• Journal of Ginseng Research
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• v.47 no.1
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• pp.123-132
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• 2023

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

• Natural Product Sciences
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• v.29 no.2
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• pp.98-103
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• 2023

Sphaeranthus africanus is commonly used as a traditional remedy for sore throats and pain treatment in Vietnam. The aerial parts have been studied for its anti-inflammatory and anti-proliferative properties. However, the antioxidant and antidiabetic potential of the plant has not been explored. In this work, hydrophilic extracts of the plant's aerial parts were prepared in order to investigate its antioxidant and anti-diabetic properties. Also, the cytotoxicity of the root was evaluated and compared to that of the aerial parts. All of the extracts inhibited lipid peroxidation with IC₅₀ values ranging from 2.05 to 3.56 ㎍/mL, indicating substantial antioxidant activity. At an IC₅₀ value of 4.80 ㎍/mL, the 50% ethanol extract exhibited the most potent inhibition of α-glucosidase. The cytotoxic activity of root extracts is 2 to 5-fold less than that of the aerial parts. Nevertheless, dichloromethane and ethyl acetate extracts of the root demonstrated a selective effect on leukemia cells, with no harm towards the normal HEK-293 cell line. This work provides a scientific support for the antioxidant and antidiabetic activity of the plant. Hence, it may find a promising material for the development of novel antioxidant and antidiabetic agents. More research can be conducted on the phytochemistry and anticancer activities of the plant's root.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.4
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• pp.313-322
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• 2024

Mutations within the SCN5A gene, which encodes the α-subunit 5 (Na_V1.5) of the voltage-gated Na⁺ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the Na_V1.5 current (I_Na) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of I_Na was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the co-expression of SCN1B with p.A385T/R504T revealed significant reduction of I_Na and slower recovery from inactivation, consistent with the loss-of-function in Na⁺ channels. The SCN1B dependent reduction of I_Na was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of Na_V1.5, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.6
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• pp.920-926
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• 2010

Marine alga, Chlorella vulgaris, was extracted by chloroform-methanol (2:1, v/v) solvents for lipid extraction at $35^{\circ}C$ for five hours (HCM-35) and its process was compared with conventional lipid extraction condition such as chloroform-methanol (2:1, v/v) at $65^{\circ}C$ for one hour (CM-65). This low temperature extraction process showed that 80% of total lipid was extracted and its residues contained relatively unchanged amounts of intact proteins and other minerals as well as amino acid profiles. Interestingly enough, the weight fraction of carbohydrate in the residues slightly increased due to less denaturation at low process temperature. The biological activities of the residues such as cytotoxicity and immune cell growth activation were not much changed after being extracted. The sensory evaluation were found to be very favorable for being used as a food additive and/or food supplement. This result could also help to maintain the economic feasibility of utilizing marine resources in food and other relevant industries.

• Analytical Science and Technology
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• v.28 no.5
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• pp.331-340
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• 2015

Mutations in Fused in Sarcoma (FUS) have been identified in patients with amyotrophic lateral sclerosis (ALS) or Frontotemporal Dementia (FTD). Pathological FUS is mis-localized to cytosol and forms aggregates associated with stress granules (SG), while FUS is normally localized to nucleus. However, it is largely unknown how pathological FUS forms SG-aggregates and which domains are responsible for this process. In this study, we examined cellular localization and aggregation of ALS-linked FUS missense mutants (P525L, R521C, R521H, R521G), analyzed the domains responsible for cytosolic FUS aggregation in HEK293T cells, and confirmed this in cultured mouse neurons. To do this, we firstly generated missense mutants of FUS and then examined their cellular localization. We found that P525L was mostly mis-localized to cytosol and formed FUS-positive SG aggregates while R521C, R521H, or R521G was localized to both nucleus and cytosol. To further characterize the domains required for aggregate formation of cytosolic FUS, we generated different domain-deletion mutants using FUS-∆17 which has a deletion of nuclear localization signal. Interestingly, cytosolic FUS without SYGQ and RGG1 domain or cytosolic FUS without RGG2-ZnF-RGG3 domain did not form FUS-positive SG aggregates, while cytosolic FUS without RRM domain generated more aggregates compared to FUS-∆17. Taken together, these data suggest that SYGQ-RGG1 or RGG2-ZnF-RGG3 domain contributes to formation of cytosolic aggregate, while RRM domain might interfere with FUS aggregation. Therefore, our studies will provide important insight for understanding cellular pathogenesis of neurodegeneration associated with FUS aggregate as well as finding therapeutic targets for ALS or FTD.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.2
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• pp.219-229
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• 2020

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiRRB-REPORT^TM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

• Korean Journal of Medicinal Crop Science
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• v.16 no.5
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• pp.349-355
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• 2008

Anticancer and immuno-modulatory activities of methanol extracts from different parts, bark, wood and leaf, of Cornus macrophylla Wall. were investigated in this study. All extracts at a concentration of 1.0mg/ml showed relativity low cytotoxicities on human normal kidney cell (HEK293) by approximately 25%. Bark extract of C. macrophylla showed the highest anticancer activity on human lung cancer cell line (A549) and human breast cancer cell line (MCF-7) by 57.4% and 58.7%, respectively, at a concentration of 1.0mg/ml. All extracts enhanced the growth of human B and T cells, showing 38.7% and 65.9% increase compared to control, respectively, by 5 days incubation with bark extract. The secretions of interleukin 6 (IL6) and tumor necrosis factor alpha (TNF-$\alpha$) from human B and T cells were significantly increased by extracts, especially bark extract. B or T cell medium, which contains cytokines (IL 6 and TNF-$\alpha$) secreted by bark extract treatment for 5 days, time-dependently enhanced the growth of NK-92MI cells with the maximal effect at 5th day of incubation. These results suggest that C. macrophylla, especially bark, has the potential for anticancer and immuno-modulatory activities.

• Journal of the Korean Society of Food Science and Nutrition
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• v.38 no.9
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• pp.1243-1252
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• 2009

We investigated a method to improve anticancer activities of Acer mono by ultra high pressure extraction process. The extract yields by ultra high pressure were 9.49% and 9.87% for 5 min and 15 min processing time, respectively, which were relatively higher than 3$\sim$4% of conventional extraction processes due to their resid bark structure. The extract for 15 minutes extraction (HPE15) showed higher potent scavenging effect as 94.56% than the control, BHA as 93.24%. On SOD-like test, HPE15 also showed the highest activity as 38.6% at 1.0 mg/mL concentration. The cytotoxicity of HPE15 on normal human lung and kidney cell were below 23.54% in adding 1.0 mg/mL. Generally, human cancer cell growth stomach adenocarcinoma (AGS), lung adenocarcinoma (A549), breast adenocarcinoma (MCF-7), colon adenocarcinoma (Caco-2) and liver adenocarcinoma (Hep3B) were inhibited up to 75% with higher selectivity of above 4.0. High antioxidant activity of HPE15 resulted in high anticancer activity, and its activity was also due to higher yields of Acer mono by ultra high pressure extraction process. It was also proved by HPLC comparison analysis.

• 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.

• 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.