• Biomedical Science Letters
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• v.29 no.2
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• pp.81-87
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• 2023

Pan-Enterovirus (Pan-EV) infects millions of children and infants worldwide every year. As severe infections have recently been reported, the need for monitoring has consequently intensified. Pan-EV is a categorical name for waterborne enteroviruses belonging to the Picornaviridae family, and includes a wide range of pathogens including Coxsackievirus (CoxV), Echovirus (EcoV) and Enterovirus (EV). In this study, we proposed an optimal RT-nested PCR method for diagnosis of various types of Pan-EV in an aquatic environment and developed a positive control. Considering detection sensitivity, specific reaction, and final identification, one condition capable of amplifying 478 bp among the four candidates in the 1^st round PCR (RT-PCR) and one condition in the 2^nd round PCR (nested PCR) were selected. Through the detection of nucleic acids extracted from 123 groundwater samples and the detection sensitivity test based on artificial spiking in the sample, the methods are optimal for non-disinfected water samples such as groundwater. We developed a positive control for Pan-EV detection that can be amplified to different sizes under the two conditions. Accuracy could be further improved by testing for contamination from the control group. The method proposed in this study and the positive control developed are expected to be used in monitoring Pan-EV in aquatic environments including groundwater through future research using more samples.

• The Plant Pathology Journal
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• v.40 no.4
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• pp.390-398
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• 2024

The Chinese artichoke (Stachys affinis syn. S. sieboldii) is a widely cultivated crop, and its rhizome is used as a medicinal vegetable. To investigate the causes of viral diseases in Chinese artichokes, the infection rates of four virus species infecting Chinese artichoke were investigated. Since the Chinese artichoke propagates through its tuber, this study aimed to determine whether viral transmission to the progeny is possible through the tuber, by identifying the virus present in the tuber and investigating its accumulation. First, reverse transcription polymerase chain reaction analysis was performed to detect viruses using total RNA extracted from the flowers, leaves, and tubers of Chinese artichoke plants. Alfalfa mosaic virus (AMV) and Chinese artichoke mosaic virus (ChAMV) had high infectivity in Chinese artichoke and most plants were simultaneously infected with AMV and ChAMV. These viruses were present in all tissues, but their detection frequency and accumulation rates varied across different tissues of the Chinese artichoke. Also, we sequenced the coat protein (CP) genes of AMV and ChAMV to investigate genetic variations of virus between the leaf and tuber. It provides information on CP gene sequences and genetic diversity of isolates identified from new hosts of AMV and ChAMV. This study offers valuable insights into the distribution and spread of the ChAMV and AMV within Chinese artichoke plants, which have implications for the management and control of viral infections in crops.

• Journal of Microbiology and Biotechnology
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• v.34 no.8
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• pp.1688-1697
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• 2024

The current study aimed to determine whether Strongylocentrotus intermedius (S. intermedius) extract (SIE) exerts anti-obesity potentials employing 3T3-L1 cells as in vitro model. Herein we reported that treatment of SIE for 6 days reduced lipid accretion and triglyceride content whereas it increased the release of free glycerol. The inhibited lipid accumulation and induced lipolysis were evidenced by the downregulation of lipogenesis proteins, such as fatty acid synthase and lipoprotein lipase, and the upregulation of hormone-sensitive lipase expression. Furthermore, the downregulation of adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein α, and sterol regulatory element-binding protein 1, highlights that reduced lipid accumulation is supported by lowering adipocyte differentiation. Additionally, treatment activates brown adipocyte phenotype in 3T3-L1 cells by inducing expression of brown adipose tissue-specific proteins, such as uncoupling protein 1 and peroxisome proliferator-activated receptor-γ coactivator 1α. Moreover, SIE induced the phosphorylation of AMP-activated protein kinase (AMPK). The pharmacological approach using AMPK inhibitor revealed that the restraining effect of SIE on adipogenesis and promotion of adipocyte browning were blocked. In GC-MS analysis, SIE was mainly composed of cholest-5-en-3-ol (36.71%) along with saturated and unsaturated fatty acids which have favorable anti-obesity potentials. These results reveal that SIE has the possibility as a lipid-lowering agent for the intervention of obesity.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.

• Childhood Kidney Diseases
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• v.15 no.1
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• pp.38-48
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• 2011

Purpose: Focal segmental glomerulosclerosis (FSGS) is the most common glomerulopathy causing pediatric renal failure. Since specific treatment targeting the etiology and pathophysiology of primary FSGS is yet elusive, the authors explored the pathophysiology of FSGS by transcriptome analysis of the disease using an animal model. Methods: FGS/kist strain, a mouse model of primary FSGS, and RFM/kist strain, as control and the parent strain of FGS/kist, were used. Kidney tissues were harvested and isolated renal cortex was used to extract mRNA, which was run on AB 1700 mouse microarray chip after reverse transcription to get the transcriptome profile. Results: Sixty two genes were differentially expressed in FGS/kist kidney tissue compared to the control. Those genes were related to cell cycle/cell death, immune reaction, and lipid metabolism/vasculopathy, and the key molecules of their networks were TNF, IL-6/4, IFN${\gamma}$, TP53, and PPAR${\gamma}$. Conclusion: This study confirmed that renal cell death, immune system activation with subsequent fibrosis, and lipid metabolism-related early vasculopathy were involved in the pathophysiology of FSGS. In addition, the relevance of methodology used in this study, namely transcriptome profiling, and Korean animal model of FGS/kist was validated. Further study would reveal novel pathophysiology of FSGS for new therapeutic targets.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.9
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• pp.1334-1341
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• 2007

This study identified hepatic differentially expressed genes (DEGs) affecting the marbling of muscle. Most dietary nutrients bypass the liver and produce plasma lipoproteins. These plasma lipoproteins transport free fatty acids to the target tissue, adipose tissue and muscle. We examined hepatic genes differentially expressed in a differential-display reverse transcription-polymerase chain reaction (ddRT-PCR) analysis comparing high- and low-marbled Hanwoo steers. Using 60 arbitrary primers, we found 13 candidate genes that were upregulated and five candidate genes that were downregulated in the livers of high-marbled Hanwoo steers compared to low-marbled individuals. A BLAST search for the 18 DEGs revealed that 14 were well characterized, while four were not annotated. We examined four DEGs: ATP synthase F0, complement component CD, insulin-like growth factor binding protein-3 (IGFBP3) and phosphatidylethanolamine binding protein (PEBP). Of these, only two genes (complement component CD and IGFBP3) were differentially expressed at p<0.05 between the livers of high- and low-marbled individuals. The mean mRNA levels of the PEBP and ATP synthase F0 genes did not differ significantly between the livers of high- and low-marbled individuals. Moreover, these DEGs showed very high inter-individual variation in expression. These informative DEGs were assigned to the bovine chromosome in a BLAST search of MS marker subsets and the bovine genome sequence. Genes related to energy metabolism (ATP synthase F0, ketohexokinase, electron-transfer flavoprotein-ubiquinone oxidoreductase and NADH hydrogenase) were assigned to BTA 1, 11, 17, and 22, respectively. Syntaxin, IGFBP3, decorin, the bax inhibitor gene and the PEBP gene were assigned to BTA 3, 4, 5, 5, and 17, respectively. In this study, the in silico physical maps provided information on the specific location of candidate genes associated with economic traits in cattle.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.3
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• pp.328-337
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• 2017

Objective: An experiment was conducted to determine the relationship between the KAP11.1 and the regulation wool fineness. Methods: In previous work, we constructed a skin cDNA library and isolated a full-length cDNA clone termed KAP11.1. On this basis, we conducted a series of bioinformatics analysis. Tissue distribution of KAP11.1 mRNA was performed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The expression of KAP11.1 mRNA in primary and secondary hair follicles was performed using real-time PCR (real-time polymerase chain reaction) analysis. The expression location of KAP11.1 mRNA in primary and secondary hair follicles was performed using in situ hybridization. Results: Bioinformatics analysis showed that KAP11.1 gene encodes a putative 158 amino acid protein that exhibited a high content of cysteine, serine, threonine, and valine and has a pubertal mammary gland) structural domain. Secondary structure prediction revealed a high proportion of random coils (76.73%). Semi-quantitative RT-PCR showed that KAP11.1 gene was expressed in heart, skin, and liver, but not expressed in spleen, lung and kidney. Real time PCR results showed that the expression of KAP11.1 has a higher expression in catagen than in anagen in the primary hair follicles. However, in the secondary hair follicles, KAP11.1 has a significantly higher expression in anagen than in catagen. Moreover, KAP11.1 gene has a strong expression in inner root sheath, hair matrix, and a lower expression in hair bulb. Conclusion: We conclude that KAP11.1 gene may play an important role in regulating the fiber diameter.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.623-634
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• 2005

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.109-122
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• 2001

Gingival fibroblasts(GF) and periodontal ligament fibroblasts(PDLF) are the major cellular components of periodontal soft connective tissues, but the precise molecular biological differences between these cells are not yet known. In the present study, we investigated the expression of S100A4, S100A2 calcium-binding protein and osteoblast-specific factor 2(OSF-2, Periostin) mRNA in GF and PDLF in vitro through the process of reverse transcription-polymerase chain reaction(RT-PCR) and Northern blot analysis in each. Human GF and PDLF were isolated from the gingival connective tissue and the middle third of freshly extracted healthy third molars. They were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum and cells in the third passage were used in the experiments. After extracting total RNA from cultured cells, RT-PCR and Northern analysis were performed using S100A4-, S100A2- and Periostin-specific oligonucleotide primers and subcloned cDNA probes in each. In PT-PCR and Northern analysis, the expression of S100A4 and Periostin mRNA in GF was slightly detectable. Interestingly, the expression of S100A4 and periostin mRNA in PDLF was much higher than that in GF. On the other hand, S100A2 mPNA was highly expressed in both GF and PDLF. Since there was a marked difference of S100A4 and Periostin expression between GF and PDLF in vitro, these data suggest that S100A4 and periostin could be used as a useful marker for distinguishing cultured gingival fibroblasts and periodontal ligament cells.

• The Journal of Korean Society of Virology
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• v.26 no.1
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• pp.9-22
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• 1996

Respiratory Syncytial virus (RSV) is an important cause of acute lower respiratory tract infections in human, with infants and young children being particularly susceptible. In the temperate zones, sharp annual outbreaks of RSV occur during the colder months, in both the northern and the southern hemisphere. RSV is unusual in that it can repeatedly reinfect individuals throughout life and infect babies in the presence of maternal antibody. RSV isolates can be divided into two subgroups, A and B, on the basis of their reactions with monoclonal antibodies, and the two subgroups are also distinct at the nucleotide sequence level. The specific diagnosis of RSV infection was best made by isolation of virus in tissue culture, identification of viral antigen, or by specific serologic procedures. Recently, rapid detection of RSV and analysis of RSV strain variation became possible by development of methods of reverse transcription and polymerase chain reaction amplification. In this study, to determine the genetic diversity of RSV found in Korea, 173 bp and 164 bp spanning selected regions of the RSV F and SH genes were enzymatically amplified and sequenced, respectively. Eight for F gene and three for SH gene were detected in 66 nasopharyngeal swap samples tested. Two major antigenic subgroups, A and B were confirmed from Korean samples (seven for subgroup A and one for subgroup B). At the nucleotide level of the F gene region, Korean subgroup A strains showed 95-99% homologies compared to the prototype A2 strain of subgroup A and 93-100% homologies among Korean subgroup A themselves. For the SH gene region, Korean subgroup A strain showed 97.5% homology compared to the prototype A2 strain of subgroup A, and Korean subgroup B strain showed 97% homology compared to the prototype 18537 strain of subgroup B. Most of base changes were transition and occured in codon position 3, which resulted in amino acid conservation. Using the maximum parsimony method, phylogenetic analysis indicated that Korean RSV strains formed a group with other RSV strains isolated from the United States, Canada, the Great Britain and Australia.