• Tuberculosis and Respiratory Diseases
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• v.56 no.2
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• pp.187-197
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• 2004

Background : Nitric Oxide (NO) is a multi-faceted molecule with dichotomous regulatory roles in many areas of biology. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in other cell types. This study was performed to characterize NO-induced cell death in lung epithelial cells and to investigate the roles of cell death regulators including iron, bcl-2 and p53. Methods : A549 cells were used for lung epithelial cells. SNP (sodium nitroprusside) and SNAP (S-nitroso-N-acetyl- penicillamine) were used for NO donor. Cytoxicity assay was done by MTT assay and crystal violet assay. Apoptotic assay was done by fluorescent microscopy after double staining with propidium iodide and hoecst 33342. Iron inhibition study was done with RBCs and FeSO4. For bcl-2 study, bcl-2 overexpressing cells (A549-bcl-2) were used and for p53 study, Western blot analysis and p53 functionally knock-out cells (A549-E6) were used. Results : SNP and SNAP induced dose-dependent cell death in A549 cells and fluorescent microscopy revealed that SNAP induced apoptosis in low doses but necrosis in high doses while SNP induced exclusively necrotic cell death. Iron inhibition study using RBCs and FeSO4 significantly blocked SNAP-induced cell death. And also SNAP-induced cell death was blocked by bcl-2 overexpression. Finally, we found that SNAP activate p53 by Western blot analysis and that SNAP-induced cell death was decreased in the abscence of p53. Conclusion : In lung epithelial cells, NO can induce cell death, more precisely apoptosis in low doses and necrosis in high doses. And iron, bcl-2, and p53 play important roles in NO-induced cell death.

• The Journal of Internal Korean Medicine
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• v.31 no.2
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• pp.331-345
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• 2010

Objectives : This study was designed to investigate the effects of Saenggantanggami-bang (SG) on nonalcoholic fatty liver disease. Methods : HepG2 cells were used in an in vitro model. HepG2 cells were divided into three groups. The Normal group was incubated with no fatty acid. The Control group was incubated with 1mM palmitic acid to introduce fat overloading. The PA-SG group was incubated with 1mM palmitic acid and various concentrations of Saenggantanggami-bang (SG). Cell viability and cytotoxicity were analyzed by MTT assay and LDH assay. Intracellular triglyceride (TG) levels, reactive oxygen species (ROS) levels, ATP amount, and GST activity were measured. Cell death pattern and protective effect of SG on cell death were studied by DNA fragmentation and caspase-3 intensity (western blot). Results : Compared with the Control group, cell viability of the PA-SG group significantly increased (P<0.01), cytotoxicity of the PA-SG group decreased (P<0.01), and intracellular TG levels and ROS levels of the PA-SG group decreased (P<0.05). In DNA fragmentation assay, necrotic pattern was observed and DNA fragment decreased in the PA-SG group. In western blot, apoptotic pattern was observed, caspase-3 intensity of the PA-SG group was reduced significantly, but there were no significant differences in intracellular ATP amount and GST activity between the control group and the PA-SG group. Conclusion : The results suggest that Saenggantanggami-bang can be a potential candidate for the clinical treatment of nonalcoholic fatty liver disease.

• Biomolecules & Therapeutics
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• v.11 no.3
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• pp.174-177
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• 2003

The present experiment was performed to investigate the protective effects of paeonol isolated from Moutan Cortex Radicis on primary cultured rat hepatocytes exposed to Br-A23187 ($Ca^{2+}$ ionophore). Br-A23187 is frequently used as a model of cell killing as inducing both necrotic and apoptotic cell death. Hepatocytes were isolated by collagenase perfusion from livers of fasted male Sprague Dawley rats and cultured overnight. Cell viability was determined by propidium iodide using fluorocytometry in Krebs-Ringer-HEPES buffer at pH 7.4. In addition, intracellular calcium was measured by excitation at 340 and 380 nm and emission at 505 nm using a luminescence spectrophotometer. Paeonol (20-100 ${\mu}M$) inhibited cell killing induced by 10 ${\mu}M$ Br-A23187, in a dose-dependent manner. Paeonol also reduced increased intracellular calcium level when hepatocytes were exposed to Br-A23187. Therefore, the present results suggest that paeonol protects the hepatocytotoxicity induced by Br-A23187, via inhibiting the influx of calcium into into rat hepatocytes.

• Proceedings of the KSME Conference
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• 2008.11a
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• pp.1539-1542
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• 2008

Plasma is 4th state of matters, which consists of electrons, neutral, and ionized particles. In biomedical research, cold plasma, which is generated in atmospheric condition, has been applied to disinfect microorganisms such as bacteria and yeast cells. Because of its low temperature condition, the heat-sensitive medical device can be easily sterilized by the cold plasma treatment. In recent years, the effects of plasma on mammalian cells have arisen as a new issue. Generally, plasma induces intensity dependent necrotic cell death. In this research, we investigate the feasibility of cold plasma treatment for cancer therapy by conducting comparative study of plasma effects on normal and cancer cells. We use THLE-2 (human liver normal cell) and SK-Hep1 (human liver metathetic cancer cell) as our target cells. The needle type of cold plasma is generated by the Helium plasma device. Two types of cells have different onset plasma conditions for the necrosis, which may be explained by difference in electrical properties of these two cell types.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.224-228
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• 2010

The encapsulation of bacteria may be used to harness them for longer periods of time in order to make them viable, whereas antibiotic treatment would result in controlled release of therapeutic molecules. Encapsulated Escherichia coli GFP (green fluorescent protein) (E. coli GFP) was used here as a model for therapeutic substance - GFP fragments release (model of bioactive substances). Our aim was to evaluate the performance of bacteria encapsulated in hollow fibers (HFs) treated with antibiotic for induction of cell death. The polypropylene-surface-modified HFs were applied for E. coli encapsulation. The encapsulated bacteria were treated with tetracycline in vitro or in vivo during subcutaneous implantation into mice. The HF content was evaluated in a flow cytometer, to assess the bacteria cell membrane permeability changes induced by tetracycline treatment. It was observed that the applied membranes prevented release of bacteria through the HF wall. The E. coli GFP culture encapsulated in HF in vitro proved the tetracycline impact on bacteria viability and allows the recognition of the sequence of events within the process of bacteria death. Treatment of the SCID mice with tetracycline for 8 h proved the tetracycline impact on bacteria viability in vivo, raising the necrotic bacteria-releasing GFP fragments. It was concluded that the bacteria may be safely enclosed within the HF at the site of implantation, and when the animal is treated with antibiotic, bacteria may act as a local source of fragments of proteins expressed in the bacteria, a hypothetical bioactive factor for the host eukaryotic organism.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.233-240
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• 2020

Autophagy regulators are often effective as potential cancer therapeutic agents. Here, we investigated paclitaxel sensitivity in cells with knockout (KO) of ATG5 gene. The ATG5 KO in multidrug resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) was generated using the CRISPR/Cas9 technology. The qPCR and LC3 immunoblot confirmed knockout of the gene and protein of ATG5, respectively. The ATG5 KO restored the sensitivity of Ras-NIH 3T3/Mdr cells to paclitaxel. Interestingly, ATG5 overexpression restored autophagy function in ATG5 KO cells, but failed to rescue paclitaxel resistance. These results raise the possibility that low level of resistance to paclitaxel in ATG5 KO cells may be related to other roles of ATG5 independent of its function in autophagy. The ATG5 KO significantly induced a G₂/M arrest in cell cycle progression. Additionally, ATG5 KO caused necrosis of a high proportion of cells after paclitaxel treatment. These data suggest that the difference in sensitivity to paclitaxel between ATG5 KO and their parental MDR cells may result from the disparity in the proportions of necrotic cells in both populations. Thus, our results demonstrate that the ATG5 KO in paclitaxel resistant cells leads to a marked G₂/M arrest and sensitizes cells to paclitaxel-induced necrosis.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.766-770
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• 2013

Several upstream activators required for proper activation of brlA are involved in the development, vegetative growth, toxin production, and pathogenesis of Aspergillus fumigatus. In this study, we characterized the roles of two upstream developmental regulators, A. fumigatus flbB (AfuflbB) and flbE (AfuflbE), in toxin production and virulence. The deletion of AfuflbB and AfuflbE resulted in reduction of the expression of AfulaeA. Moreover, only about 8% to 10% of fumagillin was produced in the two mutants compared with that of wild type, and ${\Delta}AfuflbB$ strain produced 85% of gliotoxin compared with wild type, whereas none was produced by ${\Delta}AfuflbB$. Flow-cytometric analysis revealed decreased necrotic and apoptotic polymorphonuclear leukocytes cell death after exposure to supernatants from ${\Delta}AfuflbB$ and ${\Delta}AfuflbB$ strains compared with the wild type. These results indicate that FlbB and FlbE are necessary for the proper laeA expression, toxin production, and virulence of A. fumigatus.

• Molecules and Cells
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• v.21 no.1
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• pp.7-20
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• 2006

The major event in human immunodeficiency virus type 1 (HIV-1) infection is the death of many cells related to host immune response. The demise of these cells is normally explained by cell suicide mechanism, apoptosis. Interestingly, the decrease in the number of immune cells, such as non-CD4+ cells as well as CD4+ T cells, in HIV infection usually occurs in uninfected bystander cells, not in directly infected cells. It has, therefore, been suggested that several soluble factors, including viral protein R (Vpr), are released from the infected cells and induce the death of bystander cells. Some studies show that Vpr interacts directly with adenine nucleotide translocator (ANT) to induce mitochondrial membrane permeabilization (MMP). The MMP results in release of some apoptogenic factors such as cytochrome-c (cyt-c) and apoptosis-inducing factor (AIF). Vpr also has indirect effect on mitochondria through enhancing the level of caspase-9 transcription and suppressing nuclear factor-kappa B (NF-${\kappa}B$). The involvement of p53 in Vpr-induced apoptosis remains to be studied. On the other hand, low level of Vpr expression has anti-apoptotic effect, whereas it's high level of expression induces apoptosis. Extracellular Vpr also exhibits cytotoxicity to uninfected bystander cells through apoptotic or necrotic mechanism. The facts that Vpr has cytotoxic effect on both infected cells and bystander cells, and that it exhibits both proand anti-apoptotic activity may explain its role in viral survival and disease progression.

• The Plant Pathology Journal
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• v.17 no.3
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• pp.128-140
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• 2001

Infection of pepper leaves by Colletotrichum cocodes at the two- and eight-leaf stages caused susceptible and resistant lesions 96 h after inoculation, respectively. At the two-leaf stage, progressive symptom development occurred on the infected leaves. In contrast, localized necrotic spots were characteristic symptoms at the eight-leaf stage. Infected leaves at the two-leaf stage exhibited cell death accompanied by the accumulation of autofluorescent compounds. At the eight-leaf stage, pepper leaves infected by the anthracnose fungus displayed localized autofluorescence from the symptoms. Infection of pepper leaves by C. cocodes at the two-leaf stage resulted in its rapidand massive colonization of all the leaf tissues including the vascular tissue, together with cytoplasmic collapse, distortion of chloroplasts, and disruption of host cell walls. However, penetration of C. cocodes was very limited in the older leaf tissues of pepper plants at the eight-leaf stage. Fungal hyphae grew only in the intramural spaces of the epidermal cell walls at this stage. Occlusion of amorphous material in xylem vessels, aggregation of fibrillar material in inter-cellular spaces, and deposition of protein bodies were found as resistance responses to C. cocodes.

• Archives of Pharmacal Research
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• v.23 no.1
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• pp.1-16
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• 2000

Many natural products elicit diverse pharmacological effects. Using two classes of potential chemopreventive compounds, the phenolic compounds and the isothiocyanates, we review the potential utility of two signaling events, the mitogen-activated protein kinases (MAPKs) and the ICE/Ced-3 proteases (caspases) stimulated by these agents in mammalian cell lines. Studies with phenolic antioxidants (BHA, tBHQ), and natural products (flavonoids; EGCG, ECG, and isothiocyanates; PEITC, sulforaphane), provided important insights into the signaling pathways induced by these compounds. At low concentrations, these chemicals may activate the MAPK (ERK2, JNK1, p38) leading to gene expression of survival genes (c-Fos, c-Jun) and defensive genes (Phase II detoxifying enzymes; GST, QR) resulting in survival and protective mechanisms (homeostasis response). Increasing the concentrations of these compounds will additionally activate the caspase pathway, leading to apoptosis (potential cytotoxicity). Further increment to suprapharmacological concentrations will lead to nonspecific necrotic cell death. The wider and narrow concentration ranges between the activation of MAPK/gene induction and caspases/cell death exhibited by phenolic compounds and isothiocyanates, respectively, in mammalian cells, may reflect their respective therapeutic windows in vivo. Consequently, the studies of signaling pathways elicited by natural products will advance our understanding of their efficacy and safety, of which many man become important therapeuitc drugs of the future.