• BMB Reports
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• v.32 no.1
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• pp.45-50
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• 1999

We previously found three transcription factor-binding motifs in the rat p53 promoter. They are two recognition motifs of NF1-like protein (NF1-like element 1: -296 ~ -312, NF1-like element 2: -195 ~ -219) and a bHLH protein binding element (-142 ~ -146). In this study, we investigated the DNA-protein complex formation of the three elements with nuclear extracts from both normal and regenerating liver to find the element involved in the induced transcription of p53. The level of each DNA-protein complex on NF1-like and bHLH motifs was not changed. Instead, a new element located at -264 ~ -284 was detected in the DNase I footprinting assay with regenerating nuclear extract. This element has partial homology to the AP1 consensus motif. However, the competition studies with diverse oligonucleotides suggest that the binding protein is not AP1. An in vitro transcription assay shows that this element is important for the transcriptional activation of the rat p53 promoter. Therefore, for the induced transcription of the rat p53 promoter, the-264 ~ -284 region is required in addition to two NF1-like and one bHLH motif.

• Molecules and Cells
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• v.20 no.3
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• pp.446-451
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• 2005

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It was not clear that how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. We found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in the reconstitution assay. However Grim did not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2.

• Biomolecules & Therapeutics
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• v.29 no.5
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• pp.519-526
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• 2021

In a search for effective PPAR-γ agonists, 110 clinical drugs were screened via molecular docking, and 9 drugs, including parecoxib, were selected for subsequent biological evaluation. Molecular docking of parecoxib to the ligand-binding domain of PPAR-γ showed high binding affinity and relevant binding conformation compared with the PPAR-γ ligand/antidiabetic drug rosiglitazone. Per the docking result, parecoxib showed the best PPAR-γ transactivation in Ac2F rat liver cells. Further docking simulation and a luciferase assay suggested parecoxib would be a selective (and partial) PPAR-γ agonist. PPAR-γ activation by parecoxib induced adipocyte differentiation in 3T3-L1 murine preadipocytes. Parecoxib promoted adipogenesis in a dose-dependent manner and enhanced the expression of adipogenesis transcription factors PPAR-γ, C/EBPα, and C/EBPβ. These data indicated that parecoxib might be utilized as a partial PPAR-γ agonist for drug repositioning study.

• Environmental Mutagens and Carcinogens
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• v.22 no.3
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• pp.175-182
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• 2002

Endocrine disruptors (EDs) are the chemicals that affect endocrine systems through activation or inhibition of steroid hormone response. It is necessary to have a good system to evaluate rapidly and accurately endocrine-disrupting activities of suspected chemicals and their degradation products. The key targets of EDs are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We constructed a co-expression system of Gal4p DNA binding domain (DBD)- ligand binding domain of human estrogen receptor $\alpha$ or $\beta$, and Gal4p transactivation domain (TAD)-co-activator AIB-1, SRC-1 or TIF-2 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of CYC1 promoter and Gal4p binding site (GAL4 upstream activating sequence, GAL4$_{UAS}$). Expression of this reporter gene was dependent on the presence of estrogen or EDs in the culture medium. We found that the two-hybrid system with combination of the hER$\beta$ LBD and co-activator SRC-1 was most effective in the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by those chemicals correlated with their estrogenic activities measured by other assay systems, indicating that this assay system is efficient and reliable for measuring estrogenic activity. The data in this research demonstrated that the yeast detection system using steroid hormone receptor and co-activator is a useful tool for identifying chemicals that interact with steroid receptors.s.

• Archives of Pharmacal Research
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• v.19 no.5
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• pp.381-385
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• 1996

Rat ventricles respond with a biphasic positive inotropic effect to ouabain, low-dose and high-dose effects but rat atria with only a monophasic high dose effect. In an effect to understand the difference in response to ouabain of two tissues between rat atria and ventricles the levels of the $a_{2}$ -isoform of the $Na^{+}$, $K^{+}$-ATPase which has higher affinity for ouabain than the $a_{1}$-iso-form were determined by a $[^{3}H]$ouabain binding assay. The yield of protein per gram wet weight was about 47 mg for atria and 100 mg for ventricles. The $K_{d}$ values of ouabain for the high-affinity ouabain binding site $(a_{2} -isoform)$ were nearly the same (230 nM) in the atria and ventricles. However, the numbers of the $a_{2}$-isoform $(B_{max})$ per mg protein were approximately half in the atria. When the binding data were expressed in unit per gram tissue wet weight, the numbers of $a_{2}$ -isoform in the atria was about 25% of that in the ventricles. THese results demonstrate that the $a_{2}$ -isoform of the $Na^{+}$, $K^{+}$-ATPase in the rat atria could be detected by $[^{3}H]$ouabain binding assay and the levels of this isoform are too low to show the low-dose effect of ouabain.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.315-319
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• 2003

A novel method is developed to yield virus titers in 10 h, is easy to .perform using 96-well plates, and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immune-staining. The titer is determined by counting foci produced due to infection of virus under a fluorescent microscopy. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post infection time of 4 h. Therefore, 10 hours were enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer.

• YAKHAK HOEJI
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• v.45 no.3
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• pp.302-309
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• 2001

Wheat germ agglutinin (WGA) pectin, which binds to human melanoma cell line, was conjugated with Praecoxin A using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a cross-linking agent. Physical mixture (PM) of WGA and Praecoxin A was also prepared by using a non-specific binding property of Praecoxin A to WGA. The WGA:Praecoxin A ratio in the conjugate and PM was approximately 1:18 and 1:20, respectively. The results of hemagglutination assay and enzyme-linked lectin assay indicated that the conjugate and PM maintained the lectin-like properties of the WGA. The binding ratio of conjugate was about 70% during 4-24 hr, but the most of Praecoxin A was released within 24 hr in the case of PM. These results lead to the conclusion that the conjugate is potentially useful for the formulation of injection that requires targeting for melanoma as well as sustained release at the site.

• Bulletin of the Korean Chemical Society
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• v.33 no.1
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• pp.261-269
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• 2012

The cyclic dimers of enkephalin were isolated as minor components during the solution synthesis of the corresponding cyclic monomers. The ratio of cyclic dimer to monomer was approximately 1:4 from the percent of yields. In the receptor binding assay of two cyclic dimmers, ($Tyr_2-C[D-Glu-Phe-gPhe]_2$ 6, $Tyr_2-C[D-Asp-Phe-gPhe-rLeu]_2$ 8), both analogs exhibited the high preference for ${\delta}$ receptor compared to monocyclic counterparts. In the nociceptive activity, both showed about 5 times less potent than the cyclic monomers. The repeated synthesis of 14-membered cyclic analog, Tyr-C[D-Glu-Phe-gPhe-D-rLeu] 14, which was known as having three distinct cis-trans isomers, gave rise to apparently different conformational analog arousing only trans isomer. In the receptor binding assay, it showed tremendously high selectivity toward ${\mu}$ receptor $({\delta}/{\mu}=160)$.

• Animal cells and systems
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• v.6 no.4
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• pp.311-315
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• 2002

The RAD4 gene is essential for nucleotide excision repair in Saccharomyces cerevisiae. It has been known that the deduced amino acid sequence of Rad4 protein contains three DNA-dependent ATPase/helicase motifs. To determine the biochemical activities and functional role of RAD4 the Rad4 protein was expressed and purified. Immunoblot analysis showed a specific band of 21 kDa, which was well-matched with the size of open reading frame of the RAD4 gene. The purified Rad4 protein had no detectable helicase activity. However, the protein could interact with double stranded oligonucleotides, as judged by mobility shift assay. This result suggests that the Rad4 protein is a DNA binding protein.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.102-108
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• 1994

Autonomously replicating sequence Binding Factor 1(ABF1) is a DNA-binding protein that specifically recognizes the $RTCRYN_5ACG$ at many sites in the yeast genome including the promoter element, mating-type silencer and ARS. To express the intact full-length ABF1 gene in E. coli, the ABF1 gene has been cloned into pMAL-c2 and His-61, Leu-353 and Leu-360 were substituted with other amino acid. ABF1 fusion proteins of wild type ABF1 and H61A, L353R and L360R nutants were purified by amylose resin affinity chromatography. Fusion protein of MBP and ABF1 was digested by Factor Xa and Characterized by gel retardation assay and complementation test. As aresult, we suggested that other DNA binding motif except atypical inc-finger motif is in the middle region of ABF1.