• Archives of Pharmacal Research
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• 제29권7호
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• pp.570-576
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• 2006

Phytoestrogen biochanin A is an isoflavone derivative isolated from red clover Trifolium pratense with anticarcinogenic properties. This study examined the action of biochanin A with the carcinogen activation pathway that is mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 breast carcinoma cells. Treating the cells with biochanin A alone caused the accumulation of CYP1A1 mRNA and an increase in CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose dependent manner. A concomitant treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and biochanin A markedly reduced the DMBA-inducible EROD activity and CYP1A1 mRNA level. In addition, the biochanin A treatment alone activated the DNA-binding capacity of the AhR for the dioxin-response element (DRE) of CYP1A1, as measured by the electrophoretic-mobility shift assay (EMSA). EMSA revealed that biochanin A reduced the level of the DMBA-inducible AhR-DRE binding complex. Furthermore, biochanin A competed with the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), for binding to the AhR in an isolated rat cytosol. The biochanin A competitively inhibited the metabolic activation of DMBA, as measured by the formation of the DMBA-DNA adducts. These results suggest that biochanin A may thus be a natural ligand to bind on AhR. Therefore, biochanin A may be due to act an antagonist/agonist of the AhR pathway.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2001년도 춘계심포지움 및 학술발표회
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• pp.126-126
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• 2001

Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wild range of environmental and industrial chemicals. It is clear that in vivo methods will be required to identify adverse effects produced by these chemicals. (omitted)

• Archives of Pharmacal Research
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• 제28권12호
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• pp.1365-1375
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• 2005

The antiestrogenic effects of marijuana smoke condensate (MSC) and three major cannabinoids, i.e., $\bigtriangleup^{9}$-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), were evaluated using in vitro bioassays, viz., the human breast cancer cell proliferation assay, the recombinant human estrogen receptor (ER) competitive binding assay, and the reporter gene assay. The inhibitory effects on estrogen were also examined using the ethoxyresorufin-O­deethylase (EROD) assay, the aromatase assay, and the 17$\beta$-estradiol ($E_{2}$) metabolism assay. The results showed that MSC induced the antiestrogenic effect via the ER-mediated pathway, while THC, CBD, and CBN did not have any antiestrogenic activity. This suggests that the combined effects of the marijuana smoke components are responsible for the antiestrogenicity of marijuana use. In addition, MSC induced the CYP1A activity and the $E_{2}$ metabolism, but inhibited the aromatase activity, suggesting that the antiestrogenic activity of MSC is also related to the indirect ER-dependent pathway, as a result of the depletion of the in situ $E_{2}$ level available to bind to the ER. In conclusion, pyrogenic products including polycyclic aromatic hydrocarbons (PAHs) in the non-polar fraction, which is the most biologically active fraction among the seven fractions of MSC, might be responsible for the antiestrogenic effect.

• The Korean Journal of Physiology and Pharmacology
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• 제1권6호
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• pp.769-774
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• 1997

Non-neuronal high affinity binding sites for benzodiazepines have been found in many peripheral tissues including cardiac muscle and vascular smooth muscle, and have been designated as 'peripheral benzodiazepine receptor'. Benzodiazepines have been shown to induce relaxation of the ileal, vesical, and uterine smooth muscles. However, it is still unclear about possible involvement of peripheral benzodiazepine receptor on the contractility of trachealis muscle. This study was performed to investigate the role of the peripheral benzodiazepine receptor on the contractility of canine trachealis muscle. Canine trachealis muscle strips of 15 mm long were suspended in an isolated organ bath containing 1 ml of physiological salt solution maintained at $37^{\circ}C$, and aerated with $95%\;O_2/5%\;CO_2$. Isometric myography was performed, and the results of the experiments were as follows: Ro5-4684, FGIN-1-27 and clonazepam reduced a basal tone of isolated canine trachealis muscle strip concentration dependently, relaxant actions of RoS-4684 and FGIN-1-27 were antagonized by PK11195, a peripheral benzodiazepine receptor antagonist. Flumazenil, a central type antagonist, did not antagonize the relaxant action of Peripheral type agonists. Saturation binding assay of [3H]Ro5-4864 showed a high affinity$(Kd=5.33{\pm}1.27nM,\;Bmax=\;867.3{\pm}147.2\;fmol/mg\;protein)$ binding site on the canine trachealis muscle. Ro 5-4684 suppressed the bethanechol-, 5-hydroxyoyptamine- and histamine- induced contractions. Platelet activating factor (PAF) exerted strong and prolonged contraction in trachealis muscle strip. Strong tonic contraction by PAE was attenuated by Ro 5-4684, but not by WEB 2086, a PAF antagonist. Based on these results, it is concluded that the peripheral benzodiazepine receptor mediates the inhibitory regulation of contractilty of canine trachealis muscle.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1508-1514
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• 2016

A series of antagonists specifically targeting growth hormone receptors (GHR) in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH). Therefore, in this study, we developed and characterized a porcine GHR (pGHR) antibody antagonist (denoted by AN98) via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1) secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

• Animal cells and systems
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• 제2권1호
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• pp.133-137
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• 1998

The present study aims to characterize a cDNA encoding zebrafish thyroid hormone receptor $\alpha{1}$ $(zTR\alpha{1)}$ in order to investigate its possible role in the early stage of embryonic development. A mobility shift assay showed that $zTR\alpha{1}$ overexpressed in COS7 cells specifically bound to thyroid hormone response element (TRE). In addition, the specific interaction of anti-rat $TR\alpha{1}$ antibodies with $zTR\alpha1$/TRE complexes demonstrated that the cDNA clone encoded zebrafish thyroid hormone receptor $\alpha{1}$. Transient cotransfection assays showed that $zTR\alpha{1}$ repressed the transcription which was induced by retinoic acid (RA), a well-characterized embryonic morphogen. These results suggest that zTRal may be involved in regulating the RA-induced gene transcription during early embryonic development.

• Archives of Pharmacal Research
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• 제27권8호
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• pp.857-862
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• 2004

Naringenin, dietary flavonoid, is antioxidant constituents of many citrus fruits. In the present study, we investigated the effect of naringenin on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible CYP1 A 1 gene expression in mouse hepatoma Hepa-1c1c7 cells. Naringenin alone did not affect CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. In contrast, the TCDD-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and naringenin in a dose dependent manner. TCDD-induced CYP1A1 mRNA level was also markedly suppressed by naringenin. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and electrophoretic mobility shift assay revealed that naringe-nin reduced transformation of the aryl hydrocarbons receptor(AhR) to a form capable of specif-ically binding to the DRE sequence in the promoter of the CYP1A1 gene. These results suggest the down regulation of the CYP1A1 gene expression by either naringenin in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear AhR.

• Journal of Periodontal and Implant Science
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• 제23권1호
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• pp.48-55
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• 1993

Macrophage inflammatory $protein-1{\alpha}(MIP-1{\alpha})$ from activated T cell or macrophage, which is small inducible cytokine of unkown biological function, has been shown to display inflammation chemokinetic activities, as well as myelosuppressive effect on more immature progenitor cells. In this paper we show the $MIP-1{\alpha}$ mRNA expression and the presence of $MIP-1{\alpha}$ binding sites from murine macrophage cell line RAW 264.7, and primary cells of mouse bone marrow and spleen. $MIP-1{\alpha}$ mRNA was induced from LPS-stimulated RAW 264.7, but not inhibited by cyclosporin A treatment, and also was expressed from mouse splenocyted and bone marrow cell which were not increased by ferritin or lactoferrin treatment. The results of receptor binding assay showed that radiolabeled RAW 264.7 cell with kd value of 0.91 nM, and binding sites per cell of 378. bone marrow cell and splenocyte also appeared to have $MIP-1{\alpha}$ binding sites 33 and 11 per cell, respectiviely.

• 대한의생명과학회지
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• 제16권4호
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• pp.221-227
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• 2010

To elucidate the invasion mechanism of pathogenic Naegleria fowleri, especially a receptor-ligand recognition, we investigated the in vitro cytotoxicity of pathogenic N. fowleri and nonpathogenic N. gruberi to murine macrophages, RAW 264.7, by adding four kinds of saccharides, ${\alpha}$-fucose, ${\beta}$-galactose, ${\alpha}$-D-mannopyranoside (${\alpha}$-mannose) and xylose. There was not enough of a difference in the cytotoxicity of N. fowleri treated with 10 mM of each saccharide. In particular, the cytotoxicity of N. fowleri was highly inhibited by 100 mM ${\alpha}$-mannose, which was 62.3% inhibition calculated by the analysis of lactate dehydrogenase (LDH) release assay. Although murine macrophages were not significantly destroyed by nonpathogenic N. gruberi under hematoxylin staining, the cytotoxicity of N. gruberi was inhibited from 31.5% to 14.5% (P<0.01) by 100 mM ${\alpha}$-mannose treatment. The binding of N. fowleri to macrophages was inhibited from 33% to 50% by 100 mM ${\alpha}$-mannose. Furthermore, as results of the adhesion assays which were performed to determine whether binding of Naegleria is mediated by saccharides-binding protein, the binding ability of N. fowleri as well as N. gruberi was inhibited by 100 mM ${\alpha}$-mannose.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1527-1535
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• 2009

Polyphosphate (polyP) plays diverse physiological functions in prokaryotes and eukaryotes, but most of their detailed mechanisms are still obscure. Here, we show that deletion of polyphosphate kinase (PPK), the principal enzyme responsible for synthesis of polyP, resulted in augmented expression of cAMP receptor protein (CRP) and rpoS and lowered $H_2O_2$ sensitivity in Salmonella Typhimurium ATCC14028. The binding of cAMP-CRP complex to rpoS promoter and further stimulation of its transcription were proved through electrophoretic mobility shift assay, lacZ fusion, and exogenous cAMP addition, respectively. The rpoS expression increased in cpdA (cAMP phosphodiesterase coding gene) mutant, further suggesting that cAMP-CRP upregulated rpoS expression. These results demonstrate that PPK affects oxidative stress response by modulating crp and rpoS expression in S. Typhimurium.