• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.241-249
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• 2017

Plasma membrane hyperpolarization associated with activation of $Ca^{2+}$-activated $K^+$ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary ${\gamma}^2$-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific $K^+$ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical $BK_{Ca}$ activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting ${\gamma}^2$ subunit, hLRRC52. As previously reported, Slo3 $K^+$ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 $K^+$ current, and internal alkalinization and $Ca^{2+}$ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 $K^+$ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular $Ca^{2+}$. In contrast, elevation of intracellular $Ca^{2+}$ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate $Ca^{2+}$-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.3
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• pp.413-418
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• 2016

Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

• Korean Journal of Medicinal Crop Science
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• v.17 no.5
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• pp.321-327
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• 2009

Sunlight, and in particular its UV component, is the major environmental trigger that underlies the major signs of human skin and skin cancer in general. Therefore, this study was carried out to investigate the UV protection effects of R. coreanus. R. coreanus was extracted by ultra high pressure extraction process at 500 MPa and $30^{\circ}C$ for 5 and 15 minutes. The cytotoxicity of the extracts extracted by ultra high pressure process on human dermal fibroblast cell CCD-986sk, human kidney normal cell HEK293, and human lung normal cell HEL299 was measured as 17.5%, 16.5% and 14.0%, respectively in adding $1.0\;mg/m{\ell}$ of the samples, which was much lower than that from conventional water extraction method at $100^{\circ}C$ as 23.2%, 22.5%, 21.2%. The secretion of $NO^-$ from macrophage showed $15.9\;{\mu}M$ on the R. coreanus extract from this process, which was higher than others. Prostaglandin $E_2$ ($PGE_2$) production from UV-induced human skin cells was also greatly decreased down to $510\;pg/m{\ell}$, compared to the control. From the results, we considered that the extracts from R. coreanus could be potent natural materials for skin anti-inflammation agent, and could be used as a potential anti-aging for the photo-damaged skin.

• Korean Journal of Medicinal Crop Science
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• v.17 no.4
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• pp.280-285
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• 2009

Both fresh Codonopsis lanceolata and lactic acid bacteria fermented Codonopsis lanceolata were extracted with water at $100^{\circ}C$, and tested for anticancer activity using several human cancer cell lines. The fermented extracts inhibited the growth of hepatocellular carcinoma cells up to 90%, compared to 75% for fresh Codonopsis lanceolata. The extracts of cytotoxicity on human normal lung cells (HEK293) were as low as 15%. Especially, human hepatocellular carcinoma cell were more efficiently inhibited than other cells. This extract also inhibited $\alpha$-glucosidase activity up to 60% at 1.0mg/$m{\ell}$. This fermented extracts showed the inhibition potency on tyrosinase by 25% at 1.0mg/$m{\ell}$. From the results, the fermented Codonopsis lanceolata enhanced several biological activities up to $20{\sim}30%$, compared to those from fresh Codonopsis lanceolata. It implies that fermentation process could be one of useful methods of utilizing low quality Codonopsis lanceolata. Because this process could yield high amounts of biologically active compounds by the help of microbial growth.

• Molecular & Cellular Toxicology
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• v.3 no.4
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• pp.292-298
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• 2007

Zinc plays indispensable roles in metabolism, including cell growth, apoptosis, proliferation and differentiation. Kidneys are target organs for various regulators of mineral metabolism, and play a key role in zinc balance. To investigate the zinc uptake efficiency, we examined the zinc uptake and accumulation level in vivo and in vitro study. Plasma zinc concentration was peaked out at 1 hr after oral zinc administration. The renal zinc level was peaked out at 12 hr after oral zinc administration, and it was the highest in 40 mg/kg Zn-Asp administrated group in comparison with other groups. In addition, the m-RNA expression level of zinc transporter-1 (ZnT-1), zinc transporter-2 (ZnT-2) and high-affinity L-aspartate transporter (EAAT-3) in Zn-Asp administered group were increased compared with control groups and $ZnSO_4$ group. In order to investigate the intracellular zinc uptake mechanism, we performed the in vitro study by using human embryonic kidney cell line, HEK 293. Intracellular zinc level was peaked out at 3 hr after zinc treatment. In the same way, the mRNA expression level of ZnT-1 and EAAT-3 were increased compared with control group. This study showed that Zn-Asp is effective the zinc uptake into the kidney by increasing the zinc transporter expression.

• International Journal of Oral Biology
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• v.39 no.3
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• pp.145-151
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• 2014

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.

• Mass Spectrometry Letters
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• v.7 no.1
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• pp.1-11
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• 2016

Various efforts have been developed to improve sample preparation steps, which strongly depend on hands-on processes for accurate and sensitive quantitative proteome analysis. In this study, we carried out heating the sample prior to trypsin digestion using an instrument to improve the tryptic digestion process. The heat shock generated by the system efficiently denatured proteins in the sample and increased the reproducibility in quantitative proteomics based on peptide abundance measurements. To demonstrate the effectiveness of the protocol, three cell lines (A human lung cancer cell line (A549), a human embryonic kidney cell line (HEK293T), and a human colorectal cancer cell line (HCT-116)) were selected and the effect of heat shock was compared to that of normal tryptic digestion processes. The tryptic digests were desalted and analysed by LC-MS/MS, the results showed 57 and 36% increase in the number of identified unique peptides and proteins, respectively, than conventional digestion. Heat shock treated samples showed higher numbers of shorter peptides and peptides with low inter-sample variation among triplicate runs. Quantitative LC-MS/MS analysis of heat shock treated sample yielded peptides with smaller relative error percentage for the triplicate run when the peak areas were compared. Exposure of heat-shock to proteomic samples prior to proteolysis in conventional digestion process can increase the digestion efficiency of trypsin resulting in production of increased number of peptides eventually leading to higher proteome coverage.

• Korean Journal of Medicinal Crop Science
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• v.14 no.6
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• pp.317-321
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• 2006

This study was performed to compare effect of anticancer activities on Rhodiola sachalinensis by ultrasonifi-cation process and solvent. Compared the yield to the water extracts (WE), wafer extracts with ultrasonification (WEU) at60, loot and ethyl alcohol extracts (EE), ethyl alcohol extracts with ultrasonification (EEU) at 60 ${\circ}$C from Rhodiola sachalinensis. Experimental studies were progressed through the anticancer activities such as cell cytotoxicity, inhibition activities of cell growth. The cell cytotoxicity using human embryonic kidney cell (HEK293) was showed cytotoxicity of below 26.26%by extracts of Rhodiola sachalinensis in 1.0 mg/ml concentration, The anticancer activities were increased in over 70% by extracts of Rhodiola sachalinensis in A549, AGS, Hep3B and MCF-7 cells. From the results, the extract Rhodiola sachalinensis were showed useful anticancer activities.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.203-212
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• 2020

Promyelocytic leukemia (PML) gene, through alternative splicing of its C-terminal region, generates several PML isoforms that interact with specific partners and perform distinct functions. The PML protein is a tumor suppressor that plays an important role by interacting with various proteins. Herein, we investigated the effect of the PML isoforms on oncostatin M (OSM)-induced signal transducer and activator of transcription-3 (STAT-3) transcriptional activity. PML influenced OSM-induced STAT-3 activity in a cell type-specific manner, which was dependent on the p53 status of the cells but regardless of PML isoform. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. In conclusion, PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in the aberrant activation of STAT-3 in cancer cells bearing mutant p53 probably might occur through the interaction of mutant p53 with PML.

• Journal of Pharmaceutical Investigation
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• v.35 no.1
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• pp.17-23
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• 2005

Polyethylenimine (PEI) has been used as cationic polymers for efficient gene transfer without the need for endosomolytic agents. Various kinds of PEIs with different molecular weight were tested in order to investigate the effects of the molecular weight of PEI on the transfection efficiency and cell cytotoxicity. The ${\beta}-galactosidase$ expression $(pCMV-{\beta}-gal)$ plasmid was used as a model DNA. Complex formation between PEI and pDNA was assessed by 1% agarose gel electrophoresis method. Particle size and zeta-potential of complexes were determined by electrophoretic light scattering spectrometer. In vitro transfection efficiency was assayed by measuring ${\beta}-galactosidase$ activity. Cell cytotoxicity was determined by MTT assay. Particle sizes of the complexes became smaller on increasing molecular weights of PEI and N/P ratios. Surface potential of complexes was increased as the molecular weight of PEI increased. Transfection efficiency of $pCMV-{\beta}-ga1$ on the HEK 293 cells was greatest with PEI 25 K system but having the lowest cell viability. PEI with high molecular weight showed higher transfection efficiency and cell viability than PEI with low molecular weight.