의한 NF-L의 산화적 손상을 막지 못했으나 KP-HE는 NF-L의 변형을 효과적으로 억제하였다. KP-HE는 peroxyl radical에 의한 NF-L 변형에서 나타나는 dityrosine 형성을 효과적으로 억제하였고 peroxyl radical 소거활성도 증가시켰다. 파킨슨병 환자에서 발견되는 신경독성물질인 tetrahydropapaveroline(THP)에 의한 NFL의 변형에 KP, HE, KP-HE가 미치는 영향을 관찰하였다. 그 결과 KP-HE는 KP와 HE에 비해서 THP에 의한 NF-L의 변형을 효과적으로 억제하는 것으로 관찰되었다. 또한 KP-HE는 THP에 의한 NF-L 변형에서 나타나는 dityrosine 형성도 효과적으로 억제하였고 THP에 의해 유도되는 활성산소 생성도 현저히 감소시켰다. 이와 같은 결과들은 KP-HE가 활성산소와 신경독성물질에 의한 산화적 스트레스로부터 세포를 보호해 줄 수 있을 것으로 사료되었다. 그러므로 엄나무 발효물은 신경퇴행성질환을 예방할 수 있는 식품소재로 이용될 수 있을 것으로 사료된다.

• Bulletin of the Korean Chemical Society
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• 제32권9호
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• pp.3421-3424
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• 2011

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 this study, we examined oxidative modification of neurofilament-L (NF-L) induced by salsolinol. When disassembled NF-L was incubated with salsolinol, the aggregation of protein was increased with the concentration of sasolinol. The formation of carbonyl compound was obtained in salsolinol-mediated NF-L aggregates. This process was protected by free radical scavengers, such as N-acetyl-L-cysteine and glutathione. These results suggest that the aggregation of NF-L is mediated by salsolinol via the generation of free radicals. We also investigated the effects of copper ion on salsolinol-mediated NF-L modification. In the presence of copper ions, salsolinol enhanced the modification of NF-L. We suggest that salsolinol might be related to abnormal aggregation of NF-L which may be involved in the pathogenesis of neurodegenerative diseases and related disorders.

• 생명과학회지
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• 제8권4호
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• pp.410-415
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• 1998

HPLC 및 electrospary mass spectrum으로부터 L-L dipeptide의 존제하에서 pepsin은 hexapeptide인 L-S-pNF-Nle-A-OMe를 가수분해하여 가수분해물외의 새로운 생성물을 합성하는 것이 확인되었다. 이 생성물은 254nm에서 p-nitro-Phe 잔기를 포함하는 peptide였다. 실험결과로부터 E(L-S-pNF)와 L-L 사이의 acyl transpeptidation에 의해 L-S-pNF-L-L가 생성됨을 뒷받침한다. 이러한 transpeptidation 결과는 product 저해실험에 의한 결과에 기초한 것과는 반대로 L-S-pNF가 해리되기전에 Nle-A-L-OMe가 먼저 한다는 것을 보여준다. 그리고, electrospray mass spectrum 으로부터 위에서 검출된 새로운 펩티드에 해당하는 peak (MW 636.1)을 얻었는데, 이는 새 펩티드의 생성을 확실히 증명하는 증거이다. 한편, Nle-A-L-OMe 생성에 대한 solvent isotope effect는 1.736$\pm$0.121이며 L-S-pNF는 2.28$\pm$0.184 그리고 L-S-pNF-L-L의 생성에는 inverse isotope effect로서 0.576$\pm$0.045였는데, 이는 상기 생성물 해리 순서를 확인시켜 준다. D$_{2}$에서 transpeptidation은 더 빠르기 때문에 isotopically-sensitive단계는 Nle-A-L-OMe해리후에 존재하는 것을 알 수 있다. 본 실험결과는, Rebholz and Northrop$^{1)}$ 및 Cho등의 $^{2)} iso-mechanism이론의 타당성을 제시한다.

• 대한의생명과학회지
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• 제12권4호
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• pp.413-418
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• 2006

Charcot-Marie-Tooth disease (CMT) is blown as one of the inherited disorder of peripheral nervous system. Recently, it was found that point mutations in the neurofilament light subunit (NF-L) gene cause CMT. Neurofilaments (NFs) are heteropolymers consist of NF-L, NF-M and NF-H. To assess the relationship between CMT and NF-L mutation in cellular level, we performed phenotypic analysis of the mutant NF-L (E397K) using cultured cell lines. Vimentin-deficient human adrenal carcinoma SW13 (Vim-) cells have a potential to form the intermediate filaments when the cells are expressing both NF-L and NF-M. Our results show that co-expression of wild type NF-L with NF-M showed intermediate filament formation in SW13 (Vim-) cells, while E397K with NF-M did not. This result means that E397K mutant lost its ability to form the intermediate filament in vivo, and further suggests that the E397K mutation is closely related to CMT.

• BMB Reports
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• 제45권2호
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• pp.114-119
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• 2012

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegeneration. Oxidative modification of neurofilament proteins has been implicated in the pathogenesis of neurodegenerative disorders. In this study, oxidative modification of neurofilament-L (NF-L) by salsolinol and the inhibitory effects of histidyl dipeptides on NF-L modification were investigated. When NF-L was incubated with 0.5 mM salsolinol, the aggregation of protein was increased in a time-dependent manner. We also found that the generation of hydroxyl radicals (${\bullet}OH$) was linear with respect to the concentrations of salsolinol as a function of incubation time. NF-L exposure to salsolinol produced losses of glutamate, lysine and proline residues. These results suggest that the aggregation of NF-L by salsolinol may be due to oxidative damage resulting from free radicals. Carnosine, histidyl dipeptide, is involved in many cellular defense processes, including free radical detoxification. Carnosine, and anserine were shown to significantly prevent salsolinol-mediated NF-L aggregation. Both compounds also inhibited the generation of ${\bullet}OH$ induced by salsolinol. The results indicated that carnosine and related compounds may prevent salsolinol-mediated NF-L modification via free radical scavenging.

• BMB Reports
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• 제41권12호
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• pp.868-874
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• 2008

Neurofilaments (NFs) are neuronal intermediate filaments composed of light (NF-L), middle (NF-M), and heavy (NF-H) subunits. NF-L self-assembles into a "core" filament with which NF-M or NF-H co-assembles to form the neuronal intermediate filament. Recent reports show that point mutations of the NF-L gene result in Charcot-Marie-Tooth disease (CMT). However, the most recently described rod domain mutant of human NF-L (A148V) has not been characterized in cellular level. We cloned human NF-L and used it to engineer the A148V. In phenotypic analysis using SW13 cells, A148V mutation completely abolished filament formation despite of presence of NF-M. Moreover, A148V mutation reduced the levels of in vitro self-assembly using GST-NF-L (H/R) fusion protein whereas control (A296T) mutant did not affect the filament formation. These results suggest that alanine at position 148 is essentially required for NF-L self-assembly leading to subsequent filament formation in neuronal cells.

• 한국식품과학회지
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• 제44권1호
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• pp.135-139
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• 2012

본 연구에서는 증숙 및 발효에 의한 더덕(Codonopsis lanceolata)의 이화학적 특성과 생리활성의 변화에 미치는 영향에 대해 조사하였다. 건조된 더덕 시료를 증숙처리한 후 Lactobacillus rhamnosus를 접종하여 $37^{\circ}C$에서 7일간 혐기적 조건에서 발효시켰다. 비증숙과 비발효 더덕추출물(NS-NF)을 대조군으로 하여 처리군에는 비증숙과 발효 더덕추출물(NS-LF), 증숙과 비발효 더덕 추출물(S-NF), 증숙과 발효 더덕추출물(S-LF)을 포함한다. 총 폴 리페놀성 화합물 함량은 NS-NF(8.9 mg GAE/g)와 비교하여 S-NF와 S-LF에서 유의적으로 증가되었다(26 mg GAE/g). 총 플라보노이드 함량은 S-NF(8.1 mg RE/mL)와 S-LF(7.8 mg RE/mL)에서 가장 높았다. S-NF와 S-LF의 $EC_{50}$ 값이 각각 0.8과 0.6 mg/mL으로 높은 항산화능을 보였다. Staphylococcus aureus, Listeria monocytogenes, Salmonella Typhimurium, Shigella boydii의 생육은 S-LF에 의해 효과적으로 저해되었다(MIC<9 mg/mL). NS-NF($EC_{50}$ <17 mg/mL)에 비교하여 NS-LF와 S-LF($EC_{50}$ <6 mg/mL)가 가장 높은 ${\alpha}$-glucosidase와 tyrosinase 저해효과를 나타냈다. Aacetylcholinesterase(AChE)는 S-LF($EC_{50}$ <32 mg/mL)에 의해 효과적으로 저해되었다. 따라서 본 연구의 결과는 증숙에 의해 더덕의 총 페놀 함량, 플라보노이드 함량, 항산화능이 향상되었으며 발효에 의해 특이적으로 항미생물 효과, 효소활성저해 및 인지기능 개선 등과 같은 생리활성을 효과적으로 증진시켰다.

• Bulletin of the Korean Chemical Society
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• 제28권1호
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• pp.77-80
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• 2007

As neurofilament proteins are major cytoskeletal components of neuron, abnormality of neurofilament is proposed in brain with neurodegenerative disorders such as Parkinson's disease (PD). Since oxidative stress might play a critical role in altering normal brain proteins, we investigated the oxidative modification of neurofilament-L (NF-L) induced by the reaction of cytochrome c with H2O2. When NF-L was incubated with cytochrome c and H2O2, the protein aggregation was increased in cytochrome c and H2O2 concentrationsdependent manner. Radical scavengers, azide, formate and N-acetyl cysteine, prevented the aggregation of NFL induced by the cytochrome c/H2O2 system. The formations of carbonyl group and dityrosine were obtained in cytochrome c/H2O2-mediated NF-L aggregates. Iron specific chelator, desferoxamine, prevented the cytochrome c/H2O2 system-mediated NF-L aggregation. These results suggest that the cytochrome c/H2O2 system may be related to abnormal aggregation of NF-L which may be involved in the pathogenesis of PD and related disorders.