• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1343-1349
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• 2021

Cockroaches live in places where various pathogens exist and thus are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the Periplaneta americana transcriptome and detected periplanetasin-5 using an in silico antimicrobial peptide prediction method. In this study, we investigated whether periplanetasin-5 has anticancer activity against the human leukemia cell line K562. Cell growth and survival of K562 cells treated with periplanetasin-5 were decreased in a dose-dependent manner. By using flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation, we found that periplanetasin-5 induced apoptotic and necrotic cell death in leukemia cells. In addition, these events were associated with increased levels of the pro-apoptotic proteins Fas and cytochrome c and reduced levels of the anti-apoptotic protein Bcl-2. Periplanetasin-5 induces the cleavage of pro-caspase-9, pro-caspase-8, pro-caspase-3, and poly (ADP-ribose) polymerase (PARP). The above data suggest that periplanetasin-5 induces apoptosis via both the intrinsic and extrinsic pathways. Moreover, caspase-related apoptosis was further confirmed by using the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), which reversed the periplanetasin-5-induced reduction in cell viability. In conclusion, periplanetasin-5 caused apoptosis in leukemia cells, suggesting its potential utility as an anticancer therapeutic agent.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.3
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• pp.185-195
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• 2022

Objective: This study aimed to determine the effect of sperm DNA fragmentation (SDF) on the cumulative live birth rate (CLBR) in intracytoplasmic sperm injection (ICSI) cycles in couples with unexplained infertility. Methods: We conducted a prospective study of 145 couples who underwent ICSI cycles for unexplained infertility. Based on the SDF rate, patients were categorized into a low SDF group (SDF ≤30%, n=97) and a high SDF group (SDF >30%, n=48). SDF was assessed using the acridine orange test on density gradient centrifugation prepared samples. The CLBR was calculated as the first live birth event per woman per egg collection over 2 years. Results: The high SDF group (SDF >30%) showed a significantly lower CLBR (p<0.05) and a significantly higher miscarriage rate (p<0.05) than the low SDF group (SDF ≤30%). No significant difference was observed in the implantation and cumulative pregnancy rates between the two SDF groups. The total number of embryo transfers was stratified further into fresh and frozen embryo transfers. In the fresh embryo transfers, there were significant differences in the implantation rates, clinical pregnancy rates, and live birth rates (p<0.05) between the low SDF and high SDF groups. However, in the frozen embryo transfers, there were no significant differences in clinical outcomes between the two groups. In the multivariable logistic regression analysis, SDF was a predictor of CLBR (p<0.05) when adjusted for possible confounding factors. Conclusion: High SDF was associated with a lower CLBR and a higher miscarriage rate in the ICSI cycles of couples with unexplained infertility.

• Biomolecules & Therapeutics
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• v.30 no.4
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• pp.348-359
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• 2022

Gastric adenocarcinoma is among the top causes of cancer-related death and is one of the most commonly diagnosed carcinomas worldwide. Benzyl isothiocyanate (BITC) has been reported to inhibit the gastric cancer metastasis. In our previous study, BITC induced apoptosis in AGS cells. The purpose of the present study was to investigate the effect of BITC on autophagy mechanism in AGS cells. First, the AGS cells were treated with 5, 10, or 15 μM BITC for 24 h, followed by an analysis of the autophagy mechanism. The expression level of autophagy proteins involved in different steps of autophagy, such as LC3B, p62/SQSTM1, Atg5-Atg12, Beclin1, p-mTOR/mTOR ratio, and class III PI3K was measured in the BITC-treated cells. Lysosomal function was investigated using cathepsin activity and Bafilomycin A1, an autophagy degradation stage inhibitor. Methods including qPCR, western blotting, and immunocytochemistry were employed to detect the protein expression levels. Acridine orange staining and omnicathepsin assay were conducted to analyze the lysosomal function. siRNA transfection was performed to knock down the LC3B gene. BITC reduced the level of autophagy protein such as Beclin 1, class III PI3K, and Atg5-Atg12. BITC also induced lysosomal dysfunction which was shown as reducing cathepsin activity, protein level of cathepsin, and enlargement of acidic vesicle. Overall, the results showed that the BITC-induced AGS cell death mechanism also comprises the inhibition of the cytoprotective autophagy at both initiation and degradation steps.

• Journal of Ginseng Research
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• v.47 no.3
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• pp.479-491
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• 2023

Background: Hepatocellular carcinoma (HCC) has a high incidence and is one of the highest mortality cancers when advanced stage is proceeded. However, Anti-cancer drugs available for treatment are limited and new anti-cancer drugs and new ways to treat them are minimal. We examined that the effects and possibility of Red Ginseng (RG, Panax ginseng Meyer) as new anti-cancer drug on HCC by combining network pharmacology and molecular biology. Materials and Methods: Network pharmacological analysis was employed to investigate the systems-level mechanism of RG focusing on HCC. Cytotoxicity of RG was determined by MTT analysis, which were also stained by annexin V/PI staining for apoptosis and acridine orange for autophagy. For the analyze mechanism of RG, we extracted protein and subjected to immunoblotting for apoptosis or autophagy related proteins. Results: We constructed compound-target network of RG and identified potential pathways related to HCC. RG inhibited growth of HCC through acceleration of cytotoxicity and reduction of wound healing ability of HCC. RG also increased apoptosis and autophagy through AMPK induction. In addition, its ingredients, 20S-PPD (protopanaxadiol) and 20S-PPT (protopanaxatriol), also induced AMPK mediated apoptosis and autophagy. Conclusion: RG effectively inhibited growth of HCC cells inducing apoptosis and autophagy via ATG/AMPK in HCC cells. Overall, our study suggests possibility as new anti-cancer drug on HCC by proof for the mechanism of the anti-cancer action of RG.

• Tuberculosis and Respiratory Diseases
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• v.69 no.1
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• pp.16-23
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• 2010

Background: Most lung cancer patients receive systemic chemotherapy at an advanced stage disease. Cisplatin-based chemotherapy is the main regimen for treating advanced lung cancer. Recently, autophagy has become an important mechanism of cellular adaptation under starvation or cell oxidative stress. The purpose of this study was to determine whether or not autophagy can occurred in cisplatin-treated lung cancer cells. Methods: H460 cells were incubated with RPMI 1640 and treated in $5{\mu}M$ or $20{\mu}M$ cisplatin concentrations at specific time intervals. Cells surviving cisplatin treatment were measured and compared using an MTT cell viability assay to cells that underwent apoptosis with autophagy by nuclear staining, apoptotic or autophagic related proteins, and autophagic vacuoles. The development of acidic vascular organelles was using acridine orange staining and fluorescent expression of GFP-LC3 protein in its transfected cells was observed to evaluate autophagy. Results: Lung cancer cells treated with $5{\mu}M$ cisplatin-treated were less sensitive to cell death than $20{\mu}M$ cisplatin-treated cells in a time-dependent manner. Nuclear fragmentation at $5{\mu}M$ was not detected, even though it was discovered at $20{\mu}M$. Poly (ADP-ribose) polymerase cleavages were not detected in $5{\mu}M$ within 24 hours. Massive vacuolization in the cytoplasm of $5{\mu}M$ treated cells were observed. Acridine orange stain-positive cells was increased according in time-dependence manner. The autophagosome-incorporated LC3 II protein expression was increased in $5{\mu}M$ treated cells, but was not detected in $20{\mu}M$ treated cells. The expression of GFP-LC3 were increased in $5{\mu}M$ treated cells in a time-dependent manner. Conclusion: The induction of autophagy occurred in $5{\mu}M$ dose of cisplatin-treated lung cancer cells.

• Journal of Microbiology
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• v.42 no.4
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• pp.285-291
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• 2004

The soil bacterial community and some inoculated bacteria were monitored to assess the microbial responses to prescribed fire in their microcosm. An acridine orange direct count of the bacteria in the unburned control soil were maintained at a relatively stable level $(2.0\~2.7\times10^9\;cells/g^{-1}{\cdot}soil)$ during the 180 day study period. The number of bacteria in the surface soil was decreased by fire, but was restored after 3 months. Inoculation of some bacteria increased the number of inoculated bacteria sev­eral times and these elevated levels lasted several months. The ratios of eubacteria detected by a flu­orescent in situ hybridization (FISH) method to direct bacterial count were in the range of $60\~80\%$ during the study period, with the exception of some lower values at the beginning, but there were no definite differences between the burned and unburned soils or the inoculated and uninoculated soils. In the unburned control soil, the ratios of $\alpha-,\beta-\;and\;\gamma-subgroups$ of the proteobacteria, Cytophaga-Fla­vobacterium and other eubacteria groups to that of the entire eubacteria were 13.7, 31.7, 17.1, 16.8 and $20.8\%,$ respectively, at time 0. The overall change on the patterns of the ratios of the 5 subgroups of eubacteria in the uninoculated burned and inoculated soils were similar to those of the unburned con­trol soil, with the exception of some minor variations during the initial period. The proportions of each group of eubacteria became similar in the different microcosms after 6 months, which may indicate the recovery of the original soil microbial community structure after fire or the inoculation of some bac­teria. The populations of Azotobacter vinelandii, Bacillus megaterium and Pseudomonas fluorescens, which had been inoculated to enhance the microbial activities, and monitored by FISH method, showed similar changes in the microcosms, and maintained high levels for several months.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Korean Journal of Veterinary Research
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• v.37 no.2
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• pp.409-416
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• 1997

Bovine theileriosis caused by Theileria sergenti is the tick-borne intraery- throcytic piroplasmosis, that occurs in most regions of Korea. It results in severe economic losses on a farm caused by anemia, milk production loss, abortion and death. This study was undertaken to confirm the effects of the growth hormone and the insulin-like growth factor-I which are associated in the growth of cattle infected by T sergenti. The blood of one hundred and twenty ten-month Holstein was collected and the prepared blood smear was stained with acridine orange to investigate their parasitemia. And the hematological profiles were observed. According to the value of the hematocrit, they were categorized into four groups : Group 1 was under 20 percent, groups 2 and 3 were from over 21 to under 30 percent and from over 31 to under 35 percent and group 4 was over 36 percent. As the value of the hematocrit decreased, parasitemia(%) in erythrocytes was observed to increase(Y=-1.064X + 30.537, r=0.660). The amounts of the growth hormone and the insulin-like growth factor-I in the serum were measured by the radioimmunoassay. The growth hormone in serum of the group 1, group 2, group 3 and group 4 were observed as $0.238{\pm}0.043nmol/l$, $0.21{\pm}0.024nmol/l$, $0.366{\pm}0.035nmol/l$ and $0.646{\pm}0.223nmol/l$, respectively. The quantitative of the insulin-like growth factor-I in the same groups were observed also as $209.686{\pm}18.94ng/ml$, $250.9{\pm}12.609ng/ml$, $279.3{\pm}8.883ng/ml$ and $365.9{\pm}22.45ng/ml$, respectively. It can be concluded that the growth hormone and the insulin-like growth factor-I were observed to decrease in severe anemia due to theileriosis.

• Korean Journal of Acupuncture
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• v.26 no.1
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• pp.79-84
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• 2009

Objective : In 1960's Bonghan Kim's team found BongHan(BH) ducts which were presumed as acupuncture meridians and BH corpuscles. They asserted Bonghan theory and SanAl theory which was involved in cell division and cell restoration. However, many other experiments which had been operated to demonstrate and find the existence of BH ducts had failed because of the secret of blue stain drugs. During the last several years, BongHan theory has been revived through experimental researches to find the anatomical structures of BH ducts and corpuscles by Soh's Biomedical Physics Lab. Soh's research team used the staining with Janus Green B, Alcian blue, nanoparticles and Acridine Orange. We used DAPI staining to find the existence of BH ducts and the corpuscles and to observe nuclear arrangement. Methods : We used japan white rabbits as experimental animals. BH ducts and corpuscles were stained with DAPI. The nucleus configuration in BH ducts stained with DAPI were observed with microscope. Results : In this study, we found thread like structures in silver white color distinguished from the blood vessels, nerves and lymph vessels. These thread like vessels in the linear duct shape were connected to same colored mass in the ball shape. Thread like structures we found could be separated easily from the surrounding other organ mass. The nuclei of the thread like structure in DAPI staining, are about 10${\sim}$20${\mu}m$ length, in rod shape and linear arrangement. Conclusion : We concluded that the thread like structure we found was same vessel reported by Soh's research team, BongHan ducts and corpuscle.

• Biomolecules & Therapeutics
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• v.28 no.2
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• pp.202-210
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• 2020

Fluoxetine is used widely as an antidepressant for the treatment of cancer-related depression, but has been reported to also have anti-cancer activity. In this study, we investigated the cytotoxicity of fluoxetine to human gastric adenocarcinoma cells; as shown by the MTT assay, fluoxetine induced cell death. Subsequently, cells were treated with 10 or 20 µM fluoxetine for 24 h and analyzed. Apoptosis was confirmed by the increased number of early apoptotic cells, shown by Annexin V- propidium iodide staining. Nuclear condensation was visualized by DAPI staining. A significant increase in the expression of cleaved PARP was observed by western blotting. The pan-caspase inhibitor Z-VAD-FMK was used to detect the extent of caspase-dependent cell death. The induction of autophagy was determined by the formation of acidic vesicular organelles (AVOs), which was visualized by acridine orange staining, and the increased expression of autophagy markers, such as LC3B, Beclin 1, and p62/SQSTM 1, observed by western blotting. The expression of upstream proteins, such as p-Akt and p-mTOR, were decreased. Autophagic degradation was evaluated by using bafilomycin, an inhibitor of late-stage autophagy. Bafilomycin did not significantly enhance LC3B expression induced by fluoxetine, which suggested autophagic degradation was impaired. In addition, the co-administration of the autophagy inhibitor 3-methyladenine and fluoxetine significantly increased fluoxetine-induced apoptosis, with decreased p-Akt and markedly increased death receptor 4 and 5 expression. Our results suggested that fluoxetine simultaneously induced both protective autophagy and apoptosis and that the inhibition of autophagy enhanced fluoxetine-induced apoptosis through increased death receptor expression.