• Journal of Ginseng Research
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• v.43 no.1
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• pp.154-160
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• 2019

Background: Compound K (CK) is an active metabolite of ginseng saponin, ginsenoside Rb1, that has been shown to have ameliorative properties in various diseases. However, its role in inflammation and the underlying mechanisms are poorly understood. In this report, the antiinflammatory role of CK was investigated in macrophage-like cells. Methods: The CK-mediated antiinflammatory mechanism was explored in RAW264.7 and HEK293 cells that were activated by lipopolysaccharide (LPS) or exhibited overexpression of known activation proteins. The mRNA levels of inflammatory genes and the activation levels of target proteins were identified by quantitative and semiquantitative reverse transcription polymerase chain reaction and Western blot analysis. Results: CK significantly inhibited the mRNA expression of inducible nitric oxide synthase and tumor necrosis factor-${\alpha}$ and morphological changes in LPS-activated RAW264.7 cells under noncytotoxic concentrations. CK downregulated the phosphorylation of AKT1, but not AKT2, in LPS-activated RAW264.7 cells. Similarly, CK reduced the AKT1 overexpression-induced expression of aldehyde oxidase 1, interleukin-$1{\beta}$, interferon-${\beta}$, and tumor necrosis factor-${\alpha}$ in a dose-dependent manner. Conclusion: Our results suggest that CK plays an antiinflammatory role during macrophage-mediated inflammatory actions by specifically targeting the AKT1-mediated signaling pathway.

• Journal of Veterinary Science
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• v.19 no.6
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• pp.735-743
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• 2018

We investigated the effect of transforming growth factor beta 1 ($TGF-{\beta}1$) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with $TGF-{\beta}1$ at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of $TGF-{\beta}1$. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by $TGF-{\beta}1$ stimulation was dose and time dependent. $TGF-{\beta}1$ was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in $TGF-{\beta}1$ treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the $TGF-{\beta}1$-treated scaffolds. Together, our results suggest that $TGF-{\beta}1$ has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

• Journal of Apiculture
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• v.34 no.1
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• pp.39-46
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• 2019

BQCV multi-point PCR was developed as a rapid multiplex detection method for BQCV, one of the viral pathogens of honeybees. It could detect BQCV specific genes qualitative as well as quantitative detection based on ultra-rapid PCR. Three primer pairs (RNA dependent RNA polymerase, capsid protein, 3C like protease) were specifically designed for accurate the detection and were optimized for minimizing the detection time and increasing the sensitivity. Our advanced diagnostic system have the accuracy by lowering the concern about the variation in the BQCV detection site. In addition, it should be an opportunity to identify mutations that are mixed with other viruses.

• Journal of Veterinary Science
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• v.22 no.1
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• pp.12.1-12.11
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• 2021

Background: Bats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV). Objectives: The objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensin-converting enzyme 2 (ACE2) receptor. Methods: The phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA. Results: Phylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%-67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2. Conclusions: These results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.

• Journal of Microbiology and Biotechnology
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• v.32 no.7
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• pp.911-917
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• 2022

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

• Biomedical Science Letters
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• v.29 no.4
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• pp.321-329
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• 2023

Anaplastic thyroid cancer has the highest mortality rate of all thyroid cancers and shows low responsiveness to most treatments. Hongyoung, a reddish-colored potato, is an excellent source of dietary polyphenol containing a large amount of anthocyanins, which has anti-cancer and anti-inflammatory effects. This study investigated the effects of Hongyoung extract on apoptosis and invasiveness in SNU-80 anaplastic thyroid cancer cells. The quantification of the total polyphenol content was done by spectrophotometric measurement. Cell growth was measured by using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) 2H tetrazolium, monosodium salt (MTS) assay. Cell cycle was analyzed through FACS analysis. Induction of apoptosis in cells was investigated by annexin V staining using flow cytometer and the expression of caspase-3 and Poly (ADP-ribose) polymerase (PARP) through western blot. mRNA expression and protein activation of matrix metalloproteinases (MMP)-2/-9 were examined by RT-PCR and zymography. As a result, the TPC of Hongyoung was 292.43±8.42 mg gallic acid equivalent (GAE)/100 g dry extract. Hongyoung showed a dose-dependent cell growth inhibition, and the IC₅₀ values was 1,000 ㎍/mL. sub-G1 phase was more than doubled compared to the control group, and S and G2/M phase arrest were also induced. Hongyoung induced apoptosis by increasing FITC-Annexin V-positive cells and increased the activation of caspase-3 (cleaved caspase-3) and PARP (fragmented PARP). Hongyoung significantly inhibited mRNA expression and protein activation of MMP-2/-9 in phorbol 12-myristate 13-acetate (PMA)-treated SNU-80 cells. Therefore, this study suggests the possibility of development of Hongyoung extract as an anti-cancer agent.

• Development and Reproduction
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• v.27 no.4
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• pp.185-193
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• 2023

Although increasing evidence of cause-and-effect relationship between BPA exposure and female reproductive disorders have been suggested through many studies, the precise biochemical and molecular mechanism(s) by which BPA interferes with steroidogenesis in the ovarian cells still remain unclear. Therefore, the purpose of this study was to discover the steroidogenic biomarker(s) associated with BPA treatment in human granulosa cell line, KGN. In this study, our results obtained via the analysis of steroidogenesis-related protein expression in KGN cells using quantitative polymerase chain reaction (qPCR) and western blot analyses revealed that the expression levels of steroidogenic acute regulatory (StAR) and aromatase decreased considerably and gradually after BPA treatment in a dose-dependent manner under BPA treatment. Further, remarkable decreases in their expression levels at the cellular levels were also confirmed via immunocytochemistry, and subsequent StAR and aromatase mRNA expression levels showed profiles similar to those observed for their proteins, i.e., both StAR and aromatase mRNA expression levels were significantly decreased under BPA treatment at concentrations ≥0.1 μM. We observed that follicle stimulating hormone upregulated StAR and aromatase protein expression levels; however, this effect was suppressed in the presence of BPA. Regarding the steroidogenic effects of BPA on KGN cells, controversies remain regarding the ultimate outcomes. Nevertheless, we believe that the results here presented imply that KGN cells have a good cellular and steroidogenic machinery for evaluating endocrine disruption. Therefore, StAR and aromatase could be stable and sensitive biomarkers in KGN cells for the cellular screening of the potential risk posed by exogenous and environmental chemicals to female reproductive (endocrine) function.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.30 no.6
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• pp.474-481
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• 2004

Squamous cell carcinoma is the most prevalent oral cancer, which is characterized by its low survival rate, high malignancy, mortality with facial defects, and poor prognosis. Exact cause and pathogenesis of the squamous cell carcinoma is still unknown. Various routes including smoking, radiation, and viral infections predispose its genesis, and recent studies revealed that genetic defects which fail to prevent cancer proliferation play a role. Generally, a cancer develops from the decreased rate of apoptosis which is an active and voluntary cell death, and from the altered cell cycles. Anticancer effect can be obtained by recovering the apoptotic process, and by suppressing the cell cycles. Among the apoptosis related factors, bcl-2, caspase-9, and VDAC (voltage-dependent anion channel)are produced in mitochondria of the cell. Cyclosporin-A is known to induce apoptosis through its activation with VDAC. This study was to reveal the anticancer effect of Cyclosporin A to the oral squamous cell carcinoma. The inverted microscope was used to find alterations in the tissue, and sensitivity test to the anticancer cells was performed with MTT (Tetrazolium-based colorimetric) assay. Following cell line culture of primary and metastastic oral squamous cell carcinoma, electrophoresis was performed with extracted total RNA. Finally, semi-quantitative study was carried out through RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The results of this study are as follows: 1. The inverted microscopic observation revealed a poorly defined cytoplasm at $2000ng{\sim}3000ng/ml$, indistinct nucleus, and apoptosis. 2. The Growth of cancer cells was decreased at 1000ng/ml of cyclosporin-A. No cancer cell growth was observed at over 2000ng/ml concentration of cyclosporin-A, and at one week, growth of cancer cells was ceased. 3. The MTT assays were decreased as cyclosporin-A concentration was increased. This means that the activation of succinyl dehydrogenase in mitochondria was decreased following administration of cyclosporin A. 4. A result of RT-PCR showed that amount of mRNA of VDAC-2 was decreased half times at a cyclosporine-A concentration of 2000ng/ml. In bcl-2, amount of mRNA was significantly decreased 1/5 times at 2000ng/ml. caspase-9, however, showed slight increase compared to the control group. From the results obtained in this study, administration of cyclosporin-A to the cell lines of oral squamous cell carcinoma induced alterations in morphology and growth of the cells as its concentration increased. Since apoptosis related factors such as VDAS-2, bcl-2, and caspase-9 also showed distinct alterations on their mRNAs, further research on cyclosporin A as an anti-cancer agent will be feasible.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.3
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• pp.398-407
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• 2020

Objective: The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3. Methods: Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3. Results: Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis. Conclusion: The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.

• Journal of Korean Neurosurgical Society
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• v.37 no.6
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• pp.443-448
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• 2005

Objective: A variety of genetic alterations in human glioblastoma comprises signal transduction and cell cycle arrest control of cellular processes. Subtractive hybridization is potentially a faster method for identifying differentially expressed genes associated with a particular disease state. Using the technique of subtraction, we isolated novel genes that are overexpressed in glioblastoma tissue as compared to normal brain tissue. Methods: We evaluated the differential expression of genes in each of hybridizing tester and driver cDNAs to digested 130 clones. After sequencing of 130 clones and homology search, this study performed to determine mRNA expression of the unknown gene, "clone 47", in brain tissue, glioblasoma, and several cancer cell lines by reverse transcription-polymerase chain reaction (RT-PCR). To test the time course for Go-phase arrest, serum stimulation and expression at various times for RT-PCR performed. Results: We identified 23 novel genes by BLAST of the digested 130 clones. The expressions of "clone 47" mRNA of glioblastoma and several cancer lines were significantly higher than normal brain tissues and several normal cell lines. We confirmed the mRNA expression of "clone 47" was up-regulation for $0.5{\sim}1hr$ of WI-38 cell differentiation. Conclusion: The novel gene, "Clone 47" is upregulated in glioblastoma tissue and several cancer cell lines. This gene is time dependent activation during time course of serum stimulation. This result suggests that "clone 47" playa role in brain tumorigenesis and the activation of this "clone 47" may be necessary for the development of cancer.