• IMMUNE NETWORK
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• v.2 no.3
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• pp.150-157
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• 2002

Background: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. Methods: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. Results: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. Conclusion: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.114-114
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• 2003

Ginseng is a medicinal herb widely used in Asian countries, and its pharmacological effects has been demonstrated in various systems such as cardiovascular, central nervous, and endocrine systems. Its effects are mainly attributed to the ginsenosides. We hypothesize that a component of Panax ginseng, ginsenoside-Rbl, acts by binding to estrogen receptor. We have investigated the estrogenic activity of ginsenoside-Rbl in a transient transfection system using estrogen receptors ${\alpha}$ or ${\beta}$ with estrogen -responsive luciferase plasmids in COS monkey kidney cells. Ginsenoside-Rbl activated both estrogen receptors ${\alpha}$ and ${\beta}$ in a dose-dependent manner (0.5 -100 M ). Activation was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the estrogenic effect of ginsenoside-Rbl is estrogen receptor dependent. Next, we evaluated the ability of ginsenoside-Rbl to induce estrogen-responsive progesterone receptor gene by semi-quantitative RT-PCR assays. MCF-7 cells treated with l7${\beta}$-estradiol or ginsenoside- Rb1 exhibited an increased expression of progesterone receptor mRNA. However, ginsenoside-Rbl failed to displace the specific binding of ［3H］17${\beta}$-estradiol to estrogen receptor in MCF-7 cells as examined by whole cell ligand binding assays, suggesting that there is no direct interaction of ginsenoside-Rbl with estrogen receptor. Our results indicate that estrogen-like activity of ginsenoside-Rbl is independent of direct estrogen receptor association.

• Bulletin of the Korean Chemical Society
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• v.30 no.5
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• pp.1101-1106
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• 2009

Multivalent and multi-specific antibodies can provide valuable tools for bio-medical research, diagnosis and therapy. In antigen-antibody interactions, the avidity of antibodies depends on the affinity and the number of binding sites.$^1$ As artificial multivalent antibody agents, single chain Fv-streptavidin fusion tetramer proteins $(scFv-SA)_4$ have been previously tested.$^{1,\;2}$ Although, the Fab domain is known to be more stable than scFv in animal models,$^{3,\;4}$ it has never been used to make a multivalent agent with a streptavidin fusion. In this study, we prepared tetra-valent $(Fab-cSA)_4$ by fusing Fab with core streptavidin (cSA). This molecule was made using inclusion body production, refolding and chromatography purification. Affinities of the Fab-cSA tetramer and a scFv-cSA tetramer to a cell surface antigen were compared by ELISA using biotin-HRP. The Fab-cSA tetramer showed higher binding avidity than the scFv-cSA tetramer. The higher binding avidity of the Fab-cSA tetramer demonstrates its potential as a therapeutic agent for target-specific antibody therapy.

• BMB Reports
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• v.41 no.8
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• pp.575-580
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• 2008

Thymocyte-specific transcriptional regulatory systems can be used to better understand the relationship between transcription and V(D)J recombination during early T cell development. In this study, we generated transgenic mice expressing the transactivator Gal4-VP16 or the Gal4 DNA binding domain (Gal4-DBD) under the control of the lck proximal promoter, which is only active in immature thymocytes. From these studies Gal4-VP16 and Gal4-DBD expression was shown to significantly alter thymic cellularity and differentiation without significantly changing the $CD3^+$ thymocyte distribution. Furthermore, the presence of Gal4-VP16 or Gal4-DBD in the transgenic thymocytes retarded the mobility of the Gal4 DNA binding motif as determined by a gel mobility shift assay, suggesting that the developmental alteration did not affect the functional property of the transgenic proteins. These results indicated that lck promoter-driven Gal4-VP16 or Gal4-DBD expression did not affect $CD3^+$ mature thymocytes, thus this system can be applied to study transcriptional regulation of transresponder genes in bigenic mouse model thymocytes.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.182-182
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• 1996

Annexin I is a member of the annexin family of calcium dependent phospholipid binding proteins and has anti-inflammatory activity by inhibiting phospholipase A$_2$ (PLA$_2$). Recent X-ray crystallographic study of annexin I identified six Ca$\^$2+/ binding bites, which was different types (type II, III) from the well-known EF-hand motif (type I). In this work, the structure of annexin I was studied at atomic level by using $^1$H, $\^$15/N and $\^$l3/C NMR(nuclear magnetic resonance) spectroscopy, and the effect of Ca$\^$2+/ binding on the structure of annexin I was studied, and compared with that of Mg$\^$2+/ binding, When Ca$\^$2+/ was added to annexin I, NMR peak change was occured in high- and low-field regions of $^1$H-NMR spectra. NMR peak change by Ca$\^$2+/ binding was different from that by Mg$\^$2+/ binding. Because annexin I is a larger protein with 35 kDa molecular weight, site-specific (amide-$\^$15/N, carbonyl-$\^$l3/C) labeling technique was also used. We were able to detect methionine, tyrosine and phenylalanine peaks respectively in $\^$13/C-NMR spectra, and each residue was able to be assigned by the method of doubly labeling annexin I with [$\^$13/C] carbonyl-amino acid and [$\^$15/N] amide-amino acid. In $\^$l3/C-NMR spectra of [$\^$13/C] carbonyl-Met labeled annexin I, we observed that methionine residues spatially located near Ca$\^$2+/ binding Sites Were Significantly effected by Ca$\^$2+/ binding. From UV spectroscopic data on the effect of Ca$\^$2+/ binding, we knew that Ca$\^$2+/ binding sites of annexin I have cooperativity in Ca$\^$2+/ binding. The interaction of annexin I with PLA$_2$ also could be detected by using heteronuclear NMR spctroscopy. Consequently, we expect that the anti-inflammatory action mechanism of annexin I may be a specific protein-protein interaction. The residues involved in the interaction with PLA$_2$ can be identified as active site by assigning NMR peaks effected by PLA$_2$ binding.

• Development and Reproduction
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• v.16 no.4
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• pp.379-384
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• 2012

Lhx8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian folliculogenesis. However, Lhx8 DNA binding sequences are not characterized yet. We aimed to identify and characterize a cis-acting sequence of germ-cell specific transcriptional factor, Lhx8. To identify Lhx8 DNA binding element, Cyclic Amplification of Sequence Target (CAST) Analysis was performed. Electrophoretic Mobility Shift Assay (EMSA) was processed for the binding specificity of Lhx8. Luciferase assay was for the transcriptional activity of Lhx8 through identified DNA binding site. We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE). In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element. These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

• Korean Journal of Pharmacognosy
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• v.32 no.3 s.126
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• pp.212-218
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• 2001

The water extracts of 82 Korean medicinal herbs were prepared and were examined for the binding affinity on the glycine binding site of NMDA (N-methyl-D-aspartate) receptor prepared by the synaptic membranes from the forebrains of male Sprague-Dawley rats. Among the tested, the extracts of Dioscoreae Rhizoma, Hoveniae Semen cum Fructus, Astragali Radix, Armeniacae Semen, Huttuynia cordata Herba, Acanthopanacis Cortex, Aurantii nobilis Pericarpium, Phellinus linteus, Amomi Fructus, Artemisiae capillaris Herba, Polyporus, Agastachis Herba and of Galli Stomachichum Corium were found to exhibit significant competitions with $[^3H]-MDL$ 105,519 for the glycine specific binding site of NMDA receptor in a dose dependent manner, respectively.

• Microbiology and Biotechnology Letters
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• v.51 no.3
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• pp.243-249
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• 2023

We recently found that the insect-derived antimicrobial peptide CopA3 (LLCIALRKK) directly binds to and inhibits the proteolytic activation of caspases, which play essential roles in apoptotic processes. However, the mechanism of CopA3 binding to caspases remained unknown. Here, using recombinant GST-caspase-3 and -6 proteins, we investigated the mechanism by which CopA3 binds to caspases. We showed that replacement of cysteine in CopA3 with alanine caused a marked loss in its binding activity towards caspase-3 and -6. Exposure to DTT, a reducing agent, also diminished their interaction, suggesting that this cysteine plays an essential role in caspase binding. Experiments using deletion mutants of CopA3 showed that the last N-terminal leucine residue of CopA3 peptide is required for binding of CopA3 to caspases, and that C-terminal lysine and arginine residues also contribute to their interaction. These conclusions are supported by binding experiments employing direct addition of CopA3 deletion mutants to human colonocyte (HT29) extracts containing endogenous caspase-3 and -6 proteins. In summary, binding of CopA3 to caspases is dependent on a cysteine in the intermediate region of the CopA3 peptide and a leucine in the N-terminal region, but that both an arginine and two adjacent lysines in the C-terminal region of CopA3 also contribute. Collectively, these results provide insight into the interaction mechanism and the high selectivity of CopA3 for caspases.

• BMB Reports
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• v.31 no.5
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• pp.427-431
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• 1998

In the previous report (Choi et al., 1997), the angiogenin binding peptides identified from a phage-peptide library were analyzed by using the fusion proteins composed of the Escherichia coli maltose binding protein and its corresponding peptides. However, it was difficult to obtain a sufficient amount of the fusion proteins required for further analysis because of the low expression level. We now report a high level expression of the fusion protein and analysis of its anti-angiogenin activity. The use of strong T7 promoter and removal of signal sequence allowed about a 20-fold increase in the expression efficiency of the fusion protein. We were able to obtain about 10 mg of purified fusion protein from one liter of culture. The purified fusion protein showed angiogenin-specific affinity and inhibited the binding of biotinylated actin to human angiogenin at $IC_{50}$ of 0.6 mM. Its anti-angiogenin activity was also revealed by the chorioallantoic membrane assay.

• YAKHAK HOEJI
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• v.28 no.3
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• pp.129-138
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• 1984

Specific [$^{3}H$] ouabain binding and $Ca^{2+}$ -uptake were measured to elucidate the role of high affinity [$^{3}H$] ouabain binding site in rat cardiac myocytes which contain 65% of rod cells. High affinity [$^{3}$H] ouabain binding site, which is about 3% of total pump sites, with apparent dissociation constant ($K_{D}$) of $1.1{\times}10^{-7}M$ and maximum binding site concentration (Bmax) of 1.2 pmol/mg protein ($1.754{\times}10^{5}cells$) were identified. At the concentration of $10^{-7}M$ to $10^{-4}M$, ouabain produced concentration dependent increase in $Ca^{2+}$-uptake of myocytes. The effect of ouabain on $Ca^{2+}$-uptake was not effected by membrane depolarization (elevated K+ in incubation medium) or verapamil. These results suggest that in rat ventricular myocytes the ouabain receptor complex to high affinity site may increase Na+ - $Ca^{2+}$ exchange across the sarcolemmal membrane by inhibition of Na+, K+ - ATPase.