• Title/Summary/Keyword: IS-PCR

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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.

Insulin-like Growth Factor-I Induces Plectin and MACF1 Expression in C2C12 Myotubes (C2C12 myotube에서 insulin-like growth factor-I이 plectin과 MACF1 발현에 미치는 영향)

  • Kim, Hye Jin;Hwang, Ji Sun;Kwak, Yi-Sub;Lee, Won Jun
    • Journal of Life Science
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    • v.22 no.12
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    • pp.1651-1657
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    • 2012
  • Plectin and microtubule actin cross-linking factor 1 (MACF1) are architectural proteins that contribute to the function of skeletal muscle as generators of mechanical force. However, the influence of insulin- like growth factor-I (IGF-I), a master regulator of skeletal muscle cells, on plectin and MACF1 in skeletal muscle cells has not been demonstrated. The effect of IGF-I on plectin and MACF1 gene expression was investigated by treating differentiated C2C12 murine skeletal muscle cells with 20 ng/ml of IGF-I at different time points. The IGF-I treatment increased plectin protein expression in a dose-dependent manner. The mRNA level of plectin was measured by real-time quantitative PCR to determine if plectin induction was regulated pretranslationally. IGF-I treatment resulted in a very rapid induction of plectin mRNA transcript in C2C12 myotubes. Plectin mRNA increased by 140 and 180% after 24 and 48 hours of IGF-I treatment, respectively, and returned to the control level after 72 hours of IGF-I treatment. MACF1 mRNA increased 86 and 90% after 24 and 48 hours of IGF-I treat-ment, respectively, and returned to the control level after 72 hours of IGF-I treatment. These results suggested that the plectin gene is regulated pretranslationally by IGF-I in skeletal muscle cells. In conclusion, IGF-I induces a rapid transcriptional modification of the plectin and MACF1 genes in C2C12 skeletal muscle cells and has modulating effects on a cytolinker protein as well as on contractile proteins.

Apoptotic Effect of Sasa quelpaertensis Nakai in Human Colon Cancer HT-29 Cells (인간 대장암 HT-29 세포에서 제주조릿대의 세포사멸 효과)

  • Byun, Ji Hee;Kim, Min Young
    • Journal of Life Science
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    • v.24 no.9
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    • pp.1012-1018
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    • 2014
  • Sasa quelpaertensis Nakai (Korean name, Jeju-Joritdae) is one of the most abundant plants on Mt. Halla, Jeju Island, and it has long been used in traditional medicines. Recent studies have reported it as possessing various beneficial functions, including anti-inflammatory, anti-diabetic, anti-hypertension, anti-gastritis, anti-oxidant, and anti-cancer effects. However, the molecular mechanisms of its anti-cancer activity have not been clearly elucidated. In this study, we investigated the anti-cancer effects and mechanism of S. quelpaertensis on human colon cancer HT-29 cells. Cell growth inhibition by S. quelpaertensis was determined by MTT assay. Apoptosis was performed by DNA fragmentation, flow cytometry with propidium iodide staining (PI), and reverse transcription-polymerase chain reaction (RT-PCR) to confirm the anti-apoptotic factors, such as inhibitor of apoptosis (IAP) family members. $NO^{\bullet}$ production was determined by Griess assay. S. quelpaertensis treatment resulted in the time- and dose-dependent inhibition of the cell viability of HT-29 cells by inducing apoptosis, as evidenced by the accumulation of the sub-G1 cell population stained by PI, as well as the ladder-like DNA fragmentation in a dose-dependent manner. S. quelpaertensis-inducing apoptosis was accompanied by the induction of S cell cycle arrests, increasing $NO^{\bullet}$ concentrations, and the down-regulation of IAPs, including X-chromosome-linked IAP (XIAP), cellular IAP-1 (cIAP-1), cIAP-2, and survivin. Taken together, these findings have important implications for future clinical developments of S. quelpaertensis in colon cancer treatment.