• Proceedings of the Korean Society of Toxicology Conference
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• 2006.11a
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• pp.46-54
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• 2006

The methylmercury (MeHg) is a toxic environmental pollutant, causing serious neurological and developmental effects in humans. Recent epidemiological studies have indicated that ingestion of MeHg in fish during pregnancy can result in neuroethological effects in the offspring. However, the mechanism underlying the MeHg-toxicity is not fully understood. To elucidate the mechanisms of toxicity of MeHg and of defense against MeHg, we searched for factors that determine the sensitivity of yeast cells to MeHg, and found that overexpression of Cdc34, a ubiquitin-conjugating enzyme (E2) that is a component of the ubiquitin-proteasome (UP) system, induces a resistance to MeHg toxicity in both yeast and human cells. The UP system is involved in the intracellular degradation of proteins. When Cdc34 is overexpressed in cells, ubiquitination reactions are activated and the degradation of certain proteins by the UP system is enhanced. Therefore, it seems likely that certain as-yet-unidentified proteins that increase MeHg toxicity might exist in cons and that toxicity might be reduced by the enhanced degradation of such proteins, mediated by the UP system, when Cdc34 is overexpressed. SCF ubiquitin-ligase is a component of UP system and consists of Skpl, the scaffold protein Cdc53, the RING-finger protein Hrt1, and one member of the family of F-box proteins. The F-box proteins directly bind to the substrates and are the determinants of substrate specificity of SCF. Therefore, we searched for the f-box protein that cofers resistance to MeHg, and found that overexpression of Hrt3 or Yi1224w induced resistance to MeHg toxicity in yeast cells. Since the protein(5) that enhance toxicity of MeHg might plausibly be induced in substrates of both f-box proteins, we next searched for substrate proteins that are recognized by Hrt3 or Y1r224w using two-hybrid screen. We found that Did3 or Crsl interacts with Hrt3; and Eno2 interacts with Yir224w. The yeast cells that overexpressed each those proteins showed hypersensitivity to MeHg, respectively, indicating that those proteins enhance the MeHg toxicity. Both Dld3 and Eno2 are proteins involved in the synthesis of pyruvate, and overexpression of both proteins might induce increase in interacellular levels of pyruvate. Deletion of Yi1006w that transports pyruvate into the mitochondria induced aresistance to MeHg. These results suggest that the promotion of the pyruvate irdlowinto the mitochondria might enhance MeHg toxicity. This study providesimportant keyfor the elucidauon of the molecular mechanism of MeHg toxicity.

• Korean Journal of Microbiology
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• v.34 no.4
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• pp.219-224
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• 1998

The cyclic metabolism of poly(3-hydroxyhutyrate-co-3-hydroxyvalerate) synthesized from glucose by Alcaligenes sp. SH-69 in the presence or absence of new carbon substrate was investigated. In batch culture, the content and weight average molecular weight of the copolymer already stored in the cell decreased rapidly when there was no other carbon source available. After the depletion of carbon source, the amount of high molecular weight copolymer decreased more rapidly than that of low molecular weight copolymer, and as a result, average molecular weight distribution shifted to the lower value. The addition of a mixture of glucose and levulinic acid when the initial carbon substrate, glucose, was nearly depleted supported the continual increase in cell mass and the accumulation of poly(3HB-co-3HV) with high molar fraction of 3HV. However, solvent fractionation of the polymer with acetone revealed the degradation of pre-existing polyhydroxyalkanoale (PHA) in parallel with the synthesis of PHA from new carbon substrate. Even though PHAs obtained from each substrate alone were the copolymer of 3HB and 3HV, it was found that the polymer accumulated in the cells grown by sequential feeding was mainly physical mixture of two poly(3HB-co-3HV) copolymers containing different molar fractions of 3HV.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.46 no.1
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• pp.11-22
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• 2020

In this study, we investigated anti-pigmentation effect of a hexapeptide. The peptide significantly reduced melanin contents and inhibited tyrosinase activity in a dose-dependent manner, in which tyrosinase is a key enzyme in melanogenesis. The peptide also significantly reduced the expression levels of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and their upstream transcription factor, microphthalmia-associated transcription factor (MITF). Furthermore, the peptide suppressed the phosphorylation level of cAMP-response element binding protein (CREB), a transcription factor of MITF, and increased the phosphorylation level of extracellular signal-regulated kinase (ERK), a kinase mediates MITF phosphorylation and proteasomal degradation. The peptide significantly inhibited the expression of Rab27A, Melanophilin, and MyosinVa, the components of motor complex involved in intracellular movement of melanosome. These results suggest that Hexapeptide could be used as an effective whitening agent that has inhibitory effect on melanin production and melanosome transport by regulating expression and degradation of MITF in melanocytes.

• The Korean Journal of Zoology
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• v.29 no.3
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• pp.215-233
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• 1986

The effect of tryptophan on brain formation at the early stage of chick embryo has been investigated morphologically using electron microscope. The electron micrographs of cerebral cortex cells of $5\\sim10$ day old chick embryo, which received 1.0mg of tryptophan showed that the irregularity, evagination and disruption of nuclear membrane and nuclear chromatin condensation, nucleolar chromatin margination and segragation. Hypertrophy of stalks, vesicles, and vacuoles can be seen and dilation and vesiculation of rough endoplamic reticulum and polysome disaggregation occured. Protein and RNA levels and the activity of several enzymes such as lactate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and glucose 6-phosphate dehydrogenase of tryptophan administered group were significantly lower than those of control group suggesting that the tryptophan administration depressed protein biosynthesis resulting in the decrease of enzyme activity. It was found that serotonin content of egg yolk which has been incubated for 10 days were as much as three times that of control egg yolk. It is not clear whether the increase of serotonin content might inhibit intracellular yolk granule degradation which might result in malformation of chick embryo, but it is likely that tryptophan administration might depress protein biosynthesis, consequently, the enzyme biosynthesis would be impaired. This might give rise to improper development of chick embryo.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8503-8512
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• 2016

The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline, WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of WP631 and DOX suggest that this bisanthracycline can be an effective alternative in ovarian cancer treatment.

• Biomolecules & Therapeutics
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• v.24 no.5
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• pp.543-551
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• 2016

This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin $E_2$ ($PGE_2$), tumor necrosis factor-alpha (TNF-${\alpha}$), interleukin-1 beta (IL-$1{\beta}$), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-${\kappa}B$ p65). VBME significantly inhibited LPS-induced production of NO and $PGE_2$ and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-${\alpha}$, IL-$1{\beta}$, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-${\kappa}B$ p65 translocation by blocking $I{\kappa}B-{\alpha}$ phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-${\kappa}B$ signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.8
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• pp.3213-3222
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• 2015

Background: Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative stress have long been associated with cancer. ROS play a vital role in regulating actin dynamics that are sensitive to oxidative modification. The current work aimed at studying the effects of sub-lethal metabolic oxidative stress on actin cytoskeleton, focal adhesion and cell migration. Materials and Methods: T47D human breast cancer cells were treated with 2-deoxy-D-glucose (2DG), L-buthionine sulfoximine (BSO), or doxorubicin (DOX), individually or in combination, and changes in intracellular total glutathione and malondialdehyde (MDA) levels were measured. The expression of three major antioxidant enzymes was studied by immunoblotting, and cells were stained with fluorescent-phalloidin to evaluate changes in F-actin organization. In addition, cell adhesion and degradation ability were measured. Cell migration was studied using wound healing and transwell migration assays. Results: Our results show that treating T47D human breast cancer cells with drug combinations (2DG/BSO, 2DG/DOX, or BSO/DOX) decreased intracellular total glutathione and increased oxidized glutathione, lipid peroxidation, and cytotoxicity. In addition, the drug combinations caused a reduction in cell area and mitotic index, prophase arrest and a decreased ability to form invadopodia. The formation of F-actin aggregates was increased in treated T47D cells. Moreover, combination therapy reduced cell adhesion and the rate of cell migration. Conclusions: Our results suggest that exposure of T47D breast cancer cells to combination therapy reduces cell migration via effects on metabolic oxidative stress.

• Journal of Life Science
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• v.29 no.8
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• pp.871-878
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• 2019

The aim of this study was to determine the anticancer activities of an 80% methanol extract and various fractions of Rhododendron brachycarpum (RB). The n-hexane fraction of RB showed the highest inhibitory activity (Inhibit concentration $50%=20.2{\pm}1.2{\mu}g/ml$) in HT-29 cells. Colony- and sphereforming abilities were significantly correlated with a decrease in the cell count and size. A TOP/FOP flash reporter assay revealed that the inhibitory activity of the n-hexane fraction of RB ($0.22{\pm}0.02$ fold change) was lower than that of the 80% methanol extract and that of other fractions. The n-hexane and ethyl acetate fractions of RB were predominantly dependent on the expression levels of intracellular ${\beta}-catenin$. Western blotting using $p-GSK3{\beta}$ with only the n-hexane fraction of RB was conducted to examine whether these secondary metabolites reduced ${\beta}-catenin$ degradation. Intracellular ${\beta}-catenin$ regulation resulted in quantitative changes in the nucleus. In summary, these results demonstrate the potential of the n-hexane fraction of RB as a natural anticancer agent.

• Nutrition Research and Practice
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• v.17 no.5
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• pp.899-916
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• 2023

BACKGROUND/OBJECTIVES: Oxidative stress is a fundamental neurodegenerative disease trigger that damages and decimates nerve cells. Neurodegenerative diseases are chronic central nervous system disorders that progress and result from neuronal degradation and loss. Recent studies have extensively focused on neurodegenerative disease treatment and prevention using dietary compounds. Heseperetin is an aglycone hesperidin form with various physiological activities, such as anti-inflammation, antioxidant, and antitumor. However, few studies have considered hesperetin's neuroprotective effects and mechanisms; thus, our study investigated this in hydrogen peroxide (H₂O₂)-treated SH-SY5Y cells. MATERIALS/METHODS: SH-SY5Y cells were treated with H₂O₂ (400 µM) in hesperetin absence or presence (10-40 µM) for 24 h. Three-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assays detected cell viability, and 4',6-diamidino-2-phenylindole staining allowed us to observe nuclear morphology changes such as chromatin condensation and apoptotic nuclei. Reactive oxygen species (ROS) detection assays measured intracellular ROS production; Griess reaction assays assessed nitric oxide (NO) production. Western blotting and quantitative polymerase chain reactions quantified corresponding mRNA and proteins. RESULTS: Subsequent experiments utilized various non-toxic hesperetin concentrations, establishing that hesperetin notably decreased intracellular ROS and NO production in H₂O₂-treated SH-SY5Y cells (P < 0.05). Furthermore, hesperetin inhibited H₂O₂-induced inflammation-related gene expression, including interluekin-6, tumor necrosis factor-α, and nuclear factor kappa B (NF-κB) p65 activation. In addition, hesperetin inhibited NF-κB translocation into H₂O₂-treated SH-SY5Y cell nuclei and suppressed mitogen-activated protein kinase protein expression, an essential apoptotic cell death regulator. Various apoptosis hallmarks, including shrinkage and nuclear condensation in H₂O₂-treated cells, were suppressed dose-dependently. Additionally, hesperetin treatment down-regulated Bax/Bcl-2 expression ratios and activated AMP-activated protein kinase-mammalian target of rapamycin autophagy pathways. CONCLUSION: These results substantiate that hesperetin activates autophagy and inhibits apoptosis and inflammation. Hesperetin is a potentially potent dietary agent that reduces neurodegenerative disease onset, progression, and prevention.

• Journal of Life Science
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• v.28 no.9
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• pp.1016-1021
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• 2018

Clostridium difficile toxin A causes pseudomembranous colitis. The pathogenesis of toxin A-induced colonic inflammation includes toxin A-dependent epithelial cell apoptosis, resulting in the loss of barrier function provided by epithelial cells against luminal pathogens. Toxin A-dependent epithelial cell apoptosis has been linked to toxin A-induced production of reaction oxygen species and subsequent p38MAPK activation; $p21^{CIP1/WAF1}$ upregulation-dependent cell cycle arrest; cytoskeletal disaggregation; and/or the induction of Fas ligand on epithelial cells. However, the molecular mechanisms underlying toxin A-induced apoptosis remain poorly understood. This study tested whether toxin A could block the Wnt signaling pathway, which is involved in gut epithelial cell proliferation, differentiation and antiapoptotic progression. Toxin A treatment of nontransformed human colonocytes (NCM460) rapidly reduced ${\beta}$-catenin protein, an essential component of the Wnt signaling pathway. Exposure of mouse ileum to toxin A also significantly reduced ${\beta}$-catenin protein levels. MG132 inhibition of proteasome-dependent protein degradation resulted in the recovery of toxin A-mediated reduction of ${\beta}$-catenin, indicating that toxin A may activate intracellular processes, such as $GSK3{\beta}$, to promote degradation of ${\beta}$-catenin. Immunoblot analysis showed that toxin A increased active phosphorylation of $GSK3{\beta}$. Because the Wnt signaling pathway is essential for gut epithelial cell proliferation and anti-apoptotic processes, our results suggest that toxin A-mediated inhibition of the Wnt signaling pathway may be required for maximal toxin A-induced apoptosis of gut epithelial cells.