• Korean Journal of Microbiology
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• v.48 no.2
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• pp.79-86
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• 2012

ErmSF is one of the Erm family proteins which catalyze S-adenosyl-$_L$-methionine dependent modification of a specific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B ($MLS_B$) antibiotics. $^{222}FXPXPXVXS^{230}$ (ErmSF numbering) sequence appears to be a consensus sequence among the Erm family. This sequence was supposed to be involved in direct interaction with the target adenine from the structural studies of Erm protein ErmC'. But in DNA methyltarnsferase M. Taq I, this interaction have been identified biochemically and from the complex structure with substrate. Arginine 223 and 227 in this sequence are not conserved among Erm proteins, but because of the basic nature of residues, it was expected to interact with RNA substrates. Two amino acid residues were replaced with Ala by site-directed mutagenesis. Two mutant proteins still maintained its activity in vivo and resistant to the antibiotic erythromycin. Compared to the wild-type ErmSF, R223A and R227A proteins retained about 50% and 88% of activity in vitro, respectively. Even though those arginine residues are not essential in the catalytic step, with their positive charge they may play an important role for RNA binding.

• Journal of Life Science
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• v.25 no.5
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• pp.585-593
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• 2015

Stress fiber (SF) alteration is mediated by cellular receptors, which, upon interaction with the extracellular counterpart, signal to the actin cytoskeleton for remodeling. This association is mediated by a variety of scaffold and signaling factors, which control the mechanical and signaling activities of the interaction site. The heterotrimeric transmembrane lymphotoxin α1β2 (LTα1β2), a member of the tumor necrosis factor (TNF) family of cytokines, including soluble homotrimeric lymphotoxin (LT α), plays an important role in lymphoid tissue architecture. Ligation between LTα1β2 and the lymphotoxin β receptor (LTβR) activates signal-cascade in fibroblastic reticular cells (FRCs). We found LTβR stimulation using an agonistic anti-LTβR antibody alone or combined with LTα or TNFα induced changes in the actin and plasticity of cells. To clarify the involvement of myosin underlying the alteration, we analyzed the effect of myosin light chain kinase (MLCK) with an MLCK inhibitor (ML7), the phosphorylation level of myosin light chains (MLC), and the level of phospho-myosin phosphatase target subunit 1 (MYPT1) after treatment with an agonistic anti-LTβR antibody for cytoskeleton reorganization in FRCs. The inhibition of MLCK activity induced changes in the actin cytoskeleton organization and cell morphology in FRC. In addition, we showed the phosphorylation of MLC and MYPT1 was reduced by LTβR stimulation in cells. A DNA chip revealed the LTβR stimulation of FRC down-regulated transcripts of myosin and actin components. Collectively, these results suggest LTβR stimulation is linked to myosin regarding SF alteration in FRC.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.12 no.1
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• pp.312-317
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• 2011

Vanilloid receptor TRPV1 (known as capsaicin channel, transient receptor potential vanilloid 1) is known to be a key protein in the pain signal transduction. However, the proteins controlling the activity of the channel are not much known yet. Recently mouse Rab11-FIP3 (Rab11-family interaction protein 3) was found and reported to interact with rat TRPV1. Rab11 has been shown to play a key role in a variety of cellular processes including plasma membrane recycling, phagocytosis, and transport of secretory proteins from the trans-Golgi network. Therefore, Rab11-FIP3 was proposed to be involved in the membrane trafficking of TRPV1. In this study, the unreported rat Rab11-FIP3 was yet cloned in order to show the specific interaction of the TRPV1 and Rab11-FIP3 in the same species of rat and to examine the membrane trafficking of TRPV1. The result showed that rat Rab11-FIP3 is expected to have 489 amino acids and showed 80% identity with that of human and over 90% identity with that of mouse. Rab11-FIP3 was found to be expressed in heart, brain, kidney, testis using northern and western blot analyses. We also found that rat Rab11-FIP3 was colocalized with rat TRPV1 but not with TRPV2 of same family in the rat brain by using immunohistochemistry showing that two proteins interact specifically, suggesting the role of Rab11-FIP3 in the membrane trafficking.

• The Korean Journal of Mycology
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• v.40 no.4
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• pp.224-228
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• 2012

In order to screen interactor(s) of the Aspergillus nidulans ${\alpha}$-COP of COPI vesicle, we performed the yeast two hybrid screening by using the gene for A. nidulans ${\alpha}$-COP as a bait and identified ${\varepsilon}$-COP of the COPI vesicle as an interacting protein. The A. nidualns gene for the ${\varepsilon}$-COP was designated $aneA^+$ ($\underline{A.}$ $\underline{n}$idulans $\underline{e}$psi-lone-COP), which encoded 296 amino acid residues with high level of identity with orthologs from other fungi. Domain analyses with yeast two-hybrid system suggested that the interaction between ${\alpha}$-COP and ${\varepsilon}$-COP relied on the C-terminus of both proteins, and that the N-terminal WD domian of ${\alpha}$-COP and the TPR region of ${\varepsilon}$-COP were not essential but required for the enhancement of the interaction. These results indicate that the interaction mode between ${\alpha}$-COP and ${\varepsilon}$-COP of COPI vesicle is evolutionarily well conserved in eukaryotes.

• Journal of Yeungnam Medical Science
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• v.2 no.1
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• pp.175-181
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• 1985

Agrobacterium tumefaciens induces cancerous growths called crown galls at wound sites on dicotyledonous plants. A large plasmid called Ti plasmid is responsible for virulence. Upon tumor induction, part of the plasmid, termed T-DNA, becomes integrated into plant genome and its genetic sequences are expressed. These properties allow Ti plasmids to be used as gene vectors in plants. Several in vitro methods for the transfer of Ti plasmid into plant cell have been developed. One of them is the treatment of bacterial spheroplasts and plant protoplasts mixture with polyethylene glycol that is generally used as fusogen in cell-to-cell fusion. Several workers investigated the interaction of bacterial spheroplasts with plant protoplasts in the presence of polyethylene glycol and suggested that the interaction is not fusion but endocytosis. In this report we observed the interaction of Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts by electron microscope. Agrobacterium tumefaciens spheroplasts and Nicotiana tabacum protoplasts were prepared and mixed in the presence of polyethylene glycol and high pH-high $Ca^{2+}$ buffer. Then the interaction of the spheroplasts with the protoplasts was examined by transmission electron microscope. After the treatment of polyethylene glycol the spheroplasts adhered to the surface of the protoplasts and then they were engulfed by the protoplasts. After the high pH-high $Ca^{2+}$ buffer treatment the engulfed spheroplasts lost their cell integrity. No fusion process was observed. Thus all these observations suggest that the introduction process of Agrobacterium tumefaciens spheroplasts into Nicotiana tabacum protoplasts with the aid of polyethylene glycol is endocytosis.

• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1279-1284
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• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

• Toxicological Research
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• v.34 no.4
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• pp.281-290
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• 2018

Exposure to chemical agents is an inevitable consequence of modern society; some of these agents are hazardous to human health. The effects of chemical carcinogens are of great concern in many countries, and international organizations, such as the World Health Organization, have established guidelines for the regulation of these chemicals. Carcinogens are currently categorized into two classes, genotoxic and non-genotoxic carcinogens, which are subject to different regulatory policies. Genotoxic carcinogens are chemicals that exert carcinogenicity via the induction of mutations. Owing to their DNA interaction properties, there is thought to be no safe exposure threshold or dose. Genotoxic carcinogens are regulated under the assumption that they pose a cancer risk for humans, even at very low doses. In contrast, non-genotoxic carcinogens, which induce cancer through mechanisms other than mutations, such as hormonal effects, cytotoxicity, cell proliferation, or epigenetic changes, are thought to have a safe exposure threshold or dose; thus, their use in society is permitted unless the exposure or intake level would exceed the threshold. Genotoxicity assays are an important method to distinguish the two classes of carcinogens. However, some carcinogens have negative results in in vitro bacterial mutation assays, but yield positive results in the in vivo transgenic rodent gene mutation assay. Non-DNA damage, such as spindle poison or topoisomerase inhibition, often leads to positive results in cytogenetic genotoxicity assays such as the chromosome aberration assay or the micronucleus assay. Therefore, mechanistic considerations of tumor induction, based on the results of the genotoxicity assays, are necessary to distinguish genotoxic and non-genotoxic carcinogens. In this review, the concept of threshold of toxicological concern is introduced and the potential risk from multiple exposures to low doses of genotoxic carcinogens is also discussed.

• Parasites, Hosts and Diseases
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• v.44 no.4 s.140
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• pp.303-312
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• 2006

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the $X-\alpha-gal$ positive and PCR, 157 colonies of the $X-\beta-gal$ assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.

• BMB Reports
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• v.51 no.7
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• pp.338-343
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• 2018

Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called 'Sal box' composed of an 11-base pair motif. The interaction of TTF-I with the 'Sal box' is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity.

• Biomedical Science Letters
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• v.13 no.1
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• pp.1-10
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• 2007

Nebulin, a giant modular protein from muscle, is thought to act as molecular ruler in sarcomere assembly. In skeletal muscle, the C-terminal ${\sim}50 kDa$ region of nebulin extends into the Z-line lattice. The most recent studies implicated highlighting its extensive isoform diversity and exciting reports revealed its expression in cardiac and non-muscle tissues containing brain. Also these novel findings are indicating that nebulin is actually a multifunctional filament system, perhaps playing roles in signal transduction, contractile regulation, and myofibril force generation, as well as other not yet defined functions. However the binding protein of nebulin and function in brain is still unknown. A novel binding partner of nebulin C-terminal region was identified by screening a human brain cDNA library using yeast two-hybrid system. Nebulin C-terminus binding protein 51 (NCBP51) was contained a RING-finger domain and identified a new isoform of RING finger protein 125 (RNF125). The interaction was confirmed using the GST pull-down assay. NCBP51 belongs to a family of the RING finger proteins and its function remains to be identified in brain. The role of nebulin and NCBP51 will be studied by loss-of-function using siRNA technique in brain.