• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.503-516
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• 2020

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC₅₀, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the β ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 μM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQ-specific agonist in the tissue.

• Korean Journal of Medicinal Crop Science
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• v.17 no.3
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• pp.210-216
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• 2009

This study was performed to compare the effects of immuno-modulating activities of Rhodiola sachalinensis A. Bor. fractionized by consecutive solvent separation. The Cytotoxicity of all fractionized extracts on human kidney cell (HEK293) was lower than crude extracts. Generally, the butanol and chloroform extracts showed less cytotoxicity on about 10.07% and 9.67% than the crude extracts. For human immune B and T cell growth, chloroform fraction showed the highest cell growth compared to the control. The secretion of cytokines (IL-6, $TNF-\alpha$) on human B and T cells were increased by adding chloroform extracts. Also, NK cell growth was significantly improved up to nearly 30% by adding the supernatant of B cell medium grown with the chloroform fraction. It was also found that chloroform fraction could yield higher nitric oxide production from macrophage than untreated control cells. Differentiation of HL-60 cells was increased up to 131.9% after treatment with chloroform fraction extracts, compared to the control. These results indicate that the chloroform fraction of R. sachalinensis have high immune activation activity than others fractions and the crude extracts, implying that this chloroform fractions could be used a new functional material.

• Korean Journal of Food Science and Technology
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• v.41 no.5
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• pp.552-559
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• 2009

In this study, the anticancer activity of the water extract at $100^{\circ}C$ was compared to that of the callus extracts via a ultrasonification extraction process. All the extracts were utilized to evaluate cytotoxicity, antioxidant and immune activities. The callus extracted via ultrasonification extraction showed relatively low cytotoxicity on normal human cell lines, HEK293 and HEL299, showing 13.17% and 21.78%, respectively. The callus extract has 59.82% which was similar to 61.70% for water extracts. It was also found that callus extract yielded higher nitric oxide secretion form macrophage than other extracts. The growths of both human stomach adenocarcinoma (AGS) cell and human lung carcinoma (A549) were inhibited up to 70% by adding 1.0 mg/mL of the callus extracts with ultrasonification extraction. This inhibition ratio (70%) was almost close to that of water extract. Human hepatoma carcinoma (HEP3B) cell growth was most significantly inhibited up to 75% by adding 1.0 mg/mL of callus extracts, and its selectivity was highest compared to other extracts. It indicates that the callus extracts could selectively inhibit growth of digestive system-related cancer cells. It can be also concluded from the results of this study that the callus extracts associated with ultrasonification extraction process have the potential for anticancer activity.

• Journal of Life Science
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• v.16 no.4
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• pp.571-578
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• 2006

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

• Journal of Life Science
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• v.33 no.1
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• pp.1-7
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• 2023

Intracellular cargo transport is mediated by molecular motor proteins, such as kinesin and cytoplasmic dynein. Kinesins make up a large subfamily of molecular motors. Kinesin-1 is a plus-end-directed molecular motor protein that moves various cargoes, such as organelles, protein complexes, and mRNAs, along a microtubule track. It consists of the kinesin superfamily protein (KIF) 5A, 5B, and 5C (also called kinesin heavy chains) and kinesin light chains (KLCs). Kinesin-1 interacts with many different binding proteins through its carboxyl (C)-terminal region of KIF5s and KLCs, but their binding proteins have not yet been fully identified. In this study, a yeast two-hybrid assay was used to identify the proteins that interact with the KIF5A specific C-terminal region. The assay revealed an interaction between KIF5A and glutamate-rich 4 (ERICH4). ERICH4 bound to the KIF5A specific the C-terminal region but did not interact with the C-terminal region of KIF5B or KIF3A (a motor protein of kinesin-2). In addition, KIF5A did not interact with another isoform, ERICH1. Glutathione S-transferase (GST) pull-downs showed that KIF5A interacts with GST-ERICH4 and GST-ERICH4-amino (N)-terminal but not with GST-ERICH4-C or GST alone. When co-expressed in HEK-293T cells, ERICH4 co-localized with KIF5A and co-immunoprecipitated with KIF5A and KLC but not KIF3B. Together, our findings suggest that ERICH4 is capable of binding to KIF5A and that it may serve as an adaptor protein that links kinesin-1 with cargo.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.5
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Analytical Science and Technology
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• v.29 no.1
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• pp.10-18
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• 2016

Phosphodiesterase (PDE) plays an important role in cAMP-mediated signaling within cells. We previously showed that the long-form of Aplysia PDE4 (ApPDE4) was localized in the plasma membrane and the presynaptic terminal in Aplysia sensory neurons, and the 16 N-terminal amino acid was sufficient for this targeting process. In this study, we characterized the cellular localization of various ApPDE4 mutants. We first identified the roles of each amino acid within the group of 16 N-terminal amino acids of long-form ApPDE4. As a result, we were able to identify various mutants that were localized to both the plasma membrane and the Golgi complex, Golgi only, or both the endoplasmic reticulum (ER) and the Golgi complex. To examine the role of palmitoylation on the cellular localization of ApPDE4 mutants, 2-bromo palmitate (2-BR) was used as a treatment. As a result, in the presence of 2-BR, the plasma membrane targeting of many mutants was impaired, indicating that palmitoylation was involved in the plasma membrane targeting of the mutants. We also found that PI4P play crucial roles in the Golgi targeting of (N16,C3S/VV/G)-mRFP, L(N16,C3S/LFS/R)-mRFP, and L(N16,EPL/R)-mRFP.

• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2119-2128
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• 2017

Citrinin (CIT) is a toxic secondary metabolite produced by fungi belonging to the Penicillium, Aspergillus, and Monascus spp. This toxin has been detected in many agricultural products. In this study, a strain Y3 with the ability to eliminate CIT was screened and identified as Cryptococcus podzolicus, based on the sequence analysis of the internal transcribed spacer region. Neither uptake of CIT by cells nor adsorption by cell wall was involved in CIT elimination by Cryptococcus podzolicus Y3. The extracellular metabolites of Cryptococcus podzolicus Y3 stimulated by CIT or not showed no degradation for CIT. It indicated that CIT elimination was attributed to the degradation of intracellular enzyme(s). The degradation of CIT by C. podzolicus Y3 was dependent on the type of media, yeast concentration, temperature, pH, and initial concentration of CIT. Most of the CIT was degraded by C. podzolicus Y3 in NYDB medium at 42 h but not in PDB medium. The degradation rate of CIT was the highest (94%) when the concentration of C. podzolicus Y3 was $1{\times}10^8cells/ml$. The quantity of CIT degradation was highest at $28^{\circ}C$, and there was no degradation observed at 3$5^{\circ}C$. The study also showed that acidic condition (pH 4.0) was the most favorable for CIT degradation by C. podzolicus Y3. The degradation rate of CIT increased to 98% as the concentration of CIT was increased to $20{\mu}g/ml$. The toxicity of CIT degradation product(s) toward HEK293 was much lower than that of CIT.

• Journal of Yeungnam Medical Science
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• v.21 no.2
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• pp.177-190
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• 2004

Background: Secretory phospholipase $A_2$ ($sPLA_2$) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA $sPLA_2$ ($sPLA_2$-IIA) has been detected in the inflammatory fluids, and its plasma level increases in the inflammatory disease. This study examined the effect of $sPLA_2$-IIA on mouse macropahges in order to investigate the potential mechanism of $sPLA_2$-induced inflammation. Materials and Methods: Wild type $PLA_2$ and mutant H48Q $PLA_2$ were purified from HEK293 cells transfected with the corresponding plasmids, and the $PLA_2$ activities were measured using 1-palmitoyl-2-[1-$^{14}C$]linoleoyl-3-phosphatidylethanolamine as substrates. The TNF-${\alpha}$ and IL-6 released in the supernatants were determined by ELISA. In addition, the TNF-${\alpha}$ and IL-6 mRNA were analyzed by RT-PCR. Results: $sPLA_2$-IIA stimulated the production of TNF-${\alpha}$ and IL-6 in a dose- and time-dependent manner. In addition, the effect of $sPLA_2$-IIA on cytokine production from the macrophage was found to be associated with the accumulation of their specific mRNA. The mRNA levels of TNF-${\alpha}$ and IL-6 peaked at 2 and 6 hours in a time-dependent manner, respectively. Conclusion: In conclusion, the production of proinflammatory cytokine might be mediated by the binding of $sPLA_2$-IIA to the receptors.

• Korean Journal of Food Science and Technology
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• v.41 no.3
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• pp.313-319
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• 2009

The marine microalga Spirulina maxima was extracted using water or ethanol at 100 or $80^{\circ}C$ and by ultrasonification in water at $60^{\circ}C$. The ultrasonification technique generated the highest yield (19.8%). To be therapeutically useful, the extraction should yield a product with low cytotoxicity and high immunity against skin infections. The cytotoxicity of all extracts (1.0 mg/mL) was below 25%. Moreover, the cytotoxicity of the extract generated by ultrasonification was 5%. Extracts prepared in the described manners could inhibit hyaluronidase activity by up to 40% compared to the control. Increased growth of human B, T and NK cells and an increase in cytokine secretion were observed, confirming the interrelationship between both human immune and skin immune activity. The extract prepared by ultrasonification increased the growth of human B, T and NK cells up to $10.3{\times}10^4$ cells/mL, $11.3{\times}10^4$ cells/mL and $19.1{\times}10^4$ cells/mL, respectively. The extract prepared by ultrasonification also greatly increased the secretion of both IL-6 and $TNF-{\alpha}$. Moreover, it was estimated that protein, Na and leucine occupy a high ratio. Accordingly, this study has confirmed that extracts prepared as described have the potential to effectively increase skin immunity.