• Title/Summary/Keyword: ITS-PCR

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Gaseous signal molecule SO2 regulates autophagy through PI3K/AKT pathway inhibits cardiomyocyte apoptosis and improves myocardial fibrosis in rats with type II diabetes

  • Zhao, Junxiong;Wu, Qian;Yang, Ting;Nie, Liangui;Liu, Shengquan;Zhou, Jia;Chen, Jian;Jiang, Zhentao;Xiao, Ting;Yang, Jun;Chu, Chun
    • The Korean Journal of Physiology and Pharmacology
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    • v.26 no.6
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    • pp.541-556
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    • 2022
  • Myocardial fibrosis is a key link in the occurrence and development of diabetic cardiomyopathy. Its etiology is complex, and the effect of drugs is not good. Cardiomyocyte apoptosis is an important cause of myocardial fibrosis. The purpose of this study was to investigate the effect of gaseous signal molecule sulfur dioxide (SO2) on diabetic myocardial fibrosis and its internal regulatory mechanism. Masson and TUNEL staining, Western-blot, transmission electron microscopy, RT-qPCR, immunofluorescence staining, and flow cytometry were used in the study, and the interstitial collagen deposition, autophagy, apoptosis, and changes in phosphatidylinositol 3-kinase (PI3K)/AKT pathways were evaluated from in vivo and in vitro experiments. The results showed that diabetic myocardial fibrosis was accompanied by cardiomyocyte apoptosis and down-regulation of endogenous SO2-producing enzyme aspartate aminotransferase (AAT)1/2. However, exogenous SO2 donors could up-regulate AAT1/2, reduce apoptosis of cardiomyocytes induced by diabetic rats or high glucose, inhibit phosphorylation of PI3K/AKT protein, up-regulate autophagy, and reduce interstitial collagen deposition. In conclusion, the results of this study suggest that the gaseous signal molecule SO2 can inhibit the PI3K/AKT pathway to promote cytoprotective autophagy and inhibit cardiomyocyte apoptosis to improve myocardial fibrosis in diabetic rats. The results of this study are expected to provide new targets and intervention strategies for the prevention and treatment of diabetic cardiomyopathy.

Cloning and Functional Studies of Pro-Apoptotic MCL-1ES BH3M (세포사멸을 유도하는 새로운 단백질인 MCL-1ES BH3M의 클로닝 및 기능연구)

  • Kim, Jae-Hong;Park, Mira;Ha, Hye-Jeong;Lee, Kangseok;Bae, Jeehyeon
    • Development and Reproduction
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    • v.12 no.3
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    • pp.297-303
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    • 2008
  • BCL-2 family members are essential protein for the regulation of cell death and survival consisting both antiapoptotic and pro-apoptotic proteins. In the present study, we designed and cloned a new apoptotic molecule MCL-1ES BH3M coding a modified protein of MCL-1L. Compared to MCL-1L protein, MCL-1ES BH3M lacks the PEST motifs known to be involved in MCL-1L protein degradation and has seven mutated residues in BH3 domain critical for dimerization with BCL-2 family members. Overexpression of MCL-1ES BH3M induced death of different cells, and its cell killing effect was not blocked by forced expression of the pro-survival protein MCL-1L. Expression of MCL-1ES BH3M protein led to the activation of caspase 9 and caspase 3, suggesting apoptotic cell death, and confocal fluorescent microscopic analyses showed that MCL-1ES BH3M was partially localized in mitochondria. In conclusion, we reported a new apoptotic molecule and determined its cell death activity in cells.

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The Effect of Ampelopsis japonica (Thunb.) Makino on Osteoclastogenesis and Expression of Osteoclast-Related Gene (백렴(白蘞)의 파골세포 분화 및 관련 유전자 발현 억제에 미치는 영향)

  • Hongsik Kim;Sumin Lee;Minsun Kim;Jae-Hyun Kim;Yejin Kang;Seoung Jun Kwon;Youngwoo Nam;Seungwoo Yoo;Hong-Seok Choi;SeonJin Huh;Youngjoo Sohn;Hyuk-Sang Jung
    • The Korea Journal of Herbology
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    • v.38 no.5
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    • pp.9-19
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    • 2023
  • Objectives : Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density and increased risk of fractures. Bisphosphonates and selective estrogen receptors, which are bone resorption inhibitors that are currently widely used as osteoporosis treatments, show serious side effects when administered for a long time. Research on bone resorption inhibitors that complement the problems of existing treatments is needed. The purpose of this study was to investigate the effect of inhibiting osteoclast differentiation and activity on the tuberous root of Ampelopsis japonica (Thunb.) Makino (AM). Methods : After extracting AM using distilled water and ethanol, the inhibitory effects of the two solvents on osteoclast differentiation were compared using the RANKL-induced in vitro experimental model and the TRAP assay kit. The impact of AM on bone resorption was investigated through the pit formation assay, and its effect on F-actin formation was assessed through fluorescent staining. Additionally, protein and mRNA expression levels of osteoclast differentiation markers (NFATc1, c-Fos, TRAP and ATP6v0d2) and resorption markers (MMP-9, CTK, and CA2) were analyzed via western blot and RT-PCR. Results : AM treatment significantly decreased the number of TRAP-positive cells and pit formation area. Furthermore, AM suppressed both the protein and mRNA expression of NFATc1 and c-Fos, key transcription factors involved in osteoclast differentiation and it downregulated the expression of osteoclast-associated genes such as TRAP, CTK, MMP-9, CA2, and ATP6v0d2. Conclusions : These results suggest that AM can inhibit bone resorption and osteoclast differentiation, indicating its potential for use in the treatment and prevention of osteoporosis.

Development of Method using LC-ESI-MS/MS and KASP for Identification of Gymnema sylvestre in Food (식품에서 당살초 판별을 위한 LC-ESI-MS/MS 분석법과 KASP 마커 개발)

  • Park, Boreum;Lee, Sun Hee;Eom, Kwonyong;Noh, Eunyoung;Moon Han, Kyoung;Hwang, Jinwoo;Kim, Hyungil;Baek, Sun Young
    • Journal of Food Hygiene and Safety
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    • v.37 no.2
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    • pp.46-54
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    • 2022
  • Known for its effectiveness in weight loss and diabetes prevention, Gymnema sylvestre products can be found in the US, Japanese, and Indian markets. However, the recommended dosage or safety of these products has not yet been proven. Therefore, development of an analytical method for detecting the content of Gymnema sylvestre in food products is required. Accordingly, this study proposes an analysis method that can examine Gymnema sylvestre in food using LC-ESI-MS/MS and KASP (Kompetitive Allele-Specific PCR) markers. In LC-ESI-MS/MS, a simultaneous analysis method for gymnemic acid and deacylgymnemic acid was optimized using negative ionization mode, and its validation test was completed for solid and liquid samples. In addition, KASP markers were prepared by finding the specific SNP of G. sylvestre in ITS2 and matK through DNA barcodes. The two KASP markers returned positive FAM fluorescence result when combined with G. sylvestre, and this aspect was confirmed in raw G. sylvestre as well. The applicability of the method was tested on 21 different food and healthy functional products containing G. sylvestre purchased on the internet. As a result, although there was a difference in the ratios of gymnemic acid and deacylgymnemic acid in LC-ESI-MS/MS, the index component was detected in all 21 products samples. In the KASP analysis, 9 products returned positive FAM result, and the rest of the products were found to be containing G. sylvestre extract. This study is the first study to use the dual system of LC-ESI-MS/MS and KASP for the analysis of G. sylvestre. The study has confirmed that these two methods are applicable to the examine G. sylvestre content in food products.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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Expression of lewis antigen in gastric mucosa of children with Helicobacter pylori infection (Helicobacter pylori에 감염된 소아의 위점막에서 Lewis 항원의 발현)

  • Chung, Ju-Young;Lim, Seong Jig;Han, Tae Hee
    • Pediatric Infection and Vaccine
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    • v.14 no.1
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    • pp.97-103
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    • 2007
  • Purpose : Lewis antigen has been known to have a role in the attachment of H. pylori to the gastric mucosa, but its expression pattern in children with H. pylori infection is still unclear. The recently described blood group antigen-binding adhesin BabA is known to mediate adherence of H. pylori to Lewis B receptors on gastric epithelium. We investigated the expression of Lewis antigen in gastric mucosa of Korean children with H. pylori infection. Methods : The expression of Lewis A ($Le^a$), B ($Le^b$), X ($Le^x$), and Y ($Le^y$) was evaluated by immunohistochemistry in H. pylori positive biopsy specimens from 35 children (antral gastritis in 30, peptic ulcer in 5) and in H. pylori negative specimens from 19 children. PCR assays for cagA and babA2 gene of H. pylori were performed. Results : We confirmed the expression of $Le^a$ in 60%, $Le^b$ in 97%, $Le^x$ in 100%, and $Le^y$ in 100% of the superficial epithelium of the 35 H. pylori positive children. In H. pylori negative patients, $Le^a$, $Le^b$, $Le^x$, and $Le^y$ expression was 52%, 100%, 89%, and 100%, respectively. The cagA gene was detected in 65% and babA2 gene in 25% of 35 patients. No differences in neutrophil activity and chronic inflammation were found according to the presence of cagA and babA2 genes in H. pylori. Conclusion : $Le^b$, $Le^x$ and $Le^y$ antigen were highly expressed in gastric mucosa of Korean children, but they were not associated with the status of H. pylori infection and the positivity of babA2 gene. Further studies for other mucosal receptors and toxins are needed to define the immune responses to H. pylori infection in gastric mucosa of Korean children.

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Anti-inflammatory Activity of Antimicrobial Peptide Zophobacin 1 Derived from the Zophobas atratus (아메리카왕거저리 유래 항균 펩타이드 조포바신 1의 항염증활성)

  • Shin, Yong Pyo;Lee, Joon Ha;Kim, In-Woo;Seo, Minchul;Kim, Mi-Ae;Lee, Hwa Jeong;Baek, Minhee;Kim, Seong Hyun;Hwang, Jae Sam
    • Journal of Life Science
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    • v.30 no.9
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    • pp.804-812
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    • 2020
  • The giant mealworm beetle, Zophobas atratus (Coleoptera: Tenebrionidae) has been used as a protein source for small pets and mammals. Recently, it was temporarily registered in the list of the Food Code. We previously performed an in silico analysis of the Zophobas atratus transcriptome to identify putative antimicrobial peptides and identified several antimicrobial peptide candidates. Among them, we assessed the antimicrobial and anti-inflammatory activities of zophobacin 1 that was selected bio-informatically based on its physicochemical properties against microorganisms and mouse macrophage Raw264.7 cells. Zophobacin 1 showed antimicrobial activities against microorganisms without inducing hemolysis and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Moreover, ELISA and Western blot analysis revealed that zophobacin 1 reduced expression levels of pro-inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We also investigated expression of pro-inflammatory cytokines (interleukin-6 and interleukin-1β) production through quantitative real time-PCR and ELISA. Zophobacin 1 markedly reduced the expression level of cytokines through the regulation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling. We confirmed that zophobacin 1 bound to bacterial cell membranes via a specific interaction with lipopolysaccharides. These data suggest that zophobacin 1 could be promising molecules for development as antimicrobial and anti-inflammatory therapeutic agents.

Expression Properties and Skin Permeability of Human Basic Fibroblast Growth Factor with or without PTD Fused to N- or C-terminus in Escherichia coli (대장균 발현시스템에서 단백질 전달 도메인 PTD가 인간 섬유아세포 성장인자(FGF2)의 N- 또는 C-말단에 결합 되었을 때 미치는 재조합 단백질 복합체의 발현 특성과 피부 투과능력)

  • Park, In-Sun;Choe, Chung-Hyeon;Kwon, Bo-Ra;Choi, Young-Ji;Kwon, Tae-Ho;Yu, Kang-Yeol;Lee, Juhyung;Choo, Young-Moo
    • Journal of Life Science
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    • v.28 no.3
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    • pp.275-283
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    • 2018
  • Human fibroblast growth factor (FGF) has the potential to be a commercially important therapeutic or cosmeceutical agent due to its ability to generate tissue and heal wounds. Granting permeability into skin tissues increases the therapeutic effects of FGF. Thus, several researchers have attempted the fusion of FGF conjugates with protein transduction domains (PTDs) to investigate the transduction ability and therapeutic effects of FGF. Less is known, however, about whether the location of PTD fused to the N- or C-terminus of FGF proteins has a significant impact on the folding and stability in Escherichia coli, and eventually, on transduction. Here, we report cloning of human basic fibroblast growth factor (FGF2) as a control and FGF2 with PTD fused to the N- or C-terminal ends of FGF proteins by an overlap extension PCR. We performed expression, verified expression properties of recombinant FGF2 without or with PTD fused to the N-terminus and the C-terminus, and investigated transduction ability into tissue by treating the dorsal skin of mice subjects. As a result, FGF2 and FGF2-PTD (fused to C-terminus) fusion protein were expressed as soluble forms suitable for straight-forward purification, unlike insoluble PTD-FGF2 (fused to N-terminus), but only FGF2-PTD fusion protein could transduce into the dorsal skin tissue of the mice subjects. Our results suggest that FGF2 with PTD fused to the C-terminus is more efficient than other options in terms of expression, purification, and delivery into skin tissue, as it does not require labor-intensive, costly, and time-consuming methods.

Effects of mechanical stress and interleukin-$1{\beta}$ on collagenase and TIMP-1 expression in human periodontal ligament fibroblasts (기계적 자극과 interleukin-$1{\beta}$가 치주인대 섬유아세포의 collagenase와 TIMP-1의 발현에 미치는 영향)

  • Kim, Myung-Lip;Bae, Chang
    • The korean journal of orthodontics
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    • v.28 no.1 s.66
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    • pp.165-174
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    • 1998
  • The turnover of collagen is controlled by the balance between collagen synthesis and degradation. The production of collagenase (matrix metalloproteinase-1) and its inhibitor, tissue inhibitor of matrix metallopmteinase-1 (TIMP-1) are one of the substances which regulate this balance. The periodontal ligament fibroblast plays an important role in collagen metabolism during orthodontic treatment and is believed to be an origin of the osteoblast in the alveolar bone. The collagenase secreted by the periodontal ligament fibroblast and the osteoblast initiates the bone resorption by removing the osteoid layer in the alveloar bone. The interleukin-$1{\beta}$ is secreted by the macrophage during orthodontic treatment. The present study was undertaken to assess the effect of mechanical stress and interleukin-$1{\beta}$ on the expression of collagenase and TIMP-1 in the periodontal ligament fibroblasts using reverse transcription polymerase chain reaction and immunohistochemical staining. The periodontal ligament fibroblasts were stitched by placing the $Petriperm dish^{\circledR}$ dish on the top of spheroidal convex watch glass ($5\%$ surface increase) and tented with interleukin-$1{\beta}$ (1.0 ng/ml), or treated with both of them. Treatment with mechanical stress and/or interleukin-$1{\beta}$ resulted in increased collagenase mRNA expression. The mechanical stress treated group (1.61, 1.62, 1.37 fold increase), the interleukin-$1{\beta}$, tented group (1.68, 1.60, 3.78 fold increase), the mechanical stress and interleukin-$1{\beta}$ treated group (1.89, 1.72, 5.48 fold increase) induced increases in collagenase mRNA compared with the control group after 2, 4, 8 hours respectively. But TIMP-1 mRNA expressions at experimental groups were decreased after 2, 4 hours and increased after 8 hours. The mechanical stress treated group (0.16, 0.49 fold decrease and 3.77 fold increase), the interleukin-$1{\beta}$ treated group (0.15,0.44 fold decrease and 4.46 fold increase), the mechanical stress and interleukin-$1{\beta}$ tented group (0.15, 0.69 fold decrease and 4.81 fold increase) induced changes in TIMP-1 mRNA compared with the control group after 2, 4, 8 hours, respectively. Immunohistochemical stain showed that increased collagenase and TIMP-1 staining of the mechanical stress tented group, the interleukin-$1{\beta}$ treated group, and the mechanical stress and interleukin-$1{\beta}$ treated group compared with that of the control group after 8 hours. These findings suggest that mechanical stress and interleukin-$1{\beta}$ regulate expression of collagenase and TIMP-1.

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Estimation of Anti-proliferative Activity of Saccharin against Various Cancer Cell Lines and MSCs (다양한 암세포 주와 MSCs에 대한 Saccharin의 항증식성 평가)

  • Choi, Jeong Su;Park, Sang Yong;Yang, Man Gil;Lee, Dong Beom;Lee, Tae Bok;Heo, Ji Hye;Lee, Min Woo;Kim, Suhng Wook
    • Korean Journal of Clinical Laboratory Science
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    • v.48 no.3
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    • pp.169-175
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    • 2016
  • Saccharin (o-benzoic sulfimide) is the first artificial and non-caloric sweetener that was first synthesized in 1879. In this study, we examined the biological activity of saccharin against various human cancer cell lines and human bone marrow-derived mesenchymal stem cells. A viability assay based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed to test for the cytotoxicity of saccharin about the four human cancer cell lines (H460, H157, A549 and SKOV3), one murine cancer cellline (Raw264.7), and MSCs. In order to find the differentially expressed gene in saccharin-treated MSCs against untreated MSCs, we performed annealing control primer (ACP)-based differential display reverse transcriptionp-olymerase chain reaction (DDRT-PCR). All tested cells were treated with saccharin at various concentrations (0.0, 4.8, 7.2, 9.6, 12.0, 14.4 mg/mL) for 48 hr. The number of metabolically active cancer cells decreased when treated with the saccharin at various concentrations for 48 hr as compared with the untreated cells. The decrease in cell survival was more evident with increasing concentrations of saccharin. Moreover, novel candidate genes, which were differentially expressed in MSCs in response to saccharin, were identified in 16 bands on 2% agarose gel. This revealed 16-7 up-regulated and 9 down-regulated-differentially expressed genes indicated by arrows. One of these candidate genes was a FK506-binding protein gene. The functional roles of FK506 binding proteins, with respect to the activities of stem cell proliferation, were not characterized. Further studies are required to get a better understanding of FK506-binding proteins in its roles in increasing stem cell proliferative activities from using saccharin.