• Journal of Embryo Transfer
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• v.31 no.1
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• pp.27-32
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• 2016

This study was conducted to evaluate the effects of different volume ($100{\mu}l$ vs. 2 ml) of microdrop culture on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. Blastulation rate was not different between groups ($58.4{\pm}2.9%$ vs. $61.2{\pm}4.8%$). Zona hatched rate ($38{\pm}15.4%$ vs. $27{\pm}3.4%$) and attached rate ($55{\pm}13.9%$ vs. $46{\pm}3.9%$) did not differ by the volume of culture media. Total cell numbers ($59.8{\pm}9.7$ vs. $70.3{\pm}8.7$), ICM cell numbers ($15.8{\pm}0.6$ vs. $16.8{\pm}1.5$), TE cell numbers ($44.0{\pm}9.7$ vs. $53.6{\pm}7.3$), % ICM ($26.4{\pm}2.9%$ vs. $23.8{\pm}3.3%$) and ICM:TE ratio ($1:2.8{\pm}0.4$ vs. $1:3.2{\pm}0.6$) were not different between groups (i.e., $100{\mu}l$ vs. 2 ml). These results show that the capacity of the culture medium did not effect the cell numbers of B6D2F1 mice blastocysts. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• The Korean journal of internal medicine
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• v.34 no.1
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• pp.146-155
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• 2019

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of $NF-{\kappa}B$ was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. Results: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt in HK-2 cells. $NF-{\kappa}B$ promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, $NF-{\kappa}B$, and Akt signaling pathway in HK-2 cells.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.5533-5537
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• 2013

MicroRNA-10b (miR-10b) has been reported to play an important role in some types of cancer, but the effects and possible mechanisms of action of miR-10b in the metastasis of nasopharyngeal carcinoma cells (NPC) have not been explored. The aim of the present study was to investigate the function of miR-10b in nasopharyngeal carcinoma and to determine the molecular mechanisms underlying its action. The MTT assay was used to assess proliferation of CNE-2Z cells. Wound healing and transwell migration assays were applied to assess cell migration and invasion, while and expression of E-cadherin and MMP-9 were detected using Western blot analysis. Real-time PCR was employed to detect the expression of genes related to migration and invasion and the $2^{-{\Delta}{\Delta}Ct}$ method was used to calculate the degree of expression. MTT assay showed the expression of miR-10b to have no effect on the proliferation of NPC cell lines. The wound healing assay showed that miR-10b mimics promoted the mobility and invasion of NPC cell lines. Inhibitors of miR-10b reduced the ability of NPC cell lines to migrate and invade. In addition, the expression of genes related to migration and invasion, such as E-cadherin, vimentin, and MMP-9, were confirmed to be different in the CNE-2Z NPC cell line transfected with miR-10b mimics and with miR-10b inhibitors. In the present study, miR-10b was found to upregulate the expression of MMP-9 and knockdown of miR-10b was found to significantly downregulate the expression of E-cadherin. On the whole, these results showed that miR-10b plays an important role in the invasion and metastasis of NPC cells.

• Nutrition Research and Practice
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• v.7 no.2
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• pp.89-95
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• 2013

Dietary inorganic sulfur is the minor component in our diet, but some studies suggested that inorganic sulfur is maybe effective to treat cancer related illness. Therefore, this study aims to examine the effects of inorganic sulfur on cell proliferation and gene expression in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells were cultured the absence or presence of various concentrations (12.5, 25, or 50 ${\mu}mol/L$) of inorganic sulfur. Inorganic sulfur significantly decreased proliferation after 72 h of incubation (P < 0.05). The protein expression of $ErbB_2$ and its active form, $pErbB_2$, were significantly reduced at inorganic sulfur concentrations of 50 ${\mu}mol/L$ and greater than 25 ${\mu}mol/L$, respectively (P < 0.05). The mRNA expression of $ErbB_2$ was significantly reduced at an inorganic sulfur concentration of 50 ${\mu}mol/L$ (P < 0.05). The protein expression of $ErbB_3$ and its active form, $pErbB_3$, and the mRNA expression of $pErbB_3$ were significantly reduced at inorganic sulfur concentrations greater than 25 ${\mu}mol/L$ (P < 0.05). The protein and mRNA expression of Akt were significantly reduced at an inorganic sulfur concentration of 50 ${\mu}mol/L$ (P < 0.05), but pAkt was not affected by inorganic sulfur treatment. The protein and mRNA expression of Bax were significantly increased with the addition of inorganic sulfur concentration of 50 ${\mu}mol/L$ (P < 0.05). In conclusion, cell proliferation was suppressed by inorganic sulfur treatment through the ErbB-Akt pathway in MDA-MB-231 cells.

• Molecules and Cells
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• v.45 no.10
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• pp.749-760
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• 2022

Osteoclast generation from monocyte/macrophage lineage precursor cells needs to be tightly regulated to maintain bone homeostasis and is frequently over-activated in inflammatory conditions. PARK2, a protein associated with Parkinson's disease, plays an important role in mitophagy via its ubiquitin ligase function. In this study, we investigated whether PARK2 is involved in osteoclastogenesis. PARK2 expression was found to be increased during the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. PARK2 gene silencing with siRNA significantly reduced osteoclastogenesis induced by RANKL, LPS (lipopolysaccharide), TNFα (tumor necrosis factor α), and IL-1β (interleukin-1β). On the other hand, overexpression of PARK2 promoted osteoclastogenesis. This regulation of osteoclastogenesis by PARK2 was mediated by IKK (inhibitory κB kinase) and NF-κB activation while MAPK (mitogen-activated protein kinases) activation was not involved. Additionally, administration of PARK2 siRNA significantly reduced osteoclastogenesis and bone loss in an in vivo model of inflammatory bone erosion. Taken together, this study establishes a novel role for PARK2 as a positive regulator in osteoclast differentiation and inflammatory bone destruction.

• The Journal of Internal Korean Medicine
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• v.27 no.1
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• pp.149-165
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• 2006

Objectives: This study was done to evaluate the effects of Saengkankunbi-tang on cell-viability, proliferation, cell-cycle, apoptosis and DNA replication on HepG2 cell and to find out by which molecular-biological mechanism by which Saengkankunbi-tang operates. Methods : The MTT assay, cell counting assay, [3H]-thymidine incorporation assay, flow cytometric analysis, tryphan blue exclusion assay, western blot analysis, quantative RT-PCR were taken. Results : Saengkankunbi-tang had no effect on proliferation, cell-cycle and DNA replications of HepG2 cells, while it improved cell viability and reduced apoptosis, and it activated Akt and NFKB. But, it did not produce an effect on cell viability and apoptosis when P13K/Akt pathway was blocked by LY294002 nor when $NF{\kapa}B$ activation was blocked by DN-$I{\kapa}B$. Conclusion : These results suggests that Saengkankunbi-tang improves cell viability and reduces apoptosis of HepG2 cells, by activating $NF{\kapa}B$ through PI3K/Akt pathway.

• Journal of Microbiology and Biotechnology
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• v.1 no.2
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• pp.102-110
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• 1991

Alkalophilic Bacillus sp. YJ-451, which was isolated from soil at several area in Korea, produced a novel type of bacteriolytic enzyme (cell wall peptidoglycan hydrolase) extracellulary. The cell wall hydrolytic activity was identified as a clear zone on sodium dodecyl sulfate polyacrylamide gel electrophoresis containing 0.2% (w/v) cell wall of Bacillus sp. as substrate. This enzyme was successively purified 66 fold with 3.2% yield in culture broth by ammonium sulfate precipitation, CM-cellulose column chromatography, and gel filtration, followed by hydroxylapatite column chromatography. The molecular weight of the purified enzyme was estimated to be 27,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration column chromatography. The optimum pH and temperature for the activity of the enzyme were pH 10.0 and $50^{\circ}C$, respectively. The enzyme was stable between pH 5.0 and 10.0 and up to $40^{\circ}C$. Among the microorganisms used in this experiment the enzyme was active against most of gram negative strains and the genus Bacillus such as B. megaterium, B. licheniformis, B. circulans, B. pumilus, B. macerans, B. polymyxa. The release of dinitrophenylglutamic acid but not reducing group from cell wall peptidoglycan digested by the enzyme suggested that the enzyme is a kind of peptidase which hydrolyzes the peptide bond at the amino group of D-glutamic acid in the peptidoglycan.

• Environmental Engineering Research
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• v.23 no.3
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• pp.258-264
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• 2018

The objectives of this research were to 1) isolate and identify thermophilic bacteria for food waste treatment; 2) investigate the capability of food waste treatment using Bacillus species; and 3) develop air-dried Bacillus starters for food waste treatment. Five Bacillus species were isolated from food wastes and identified as Bacillus licheniformis (B. licheniformis) G1, Bacillus circulans C2, Bacillus subtilis (B. subtilis) E1, Bacillus vanillea F1, and Bacillus atrophaeus G2 based on 16S rDNA sequencing. Each identified Bacillus and the mixture of Bacillus species were cultivated in the standard food waste at $45^{\circ}C$ for 8 d. Changes in cell count, solid contents, and pH of the food waste were monitored during cultivation. Air-dried Bacillus cell powders were prepared using wheat flour and lactomil as excipients, and the cell count and survival rate were determined. The cell count of B. licheniformis G1 exhibited the highest number among the tested Bacillus (${\sim}10^8CFU/mL$). The greatest reduction in solid contents of food waste was achieved by B. subtilis E1 (22.6%). The mixture of B. licheniformis G1 and B. subtilis E1 exhibited a synergistic effect on the reduction of solid contents. Lactomil was determined as better excipient than wheat flour based on the greatest survival rate of 95%.

• Journal of Life Science
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• v.7 no.2
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• pp.79-87
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• 1997

To determine the cause of Vibrio septicemia by understanding the characteristics endotoxin from Vibrio vulnificus, lethal dose, heat resistance and vascular permeability enhancing activity were svaluated using vegestative cell and cell homogenate and the result is as follows: 1. Vibrio vulnificus CDC B3547 of patient origin did not exihibit any significant difference in toxicity compared to Vibrio vulnificus B57 of enviroment origin. 2. Strong toxicity was observed when viable cell count of Vibrio vulnificus CDC B3547 was more than 10$^{7}$/ml. 3. Toxicity of cell homogenate was completely inactivited upon geating at 80$^{\circ}$C for 20min. 4. Cell homogenate did not show hemolyic activity but was acknowleged to have cytotoxicity. 5. Major lethal toxin against mouse was existed in Vibrio vulnificus CDC B3547; however, separation of LPS and LPS-protein complex was not successful using the current technique.

• Tuberculosis and Respiratory Diseases
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• v.55 no.5
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• pp.488-498
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• 2003

Background : Activation of the transcription factor NF-${\kappa}B$ has been shown to protect cells from tumor necrosis factor-alpha, chemotherapy, and radiation-induced apoptosis. NF-${\kappa}B$-dependent cIAP expression is a major antiapoptotic mechanism for that. NF-${\kappa}B$ activation and cIAP expression in A549 lung cancer cells which is relatively resistant to radiation-induced cell death were investigated for the mechanism of radioresistance. Materials and methods : We used A549 lung cancer cells and Clinac 1800C linear accelerator for radiation. Cell viability test was done by MTT assay. NF-${\kappa}B$ activation was tested by luciferase reporter gene assay, Western blot for $I{\kappa}B{\alpha}$ degradation, and electromobility shift assay. For blocking ${\kappa}B$, MG132 and transfection of $I{\kappa}B{\alpha}$-superrepressor plasmid construct were used. cIAP expression was analyzed by RT-PCR and cIAP2 promoter activity was performed using luciferase assay system. Results : MTT assay showed that cytotoxicity even 48 hr after radiation in A549 cells were less than 20%. Luciferas assay demonstrated weak NF-${\kappa}B$ activation of $1.6{\pm}0.2$ fold compared to PMA-induced $3.4{\pm}0.9$ fold. Radiation-induced $I{\kappa}B{\alpha}$ degradation was observed in Western blot and NF-${\kappa}B$ DNA binding was confirmed by EMSA. However, blocking NF-${\kappa}B$ using MG132 and $I{\kappa}B{\alpha}$-superrepressor transfection did not show any sensitizing effect for radiation-induced cell death. The result of RT-PCR for cIAP1 & 2 expression was negative induction while TNF-${\alpha}$ showed strong expression for cIAP1 & 2. The cIAP2 promoter activity also did not show any change compared to positive control with TNF-${\alpha}$. Conclusion : We conclude that activation of NF-${\kappa}B$ does not determine the intrinsic radiosensitivity of cancer cells, at least for the cell lines tested in this study.