• Title/Summary/Keyword: IS-PCR

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Chunghyul-dan acts as an anti-inflammatory agent in endothelial cells by regulating gene expression

  • Jung, Woo-Sang;Cho, Jin-Gu;In, Kyung-Min;Kim, Jong-Min;Cho, Ki-Ho;Park, Jung-Mi;Moon, Sang-Kwan;Kim, Kyung-Wook;Park, Seong-Uk;Pyee, Jae-Ho;Park, Sang-Gyu;Jeong, Yoon-Hwa;Park, Heon-Yong;Ko, Chang-Nam
    • Animal cells and systems
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    • v.14 no.4
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    • pp.275-282
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    • 2010
  • Chunghyul-dan (CHD) is a combinatorial drug known to exert anti-inflammatory effects in endothelial cells. In this study, we employed global transcriptional profiling using cDNA microarrays to identify molecular mechanisms responsible for the anti-inflammatory activity of CHD in endothelial cells. An analysis of the microarray data revealed that transcript levels of monocyte chemotactic protein-1 (MCP-1), vascular cell-adhesion molecule-1 (VCAM-1) and activated leukocyte cell-adhesion molecule were dramatically altered in CHD-treated endothelial cells. These changes in gene expression were confirmed by RT-PCR, Western blotting and ELISA. Chronic CHD treatment also appeared to decrease MCP-1 secretion, probably as a result of decreased MCP-1 expression. In addition, we determined that chronic CHD treatment inhibited lipopolysaccharide-stimulated adhesion of THP-1 leukocytes to endothelial cells. The inhibitory effect of CHD on LPS-stimulated adhesion resulted from downregulation of VCAM-1 expression. Transmigration of THP-1 leukocytes through endothelial cells was also inhibited by chronic CHD treatment. In conclusion, CHD controls a variety of inflammatory activities by regulating MCP-1 and VCAM-1 gene expression.

GROWTH INHIBITION OF ORAL SQUAMOUS CELL CARCINORMA CELL LINE INDUCED BY COX INHIBITOR (COX 억제제에 의해 유도되는 구강편평세포암종 세포주의 성장 억제 효과)

  • Park, Gwang-Jin;Han, Se-Jin;Lee, Jae-Hoon
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.30 no.4
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    • pp.333-344
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    • 2008
  • The objectives of this study was to explore the growth pattern of the oral squamous cell carcinoma when overexpressed COX was inhibited, explore the pathway that COX inhibitors suppressed the proliferation of cancer cells, and then hereafter investigate the potential of COX as chemopreventive target for oral squamous cell carcinoma. For confirming the COX-dependent effect and mechanisms on growth of the oral cancer cells, we treated the nonselective NSAID, Mefenamic acid and COX-2 selective inhibitor, Celecoxib in HN4 cell line. And then the cell line was evaluated with MTT assay and growth curve, the production of PGE2, total RNA extraction and RT-PCR analysis, and TEM The results were obtained as follows: 1. After administration of medication, in the result of MTT assay, Celecoxib inoculated group inhibit the cell growth rather than Mefenamic acid inoculated group. 2. The growth curve of cell line showed as time passes by there was a dramatic cell growth in the control group, and gradual growth inhibition was found in medication inoculated group and, in Celecoxib inoculated group there was more inhibition of cell growth. 3. After the administration of medication, Celecoxib tend to inhibit the synthesis of PGE2 more than Mefenamic acid. Mefenamic acid inhibit the synthesis of PGE2 more as the concentration gets high, but Celecoxib inhibited the synthesis of PGE2 even in low concentration. 4. After the administration of medication, the revelation of COX mRNA in cell line, there was a 50% decrease in COX-1, 60% decrease in COX-2 as in $50{\mu}M$ Mefenamic acid, and in Celecoxib $50{\mu}M$ there was not much difference in COX-1 and 90% decrease in COX-2 was found. 5. HN4 cell line showed broken nucleus and tangled cytoskeleton bundles in cytoplasm which meant apoptotic features after the treatment of Celecoxib in TEM view. Depending on the above results, we estimate that the inhibition of the expression of COX-2 cause the growth suppression of the oral squamous cell carcinoma, and it get achieved through pathway of reduced PGE2 production and increased apoptosis. In addition to, because COX-2 selective inhibitor specifically act to COX-2, it is considered that COX-2 selective inhibitor has the adequate potential as chemopreventive agent for oral squamous cell carcinoma.

Comparison of the Genomic Structure of the Heat Shock Protein-88(Hsp88) Genes in the Four Entomopathogenic Fungal Strains, Paecilomyces tenuipes Jocheon-1, P. tenuipes, Cordyceps militaris, and C. pruinosa

  • Liu, Ya-Qi;Park, Nam-Sook;Kim, Yong-Gyun;Kim, Keun-Ki;Park, Hyun-Chul;Son, Hong-Joo;Lee, Sang-Mong
    • International Journal of Industrial Entomology and Biomaterials
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    • v.25 no.1
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    • pp.99-110
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    • 2012
  • Comparison on the genomic structure and phylogenetic relationship of the Hsp88 genes from P. tenuipes Jochoen-1, P. tenuipes, C. militaris and C. pruinosa was described. The Hsp88 genes from the three entomopathogenic strains, P. tenuipes Jocheon-1(strain), P. tenuipes(original species), and C. militaris contain the identical genomic structure, namely 5 introns and 6 exons with the length of 13, 62, 32, 1,438, 306, 288 nucleotides encoding 713 amino acid residues, whereas in case of C. pruinosa, it contains 4 introns and 5 exons with the length of 13, 62, 32, 1,744, 288 nucleotides encoding 713 amino acid residues. The genomic DNA length of the Hsp88 genes from P. tenuipes Jocheon-1 and P. tenuipes are both 2,600 nucleotides long in size. The Hsp88 genes from C. militaris and C. pruinosa are 2,582, 2,576 nucleotides long in size, respectively. Hsp88 genes of the P. tenuipes Jochoen-1, P. tenuipes, C. militaris and C. pruinosa also contain the conserved ATP-binding domain. Phylogenetic analysis of the Hsp genes of the four strains tested in this study showed that the fungal Hsp88 is divided into two separate clades, ascomycetes and deutromycete. Within the ascomycetes fungal clade, the P. tenuipes Jochoen-1 and P. tenuipes formed a subgroup, on the other hand, C. militaris and C. pruinosa formed another subgroup. Pair-wise comparison of P. tenuipes Jocheon-1 Hsp88 with those of P. tenuipes, C. militaris and C. pruinosa Hsp88s revealed significant identity in deduced amino acid sequence among these strains. The P. tenuipes Jocheon-1 Hsp88 showed 99% identity with the P. tenuipes, 97% identity with the C. militaris, and 98% identity with the C. pruinosa.

Association of Bovine CSRP3 and ACOX1 Genes with Carcass and Meat Quality Traits (소의 도체, 육질형질과 CSRP3, ACOX1 유전자들과의 상관관계)

  • Lee, Jong-Kwan;Cho, Yong-Min;Lee, Jun-Heon
    • Korean Journal of Agricultural Science
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    • v.37 no.2
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    • pp.231-238
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    • 2010
  • There is no investigation has yet been conducted for ACOX1 and CSRP3 gene polymorphisms in Korean cattle (Hanwoo), and their associations with carcass and meat quality traits. In this study, SNPs in ACOX1 and CSRP3 genes were identified and their associations with carcass and meat quality traits were investigated in 227 Hanwoo animals. Two SNPs (g.224G> A and g.19491G>A) in ACOX1 gene and one SNP (g.14859C>T) in CSRP3 gene were identified in Hanwoo and sequence analysis indicated that these SNPs were located in the coding regions. The allele frequencies of ACOX1 g.224G>A and g.19491G>A SNPs were 0.57, 0.43, and 0.56 and 0.44, respectively, For CSRP3 g.14859C>T polymorphism, the C and T allele frequencies were 0.64 and 0.36, respectively. The Hanwoo cattle were used to detect PCR-RFLP patterns for estimating the allele frequencies. Single marker association analyses were performed between genotype of each SNP, and carcass and meat quality association traits to evaluate the relationships in Hanwoo. The g.224G>A SNP genotypes of ACOX1 gene, which was significantly associated with meat quantity grade at slaughter (P<0.03) and backfat thickness tended to be greater (P=0.06) in Hanwoo. The previously identified g.14859C>T SNP was used in this study and the obtained genotype and allele frequencies are almost similar with the previous results reported by Bhuiyan et al. (2007). However, no significant association was found between g.19491G>A SNP in the ACOX1 and g.14859C>T SNP genotypes of CSRP3 gene and considered carcass and meat quality traits. In conclusion, the information on the identified SNPs in CSRP3 and ACOX1 genes could be useful for further association study and haplotype analysis for the development of carcass and meat quality traits in Hanwoo.

A Study of Safflower Seed Extracts on Bone Formation in Vitro (홍화인 추출물이 골 형성에 미치는 영향에 관한 실험실적 연구)

  • Lee, Seong-jin;Choi, Ho-Chul;Sun, Ki-Jong;Song, Jae-Bong;Pi, Sung-Hee;You, Hyung-Keun;Shin, Hyung-Shik
    • Journal of Periodontal and Implant Science
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    • v.35 no.2
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    • pp.461-474
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    • 2005
  • The ultimate goal of periodontal therapy is the regeneration of periodontal tissue and the repair of function. For more than a decade there have been many efforts to develop materials and methods of treatment to promote periodontal tissue regeneration. Recently many efforts are concentrated on the regeneration potential of material used in traditional medicine. Safflower(Carthamus tinctorius L.) seed extract(SSE) have long clinically used in Korea to promote bone formation and prevent osteoporosis. The purpose of this study was to examine the effects of SSE on bone formation in human osteoblastic cell line. Human fetal osteoblastic cell line(hFOB 1.19) was cultured with DMEM and SSE($1{\mu}g/ml$, $10{\mu}g/ml$, $100{\mu}g/ml$, $1mg/ml$) at $34^{\cdot}C$ with 5% $CO_2$ in 100% humidity. The proliferation, differentiation of the cell was evaluated by several experiments. Cell proliferation was significantly increased at $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE after 3 and 7 days incubation(p<0.05). Cell spreading assay was significantly increased at $100{\mu}g/ml$ of SSE after 3 days and $1{\mu}g/ml$, $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE after 7 days(p<0.05). Alkaline Phosphatase(ALP) level was significantly increased in $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE(p<0.05). Collagen synthesis was significantly increased at $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE(p<0.05). A quantified calcium accumulation was significantly increased at $10{\mu}g/ml$, $100{\mu}g/ml$ of SSE(p<0.05). ALP and osteocalcin mRNA was expressed in $100{\mu}g/ml$ of SSE by RT-PCR. These results indicate that SSE are capable of increasing osteoblasts mineralization and may play an important role in bone formation.

Partial denture metal framework may harbor potentially pathogenic bacteria

  • Mengatto, Cristiane Machado;Marchini, Leonardo;de Souza Bernardes, Luciano Angelo;Gomes, Sabrina Carvalho;Silva, Alecsandro Moura;Rizzatti-Barbosa, Celia Marisa
    • The Journal of Advanced Prosthodontics
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    • v.7 no.6
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    • pp.468-474
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    • 2015
  • PURPOSE. The aim of this study was to characterize and compare bacterial diversity on the removable partial denture (RPD) framework over time. MATERIALS AND METHODS. This descriptive pilot study included five women who were rehabilitated with free-end mandibular RPD. The biofilm on T-bar clasps were collected 1 week ($t_1$) and 4 months ($t_2$) after the RPD was inserted ($t_0$). Bacterial 16S rDNA was extracted and PCR amplified. Amplicons were cloned; clones were submitted to cycle sequencing, and sequences were compared with GenBank (98% similarity). RESULTS. A total of 180 sequences with more than 499 bp were obtained. Two phylogenetic trees with 84 ($t_1$) and 96 ($t_2$) clones represented the bacteria biofilm at the RPD. About 93% of the obtained phylotypes fell into 25 known species for $t_1$ and 17 for $t_2$, which were grouped in 5 phyla: Firmicutes ($t_1=82%$; $t_2=60%$), Actinobacteria ($t_1=5%$; $t_2=10%$), Bacteroidetes ($t_1=2%$; $t_2=6%$), Proteobacteria ($t_1=10%$; $t_2=15%$) and Fusobacteria ($t_1=1%$; $t_2=8%$). The libraries also include 3 novel phylotypes for $t_1$ and 11 for $t_2$. Library $t_2$ differs from $t_1$ (P=.004); $t_1$ is a subset of the $t_2$ (P=.052). Periodontal pathogens, such as F. nucleatum, were more prevalent in $t_2$. CONCLUSION. The biofilm composition of the RPD metal clasps changed along time after RPD wearing. The RPD framework may act as a reservoir for potentially pathogenic bacteria and the RPD wearers may benefit from regular follow-up visits and strategies on prosthesis-related oral health instructions.

Cloning and Characterization of a Gene for Fibrinolytic Enzyme from Bacillus subtilis BB-1 Isolated from Black Bean Chung-kuk (흑두로 제조한 청국에서 분리된 Bacillus subtillus BB-1으로 부터 혈전용해효소 유전자 크로닝 및 특성규명)

  • Lee Young-Hoon;Lee Sung-Ho;Jeon Ju-Mi;Kim Hong-Chul;Cho Yong-Un;Park Ki-Hoon;Choi Young-Ju;Gal Sang-Wan
    • Journal of Life Science
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    • v.15 no.4 s.71
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    • pp.513-521
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    • 2005
  • A bacterium producing five fibrinolytic isozymes was isolated from black bean chung kuk. The bacterium was identified as Bacillus subtilis BB-1 by 16s rDNA sequence homology search. A gene out of five fibrinolytic genes in the Bacillus subtilis BB-1 was cloned by shot-gun method. A Cla I DNA fragment of B. subtilis BB-1 chromosome was cloned in to pBluescript II SK(-) and showed the fibrinolytic activity to bacterial cells. The Cla I DNA fragment was sequenced and the sequences did not show homology with gene for protease or fibrinolytic enzyme genes in other organisms. The Cla I DNA fragment was reduced to 2,142 bp by activity-guided PCR cloning method. The optimum pH and temperature of the enzyme were 5.0 and $35^{\circ}C$, respectively. Substrate specificity of the fibrinolytic enzyme was detected in skim milk, casein, gelatin and blood agar plates. The activity of the enzyme was not detected with these substrates. Taken together, this enzyme is a new fibrinolytic enzyme and may be used to prevent thrombosis and arteriosclerosis.

Modulation of Cell Cycle Regulators by Sulforaphane in Human Mepatocarcinoma HepG2 Cells (HepG2 인체간암세포의 세포주기조절인자 발현에 미치는 sulforaphane의 영향)

  • Bae, Song-Ja;Kim, Gi-Young;Yoo, Young-Hyun;Choi, Byung-Tae;Choi, Yung-Hyun
    • Journal of Life Science
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    • v.16 no.7 s.80
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    • pp.1235-1242
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    • 2006
  • Sulforaphane, an isothiocyanate derived from hydrolysis of glucoraphanin in broccoli and other cruciferous vegetables, was shown to induce phase II detoxification enzymes and inhibit chemically induced mammary tumors in rodents. Recently, sulforaphane is known to induce cell cycle arrest and apoptosis in human canter cells, however its molecular mechanisms are poorly understood. In tile present study, we demonstrated that sulforaphane acted to inhibit proliferation and induce morphological changes of human hepatocarcinoma HepG2 cells. Treatment of HepG2 cells with $10{\mu}M\;or\;15{\mu}M$ sulforaphane resulted in significant G2/M cell cycle arrest as determined by DNA flow cytometry. Moreover, $20{\mu}M$ sulforaphane significantly induced the population of sub-G1 cells suggesting that sulforaphane induced apoptosis. This anti-proliferative effect of sulforaphane was accompanied by a marked inhibition of ryclin A, cyclin 31 and Cdc2 protein. However, the levels of tumor suppressor p53 and Cdk inhibitor p21 mRNA and protein expression were significantly increased by sulforaphane treatment in a concentration-dependent manner. Although further studies are needed, the present work suggests that sulforaphane may be a potential rhemoprevetiveichemotherapeucc agent for the treatment of human cancer cells.

Overexpression of MicroRNA-31 as a Promising Biomarker for Prognosis and Metastasis in Human Colorectal Cancer (MicroRNA-31 과발현을 이용한 대장암의 예후예측 및 전이예측 바이오마커 발굴)

  • Hur, Keun
    • Journal of Life Science
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    • v.26 no.6
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    • pp.705-710
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    • 2016
  • Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related death worldwide. Although several diagnostic and therapeutic tools have been available, CRC remains difficult to complete cure because of insufficient understanding of the molecular mechanisms underlying this disease progression. MicroRNAs (miRNAs) are small non-coding RNA molecules that strongly regulate gene expression via transcriptional and translational control mechanisms. Many crucial cellular pathways are frequently disrupted in cancer development process due to dysregulation of several miRNAs. Mir-31 functions as an oncogene that modulate expression of multiple cancer related genes. Thus, we aimed to demonstrate clinical relevance of miR-31 in human CRC. Quantitative RT-PCR analysis of miR-31 expression was performed in 175 CRC tissues and 16 normal colonic mucosa (NM). Next, we investigated clinical significances of miR-31 expression in various clinicopathologic features in CRC patients cohort. Mir-31 was significantly up-regulated in CRC tissues compared to NM. In CRC tissues, miR-31 expression level was significantly elevated in a stage-dependent manner, which was associated with poor survival in patients with CRC. High miR-31 levels in CRC tissues significantly correlated with poor prognosis (hazard ratio [HR]=2.4) as well as distant metastasis (odds ratio [OR]=2.3). In conclusion, we identified clinical significance of miR-31 expression in CRC. High miR-31 expression may be clinically able to use as a biomarker for CRC prognosis and predicting metastasis.

Molecular Epidemiology of Metallo-β-lactamase Producing Pseudomonas aeruginosa Clinical Isolates (임상에서 분리된 Metallo-β-lactamase 생성 Pseudomonas aeruginosa의 분자역학)

  • Choi, Myung-Won
    • Journal of Life Science
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    • v.22 no.9
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    • pp.1268-1276
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    • 2012
  • The emergence and dissemination of carbapenem-resistant bacteria have resulted in limitations of antibiotic treatment and potential outbreaks of metallo-${\beta}$-lactamase (MBL) producing Pseudomonas aeruginosa resistant to carbapenems. In this study, we conducted molecular characterization of the MBL genes of the ${\beta}$-lactam drug-resistant P. aeruginosa and prepared basic data for treatment and prevention of proliferation of antimicrobial-resistant bacterial infections. Forty-two P. aeruginosa isolates of 254 were resistant to imipenem or meropenem. Among the 42 isolates, 28 isolates were positive for the Hodge test, and 23 isolates were positive for the EDTA-disk synergy test (EDST). MBLs were detected in 59.5% (25/42) of P. aeruginosa isolates. Eight isolates harbored $bla_{IMP-6}$, whereas 17 isolates harbored $bla_{VIM-2}$. The $bla_{IMP-6}$ gene was in a class 1 integron containing five gene cassettes: $bla_{IMP-6}$, qac, aacA4, $bla_{OXA-1}$, and aadA1. Some strains that produce IMP-6 and VIM-2 showed epidemiological relationships. The $bla_{IMP-6}$ gene in carbapenem-resistant P. aeruginosa showed an identical pattern to a gene cassette that was reported at a hospital in Daegu, Korea. Therefore, MBL-producing P. aeruginosa is already endemic in the community. We are concerned that the existence of carbapenem-resistant bacteria containing the blaMBL gene may increase pressure on antibiotic selection when treating infections. We believe that we should select appropriate antibiotics based on the antibiotic susceptibility test and continue the research to prohibit the emergence and spread of antibiotics resistant bacteria.