• Title/Summary/Keyword: DNA binding activity

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Screening and Purification of an Antimicrobial Peptide from the Gill of the Manila Clam Ruditapes philippinarum (바지락(Ruditapes philippinarum) 아가미로부터 항균 펩타이드의 탐색 및 정제)

  • Seo, Jung-Kil
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.49 no.2
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    • pp.137-145
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    • 2016
  • This study screened the biological activity of an acidified gill extract of the Manila clam Ruditapes philippinarum including antimicrobial, hemolytic, membrane permeabilization, and DNA-binding activity, and purified the antimicrobial material. The acidified gill extract showed potent antimicrobial activity against Bacillus subtilis and Escherichia coli without significant hemolytic activity, but showed no membrane permeabilization or DNA-binding ability. An antimicrobial material was purified from the acidified gill extract using C18 reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). Treatment of the purified material with trypsin completely abolished all of the antibacterial activity against Bacillus subtilis, suggesting that the purified material is a proteinaceous antibiotic. The molecular weight of the purified material was 2571.9 Da, but no primary structural information was obtained due to N-terminal blocking. A future study should confirm the primary structure. Our results suggest that the Manila clam gill contains proteinaceous antibiotics that have a role in first-line defense. This information could be used to better understand the Manila clam innate immune system.

HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress

  • Khanra, Kalyani;Chakraborty, Anindita;Bhattacharyya, Nandan
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.18
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    • pp.8177-8186
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    • 2016
  • The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta ($pol{\beta}{\Delta}_{208-304}$) specific for ovarian cancer. $Pol{\beta}{\Delta}_{208-304}$ has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. $Pol{\beta}{\Delta}_{208-304}$ cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards $H_2O_2$ and UV when compared with HeLa cells alone. It has been shown that cell death in $Pol{\beta}{\Delta}_{208-304}$ transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins

  • Chaikam, Vijay;Karlson, Dale T.
    • BMB Reports
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    • v.43 no.1
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    • pp.1-8
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    • 2010
  • The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs.

Midline enhancer activity of the short gastrulation shadow enhancer is characterized by three unusual features for cis-regulatory DNA

  • Shin, Dong-Hyeon;Hong, Joung-Woo
    • BMB Reports
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    • v.48 no.10
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    • pp.589-594
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    • 2015
  • The shadow enhancer of the short gastrulation (sog) gene directs its sequential expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo. Here, we characterize three unusual features of the shadow enhancer midline activity. First, the minimal regions for the two different enhancer activities exhibit high overlap within the shadow enhancer, meaning that one developmental enhancer possesses dual enhancer activities. Second, the midline enhancer activity relies on five Single-minded (Sim)-binding sites, two of which have not been found in any Sim target enhancers. Finally, two linked Dorsal (Dl)- and Zelda (Zld)-binding sites, critical for the neurogenic ectoderm enhancer activity, are also required for the midline enhancer activity. These results suggest that early activation by Dl and Zld may facilitate late activation via the noncanonical sites occupied by Sim. We discuss a model for Zld as a pioneer factor and speculate its role in midline enhancer activity.

Transcriptional regulation of soybean ${\beta}-conglycinin$ gene expression: -(II) Developmental change of soybean embryo factor 3 activity- (대두 ${\beta}-conglycinin$ 유전자 발현의 전사 조절에 관한 연구 -(II) 대두 발달과정 중의 대두 배 인자 3의 역가 변화-)

  • Lee, Kyung-Hoon;Chung, Dong-Hyo;Kim, Woo-Yeon
    • Applied Biological Chemistry
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    • v.36 no.6
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    • pp.553-556
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    • 1993
  • Soybean nuclear extracts were prepared to examine the expression of SEF3 (soybean embryo factors 3), which binds to the upstream region of soybean ${\beta}-conglycinin$ ${\alpha}'$ subunit gene and is presumed to be a trans-acting factor for the expression of the gene. The relative levels of SEF3 binding activity in nuclear extracts of maturing soybean embryos were determined using the SE3 DNA probe containing two AACCCA hexanucleotides for gel mobility shift assay. The SEF3 activity increased in developing embryos from 16 to 32 days after pollination, whereas the mobility of the SE3-SE3-SEF3 complex decreased. The mobility of the complex was increased by the treatment of nuclear extracts with alkaline phosphatese, which could be inhibited by phosphate. Formation of the SE3-SEF3 complex was not affected by the binding buffer pH between 6.8 and 8.5.

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Characterization and Transcriptional Activity of a Vitamin D Receptor Ortholog in the Ascidian Halocynthia roretzi (멍게(Halocynthia roretzi) 비타민 D 수용체 상동체 동정 및 전사활성)

  • Lee, Jung Hwan;Sohn, Young Chang
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.48 no.6
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    • pp.913-919
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    • 2015
  • In vertebrates, the vitamin D receptor (VDR), a member of the nuclear receptor superfamily, binds the biologically active ligand $1{\alpha},25-(OH)_2$-vitamin $D_3$ (1,25 $D_3$). Nearly all vertebrates, including Agnatha, possess a VDR with high ligand selectivity for 1,25 $D_3$ and related metabolites. Although a putative ancestral VDR gene is present in the genome of the chordate invertebrate Ciona intestinalis, the functional characteristics of marine invertebrate VDR are still obscure. To elucidate the ascidian Halocynthia roretzi VDR (HrVDR), we cloned full-length HrVDR cDNA and investigated the transcriptional activity of HrVDR in HEK293 cells. HrVDR consists of 1,680 nucleotides (559 amino acids [aa]), including a short N-terminal region (A/B domain; 26 aa), DNA-binding domain (C domain; 72 aa), hinge region (D domain; 272 aa), and C-terminal ligand-binding domain (E domain; 161 aa). The amino acid sequence identity of HrVDR was greatest to that of C. intestinalis VDR (56%). In the luciferase reporter assays, the transcriptional activity of HrVDR was not significantly increased by 1,25 $D_3$, whereas the farnesoid X receptor agonist GW4064 increased the transactivation of HrVDR. These results suggest the presence of a novel ligand for and a distinct ligand-binding domain in ascidian VDR.

Testing of Cancer Chemopreventive Potential of Prunella vulgaris L. Aqua-acupuncture Solution Using Biochemical Markers of Carcinogenesis (발암과정 생화학적 표식자를 이용한 하고초 약침액의 암예방 활성 측정)

  • Park, Sin-Hwa;Cho, Kyoung-Hee;Shon, Yun-Hee;Lim, Jong-Kook;Nam, Kyung-Soo
    • Korean Journal of Pharmacognosy
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    • v.32 no.2 s.125
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    • pp.163-167
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    • 2001
  • Prunella vulgaris L. aqua-acupuncture solution (PVAS) was tested for cancer chemopreventive activity using chemoprevention-associated biochemical end points. The following effects were measured. : (a) inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced cytochrome P4501A1 activity (b) inhibition of $[^3H]B[a]P-DNA$ binding (c) inhibition of phorbol 12-myristate 13-acetate (TPA)-induced free radical formation in HL-60 cells (d) inhibition of polyamine metabolism. PVAS inhibited cytochrome P4501A1-mediated ethoxyresorufin-O-deethylase activity. The binding of $[^3H]B[a]P$ metabolites to DNA of NCTC-clone 1469 cells was inhibited significantly by PVAS. There is 22% inhibition of TPA-induced free radical formation in human leukemic cells with 5 mg/ml PVAS. Proliferation of Acanthamoeba castellanii was inhibited by PAVS at concentration of 30 mg/ml. PAVS positive in these assays may inhibit the carcinogenesis process and is considered very promising cancer-preventing agent because of its multiple activities.

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Suppressive effects of Morus alba Linne Root Bark (MRAL) on activation of MC/9 mast cells (상백피에 의한 MC/9 비만세포의 활성 억제 조절 연구)

  • Lee, Ki Jeon;Kim, Bok Kyu;Kil, Ki Jung
    • The Korea Journal of Herbology
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    • v.28 no.1
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    • pp.33-42
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    • 2013
  • Objective : Morus alba Linne Root Bark (MRAL) is a medicinal herb in Korean Medicine, known for its anti-inflammatory and anti-allergic properties. However, its mechanisms of action and the cellular targets have not yet been found and the study was developed to investigate the allergic suppressive effect of MRAL. The purpose of this study is to investigate the allergic suppressive effects of MRAL on activation of MC/9 mast cells. Methods : Cytotoxic activity of MRAL (50, 100, 200, 400 ${\mu}g/mL$) on MC/9 mast cells measured using EZ-Cytox cell viability assay kit (WST reagent). The levels of interleukin-5 (IL-5), IL-13 and IL-4, IL-5, IL-6, IL-13 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and real-time PCR respectively. The expression of transcription factors such as GATA-1, GATA-2, NFAT, AP-1 and NF-${\kappa}B$ p65 DNA binding activity were measured by western blot and electrophoresis mobility shift assay (EMSA). Results : Our results indicated that MRAL (50 ${\mu}g/mL$, 100 ${\mu}g/mL$) significantly inhibited PMA/Ionomycin-induced production of IL-5 and IL-13 and the expression of IL-4, IL-5, IL-6 and IL-13 mRNA in MC/9 mast cells. Moreover, MRAL (50 ${\mu}g/mL$, 100 ${\mu}g/mL$) inhibited PMA/Ionomycin-induced GATA-1, GATA-2, NFAT-1, NFAT-2, c-Fos protein expression and NF-${\kappa}B$ p65 DNA binding activity in MC/9 mast cells. Conclusions : In conclusion, we suspect the anti-allergenic activities of MRAL, may be related to the regulation of transcription factors GATA-1, GATA-2, NFAT-1, NFAT-2, c-Fos and NF-${\kappa}B$ p65 DNA binding assay causing inhibition of Th2 cytokines IL-5 and IL-13 in mast cells.