• BMB Reports
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• v.36 no.3
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• pp.326-331
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• 2003

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSB) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, $\gamma$-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multi copy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride ($10\;{\mu}M$) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal8 amino acid-coding region were fused into the promoteriess $\beta$-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of $\beta$-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of $\gamma$-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.

• Toxicological Research
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• v.22 no.3
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• pp.275-282
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• 2006

In previous our studies, we have reported that eugenol derived from Eugenia caryophyllata(Myrtaceace) exhibits acute N-methyl-D-aspartate(NMDA)- and oxygen/glucose deprivation-induced neurotoxicity in primary cortical cultures and protects hippocampal neurons from global ischemia. In this study, we investigated whether the extracts and fractions of E. caryophyllata or eugenol shows the neuroprotective effects against delayed neuronal injury evoked by NMDA or ${\alpha}$-amino-3-hydroxy-5-methylisoxazole propionate(AMPA), and oxidative damage induced by arachidonic acid-, hydrogen peroxide-, $FeCl_2$/ascorbic acid-, and buthionine sulfoximine(BSO) in primary cortical cultures. We examined the neurotoxicity of eugenol itself in cultures and inhibitory effect of eugenol on NMDA- or kainate(KA)-induced convulsion in BALB/c mice. Each water, methanol extract and methanol fraction of E. caryophyllata was significantly attenuated NMDA-induced delayed neurotoxicity, respectively. Eugenol exhibited a significant inhibitory action against the convulsion evoked by NMDA and KA, and reduced delayed or brief neurotoxicity induced by NMDA, AMPA, and various oxidative injuries. These results suggest that eugenol derived from E. caryophyllata may contribute the neuroprotection against delayed-type excitotoxicity and excitotoxins-mediated convulsion through the amelioration of oxidative stress.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.78-78
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• 2003

Embryonic stem (ES) cells, derived from preimplantation embryo, are able to differentiate into various types of cells consisting the whole body, or pluripotency. In contrast, terminally differentiated cells do not usually alter their nature but frequently die or transform if they are exposed to inappropriate external stimulations. In addition to the plasticity, ES cells are expected to be different from terminally differentiated cells in very many ways, such as patterns of gene expressions, ability and response of the cells in confronting environmental stimulations, metabolism, and growth rate. As a model system to differentiate these two types of cells, human ES cells (MB03) and terminally differentiated cells (HeLa), we examined the ability of these two types of cells in confronting a severe oxidative insult, that is $H_2O$$_2$. Approximately 1$\times$10$^4$ cells were plated in 96 well plate and serum starved for overnight. The conditioned cells were exposed to a various concentration of $H_2O$$_2$ fur 24 hrs and loaded with neutral red (50$\mu\textrm{g}$/ml) for 4 hrs, washed with PBS for 2 min three times, and entrapped dye was dissolved out using acetic ethanol. Cytotoxicity was determined by reading the amount of dye in the medium using microplate reader. equipped with 575 nm filter. Relative amount of the dye entrapped within MB03 or HeLa were not significantly different when cells were exposed up to 0.4 mM $H_2O$$_2$. However, this sharply decreased down to 0.12% in HeLa cells when the cells were exposed to 0.8 mM $H_2O$$_2$, while it was approximately 54% in MB03 suggesting that this concentration of $H_2O$$_2$ is the defensive threshold for HeLa cells. The resistance to oxidative stimulation reversed, however, when cells were co-treated with BSO (L-buthionine- 〔S, R〕-sulfoximine) which chelates intracellular GSH. This result suggests that cellular GSH is the major defensive mechanism of human ES cells. Induction of enzymes involved in GSH metabolism and type of cell death is currently being studied.

• International Journal of Oral Biology
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• v.42 no.3
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• pp.129-135
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• 2017

The present study investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain or trigeminal neuropathic pain. Experiments were carried out on male Sprague-Dawley rats weighing between 230 and 280 g. Under anesthesia, a polyethylene tube was implanted in the atlanto-occipital membrane for intracisternal administration. IL-$1{\beta}$-induced inflammation was employed as an orofacial acute inflammatory pain model. IL-$1{\beta}$ (10 ng) was injected subcutaneously into one vibrissal pad. We used the trigeminal neuropathic pain animal model produced by chronic constriction injury of the infraorbital nerve. DL-threo-${\beta}$-benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block the spinal glutamate transporter and the glutamine synthetase activity in astroglia. Intracisternal administration of TBOA produced mechanical allodynia in naïve rats, but it significantly attenuated mechanical allodynia in rats with interleukin (IL)-$1{\beta}$-induced inflammatory pain or trigeminal neuropathic pain. In contrast, intracisternal injection of MSO produced anti-allodynic effects in rats treated with IL-$1{\beta}$ or with infraorbital nerve injury. Intracisternal administration of MSO did not produce mechanical allodynia in naive rats. These results suggest that blockade of glutamate recycling induced pro-nociception in na?ve rats, but it paradoxically resulted in anti-nociception in rats experiencing inflammatory or neuropathic pain. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic pain conditions.

• BMB Reports
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• v.34 no.6
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• pp.544-550
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• 2001

In pre-transplant total-body irradiation (TBI), the lung is a critical dose-limiting organ. Also, the possible role of oxidative stress was suggested in the development of TBI-induced lung damage. This study explores the association between TBI-induced oxidative stress and the induction of lung pathogenesis by investigating TBI-induced oxidative stress in the lungs of male C57BL/6 mice after a single dose of 10 Gy TBI. We showed significant increases of reactive oxygen species (ROS) formation and lipid peroxidation, and also a depletion and oxidation of glutathione after TBI. There is evidence that pretreatment with 1,10-phenanthroline (o-phen) significantly reduces oxidative stress in the lung. This indicates that the TBI-induced ROS generation involves a metal-catalyzed Fenton-type reaction. A pretreatment of buthionine sulfoximine (BSO) augmented the glutathione depletion and oxidation, but had no effect on the ROS formation and lipid peroxidation up to 6 h after TBI. Histopathological features that are consistent with pneumonitis were observed in the BSO pretreated-mice 1 week after irradiation. The results suggest that TBI-induced oxidative stress in the lung involves a generation of ROS through a Fenton-type reaction. Also, glutathione plays an important inhibitory role in the radiation-induced lung pathogenesis by participating in the self-amplifying cascade subsequent to the ROS generation by irradiation.

• Journal of Korean Neurosurgical Society
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• v.30 no.9
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• pp.1059-1064
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• 2001

Objective : 6R-Tetrahydrobiopterin(BH4) is a cofactor for the aromatic amino acid hydroxylases which is essential for the biosynthesis of catecholamines and serotonin. It also acts as a cofactor for nitric oxide synthase, and stimulates the release of some neurotransmitters such as dopamine, serotonin, acetylcholine and glutamate. Recently, it has been reported that BH4 could induce cellular proliferation and enhance neuronal survival. This study was performed to investigate the antioxidative effect of BH4 on the various oxidative insults in mouse cerebral cortical cell cultures. Methods : Iron ion(FeCl2), zinc ion(ZnCl2), sodium nitroprusside(SNP) and buthionine sulfoximine(BSO, a glutathione depletor) were used as oxidants. Cell death was assessed by measurement of lactate dehydrogenase efflux to bathing media at the end of exposure. Result : All 4 oxidants induced neuronal cell death associated with cell body swelling, which was markedly inhibited by trolox($100{\mu}M$), a vitamin E analog. BH4($10-100{\mu}M$) markedly inhibited the neuronal cell death induced by all 4 oxidants($20{\mu}M\;Cu^{2+}$, $20{\mu}M\;Zn^{2+}$, $1{\mu}M$ SNP or 1mM BSO). However, BH4 failed to inhibit the neuronal cell death induced by 24hr exposure to $20{\mu}M$ NMDA. Conculsion : These results suggest that BH4 has antioxidative action independently of any actions of enzyme cofactor.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.1
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• pp.265-273
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• 2004

KBPTS is the fortified prescription of Bang-pung-tong-sung-san (BPTS) by adding Spatholobi Clulis and Salviae Miltiorrzae Radix. BPTS prescription has been used in Qriental medicine for the treatments of vascular diseases including hypertension, stroke, and arteriosclerosis, and nervous system diseases. Yet, the overall mechanism underlying its activity at the cellular levels remains unknown. To investigate the protective role of KBPTS on brain functions, noxious stimulations were applied to neurons in vitro and in vivo. KBPTS pretreatment in cultured cortical neurons of albino ICR mice rescued death caused by AMPA, NMDA, and kainate as well as by buthionine sulfoximine (BSO) and ferrous chloride (Fe/sup 2+/) treatments. Furthermore, KBPTS promoted animal's recovery from coma induced by a sublethal dose of KCN and improved survival by a lethal dose of KCN. To examine its physiological effects on the nervous system, we induced ischemia in the Sprague-Dawley rat's brain by middle cerebral artery (MCA) occlusion. Neurological examination showed that KBPTS reduced the time which is required for the animal after MCA occlusion to respond in terms of forelimb and hindlimb movement$. Histological examination revealed that KBPTS reduced ischemic area and edema rate and also protected neurons in the cerebral cortex and hippocampus from ischemic damage. Thus, the present data suggest that KBPTS may play an important role in protecting neuronal cells from external noxious stimulations.

• Korean Journal of Veterinary Research
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• v.57 no.1
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• pp.1-7
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• 2017

Parkinson's disease (PD) is an irreversible neurological disorder with related locomotor dysfunction and is characterized by the selective loss of nigral neurons. PD can be experimentally induced by 6-hydroxydopamine (6-OHDA). It has been reported that reactive oxygen species, which deplete endogenous glutathione (GSH) levels, may play important roles in the dopaminergic cell death characteristic of PD. Fucoidan, a sulfated algal polysaccharide, exhibits anti-inflammatory and anti-oxidant actions. In this study, we investigated whether fucoidan can protect against 6-OHDA-mediated cytotoxicity in SH-SY5Y cells. Cytotoxicity was evaluated by using MTT and LDH assays. Fucoidan alleviated cell damage evoked by 6-OHDA dose-dependently. Fucoidan reduced the number of apoptotic nuclei and the extent of annexin-V-associated apoptosis, as revealed by DAPI staining and flow cytometry. Elevation of lipid peroxidation and caspase-3/7 activities induced by 6-OHDA was attenuated by fucoidan, which also protected against cytotoxicity evoked by buthionine-sulfoximine-mediated GSH depletion. Reduction in the glutathione/glutathione disulfide ratio induced by 6-OHDA was reversed by fucoidan, which also inhibited 6-OHDA-induced disruption of mitochondrial membrane potential. The results indicate that fucoidan may have protective action against 6-OHDA-mediated neurotoxicity by modulating oxidative injury and apoptosis through GSH depletion.

• Environmental Analysis Health and Toxicology
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• v.20 no.1
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• pp.87-95
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• 2005

Cadmium is well known as a toxic metal and has insulin mimicking effects in rat adipose tissue. This study was undertaken to investigate the effect of CdCl₂ on glucose transport and its mechanism in 3T3 - L1 adipocytes. CdCl₂ exhibits respectively 2.2 and 2.8 fold increases in the 2-deoxyglucose uptake when exposed to 10 and 25 μM of CdCl₂ for 12 hr. To investigate the stimulating mechanism of glucose transport induced by CdCl₂. Wortmannin and PD98059 were used respectively as PI3K inhibitor and MAPK inhibitor, which did not affect 2-DOG uptake. This results suggest that induced 2-deoxy-(l-3H)-D-glucose (2-DOG) uptake by CdCl₂ may not be concerned with the insulin signalling pathway. Whereas nifedipine, a calcium channel blocker inhibited the 2- DOG uptake stimulated by CdCl₂. In addition, we also measured the increased production of Reactive oxygen substances (ROS) and glutathione (GSH) level in 3T3-L1 adipocytes to investigate correlation between the glucose uptake and increased production of ROS with H2DCFDA. CdCl₂ increased production of ROS. Induced 2-DOG uptake and increased production of ROS by CdCl₂ were decreased by N-acetylcystein (NAC). And L-buthionine sulfoximine (BSO) a potent inhibitor of γ-GCS, decreased of 2-DOG uptake. Also NAC and BSO changed the cellular GSH level, but GSH/GSSG ratio remained unchanged at 10, 25 μM of CdCl₂.

• Journal of Microbiology
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• v.43 no.1
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• pp.44-48
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• 2005

In our previous study, the first structural gene (GGTI) encoding ${\gamma}-glutamyl$ transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of ${beta}-galactosidase$ from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.