• Preventive Nutrition and Food Science
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• v.15 no.4
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• pp.282-286
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• 2010

To isolate a calcium-binding peptide from chlorella protein hydrolysates, chlorella protein was extracted and hydrolyzed using Flavourzyme, a commercial protease. The degree of hydrolysis and calcium-binding capacity were determined using trinitrobenzenesulfonic acid and orthophenanthroline methods, respectively. The enzymatic hydrolysis of chlorella protein for 6 hr was sufficient for the preparation of chlorella protein hydrolysates. The hydrolysates of chlorella protein were then ultra-filtered under 5 kDa as molecular weight. The membrane-filtered solution was fractionated using ion exchange, reverse phase, normal phase chromatography, and fast protein liquid chromatography to identify a calcium-binding peptide. The purified calcium-binding peptide had a calcium binding activity of 0.166 mM and was determined to be 700.48 Da as molecular weight, and partially identified as a peptide containing Asn-Ser-Gly-Cys based on liquid chromatography/electrospray ionization tandem mass spectrum.

• BMB Reports
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• v.38 no.4
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• pp.474-480
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• 2005

Curcumin, a major active component of turmeric, has been identified as an inhibitor of the transcriptional activity of activator protein-1 (AP-1). Recently, it was also found that curcumin and synthetic curcumin derivatives can inhibit the binding of Jun-Fos, which are the members of the AP-1 family, to DNA. However, the mechanism of this inhibition by curcumin and its derivatives was not disclosed. Since the binding of Jun-Fos dimer to DNA can be modulated by redox control involving conserved cysteine residues, we studied whether curcumin and its derivatives inhibit Jun-Fos DNA binding activity via these residues. However, the inhibitory mechanism of curcumin and its derivatives, unlike that of other Jun-Fos inhibitors, was found to be independent of these conserved cysteine residues. In addition, we investigated whether curcumin derivatives can inhibit AP-1 transcriptional activity in vivo using a luciferase assay. We found that, among the curcumin derivatives examined, only inhibitors shown to inhibit the binding of Jun-Fos to DNA by Electrophoretic Mobility Shift Assay (EMSA) inhibited AP-1 transcriptional activity in vivo. Moreover, RT-PCR revealed that curcumin derivatives, like curcumin, downregulated c-jun mRNA in JB6 cells. These results suggest that the suppression of the formation of DNA-Jun-Fos complex is the main cause of reduced AP-1 transcriptional activity by curcuminoids, and that EMSA is a suitable tool for identifying inhibitors of transcriptional activation.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.351-358
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• 2007

Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that ${\alpha}$-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm ${\alpha}$-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm ${\alpha}$-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained L-fucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm ${\alpha}$-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of ${\alpha}$-L-fucose on sperm binding and penetrating into ZP caused sperm ${\alpha}$-L-fucosidase inhibited by ${\alpha}$-L-fucose.

• Animal cells and systems
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• v.16 no.2
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• pp.87-94
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• 2012

Nur77 is a member of the nuclear receptor 4A (NR4A) subgroup, which has been implicated in energy metabolism. Although Nur77 is found in adipose tissue, where TR4 plays a key role in lipid homeostasis, the role of Nur77 in adipogenesis is still controversial. Although the Nur77 responsive element (AAAGGTCA) is partially overlapped with TR4-binding sites (AGGTCA $n$ AGGTCA: $n$=0-6), the regulatory role of Nur77 in TR4 function associated with adipocyte biology remains unclear. Here, we found that Nur77 inhibits adipogenesis and TR4 transcriptional activity. Treatment with a Nur77 agonist, 1,1-bis(3'-indolyl)-1-($p$-anisyl)-methane, during 3T3-L1 adipocyte differentiation reduced adipogenesis. In reporter gene analysis, Nur77 specifically suppressed TR4 transcription activity but had little effect on $PPAR{\gamma}$ transcription activity. Consistently, Nur77 also suppressed TR4-induced promoter activity of the TR4 target gene PEPCK, which is known to be important for glyceroneogenesis in adipose tissue. Furthermore, Nur77 suppressed TR4 binding to TR4 response elements without direct interaction with TR4, suggesting that Nur77 may inhibit TR4 transcription activity via binding competition for TR4-binding sites. Furthermore, DIM-C-$pPhOCH_3$ substantially suppressed TR4-induced PEPCK expression in 3T3-L1 adipocytes. Together, our data demonstrate that Nur77 plays an inhibitory role in TR4-induced PEPCK expression in 3T3-L1 adipocytes.

• Bulletin of the Korean Chemical Society
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• v.25 no.4
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• pp.539-544
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• 2004

The ability of protoberberine alkaloids, berberine and berberrubine, to act as topoisomerase II poisons is linked to the anti-cancer activity. Minor alterations in structure have a significant effect on their relative activity. Berberine, which has methoxy group at the 19-position, is significantly less potent than berberrubine. Several observations support non-specific binding to HP14 by the berberine: (i) nonspecific upfield changes in $^1H$ chemical shift for protons of the berberine; (ii) the broadening of imino protons of HP14 upon binding of the berberine; (iii) very small increases in duplex melting temperature in the presence of the berberine. Our results reveal that substitution of a hydroxyl group to a methoxy group on the 19-position, thereby converting the berberrubine to the berberine is associated with a non-specific DNA binding affinity and a reduced topoisomerase II poisoning. The presence of a bulky 19-methoxy substituent decreases intercalating properties of berberine and makes it inactive as topoisomerase II poison.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.65-70
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• 2002

A TGF-${\beta}1$/GFP monomeric fusion protein was cloned from pPK9A and pGFP-Cl plasmid by PCR amplification. The fusion protein was expressed in a $Bac-To-Bac^{TM}$ baculovirus expression system. A 45 kDa fusion protein was purified using an Ni-NTA column with 300 mM imidazol from a cell lysate infected with recombinant viruses for 72 h post-infection. The fusion protein cross-reacted with the commercial $TGF-{\beta}1$ polyclonal Ab as well as Ab raised against a precursor, monomeric $TGF-{\beta}1$, and GFP. The binding activity of the fusion protein with a $TGF-{\beta}1$ receptor was examined. Fluorescence was observed in Mv1Lu cells, yet not in insect cells treated with the fusion protein. No fluorescence was detected in Mv1Lu cells incubated with the fusion protein treated with Ab prior to the binding reaction, or with GFP alone, thereby indicating that the binding of the fusion protein was specific to $TGF-{\beta}1$ with a receptor.

• Bulletin of the Korean Chemical Society
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• v.32 no.2
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• pp.455-458
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• 2011

In previous report, with the aid of receptor-oriented pharmacophore-based in silico screening, we characterized three novel hPNMT inhibitors (YPN010, YPN016, and YPN017) and proposed that the hydrogen bonding interaction between inhibitors and side chain of Lys57 is very important to inhibitory activity of hPNMT. To confirm the importance of Lys57, mutant with substitution of Lys57 with Ala was cloned and binding study was performed for a K57A mutant of hPNMT using STD-NMR and fluorescence experiments. The binding constants for three novel inhibitors with mutant hPNMT were dramatically decreased compared to those with wild-type protein. K57A mutant-induced conversion of noradrenaline to adrenaline was suppressed about 95 % compared to wild-type hPNMT. Mutagenic analysis using a K57A mutant confirmed the importance of the Lys57 residue in binding of the inhibitor candidate to hPNMT as well as enzymatic activity of hPNMT, implying that these results are consistent with our binding model.

• Archives of Pharmacal Research
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• v.19 no.6
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• pp.462-468
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• 1996

The present study was undertaken to investigate the effects of age on angiotensin II (AII) response and antagonistic activity of losartan using aortic rings and liver homogenates from rats ranging in age from 0.7 to 20 months. Whether the endothelium was present or not, the maximum contractile response to AII decreased with age. Removal of the endothelium enhanced AII-induced maximum contraction and these endothelial effects seemed to be due to endothelium-derived relaxing factor (EDRF) in all ages. Equilibrium binding studies demonstrated an age-related decrease in maximum binding $(B_{max})$ with little change in binding affinity $(K_d)$. In rat aorta, the extent of losartan-induced parallel shifts $(K_B)$ in AII concentration-response curves was not significantly different between ages. In addition, $IC_{50}$ value of losartan in competition binding was not changed with age in rat liver homogenates. These results suggest that the potency of losartan is not altered with age in rat aorta and liver, although AII-induced contractile response and the maximum AII binding decreased significantly with age.

• Journal of Korean Society of Water and Wastewater
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• v.20 no.6
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• pp.893-904
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• 2006

To improve sensitivity of biosensor as yeast two-hybrid detection system for estrogenic activity of suspected chemicals, we tested effects of several combinations of the bait and fish components in the two-hybrid system on Saccharomyces cerevisiae inducted a chromosome-integrated lacZ reporter gene that was under the control of CYC1 promoter and the upstream Gal4p-binding element $UAS_{GAL}$. The bait components that were fused with the Gal4p DNA binding domain are full-length human estrogen receptor ${\alpha}$ and its ligand-binding domain. The fish components that were fused with the Gal4p transcriptional activation domain were nuclear receptor-binding domains of co-activators SRC1 and TIF2. We found that the combination of the full-length human estrogen receptor ${\alpha}$ with the nuclear receptor-binding domain of co-activator SRC1 was most effective for the estrogen-dependent induction of reporter activity among the two-hybrid systems so far reported. The relative strength of transcriptional activation by representative natural and xenobiotic chemicals was well correlated with their estrogenic potency that had been reported with other assay systems.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.3
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• pp.449-456
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• 2018

Objective: Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine. Methods: To characterize the porcine Nanog promoter, the 5'-flanking region of Nanog was isolated from cells of mini-pig ears. BLAST database search showed that there are two porcine Nanog genomic loci, chromosome 1 and 5, both of which contain an exon with a start codon. Deletion mutants from the 5'-flanking region of both loci were measured using the Dual-Luciferase Reporter Assay System, and a fluorescence marker, green fluorescence protein. Results: Promoter activity was detected in the sequences of chromosome 5, but not in those of chromosome 1. We identified the sequences from -99 to +194 that possessed promoter activity and contained transcription factor binding sites from deletion fragment analysis. Among the transcription factor binding sites, a Sp1 was found to play a crucial role in basal promoter activity, and point mutation of this site abolished its activity, confirming its role in promoter activity. Furthermore, gel shift analysis and chromatin immunoprecipitation analysis confirmed that Sp1 transcription factor binds to the Sp1 binding site in the porcine Nanog promoter. Taken together, these results show that Sp1 transcription factor is an essential element for porcine Nanog basal activity the same as in human and mouse. Conclusion: We showed that the porcine Nanog gene is located on porcine chromosome 5 and its basal transcriptional activity is controlled by Sp1 transcription factor.