• Applied Microscopy
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• v.41 no.2
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• pp.109-116
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• 2011

Polycystic ovary syndrome (PCOS) is hormonal imbalance condition as the endocrine and metabolic disorder that induces the infertility and various complications in reproductive age women. Estradiol valerate (EV) is used hormone replacement therapy in menopausal women and is reported that excessive administration of EV induces the PCOS. Nerve growth factor (NGF) is the factor to regulate the survival and maturation of developing neuronal cell and is also synthesized in ovary. And NGF is overexpressed in EV-induced polycystic ovary (PCO) as previously reported. Therefore, this study examined the possibility of NGF as can be used the biological marker in diagnosis of PCOS, the hormonal imbalance condition, using PCO and CHO (chinese hamster ovarian) cell lines. The concentration of EV treatment is optimized a 1 mg as not influence on the proliferation of CHO cell but 2 mg and 3 mg of EV treatment have the inhibition effect at initial stage. The morphological change was not observed in CHO cell after dose dependent manner treatment of EV. Expression of NGF mRNA and protein is significantly increased at 30 min after EV treatment in CHO cells compared to that of control. And NGF protein expression is strongly increased in PCO tissue, which observed many follicular cysts compared to normal ovary tissue. Taken together, overexpression of NGF may be act as a molecule to induce an abnormal development of follicle, suggesting that NGF can be used as a biological marker in diagnosis of PCOS.

• Journal of Nutrition and Health
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• v.50 no.5
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• pp.415-425
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• 2017

Purpose: Many studies have suggested that neuronal cells protect against oxidative stress-induced apoptotic cell death by polyphenolic compounds. We investigated the neuroprotective effects and the mechanism of action of Momordica charantia ethanol extract (MCE) against $H_2O_2-induced$ cell death of human neuroblastoma SK-N-MC cells. Methods: The antioxidant activity of MCE was measured by the quantity of total phenolic acid compounds (TPC), quantity of total flavonoid compounds (TFC), and 2,2-Diphenyl-1-pycrylhydrazyl (DPPH) radical scavenging activity. Cytotoxicity and cell viability were determined by CCK-8 assay. The formation of reactive oxygen species (ROS) was measured using 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Antioxidant enzyme (SOD-1,2 and GPx-1) expression was determined by real-time PCR. Mitogen-activated protein kinases (MAPK) pathway and apoptosis signal expression was measured by Western blotting. Results: The TPC and TFC quantities of MCE were 28.51 mg gallic acid equivalents/extract g and 3.95 mg catechin equivalents/extract g, respectively. The $IC_{50}$ value for DPPH radical scavenging activity was $506.95{\mu}g/ml$ for MCE. Pre-treatment with MCE showed protective effects against $H_2O_2-induced$ cell death and inhibited ROS generation by oxidative stress. SOD-1,2 and GPx-1 mRNA expression was recovered by pre-treatment with MCE compared with the presence of $H_2O_2$. Pre-treatment with MCE inhibited phosphorylation of p38 and the JNK pathway and down-regulated cleaved caspase-3 and cleaved PARP by $H_2O_2$. Conclusion: The neuroprotective effects of MCE in terms of recovery of antioxidant enzyme gene expression, down-regulation of MAPK pathways, and inhibition apoptosis is associated with reduced oxidative stress in SK-N-MC cells.

• Journal of Chest Surgery
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• v.41 no.4
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• pp.447-456
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• 2008

Background: Caspase-3 is a cysteine protease that plays a major role in the process of apoptotic cell death. The dysregulated expression of c-myc contributes to the tumorigenesis in a variety of human cancers. The aim of this study was to investigate the expressions of caspase-3 and c-myc and their significances as prognosis markers in patients with completely resected non-small cell lung cancer (NSCLC). Material and Method: A total 130 consecutive patients who had undergone complete resection without pre-operative radio-therapy or chemotherapy between May 1996 and December 2003 for NSCLC were retrospectively reviewed. The median follow-up period of the patients was 50 months (range: $3{\sim}128$ months). The expressions of caspase-3 and c-myc were immuno-histochemically examined, and these were correlated with the clinico-pathologic data. Result: The prevalence of caspase-3 and c-myc expressions in the patients was 68% (88/130) and 59% (77/130), respectively. Significant association was found between the frequency of the expressions of caspase-3 and c-myc (p=0.025). The caspase-3 and c-myc expressions were not significantly associated with the prognosis in all the patients. However, according to stages, a positive caspase-3 expression was significantly correlated with a favorable prognosis for patients with stage IIIa disease (median survival period: 35 months vs. 10 months, p=0.021). Multivariate analysis showed the pathologic stage to be significantly correlated with a good prognosis in all the patients (p=0.024), and with a positive caspase-3 expression, well differentiated tumor and negative neuronal invasion in the patients with stage llla disease (p=0.005, p=0.003, p=0.004, respectively). Conclusion: Caspase-3 and c-myc were frequently expressed in NSCLC, suggesting its possible involvement in tumor development. The caspase-3 expression, as determined with performing immunohistochemical staining, may be a favorable prognostic indicator in patients with completely resected NSCLC an advanced stage (IIIa).

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.13-21
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• 1996

Intracellular calcium concentration ($[Ca^{2+}]_i$) may play a crucial role in a variety of neuronal functions. Here we report that in primary culture of mouse cerebellar granule cells nicotinic acetylcholine receptors (nAChRs) are expressed in a specific developmental stage and involved in the regulation of intracellular calcium homeostasis. Nicotine-mediated calcium responses were measured using $^{45}Ca^{2+}$ or fluorometrically using the calcium-sensitive fluorescent dye fura-2. Maximal uptake of $^{45}Ca^{2+}$ evoked by nicotine in mouse cerebellar granule cells were revealed $8{\sim}12$ days in culture. In contrast, nicotine did not alter the basal $^{45}Ca^{2+}$ uptake in cultured glial cells. In cerebellar granule cells nicotine-evoked $^{45}Ca^{2+}$ uptake was largely blocked by the NMDA receptor antagonists. Glutamate pyruvate transaminase (GPT). which removes endogenous glutamate, also prevented nicotine effects, implying the indirect involvement of glutamate in nicotine-mediated calcium responses. Fluorometric studies using fura-2 showed two phases of nicotine-evoked $[Ca^{2+}]_i$ rises: the initial rising phase and the later plateau phase. Interestingly, the NMDA receptor antagonists and GPT appeared to inhibit only the later plateau phase of nicotine-evoked $[Ca^{2+}]_i$ rises. The present results imply that nicotine mediated $^{45}Ca^{2+}$ uptake and $[Ca^{2+}]_i$ rises are attributed to the calcium fluxes through both nAchRs and NMDA receptors in a time-dependent manner. Consequently, nAChRs may play an important role in neuronal development by being expressed in a specific developmental stage and regulating the intracellular calcium homeostasis.

• Journal of Life Science
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• v.21 no.9
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• pp.1234-1243
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• 2011

Liriope platyphylla has been used in oriental medicine as an effective medical plant to improve symptoms of cough, sputum production, neurodegenerative disorders, obesity and diabetes for long time. In order to investigate the effects of novel extracts on nerve growth factors (NGF)-stimulated neuritic outgrowth, the alteration of NGF expression and NGF receptor signaling pathway were detected in neuroblastoma cells and C57BL/6 mice. Of a total of 13 novel extracts, 4 extracts (LP-E, LP-M, LP-M50, LP2E17PJ) showed high viability on MTT assay. Also, all of these extracts induced NGF secretion and NGF mRNA expression in neuroblastoma cells. However, the NGF-induced neuritic outgrowth from PC12 cells was only stimulated by LP-E, LP-M and LP-M50. Furthermore, we selected LP-M as a best candidate, based on method and amounts of extraction, in order to verify its effect in mice. C57BL/6 mice were treated with 50 mg/kg of LP-M for 2 weeks and the effects on NGF regulation were analyzed with various methods. The expression of NGF mRNA was significantly increased in LP-M treated mice compared to vehicle treated mice. Also, the signaling pathway of p75NTR was inhibited in the cortex by LP-M treatment, with no change in the hippocampus of brain. However, the signaling pathway of TrkA was dramatically activated in only hippocampus via LP-M treatment. Therefore, these results suggest that the novel four extracts of L. platyphylla may contribute to the regulation of NGF expression and secretion in neuronal cells. LP-M was especially considered to be an excellent candidate for a neurodegenerative disease-therapeutic drug.

• Journal of Life Science
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• v.19 no.1
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• pp.21-33
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• 2009

In this study, we investigated the effect of Rhei Rhizoma (RR; 大黃) water extract on gene expression in a hypoxia model of cultured rat hippocampal neurons. RR water extract $(2.5{\mu}g/ml)$ was added to the culture media on day 10 in vitro (DIV10), and a hypoxic shock (2% $O_2$/5% $CO_2$, $37^{\circ}C$, 3 h) was given on DIV13. After maintaining the cultures in normoxia for 24 hr, total RNA was isolated and used for microarray analysis. The MA-plot indicated that most genes were up- or downregulated within 2-fold. There were more downregulated genes (725 ea) than upregulated ones (472 ea) when larger than Global M value 0.2 (i.e., >15% increase) or smaller than Global M value -0.2 (i.e., >15% decrease) were considered. Antiapoptosis genes such as Tegt (2.4-fold), Nfkb1 (2.4-fold) Veg (1.8-fold), Ngfr (1.6-fold) were upregulated, while pro-apoptosis genes such as Bad (-64%), Cstb (-66%) were downregulated. Genes for combating environmental stress (stress response genes) such as Defb3 (2.7-fold), Cygb (2.2-fold), Ahsg (2.18-fold), Alox5 (2-fold) were upregulated. Genes for cell proliferation (cell cycle-related genes) such as Erbb2 (1.84-fold), Mapk12 gene (1.8-fold) was upregulated. Therefore, RR water extracts upregulate many pro-survival genes while downregulating many pro-death genes. It is interpreted that these genes, in combination with other regulated genes, can promote neuronal survival in a stress such as hypoxia.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.167-173
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• 2007

To study the transcriptional mechanisms by which expression of the murine glial cell-derived neurotrophic factor inducible transcription factor (mGIF) gene is regulated, a murine genomic clone was iso-lated using a mGIF cDNA as probe. A 13-kb genomic fragment, which comprises 4-kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Spl. The mGIF gene also has consensus sequences for AP2 binding sites. The transcriptional activity of five deletion mutants of a 2.1-kb fragment was analyzed by modulating transcription of the heterologous luciferase gene in the promoterless plasmid pGL2-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3. Transient expression assays suggested the presence of a positive regulator between -213 and -129 while a negative regulator was found in the region between -806 and -214. Relatively strong transcriptional activity was observed in neuronal NB41A3, glial C6 cells and hepatic HepG2, but very weak activity in skeletal muscle C2C12 cells. These findings confirm the tissue-specific activity of the mGIF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.

• Journal of Life Science
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• v.19 no.4
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• pp.449-456
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• 2009

Murine Neuro-2a (N2a) cells have been widely used for the investigation of neuronal differentiation, trophic interaction and neurotoxic effects of various compounds and their associated mechanisms. N2a cells have many genomic variations such as gains or losses in DNA copy number, similar to other neuroblastoma cells, and no systematic or high-resolution studies of their genome-wide chromosomal aberrations have been reported. Presently, we conducted a systematic genome-wide determination of chromosomal aberrations in N2a cells using a high-throughput, oligonucleotide array-based comparative genomic hybridization (oaCGH) technique. A hidden Markov Model was employed to assign each genomic oligonucleotide to a DNA copy number state: double loss, single loss, normal, gain, double gain and amplification. Unlike most neuroblastoma cells, Mycn amplification was not observed in N2a cells. In addition, these cells showed gain only in the neuron-derived neurotrophic factor (NF), while other neurotrophic factors such as glial line-derived NF and brain-derived NF presented normal copy numbers. Chromosomes 4, 8, 10, 11 and 15 displayed more than 1000 aberrational oligonucleotides, while chromosomes 3, 17, 18 and 19 displayed less than 20. The largest region of gain was located on chromosome 8 and its size was no less than 26.7 Mb (Chr8:8427841-35162415), while chromosome 4 had the longest region of single deletion, with a size of 15.1 Mb (Chr4:73265785-88374165).

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.5
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• pp.976-988
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• 2002

The aim of this study was to examine the memory and attention enhancement effect of YMG-1 and YMG-2, which are modified herbal extracts from Yukmijihwang-tang (YMJ). YMJ, composing six herbal medicine, has been used for restoring the normal functions of the body to consolidate the constitution, nourishing and invigorating the kidney functions for hundreds years in Asian countries. A series of studies reported that YMJ and its components enhance memory retention, protects neuronal cell from reactive oxygen attack and boost immune activities. Recently the microarray analysis suggested that YMG-1 protects neurodegeneration through modulating various neuron specific genes. A total of 55 subjects were divided into three groups according to the treatment of YMG-1 (n=20), YMG-2 (n=20) and control (C; n=15) groups. Before treatments, all of subjects were subjected to the assessments on neuropsychological tests of K-WAIS test, Rey-Kim memory test, and psychophysiological test of Event-Related Potential (ERP) during auditory oddball task and repeated word recognition task. They were repeatedly assessed with the same methods after drug treatment for 6 weeks. Although no significant effect of drug was found in Rey-Kim memory test, a significant interaction (P = .010, P < 0.05) between YMG-2 and C groups was identified in the scores digit span and block design, which are the subscales of K-WAIS. The very similar but marginal interaction (P = .064) between YMG-1 and C groups was found too. In ERP analysis, only YMG-1 group showed decreasing tendency of P300 latency during oddball task while the others tended to increase, and it caused significant interaction between session and group (p= .004). This result implies the enhancement of cognitive function in due to consideration of relationship between P300 latency and the speed of information processing. However, no evidence which could demonstrate the significant drug effect was found in neither amplitude or latency. These results come together suggest that YMG-1, 2 may enhance the attention, resulting in enhancement of memory processing. For elucidating detailed mechanism of YMG on learning and memory, the further studies are necessary.

• Journal of Life Science
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• v.20 no.12
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• pp.1820-1828
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• 2010

Cobalt(II) chloride, a chemical compound with the formula$CoCl_2$, has been widely used in the treatment of anemia, as a chemical agent for the induction of hypoxia in cell cultures, and is known to activate hypoxic signaling. However, excessive exposure to cobalt is associated with several clinical conditions, including asthma, pneumonia, and hematological abnormalities, and can lead to tissue and cellular toxicity. It is also known to induce apoptosis. One of the questions was that of whether $CoCl_2$ might induce apoptosis via endoplasmic reticulum (ER) stress in neurons. To address this question, first, the level of DNA fragmentation was measured for assay of apoptotic rates using $CoCl_2$ with neuron PC12 cells. After confirmation of apoptosis inductions, under the same conditions, the expression levels of ER stress associated factors [ER chaperones Bip, calnexin, ERp72, ERp29, PDI, and ER membrane kinases (IRE1, ATF6, PERK)] were examined by RT-PCR and Western blotting. These results indicated that apoptosis is induced through activation of ER membrane kinases via ER stress. In conclusion, during induction of apoptosis through $CoCl_2$-induced hypoxia in neuron PC12 cells, ER membrane kinase of IRE1 was dominantly up-expressed, and, consecutively, TRAF2, which has been suggested to be one of the links connecting apoptosis and ER stress, was strongly up-expressed.