• Asian Pacific Journal of Cancer Prevention
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• 제14권7호
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• pp.4347-4352
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• 2013

Aim: To investigate the molecular mechanisms underlying triggering of apoptosis by hesperetin using in silico and in vitro methods. Methods: The mechanism of binding of hesperetin with NF-${\kappa}B$ and other apoptotic proteins like BAX, BAD, $BCL_2$ and $BCL_{XL}$ was analysed in silico using Schrodinger suite 2009. In vitro studies were also carried out to evaluate the potency of hesperetin in inducing apoptosis using the human prostate cancer PC-3 cell line. Results: Hesperetin was found to exhibit high-affinity binding resulting from greater intermolecular forces between the ligand and its receptor NF-${\kappa}B$ (-7.48 Glide score). In vitro analysis using MTT assay confirmed that hesperetin reduced cell proliferation ($IC_{50}$ values of 90 and $40{\mu}M$ at 24 and 48h respectively) in PC-3 cells. Hesperetin also downregulated expression of the anti-apoptotic gene $BCL_{XL}$ at both mRNA and protein levels and increased the expression of pro-apoptotic genes like BAD at mRNA level and BAX at mRNA as well as protein levels. Conclusion: The results suggest that hesperetin can induce apoptosis by inhibiting NF-${\kappa}B$.

• 약학회지
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• 제54권6호
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• pp.500-506
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• 2010

TNF-${\alpha}$ and VCAM-1 play a pivotal role in the pathogenesis of rheumatoid arthritis, and the development of drugs targeting these molecules has extended the therapeutical approaches to rheumatoid arthritis patients. Bispecific antibodies combine the antigen-binding sites of two antibodies within a single molecule and thus they are able to bind to two different epitopes simultaneously. A specific bispecific antibody format termed "Di-diabody" was made for the efficient approach to anti-inflammation. In this study, the DNA vector construct of Di-diabody was built up against two antigens, VCAM-1 and TNF-${\alpha}$. For evaluating this Di-diabody as a bispecific antibody on the efficacy of anti-inflammation, the proteins were analyzed according to each antigen binding affinity and cell based assay related separate molecules. The 7H/Humira Di-diabody produced in this study interacted with its ligands, VCAM-1 and TNF-${\alpha}$, respectively. Also, this antibody exhibited the similar functional activities as compared to 7H-IgG in respect to inhibition of hVCAM-1-induced cell adhesion and Humira-IgG in respect to inhibition of TNF-${\alpha}$ induced cytotoxicity. Further study to elucidate the pharmacological significance of the Di-diabody is warranted using experimental animals.

• BMB Reports
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• 제34권6호
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• pp.573-577
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• 2001

The mammalian heart is known to undergo significant mechanical changes during fetal and neonatal development. The objective of this study was to define the ontogeny of the ryanodine receptor/$Ca^{2+}$ release channel and SERCA that play the major roles in excitation-contraction coupling. Whole ventricular homogenates of fetal (F) (19 and 22 days in gestation), postnatal (N) (1 and 7 days postnatal), and adult (A) (5 weeks postnatal) Sprague-Dawley rat hearts were used to study [$^3H$]ryanodine binding and oxalate-supported $^{45}Ca^{2+}$ uptake. For the ryanodine receptor, the major findings were: (1) The ryanodine receptor density, as determined by maximal [$^3H$]ryanodine binding ($B_{max}$), increased 3 fold between the F22 and A periods ($0.26{\pm}0.1$ vs. $0.73{\pm}0.07$ pmoles/mg protein, p<0.01), whereas there was no significant change during the F22 and N1 development phases ($0.26{\pm}0.1$ vs. $0.34{\pm}0.01$). (2) Affinity of the ryanodine receptor to ryanodine did not significantly change, as suggested by the lack of change in the $K_d$ during the development and maturation. For SERCA, changes started early with an increased rate of $Ca^{2+}$ uptake in the fetal periods (F19: $8.1{\pm}1.1$ vs. F22: $19.3{\pm}2.2$ nmoles/g protein/min; p<0.05) and peaked by 7 days (N7) of the postnatal age ($34.9{\pm}2.1$). Thus, we conclude that the quantitative changes occur in the ryanodine receptor during myocardial development. Also, the maturation of the $Ca^{2+}$ uptake appears to start earlier than that of the $Ca^{2+}$ release.

• 한국수산과학회지
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• 제48권1호
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• pp.71-75
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• 2015

Nicotinic acetylcholine receptors (nAChRs) are essential for neurotransmission and important therapeutic targets of diseases related to neurotransmission. A recent experimental study identified three residues (Lys185, Asp187, and Ile188) of the ${\alpha}6$ nAChR subunit as determinants of ${\alpha}$-conotoxin BuIA selectivity, yet how these residues confer toxin selectivity remains unclear. In this study, we performed all-atom molecular dynamics simulations with two toxin-bound ${\alpha}4{\beta}2$ nAChR systems: the wild-type ${\alpha}4{\beta}2$ and one in which we replaced the three ${\alpha}4$ subunit residues with three ${\alpha}6$ subunit residues identified in an experimental study (Tyr185Lys, Thr187Asp, and Arg188Ile). After mutation, Asp199 lost the salt bridge formed with Arg188 in the wild type located around loop C. Then, the loop C conformation changed and became more flexible than that of the wild type. We also detected reduced space between the toxin and the binding site in the mutant simulation, resulting in increased binding affinity to the toxin. Therefore, we propose a new Asp199 mutation that breaks the salt bridge and may produce similar selectivity to that of the Arg188 mutation.

• Journal of Microbiology and Biotechnology
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• 제25권1호
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• pp.89-97
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• 2015

Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40℃, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (k_cat/K_m) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45℃, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176^th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca²⁺, whichincreased the thermostability of M179.

• The Korean Journal of Physiology and Pharmacology
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• 제18권2호
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• pp.169-175
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• 2014

Human organic cation/carnitine transporter 1 (OCTN1) plays an important role in the transport of drugs and endogenous substances. It is known that a missense variant of OCTN1 is significantly associated with Crohn's disease susceptibility. This study was performed to identify genetic variants of the OCTN1 promoter in Korean individuals and to determine their functional effects. First, the promoter region of OCTN1 was directly sequenced using genomic DNA samples from 48 healthy Koreans. OCTN1 promoter activity was then measured using a luciferase reporter assay in HCT-116 cells. Seven variants of the OCTN1 promoter were identified, two of which were novel. There were also four major OCTN1 promoter haplotypes. Three haplotypes (H1, H3, and H4) showed decreased transcriptional activity, which was reduced by 22.9%, 23.0%, and 44.6%, respectively (p<0.001), compared with the reference haplotype (H2). Transcription factor binding site analyses and gel shift assays revealed that NF-Y could bind to the region containing g.-1875T>A, a variant present in H3, and that the binding affinity of NF-Y was higher for the g.-1875T allele than for the g.-1875A allele. NF-Y could also repress OCTN1 transcription. These data suggest that three OCTN1 promoter haplotypes could regulate OCTN1 transcription. To our knowledge, this is the first study to identify functional variants of the OCTN1 promoter.

• 생명과학회지
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• 제14권1호
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• pp.26-31
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• 2004

본 연구는 redox regulator로 알려 진 Ref-1 (Redox factor-1)과 결합하는 새로운 단백질을protein-protein interaction의 원리를 이용한 방법인 yeast two-hybrid assay로 검색, 동정하고, 검색된 단백질의 in vitro, in vivo 기능을 규명하는 데 그 목적을 두고, mouse 11-day Embryo cNA library를 prey로, full length REF-1을 bait로 하여 yeast strain 인 HF7C에 cotransformatiom시킨 후 histidine, leucine, tryptophan이 결핍된 SD plate에서 키워 자란 yeast transformants를 $\beta$-galactosidaseassay하여 screening하여 분리한 세 개의 clone중 한 clone이 DNA sequencing으로 확인한 결과 mouse thioredoxin임을 확인하였다.

• Food Science and Biotechnology
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• 제14권6호
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• pp.727-731
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• 2005

Synthesized Ile-Ala-Val-Pro (IAVP) peptide, which has the highest hypocholesterolemic effect among a number of synthesized derivatives of Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA) isolated from 11S globulin of soy protein by pepsin digestion, was selected for investigation in the present study. Using a recombinant Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), we studied in detail the inhibition of this enzyme by IAVP and compared the action of this peptide to that of lovastatin, a known competitive inhibitor of this enzyme. The concentration of IAVP required for 50% inhibition ($IC_{50}$) of HMGR activity in given experimental conditions was $340\;{\mu}M$. Kinetic analysis revealed that the studied peptide is a competitive inhibitor of HMGR with respect to both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) and nicotinamide adenine dinucleotide phosphate (NADPH), with an equilibrium constant of inhibitor binding ($K_i\;=\;[E][I]/[EI]$) of $61{\pm}1.2\;{\mu}M$ and $157{\pm}4.4\;{\mu}M$, respectively. At the same conditions, $K_i$ and $IC_{50}$ for lovastatin were $2.2{\pm}0.1\;nM$ and 12.5 nM, respectively. Thus, the given peptide interacts with HMGR as a bisubstrate, consequently blocking access of both substrates to the active sites. The achieved results suggest the design of new peptide sequences having a higher relative affinity to binding sites of this enzyme and an enhancement of their hypocholesterolemic properties.

• 생약학회지
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• 제47권1호
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• pp.29-37
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• 2016

Anti-diabetic potential of the leaves of A. elata through the inhibitory activity on PTP1B and ${\alpha}$-glucosidase has not been reported. In this study, the EtOAc fraction of methanolic extract from the leaves of A. elata showed potent inhibitory activity against the PTP1B and ${\alpha}$-glucosidase with $IC_{50}$ value of $96.29{\pm}0.3$ and $264.71{\pm}14.87{\mu}g/mL$, respectively. Three known triterpenoids, oleanolic acid, oleanolic acid-28-O-${\beta}$-D-glucopyranoside and oleanolic acid-3-O-${\beta}$-D-glucopyranoside were isolated from the most active EtOAc fraction. We determined the chemical structure of these triterpenoids through comparisons of published nuclear magnetic resonance (NMR) spectroscopic data. Furthermore, we screened these triterpenoids for their ability to inhibit PTP1B and ${\alpha}$-glucosidase over a range of concentrations ($12.5-50{\mu}M$). All three terpenoids significantly inhibited PTP1B in a concentration dependent manner and oleanolic acid effectively inhibited ${\alpha}$-glucosidase. In addition, these compounds revealed potent inhibitory activity with negative binding energies toward PTP1B, showing high affinity and tight binding capacity in the molecular docking studies. Therefore, the results of the present study clearly demonstrate that A. elata leaves and its triterpenoid constituents might be beneficial in the prevention or treatment of diabetic disease.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.86-86
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently by the use of specific anti-receptor antibodies. A 14-mer peptide (from Phe102 to Leu115 of ${\beta}$2-adrenergic receptor) was synthesized and this peptide was coupled to carrier protein Keyhole Limpet Hemocyanin(KLH) by glutaraldehyde method. A 0.5mg of KLH-coupled peptide was emulsified with equal volume of complete Freund's adjuvant and injected via popliteal lymph node to each of the three Newzealnd White rabbits. Booster injections were repeated at 4 weeks interval for three times with incomplete Freund's adjuvants. One week after the final injection, serum was prepared from ear artery. Nonspecific immunoglobulins were removed by passing the serum through KLH-Sepharose 6B affinity matrix and further by incubation with bovine lung aceton powder. The titer of the antibody for synthetic peptide which was determined by enzyme linked immunosorbent assay(ELISA) was about l/l,000. The antibody produced in this study revealed 67kDa protein band in the western blot of partially purified guinea pig lung ${\beta}$-adrenergic receptor preparation. The antibody inhibited ${\beta}$-adrenergic antaginist [3H] Dihydroalprenolol binding to soluble ${\beta}$-adrenergic receptor by 25% while control sera did not show any inhibitory effects, The result of this study suggests that the peptide sequence selected in this study may play some important roles in adrenergic receptor-ligand interaction.