• Title/Summary/Keyword: quantitative real-time PCR (qPCR)

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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 Profiling of MLO Family Genes under Podosphaera xanthii Infection and Exogenous Application of Phytohormones in Cucumis melo L. (멜론 흰가루병균 및 식물 호르몬 처리하에서 MLO 유전자군의 발현검정)

  • Howlader, Jewel;Kim, Hoy-Taek;Park, Jong-In;Ahmed, Nasar Uddin;Robin, Arif Hasan Khan;Jung, Hee-Jeong;Nou, III-Sup
    • Journal of Life Science
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    • v.26 no.4
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    • pp.419-430
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    • 2016
  • Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phyto-hormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.

Microarray Analysis of Long Non-coding RNA Expression Profile Associated with 5-Fluorouracil-Based Chemoradiation Resistance in Colorectal Cancer Cells

  • Xiong, Wei;Jiang, Yong-Xin;Ai, Yi-Qin;Liu, Shan;Wu, Xing-Rao;Cui, Jian-Guo;Qin, Ji-Yong;Liu, Yan;Xia, Yao-Xiong;Ju, Yun-He;He, Wen-Jie;Wang, Yong;Li, Yun-Fen;Hou, Yu;Wang, Li;Li, Wen-Hui
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.8
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    • pp.3395-3402
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    • 2015
  • Background: Preoperative 5-fluorouracil (5-FU)-based chemoradiotherapy is a standard treatment for locally advanced colorectal cancer (CRC). However, CRC cells often develop chemoradiation resistance (CRR). Recent studies have shown that long non-coding RNA (lncRNA) plays critical roles in a myriad of biological processes and human diseases, as well as chemotherapy resistance. Since the roles of lncRNAs in 5-FU-based CRR in human CRC cells remain unknown, they were investigated in this study. Materials and Methods: A 5-FU-based concurrent CRR cell model was established using human CRC cell line HCT116. Microarray expression profiling of lncRNAs and mRNAs was undertaken in parental HCT116 and 5-FU-based CRR cell lines. Results: In total, 2,662 differentially expressed lncRNAs and 2,398 mRNAs were identified in 5-FU-based CRR HCT116 cells when compared with those in parental HCT116. Moreover, 6 lncRNAs and 6 mRNAs found to be differentially expressed were validated by quantitative real time PCR (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the differentially expressed mRNAs indicated involvement of many, such as Jak-STAT, PI3K-Akt and NF-kappa B signaling pathways. To better understand the molecular basis of 5-FU-based CRR in CRC cells, correlated expression networks were constructed based on 8 intergenic lncRNAs and their nearby coding genes. Conclusions: Changes in lncRNA expression are involved in 5-FU-based CRR in CRC cells. These findings may provide novel insight for the prognosis and prediction of response to therapy in CRC patients.

MicroRNA 155 Expression Pattern and its Clinic-pathologic Implication in Human Lung Cancer (폐암에서 microRNA 155의 발현 양상과 임상병리학적 의의)

  • Kim, Mi Kyeong;Moon, Dong Chul;Hyun, Hye Jin;Kim, Jong-Sik;Choi, Tae Jin;Jung, Sang Bong
    • Journal of Life Science
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    • v.26 no.9
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    • pp.1056-1062
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    • 2016
  • Lung cancer is currently the most common malignant disease and the leading cause of mortality in the world and non-small cell lung cancer (NSCLC) accounts for 75-80% of lung cancer cases. miR-155 gene was found to be over expressed in several solid tumors, such as thyroid carcinoma, breast cancer, colon cancer, cervical cancer, pancreatic ductal adenocarcinoma (PDAC) and lung cancer. The aims of this study were to define the expression of miR-155 in lung cancer and its associated clinic-pathologic characteristics. Total RNA was purified from formalin-fixed, paraffin-embedded NSCLC tissues and benign lung tissues. Expression of miR-155 in human lung cancer tissues were evaluated as mean fold changes of miR-155 in cancer tissues compared to benign lung tissues by quantitative real-time reverse transcriptase polymerase chain reaction (real-time qRT-PCR) and associations of miR-155 expression with clinic-pathologic findings of cancer. Compared with the benign control group, miR-155 expression was significantly overexpressed in NSCLCs (p=<0.001). miR-155 was more overexpressed in squamous cell carcinoma than in adenocarcinoma. Poorly differentiated tumors showed significantly overexpression of miR-155 than well-differentiated tumors (p=<0.001). Overexpression of miR-155 was significantly associated with lymph node metastasis (p=<0.05). In survival analysis for all NSCLC patients, high miR-155 expression was significantly correlated with worse overall survival (p=<0.05). These results suggested that miR-155 might play an important role in lung cancer progression and metastasis.