• Journal of Marine Life Science
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• v.9 no.1
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• pp.9-21
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• 2024

Paralytic shellfish poisoning (PSP) including Saxitoxin (STX) is caused by harmful algae, and poisoning occurs when the contaminated seafood is consumed. The mouse bioassay (MBA), a standard test method for detecting PSP, is being sanctioned in many countries due to its low detection limit and the animal concerns. An alternative to the MBA is the Neuro-2a cell-based assay. This study aimed to establish various test conditions for Neuro-2a assay, including cell density, culture conditions, and STX treatment conditions, to suit the domestic laboratory environment. As a result, the initial cell density was set to 40,000 cells/well and the incubation time to 24 hours. Additionally, the concentration of Ouabain and Veratridine (O/V) was set to 500/50 μM, at which most cells died. In this study, we identified eight concentrations of STX, ranging from 368 to 47,056 fg/μl, which produced an S-shaped dose-response curve when treated with O/V. Through inter-laboratory variability comparison of the Neuro-2a assay, we established five Quality Control Criteria to verify the appropriateness of the experiments and six Data Criteria (Top and Bottom OD, EC₅₀, EC₂₀, Hill slop, and R² of graph) to determine the reliability of the experimental data. The Neuro-2a assay conducted under the established conditions showed an EC₅₀ value of approximately 1,800~3,500 fg/μl. The intra- & inter-lab variability comparison results showed that the coefficients of variation (CVs) for the Quality Control and Data values ranged from 1.98% to 29.15%, confirming the reproducibility of the experiments. This study presented Quality Control Criteria and Data Criteria to assess the appropriateness of the experiments and confirmed the excellent repeatability and reproducibility of the Neuro-2a assay. To apply the Neuro-2a assay as an alternative method for detecting PSP in domestic seafood, it is essential to establish a toxin extraction method from seafood and toxin quantification methods, and perform correlation analysis with MBA and instrumental analysis methods.

• Journal of Korean Neurosurgical Society
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• v.38 no.6
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• pp.456-464
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• 2005

Objective : The authors investigated whether rotenone induces cellular death also in non-dopaminergic neurons and high concentration of potassium ion can show protective effect for non-dopaminergic neuron in case of rotenone-induced cytotoxicity. Methods : Neuro 2A cells was treated with rotenone, and their survival as well as cell death mechanism was estimated using 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium[MTT] assay, Lactate dehydrogenase[LDH] release assay, fluorescence microscopy, and agarose gel electrophoresis. The changes in rotenone-treated cells was also studied after co-treatment of 50mM KCl. And the protective effect of KCl was evaluated by mitochondrial membrane potential assay and compared with the effects of various antioxidants. Results : Neuro 2A cells treated with rotenone underwent apoptotic death showing chromosome condensation and fragmentation as well as DNA laddering. Co-incubation of neuro 2A cells with 50mM KCl prevented it from the cytotoxicity induced by rotenone. Intracellular accumulation of reactive oxygen species[ROS] resulting by rotenone were significantly reduced by 50mM KCl. Potassium exhibited significantly similar potency compared to the antioxidants. Conclusion : The present findings showed that potassium attenuated rotenone-induced cytotoxicity, intracellular accumulation of ROS, and fragmentation of DNA in Neuro 2A cells. These findings suggest the therapeutic potential of potassium ion in neuronal apoptosis, but the practical application of high concentration of potassium ion remains to be settled.

• The Journal of Korean Medicine
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• v.26 no.4
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• pp.74-86
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• 2005

Background: There are increasing neuro-degenerative disorders with aging. Paeoniae Radix(PR) possesses various pharmacological effects such as sedative, analgesic, anti-inflammatory, anti-stress and neuro-protective actions. Also antiaging and anti-cancer effects of PR were reported. Our purpose was to investigate whether PR is useful on the treatment of Parkinson's disease, one of the neuro-degenerative disorders. Objective: We investigated whether Paeonia Radix possesses a protective effect against 1-methyl-4 phenylpyridine(MPP+)-induced cytotoxicity in neuronal cells. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, flow cytometry, DNA fragmentation assay, reverse transcription-polymerase chain reaction(RT-PCR), and Western blotting were performed on SK-N-MC neuroblastoma cells. Results: Cells treated with MPP+ exhibited several apoptotic features, while cells pre-treated with Paeonia Radix prior to MPP+ exposure showed s decrease in the occurrence of apoptotic features. Conclusions: These results suggest that Paeonia Radix may exert a protective effect against MPP+-induced apoptosis in SK-N-MC neuroblastoma cells.

• Korean Journal of Medicinal Crop Science
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• v.27 no.6
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• pp.365-375
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• 2019

Background: Oxidative stress plays an important role in neuro-degenerative disorders such as Alzheimer's disease. Oxidative stress is mediated by reactive oxygen species (ROS), which are implicated in the pathogenesis of numerous diseases, and account for the toxicity of a wide range of compounds. Methods and Results: In order to study the neuro-protective effect of the complex extracts of Aster scaber Thunberg (AS) and Chaenoleles sinensis Koehne (CSK) against hydrogen peroxide in PC12 cells, cell viability was evaluated by the MTT assay using tetrazole, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the intracellular ROS levels were determined the by 2',7'-dichlorofluorescein diacetate (DCF-DA) assay. In order to examine the anti-amnesic effects of the complex extracts of AS and CSK, behavioral tests were performed on male ICR mice. The ameliorating effect of the complex extracts against Aβ_1-42-induced learning and memory impairment was analyzed by y-maze and passive avoidance tests. The AS and CSK extracts showed neuro-protective activity both in vitro and in vivo, and the neuro-protective effect of their 60 : 40 (AS : CSK) mixture was better than that of the other mixtures. Moreover, the complex extracts synergistically inhibited acetylcholinesterase activity and rapid peroxidation. Conclusions: A mixture of the AS and CSK extracts could be used to develop functional foods and serve as raw materials for the development of therapeutics against Alzheimer's disease.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.234-241
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• 2001

The C-terminal 393 bp region of the human interleukin-16 (IL-16) gene was cloned and expressed in E. coli along with mammalian cell lines. Recombinant IL-16 expressed from E. coli was 22 kDa on SDS-PAGE and showed 260% of chemoattractant activity at a concentration of $0.1\;{\mu}g/ml$. HeLa, COS, and Neuro-2a cells were transduced by recombinant retrovirus vector pLNC/IL-16/IRES/TK and the intracellular and secreted amounts of IL-16 produced by HeLa/IL-16/TK, COS/IL-16/TK, and Neuro-2a/IL-16/TK cells were determined by enzyme-linked immunosorbent assay (ELISA). HeLa/IL-16/TK $(1{\times}10^5)$ and COS/IL-16/TK $(1{\times}10^5)$ cells secreted 36.1 and 13.3 ng of IL-16 for 48 h, respectively. Forty-nine ng and 86.4 ng of IL-16 remained in the cell lysates of HeLa/IL-16/TK and COS/IL-16/TK. Intracellular and secreted amounts of IL-16 from Neuro-2a/IL-16/TK $(5{\times}10^5)$ cells during 24 h cultivation were 50 ng and 3.3 ng, respectively. Also, HeLa and COS cells wee stably transfected with mammalian expression vector pCRIII/IL-16. Both culture media and cell lysates prepared from HeLa/IL-16 cells and COS/IL-16 cells showed chemoattractant activity ranging from 190% to 460% as compared to the control experiment. Expression of the herpes simplex virus thymidine kinase (HSV0tk) gene in pLNC/IL-16/ IRES/TK bicistronic retroviral expression vector was verified by performing a genciclovir (GCV) sensitivity assay. Finally, IL-16 repressed Tat-transactivated human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter activity.

• Yonsei Medical Journal
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• v.59 no.9
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• pp.1096-1106
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• 2018

Purpose: Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression. Materials and Methods: Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma ($PPAR-{\gamma}$) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. $PPAR-{\gamma}$ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and $NF-{\kappa}B$ activity was determined by a Caspase 3 Activity Assay Kit or $NF-{\kappa}B$ p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and $PPAR-{\gamma}$ 3'UTR. Results: MiR-128 expression was upregulated and $PPAR-{\gamma}$ expression was downregulated in plasma from AD patients and $amyloid-{\beta}$ $(A{\beta})-treated$ primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased $A{\beta}-mediated$ cytotoxicity through inactivation of $NF-{\kappa}B$ in MCN and N2a cells. Moreover, $PPAR-{\gamma}$ was a target of miR-128. $PPAR-{\gamma}$ upregulation attenuated $A{\beta}-mediated$ cytotoxicity by inactivating $NF-{\kappa}B$ in MCN and N2a cells. Furthermore, $PPAR-{\gamma}$ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and $NF-{\kappa}B$ activity in MCN and N2a cells. Conclusion: MiR-128 inhibitor decreased $A{\beta}-mediated$ cytotoxicity by upregulating $PPAR-{\gamma}$ via inactivation of $NF-{\kappa}B$ in MCN and N2a cells, providing a new potential target in AD treatment.

• Journal of Korean Neurosurgical Society
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• v.50 no.5
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• pp.426-433
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• 2011

Objective : The Histoculture Drug Response Assay (HDRA), which measures chemosensitivity using minced tumor tissue on drug-soaked gelfoam, has been expected to overcome the limitations of in vitro chemosensitivity test in part. We analyzed interim results of HDRA in malignant gliomas to see if the test can deserve further clinical trials. Methods : Thirty-three patients with malignant gliomas were operated and their tumor samples were examined for the chemosensitivity to 10 chosen drugs by HDRA. The most sensitive chemotherapy regimen among those pre-established was chosen based on the number of sensitive drugs or total inhibition rate (IR) of the regimen. The response was evaluated by 3 month magnetic resonance image. Results : Among 13 patients who underwent total resection of the tumor, 12 showed no evidence of disease and one patient revealed progression. The response rate in 20 patients with residual tumors was 55% (3 complete and 8 partial responses). HDRA sensitivity at the cut-off value of more than one sensitive drug in the applied regimen showed a sensitivity of 100%, specificity of 60% and predictability of 70%. Another cut-off value of >80% of total IR revealed a sensitivity of 100%, specificity of 69%, and predictability of 80%. For 12 newly diagnosed glioblastoma patients, median progression-free survival of the HDRA sensitive group was 21 months, while that of the non-sensitive group was 6 months ($p$=0.07). Conclusion : HDRA for malignant glioma was inferred as a feasible method to predict the chemotherapy response. We are encouraged to launch phase 2 clinical trial with chemosensitivity on HDRA.

• Molecules and Cells
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• v.45 no.2
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• pp.76-83
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• 2022

Neurons-on-a-Chip technology has been developed to provide diverse in vitro neuro-tools to study neuritogenesis, synaptogensis, axon guidance, and network dynamics. The two core enabling technologies are soft-lithography and microelectrode array technology. Soft lithography technology made it possible to fabricate microstamps and microfluidic channel devices with a simple replica molding method in a biological laboratory and innovatively reduced the turn-around time from assay design to chip fabrication, facilitating various experimental designs. To control nerve cell behaviors at the single cell level via chemical cues, surface biofunctionalization methods and micropatterning techniques were developed. Microelectrode chip technology, which provides a functional readout by measuring the electrophysiological signals from individual neurons, has become a popular platform to investigate neural information processing in networks. Due to these key advances, it is possible to study the relationship between the network structure and functions, and they have opened a new era of neurobiology and will become standard tools in the near future.

• YAKHAK HOEJI
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• v.49 no.4
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• pp.317-322
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• 2005

Isoflavones are reported to playa role in menopausal women as a phytoestrogen, which can replace estradiols in hormone replacement therapy (HRT). Recently; due to the risk of breast cancer by HRT, phytoestrogens (e.g. isoflavones) have been focused as an alternative therapy in menopause. In the study, we investigated whether isoflavones derived from Sophorae fructus (SISO) have more benefit than that of soybean isoflavones in estrogen deficient rats. We found that SISO effectively controled $H_2O_2$ comparing with the baseline (p<0.01 vs. post value of OVX-Cont), and the blood sugar and weight were also controlled with decreasing patterns. Additionally, in LDH assay for cytoprotective effect in Neuro-2a cell line, SISO protected cells from the damage by SNAP (p<0.05). In conclusion, SISO may have more beneficial effect in enhancing the menopausal signs than that of soybean isoflavones and the cytoprotective effect in neuron cells suggests that SISO can play a certain role in neuroprotection after menopause.

• Journal of Oriental Neuropsychiatry
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• v.18 no.2
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• pp.13-24
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• 2007

Objective : Recent studies indicate that the deposition of ${\beta}-amyloid$ ($A{\beta}$) is related in the pathogenesis of Alzheimer's disease (AD), but the underlying mechanism is still not clear. Method : To investigate the potential cellular functions of APP and water extract of the JangwonHwangagambang (JWHG), we use as in vitro model, neuro 2A cells were treated with either JWHG or its oriental medicines, and the effect in APP expression was determined by MTT and LDH assay. JWHG have been shown to be neuroprotective in different model systems. We asked whether JWHG treatment would influence cell survival and AD-like pathology in APP-induced neuronal cells. Result : JWHG and water extracts of some oriental medicine has attenuated high cell death in vitro. JWHG-treated cells increased percentage of cell survival more longly than controls. JWHG had significantly increas neurite outgrowth in the as compared to control cells. Conclusion : These results suggest that JWHG prevent APP-induced neurotoxicity through attenuating oxidative stress, and may be useful as potential therapeutic agents for AD.