• BMB Reports
• /
• v.36 no.5
• /
• pp.488-492
• /
• 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
• /
• v.41 no.9
• /
• pp.635-639
• /
• 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.

• Journal of the Korean Applied Science and Technology
• /
• v.35 no.1
• /
• pp.194-204
• /
• 2018

This study was to investigate the effect of the extract(KP-HE) from Kalopanax pictus(KP) fermented with Hericium erinaceum(HE) mycelium on oxidative modification of neurofilament-L(NF-L) which is closely related to neurodegenerative disorders. The oxidative modification of NF-L was induced by AAPH producing peroxyl radicals in solution, and KP, HE, and KP-HE was investigated. KP and HE did not protect NF-L against peroxyl radical-mediated NF-L modification whereas KP-HE significantly prevented NF-L modification induced by peroxyl radical. KP-HE inhibited the formation of dityrosine in oxidative modification of NF-L and stimulated the peroxyl radical scavenging activity. The effects of KP, HE, and KP-HE on the modification of NF-L by tetrahydropapaveroline(THP), a neurotoxin found in patients with Parkinson's disease was investigated. KP-HE also prevented THP-mediated NF-L modification as compared to KP and HE. In addition, KP-HE significantly inhibited the formation of dityrosine in oxidative modified NF-L and enhanced the inhibition of reactive oxygen species(ROS) was generated by THP. The results suggested that KP-HE can contribute to protected cell from oxidative stress was induced by ROS and neurotoxin. Therefore, KP-HE could potentially be used as a valuable functional food ingredient to prevent neurodegenerative disorders.

• Bulletin of the Korean Chemical Society
• /
• v.32 no.9
• /
• pp.3421-3424
• /
• 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.

• Journal of Life Science
• /
• v.8 no.4
• /
• pp.410-415
• /
• 1998

Procine pepsin hydrolysis of hexapeptide L-S-pNF-Nle-A-OMe in the presence of dipeptide L-L generates a new peak on HPLC analysis of reaction mixtures that is not seen when enzyme is incubated with either peptide alone. The peaks can be detected spectroscopically at either 214 or 254 nm, the latter consistent with a new peptide containing the p-nitro-F residue. The data suggest acyl transpeptidation between E(L-S-pNF) and L-L to form L-S-pNF-L-L. Consistent with this inference are (1) the ability of L-L-NH$_{2}$ and inability of Boc-L-L to undergo a similar transpeptidation reaction, and (2) the data from electrospray mass spectrum. This synthesis requires that Nle-A-L-OMe be released before L-S-pNF, an order opposite to that proposed on the basis of product inhibition kinetics. Consistent with this inference are reciprocal solvent isotope effects ; normal isotope effects of 1.736$\pm$0.121 on the formation of Nle-A-L-OMe and 2.281$\pm$0.184 in the formation of L-S-pNF, coupled to an inverse isotope effects of 0.576$\pm$0.045 on the formation of L-S-pNF-L-L. Because transpeptidation precedes faster in D$_{2}$O, the isotopically-sensitive step must occur after release of Nle-A-L-OMe. Isotopically-enhanced transpeptidation is consistent with the Uni-Bi iso memchanism postulated on the basis of an isotope effects on Vmax but not on Vmax/Km$^{1)}$ and confirmed by isotope effects on the onset of inhibition by pepstatin$^{2)}$.

• Biomedical Science Letters
• /
• v.12 no.4
• /
• pp.413-418
• /
• 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
• /
• v.45 no.2
• /
• pp.114-119
• /
• 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
• /
• v.41 no.12
• /
• pp.868-874
• /
• 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.

• Korean Journal of Food Science and Technology
• /
• v.44 no.1
• /
• pp.135-139
• /
• 2012

This study was designed to evaluate the physicochemical properties and biological activities of steamed and fermented Codonopsis lanceolata. The treatments included NS-NF (non-steamed and non-fermented), NS-LF (non-steamed and L. rhamnosus fermented), S-NF (steamed and non-fermented), and S-LF (steamed and L. rhamnosus fermented). Total polyphenol amounts of S-NF and S-LF were significantly increased to more than 26 mg GAE/g. The highest DPPH scavenging activities were observed for S-NF and S-LF, showing $EC_{50}$ values of 0.8 and 0.6 mg/mL, respectively. The growths of Staphylococcus aureus, Listeria monocytogenes, Salmonella Typhimurium, and Shigella boydii were effectively inhibited by S-LF (MIC < 9 mg/mL). The NS-LF and S-LF ($EC_{50}$ <6 mg/mL) effectively inhibited ${\alpha}$-Glucosidase and tyrosinase activities compared to NS-NF ($EC_{50}$ <17 mg/mL). The S-LF exhibited the highest acetylcholinesterase (AChE) inhibitory activity ($IC_{50}$ <32 mg/mL). Therefore, the results suggest that the application of the steaming process combined with probiotic fermentation can effectively enhance the biological and pharmacological activities in C. lanceolata.

• Bulletin of the Korean Chemical Society
• /
• v.28 no.1
• /
• pp.77-80
• /
• 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.