• Journal of The Korean Society of Integrative Medicine
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• v.9 no.2
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• pp.83-92
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• 2021

Purpose : Keratinocytes are the main cellular components involved in wound healing during re-epithelization and inflammation. Dysfunction of tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. The purpose of this study was to identify the various effects of a Sparassis crispa water extract (SC) on HaCaT cells and to investigate whether these effects might be applicable to human skin. Methods : We investigated the effectiveness of SC on cell HaCaT viability using MTS. The antioxidant effect of SC was analyzed by comparing the effectiveness of ABTS to that of the well-known antioxidant resveratrol. Reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method Quantitative RT-PCR analysis has shown that SC in HaCaT cells affects mRNA expression of tight-junction genes associated with skin moisturization. In addition, Wound healing is one of the most complex processes in the human body. It involves the spatial and temporal synchronization of a variety of cell types with distinct roles in the phases of hemostasis, inflammation, growth, re-epithelialization, and remodeling. wound healing analysis demonstrated altered cell migration in SC-treated HaCaT cells. Results : MTS analysis in HaCaT cells was found to be more cytotoxic in SC at a concentration of 0.5 mg/㎖. Compared to 100 µM resveratrol, 4 mg/㎖ SC exhibited similar or superior antioxidant effects. SC treatment in HaCaT cells reduced levels of claudin 1, claudin 3, claudin 4, claudin 6, claudin 7, claudin 8, ZO-1, ZO-2, JAM-A, occludin, and Tricellulin mRNA expression by about 1.13 times. Wound healing analysis demonstrated altered cell migration in SC-treated HaCaT cells and HaCaT cell migration was also reduced to 73.2 % by SC treatment. Conclusion : SC, which acts as an antioxidant, reduces oxidative stress and prevents aging of the skin. Further research is needed to address the effects of SC on human skin given the observed alteration of mRNA expression of tight-junction genes and the decreased the cell migration of HaCaT cells.

• Korean Journal of Breeding Science
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• v.41 no.4
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• pp.482-487
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• 2009

The apple rootstock M.26 (Malus pumila) is very popular apple rootstock with semi-dwarf habit and the trees on M.26 produce high quality fruit at a young age. Since it is prone to poor prop ability to soil, however, young trees require staking in windy locations. The rolC gene was introduced into M.26 by Agrobacterium tumefaciens LBA4404 harboring pBI121 to obtain its transformants with dwarfism and enhanced rooting ability. One regenerated transgenic line was confirmed by polymerase chain reaction (PCR) analysis and Southern blot analysis of genomic DNA for the existence of rolC gene. The characteristics of transgenic line in vitro were not significantly different from non-transgenic line except for the active root formation and lateral root number. The rolC transgenic line showed reduced stem length and increased root number in vitro. Rooting ability was examined in the isolated greenhouse after mound layering. Compared to non-transgenic M.26, rolC transgenic line showed significantly higher rooting ability. The transgenic line did not show any other observable variation in shoot phenotype compared with non-transgenic line excepting increased branching

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.10
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• pp.1528-1539
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• 2019

Objective: To evaluate the effects on microbial diversity and biochemical parameters of gradually increasing temperatures, from $5^{\circ}C$ to $25^{\circ}C$ on corn silage which was previously fermented at ambient or low temperature. Methods: Whole-plant corn silage was fermented in vacuum bag mini-silos at either $10^{\circ}C$ or $20^{\circ}C$ for two months and stored at $5^{\circ}C$ for two months. The mini-silos were then subjected to additional incubation from $5^{\circ}C$ to $25^{\circ}C$ in $5^{\circ}C$ increments. Bacterial and fungal diversity was assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiling and biochemical analysis from mini-silos collected at each temperature. Results: A temperature of $10^{\circ}C$ during fermentation restricted silage fermentation compared to fermentation temperature of $20^{\circ}C$. As storage temperature increased from $5^{\circ}C$ to $25^{\circ}C$, little changes occurred in silages fermented at $20^{\circ}C$, in terms of most biochemical parameters as well as bacterial and fungal populations. However, a high number of enterobacteria and yeasts (4 to $5\;log_{10}$ colony forming unit/g fresh materials) were detected at $15^{\circ}C$ and above. PCR-DGGE profile showed that Candida humilis predominated the fungi flora. For silage fermented at $10^{\circ}C$, no significant changes were observed in most silage characteristics when temperature was increased from $5^{\circ}C$ to $20^{\circ}C$. However, above $20^{\circ}C$, silage fermentation resumed as observed from the significantly increased number of lactic acid bacteria colonies, acetic acid content, and the rapid decline in pH and water-soluble carbohydrates concentration. DGGE results showed that Lactobacillus buchneri started to dominate the bacterial flora as temperature increased from $20^{\circ}C$ to $25^{\circ}C$. Conclusion: Temperature during fermentation as well as temperature during storage modulates microorganism population development and fermentation patterns. Silage fermented at $20^{\circ}C$ indicated that these silages should have lower aerobic stability at opening because of better survival of yeasts and enterobacteria.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.4
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• pp.477-484
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• 2019

Objective: We aimed to observe hair follicle (HF) development in the dorsal skin and elucidate the expression patterns of genes and proteins related to skin and HF development in Rex rabbits from birth to 8 weeks of age. Methods: Whole-skin samples were obtained from the backs of Rex rabbits at 0, 2, 4, 6, and 8 weeks of age, the morphological development of primary and secondary HFs was observed, and the gene transcript levels of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), bone morphogenetic protein 2 (BMP2), transforming growth factor ${\beta}-1$, 2, and 3 ($TGF{\beta}-1$, $TGF{\beta}-2$, and $TGF{\beta}-3$) were examined using quantitative real-time polymerase chain reaction (PCR). Additionally, Wnt family member 10b (Wnt10b) and ${\beta}$-Catenin gene and protein expression were examined by quantitative real-time PCR and western blot, respectively. Results: The results showed significant changes in the differentiation of primary and secondary HFs in Rex rabbits during their first 8 weeks of life. The IGF-I, EGF, $TGF{\beta}-2$, and $TGF{\beta}-3$ transcript levels in the rabbits were significantly lower at 2 weeks of age than at birth and gradually increased thereafter, while the BMP2 and $TGF{\beta}-1$ transcript levels at 2 weeks of age were significantly higher than those at birth and gradually decreased thereafter. ${\beta}$-Catenin gene expression was also significantly affected by age, while the Wnt10b transcript level was not. However, the Wnt10b and ${\beta}$-catenin protein expression levels were the lowest at 2 and 4 weeks of age. Conclusion: Our data showed that a series of changes in HFs in dorsal skin occurred during the first 8 weeks. Many genes, such as IGF-I, EGF, BMP2, $TGF{\beta}-1$, $TGF{\beta}-2$, $TGF{\beta}-3$, and ${\beta}$-Catenin, participated in this process, and the related proteins Wnt10b and ${\beta}$-Catenin in skin were also affected by age.

• Tuberculosis and Respiratory Diseases
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• v.82 no.2
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• pp.133-142
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• 2019

Background: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. Methods: Mouse AE II cell line MLE-12 were exposed to $1-10{\mu}g/mL$ BLM and $0.01-100{\mu}M$ baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. Results: BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor ${\alpha}$, and transforming growth factor ${\beta}1$. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. Conclusion: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

• Korean Journal of Clinical Laboratory Science
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• v.53 no.3
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• pp.217-224
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• 2021

Fluoroquinolone (FQ) resistant gram-negative pathogens have emerged worldwide, and the recent increase in FQ resistant Escherichia coli is of great concern in Korea. This study investigated FQ resistance determinants and the epidemiological relationship of 56 ciprofloxacin-resistant E. coli isolated from a tertiary hospital in Daejeon, South Korea from June to December 2018. Molecular epidemiology was investigated by multilocus sequence typing (MLST). Polymerase chain reaction (PCR) and sequence analysis were performed to identify chromosomal mutations in the quinolone resistance determining regions (QRDR) of gyrA, gyrB, parC, and parE and to describe the occurrence of the following plasmid-mediated quinolone resistance (PMQR) genes: aac(6)-Ib-cr, qepA, qnrA, qnrB, qnrC, qnrD, and qnrS. MLST analysis showed 12 sequence types (STs) and the most prevalent ST was ST131 (31/56, 55.4%), followed by ST1193 (13/56, 23.2%), and ST405 (3/56, 5.4%). In 56 ciprofloxacin-resistant E. coli isolates, Ser83→Leu and Asp87→Asn in gyrA and Ser80→Ile and Glu84→Val in parC (51.8%, 29/56) were the most frequent amino acid substitutions and aac(6)-Ib-cr (33.9%, 19/56) was the most common PMQR gene. These results of FQ resistance determinants were more frequently observed in ST131 compared with other clones. Continuous monitoring of the epidemiological characteristics of ciprofloxacin-resistant E. coli isolates and further investigation of FQ resistance determinants are necessary.

• Animal Bioscience
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• v.34 no.11
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• pp.1739-1748
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• 2021

Objective: In recent years, long noncoding RNAs (lncRNAs) have been identified in many species, and some of them have been shown to play important roles in muscle development and myogenesis. However, the differences in lncRNAs between Kazakh cattle and Xinjiang brown cattle remain undefined; therefore, we aimed to confirm whether lncRNAs are differentially expressed in the longissimus dorsi between these two types of cattle and whether differentially expressed lncRNAs regulate muscle differentiation. Methods: We used RNA-seq technology to identify lncRNAs in longissimus muscles from these cattle. The expression of lncRNAs were analyzed using StringTie (1.3.1) in terms of the fragments per kilobase of transcript per million mapped reads values of the encoding genes. The differential expression of the transcripts in the two samples were analyzed using the DESeq R software package. The resulting false discovery rate was controlled by the Benjamini and Hochberg's approach. KOBAS software was utilized to measure the expression of different genes in Kyoto encyclopedia of genes and genomes pathways. We randomly selected eight lncRNA genes and validated them by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results: We found that 182 lncRNA transcripts, including 102 upregulated and 80 downregulated transcripts, were differentially expressed between Kazakh cattle and Xinjiang brown cattle. The results of RT-qPCR were consistent with the sequencing results. Enrichment analysis and functional annotation of the target genes revealed that the differentially expressed lncRNAs were associated with the mitogen-activated protein kinase, Ras, and phosphatidylinositol 3-kinase (PI3k)/Akt signaling pathways. We also constructed a lncRNA/mRNA coexpression network for the PI3k/Akt signaling pathway. Conclusion: Our study provides insights into cattle muscle-associated lncRNAs and will contribute to a more thorough understanding of the molecular mechanism underlying muscle growth and development in cattle.

• Journal of Ginseng Research
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• v.45 no.1
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• pp.134-148
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• 2021

Background: Lung cancer has a high incidence worldwide, and most lung cancer-associated deaths are attributable to cancer metastasis. Although several medicinal properties of Panax ginseng Meyer have been reported, the effect of ginsenosides Rk1 and Rg5 on epithelial-mesenchymal transition (EMT) stimulated by transforming growth factor beta 1 (TGF-β1) and self-renewal in A549 cells is relatively unknown. Methods: We treated TGF-β1 or alternatively Rk1 and Rg5 in A549 cells. We used western blot analysis, real-time polymerase chain reaction (qPCR), wound healing assay, Matrigel invasion assay, and anoikis assays to determine the effect of Rk1 and Rg5 on TGF-mediated EMT in lung cancer cell. In addition, we performed tumorsphere formation assays and real-time PCR to evaluate the stem-like properties. Results: EMT is induced by TGF-β1 in A549 cells causing the development of cancer stem-like features. Expression of E-cadherin, an epithelial marker, decreased and an increase in vimentin expression was noted. Cell mobility, invasiveness, and anoikis resistance were enhanced with TGF-β1 treatment. In addition, the expression of stem cell markers, CD44, and CD133, was also increased. Treatment with Rk1 and Rg5 suppressed EMT by TGF-β1 and the development of stemness in a dose-dependent manner. Additionally, Rk1 and Rg5 markedly suppressed TGF-β1-induced metalloproteinase-2/9 (MMP2/9) activity, and activation of Smad2/3 and nuclear factor kappa B/extra-cellular signal regulated kinases (NF-kB/ERK) pathways in lung cancer cells. Conclusions: Rk1 and Rg5 regulate the EMT inducing TGF-β1 by suppressing the Smad and NF-κB/ERK pathways (non-Smad pathway).

• Animal Bioscience
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• v.35 no.1
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• pp.115-125
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• 2022

Objective: Intramuscular fat (IMF) is a critical economic indicator of pork quality. Studies on IMF among different pig breeds have been performed via high-throughput sequencing, but comparisons within the same pig breed remain unreported. Methods: This study was performed to explore the gene profile and identify candidate long noncoding RNA (lncRNAs) and mRNAs associated with IMF deposition among Laiwu pigs with different IMF contents. Based on the longissimus dorsi muscle IMF content, eight pigs from the same breed and management were selected and divided into two groups: a high IMF (>12%, H) and low IMF group (<5%, L). Whole-transcriptome sequencing was performed to explore the differentially expressed (DE) genes between these two groups. Results: The IMF content varied greatly among Laiwu pig individuals (2.17% to 13.93%). Seventeen DE lncRNAs (11 upregulated and 6 downregulated) and 180 mRNAs (112 upregulated and 68 downregulated) were found. Gene Ontology analysis indicated that the following biological processes played an important role in IMF deposition: fatty acid and lipid biosynthetic processes; the extracellular signal-regulated kinase cascade; and white fat cell differentiation. In addition, the peroxisome proliferator-activated receptor, phosphatidylinositol-3-kinase-protein kinase B, and mammalian target of rapamycin pathways were enriched in the pathway analysis. Intersection analysis of the target genes of DE lncRNAs and mRNAs revealed seven candidate genes associated with IMF accumulation. Five DE lncRNAs and 20 DE mRNAs based on the pig quantitative trait locus database were identified and shown to be related to fat deposition. The expression of five DE lncRNAs and mRNAs was verified by quantitative real time polymerase chain reaction (qRT-PCR). The results of qRT-PCR and RNA-sequencing were consistent. Conclusion: These results demonstrated that the different IMF contents among pig individuals may be due to the DE lncRNAs and mRNAs associated with lipid droplets and fat deposition.

• Animal Bioscience
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• v.36 no.2
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• pp.200-208
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• 2023

Objective: Muscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes. Methods: We constructed a CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes. Results: The results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPAR class="checkNonKBPoint">γ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly. Conclusion: Overexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.