• The Korean Journal of Physiology and Pharmacology
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• v.20 no.4
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• pp.399-406
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• 2016

Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.10a
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• pp.14-17
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• 2001

Positional clonging (map-based cloning) of mutations or genetic variations has been served as an invaluable tool to understand in-vivo functions of genes and to identify molecular components underlying phenotypes of interest. Mice homozygous for the cerebellar deficient folia (cdf) mutation are ataxic, with cerebellar hypoplasia and abnormal lobulation of the cerebellum. In the cdf mutant cerebellum approximately 40% of Purkinje cells are ectopically located within the white matter and the inner granule cell layer (IGL). To identify the cdf gene, a high-resolution genetic map for the cdf-gene-encompassing region was constructed using 1997 F2 mice generated from C3H/HeSnJ-cdf/cdf and CAST/Ei intercross. The cdf gene showed complete linkage disequilibrium with three tightly linked markers D6Mit208, D6Mit359, and D6Mit225. A contig using YAC, BAC, and P1 clones was constructed for the cdf critical region to identify the gene. A deletion in the cdf critical region on chromosome 6 that removes approximately 150 kb of DNA selection. cdf mutant mice with the transgenic copy of the identified gene restored the brain abnormalities of the mutant mice. The positional cloning of cdf gene provides a good example showing the identification of a gene could lead to finding a new component of important molecular pathways.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.14-17
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• 2001

Positional cloning (map-based cloning) of mutations or genetic variations has been served as an invaluable tool to understand in-vivo functions of genes and to identify molecular components underlying phenotypes of interest. Mice homozygous for the cerebellar deficient folia (cdf) mutation are ataxic, with cerebellar hypoplasia and abnormal lobulation of the cerebellum. In the cdf mutant cerebellum approximately 40% of Purkinje cells are ectopically located within the white matter and the inner granule cell layer (IGL). To identify the cdf gene, a high-resolution genetic map for the cdf-gene-encompassing region was constructed using 1997 F2 mice generated from C3H/HeSnJ-cdf/cdf and CAST/Ei intercross. The cdf gene showed complete linkage disequilibrium with three tightly linked markers D6Mit208, D6Mit359, and D6Mit225. A contig using YAC, BAC, and P1 clones was constructed for the cdf critical region to identify the gene. A deletion in the cdf critical region on chromosome 6 that removes approximately 150kb of DNA was identified. A gene associated with this deletion was identified using cDNA selection. cdf mutant mice with the transgenic copy of the identified gene restored the brain abnormalities of the mutant mice. The positional cloning of cdf gene provides a good example showing the identification of a gene could lead to finding a new component of important molecular pathways.

• Korean Journal of Biological Psychiatry
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• v.5 no.2
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• pp.219-226
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• 1998

Cytosine arabinoside(AraC) inhibits DNA synthesis and ${\beta}$-DNA polymerase, an enzyme involved in DNA repair. This, a potent antimitotic agent, is clinically used as an anticancer drug with side effect of severe neurotoxicity. Earlier reports suggested that inhibition of neuronal survival by AraC in sympathetic neuron may be due to the inhibition of a 2'-deoxycytidine-dependent process that is independent of DNA synthesis or repair and AraC induced a signal that is triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells. The present study would suggest whether caspase family(ICE/CED-3-like protease) involved in AraC-induced apoptosis pathway of PC12 cells. It was observed that treatment of PC12 cells with AraC led to decrease of viability by MTT assay and morphology changes, which did not suggest that AraC induced apoptosis in PC12 cells. The mRNA of caspase-1/caspase-3 were expressed in PC12 cells constitutively, and AraC did not activate caspase family. These results suggest that caspase-1/caspase-3 may not be required for AraC-induced cell death pathway in PC12 cells.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.1
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• pp.9-15
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• 2005

We attempted to analyze the mechanism of polychlorinated biphenyl (PCB)-induced neurotoxicity and identify the target molecules in the neuronal cells for PCBs.Since the developing neuron is particularly sensitive to PCB-induced neurotoxicity, we isolated cerebellar granule cells derived from 7-day old Sprague Dawley (SD) rats and grew cells in culture for additional 7 days to mimic PND-14 conditions. Only non-coplanar PCBs at a high dose showed a significant increase of total protein kinase C (PKC) activity at phobol 12,13-dibutyrate ([$^3M$]PDBu) binding assay, indicating that non-coplanar PCBs are more neuroactive than coplanar PCBs in neuronal cells. PKC isozymes were immunoblotted with the selected monoclonal antibodies. PKC-${\alpha}$, ${\delta}$, and ε were activated with non-coplanar PCB exposure. Receptor for activated C kinase-1 (RACK-1), anchoring protein for activated PKC, was more induced with exposure to coplanar PCBs than non-coplanar PCBs. Reverse transcription PCR (RT-PCR) analysis showed induction of neurogranin (RC-3) and growth associated protein-43 (GAP-43) mRNA with non-coplanar PCBs. The results indicate that these factors may be useful biomarkers for differentiating non-coplanar PCBs from coplanar PCBs. The present study demonstrated that non-coplanar PCBs are more neuroactive congeners than coplanar PCBs.

• Environmental Analysis Health and Toxicology
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• v.22 no.3
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• pp.279-285
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• 2007

The present study attempted to analyze the mechanism of PCB-induced neurotoxicity with respect to the PKC signaling. Since the developing neuron is particularly sensitive to PCB-induced neurotoxicity, we isolated cerebellar granule cells derived from 7-day old SD rats and grew cells in culture for additional 7 days to mimic PND-14 conditions. Only non-coplanar PCBs at a high dose showed a significant increase of total PKC activity at $[^3H]PDBu$ binding assay, indicating that non-coplanar PCBs are more neuroactive than coplanar PCBs in neuronal cells. PKC isoforms were immunoblotted with respective monoclonal antibodies. PKC-alpha and-epsilon were activated with non-coplanar PCB exposure. The result suggests that coplanar PCBs have a PKC pathway different from non-coplanar PCBs. Activation of PKC with exposure was dampened with treatment of high molecular weight of chitosan. Chilean (M.W. > 1,000 kDa) inhibited the total activity of PKC induced by the non-coplanar PCBs. Translocation of PKC isoforms was also inhibited by the high molecular weight of chitosan. The study demonstrated that non-coplanar PCBs are more potent neurotoxic congeners than coplanar PCBs and the alteration of PKC activities by PCB exposure can be blocked with the treatment of chitosan. The results suggest a potential use of chitosan as a means of nutritional intervention to prevent the harmful effects of pollutant-derived diseases.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.4
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• pp.598-601
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• 2010

This experiment was performed to investigate whether the combined-preparation of water extracts of germinated brown rice (WGR), water extracts of cultured mountain ginseng (WCG) and 70% ethanol extracts of Longanae Arillus (ELA) has hypnotic effects and/or enhances pentobarbital-induced sleep behaviors through the GABAergic system. The combined-preparation of WGR and WCG reduced sleep latency and prolonged sleep time induced by pentobarbital. ELA also reduced sleep latency and prolonged sleep time induced by pentobarbital. However, WGR or WCG itself did not induce sleep. The combined-preparation of WGR, WCG and ELA strongly reduced sleep latency and prolonged sleep time via chloride influx into primary cultured cerebellar granule cells. In conclusion, the combined-preparation of WGR, WCG and ELA augments pentobarbital-induced sleep behaviors through the modification of GABAergic system.

• Applied Microscopy
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• v.27 no.1
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• pp.57-70
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• 1997

The distribution of glutamatergic synaptic structures in the dog basilar pons was investigated at the ultrastructural level using monoclonal antibodies against fixative-modified glutamate. Electron-dense reaction product was densely localized at the perinuclear region in the neurenal somata and often observed along the microtubules located within the dendritic processes. One or more unlabelled axon terminals made asymmetric synaptic contacts with glutamate-immunoreactive dendritic profiles. In audition, reaction product was observed either within axonal processes surrounded by myelin sheath or axon terminals. Immunoreactive axon terminals made asymmetric synaptic contact either with unlabelled or labelled dendritic profiles. These observations provided an anatomic evidence of how this excitatory neural element might perform its function in a multisynaptic pathway involving glutamatergic afferents to the basilar pons, glutamate-immunoreactive pontocerebellar projection neurons, and the glutamate-positive granule cells of the cerebellar cortex.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.6
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• pp.996-999
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• 2011

This experiment was performed to investigate whether the combined-preparation of crude drugs (The $Well^{TM}$ Preparation, TW), has hypnotic effects and/or enhances pentobarbital-induced sleep behaviors. TW was mixed with water extracts of Ginseng Radix red, Germinated brown rice, cultured mountain ginseng, and 50% ethanol extracts of Longanae Arillus, Nelumbinis Folium and Chrysanthemi Flos. TW (100 mg/kg, p.o.) reduced sleep onset and prolonged sleep time induced by pentobarbital similar to muscimol (0.2 ${\mu}M$), a $GABA_A$ receptor agonist. Also, TW (2 ${\mu}g$/ml) and pentobarbital (2.5 ${\mu}M$) did not affect the chloride influx in primary cultured cerebellar granule cells, respectively, but the combined-treatment of TW (2 ${\mu}g$/ml) and pentobarbital (2.5 ${\mu}M$) increased the chloride influx onto the cells. In conclusion, TW augments pentobarbital-induced sleep behaviors; these effects may result from chloride channel activation.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.249-255
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• 2014

Programmed cell death or apoptosis is associated with changes in $K^+$ concentration in many cell types. Recent studies have demonstrated that two-pore domain $K^+$ ($K_{2P}$) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of $K_{2P}$ channels, were found to be critical for cell death. This study was performed to identify the role of $K^+$ channels in the $H_2O_2$-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to $H_2O_2$ for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited $H_2O_2$-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (${\beta}$-mercaptoethanol) with 25 mM KCl significantly decreased the rate of $H_2O_2$-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of $K^+$ channel efflux for a short-time reduces $H_2O_2$- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of $K^+$ channels which are highly expressed in a given cell type.