• Journal of Periodontal and Implant Science
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• 제41권2호
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• pp.73-78
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• 2011

Purpose: For periodontal tissue engineering, it is a primary requisite and a challenge to select the optimum types of cells, properties of scaffold, and growth factor combination to reconstruct a specific tissue in its natural form and with the appropriate function. Owing to fundamental disadvantages associated with using a two-dimensional substrate, several methods of seeding cells into three-dimensional scaffolds have been reported and the authors have asserted its usefulness and effectiveness. In this study, we explore the cell attachment of periodontal ligament fibroblasts on nanohydroxyapatite (n-HA) scaffold using avidin biotin binding system (ABBS). Methods: Human periodontal ligament fibroblasts were isolated from the health tooth extracted for the purpose of orthodontic procedure. HA nanoparticles were prepared and $Ca(NO_3)_2-_4H_2O$ and $(OC_2H_5)_3P$ were selected as precursors of HA sol. The final scaffold was 8 mm in diameter and 3 mm in height disk with porosity value of 81.55%. $1{\times}10^5$ periodontal ligament fibroblasts were applied to each scaffold. The cells were seeded into scaffolds by static, agitating and ABBS seeding method. Results: The number of periodontal ligament fibroblasts attached was greater for ABBS seeding method than for static or agitating method (P<0.05). No meaningful difference has been observed among seeding methods with scanning electron microscopy images. However, increased strength of cell attachment of ABBS could be deduced from the high affinity between avidin and biotin ($Kd=10^{-15}\;M$). Conclusions: The high-affinity ABBS enhances the ability of periodontal ligament fibroblasts to attach to three-dimensionally constructed n-HA scaffold.

• Journal of Applied Biological Chemistry
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• 제53권4호
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• pp.195-201
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• 2010

B. pseudomycoides로 부터 alanine racemase 유전자를 분리한 다음 이 효소에 존재하는 두개의 cysteine을 하나(C316A) 또는 두개 모두(C316-365A) alanine으로 치환시켰다. 치환된 alanine racemamase는 pET-21 운반체에 삽입한 다음 숙주세포로서 E. coli BL21 (DE3)를 이용하여 발현시켰다. 발현된 단백질은 6XHis이 결합된 affinity chromatography를 이용하여 분리하였으며 SDS-PAGE 분석에서 모두 약 46 kDa의 주요 단일밴드를 나타내었다. Cysteine(-) 변이체의 alanine racemase가 모두 활성도를 보여 cysteine이 catalytic 또는 binding sit에 관여하지 않는 것으로 추정되었다. 변이체 효소들은 wild type에 비하여 열 안정성이 모두 떨어져 $60^{\circ}C$ pH 8.0에서의 활성도 반감시간이 각각 26(wild type), 21(C316A) 18분(C316-365A)-을 나타내었다. 이러한 결과는 cysteine이 열안정화에 상당히 기여함을 알 수 있었다. 그러나 pH 변화에 대한 안정성은 큰 차이가 없었다.

• Bulletin of the Korean Chemical Society
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• 제31권9호
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• pp.2497-2502
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• 2010

Arg13 is a conserved active-site residue in all known Pi class glutathione S-transferases (GSTs) and in most Alpha class GSTs. To evaluate its contribution to substrate binding and catalysis of this residue, three mutants (R13A, R13K, and R13L) were expressed in Escherichia coli and purified by GSH affinity chromatography. The substitutions of Arg13 significantly affected GSH-conjugation activity, while scarcely affecting glutathione peroxidase or steroid isomerase activities. Mutation of Arg13 into Ala largely reduced the GSH-conjugation activity by approximately 85 - 95%, whereas substitutions by Lys and Leu barely affected activity. These results suggest that, in the GSH-conjugation activity of hGST P1-1, the contribution of Arg13 toward catalytic activity is highly dependent on substrate specificities and the size of the side chain at position 13. From the kinetic parameters, introduction of larger side chains at position 13 results in stronger affinity (Leu > Lys, Arg > Ala) towards GSH. The substitutions of Arg13 with alanine and leucine significantly affected $k_{cat}$, whereas substitution with Lys was similar to that of the wild type, indicating the significance of a positively charged residue at position 13. From the plots of log ($k_{cat}/{K_m}^{CDNB}$) against pH, the $pK_a$ values of the thiol group of GSH bound in R13A, R13K, and R13L were estimated to be 1.8, 1.4, and 1.8 pK units higher than the $pK_a$ value of the wild-type enzyme, demonstrating the contribution of the Arg13 guanidinium group to the electrostatic field in the active site. From these results, we suggest that contribution of Arg13 in substrate binding is highly dependent on the nature of the electrophilic substrates, while in the catalytic mechanism, it stabilizes the GSH thiolate through hydrogen bonding.

• Journal of Dairy Science and Biotechnology
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• 제15권1호
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• pp.1-9
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• 1997

본 실험에서는 한우초유를 33% ammonium sulfate 포화용액으로 처리하여 조면역글로블린을 얻은 후 gel filtration, ion-exchange chromatography를 이용하여 조면역글로블린의 분리 정도를 조사하고 affinity chromatography column을 이용하여 조면역글로블린으로 부터 Ig G의 결합정도를 알아보고 Protein G Sepharose fast flow system을 이용하여 신속하게 대량으로 Ig G의 분리를 꾀하여 얻어진 Ig G를 이용하여 ELISA방법으로 항체 생성유무를 측정하였으며, 그 결과는 다음과 같다. 1. HPLC상에서 Superose 12 column을 이 용하여 한우초유의 조면역글로블린을 분리한 결과 Holstein초유의 조면역글로블린과 유사한 분리정도를 나타냈지만 약 84%의 Ig G를 한우초유의 조면역글로블린으로 부터 분리할 수 있었다. 2. Mono Q를 이용하여 HPLC에서 한우초유의 조면역글로블린을 gel filtration방법보다 짧은 시간에 분리할 수 있었지만 그다지 분리 정도가 좋지 않은 것으로 나타났다. 3. Hi-trap protein G column이 protein A sepharose CL-4B column보다 더 많은 양의 Ig G를 한우초유의 조면역글로블린으로 부터 얻을 수가 있었다. 4. Protein G Sepharose fast flow system을 이용하여 20mg의 sample양을 주입하여도 충분히 Ig G를 분리할 수 있었으며, ml 당 약 1.25mg의 Ig G를 얻을 수가 있었다. 5. ELISA방법을 이용하여 한우초유의 Ig G에 대한 항체 생성 유무를 측정한 결과, 면역화된 토끼에서 정상 토끼의 혈청에서보다 titer가 높게 나타났으므로 항체생성이 확인되었다.

• 한국미생물·생명공학회지
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• 제39권2호
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• pp.140-145
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• 2011

돼지유행성설사 바이러스(porcine epidemic diarrhea virus: PEDV)는 자돈에게 감염 시 수양성설사를 동반한 급성 장염을 유발하며 매우 높은 폐사율을 보이는 그룹 1 코로나바이러스이다. PEDV는 다른 그룹 1 코로나바이러스와 마찬가지로 숙주 세포에 감염 시 aminopeptidase N (APN)을 세포 수용체로 이용한다고 알려져 있다. 코로나바이러스의 spike(S) 단백질은 숙주세포의 표면에 부착과 관련하여 감염 개시에 있어 중요한 역할을 하는 것으로 알려져 있으며 특히 S 단백질의 S1 도메인은 세포 수용체에 특이적인 결합을 매개하는 수용체 결합 도메인(receptor binding domain: RBD)을 포함하고 있는 것으로 알려져 있다. 이미 많은 코로나바이러스의 RBD의 위치가 확인되어져 있지만 PEDV의 RBD에 대해서는 아직까지 알려진 바가 없다. 본 연구에서는 돼지 APN 수용체와 결합을 매개하는 PEDV의 RBD를 규명하기 위해 S1 도메인을 주형으로 하는 일련의 재조합 truncated variant들을 제작하였고 각각의 truncated들이 실제로 pAPN과의 결합을 이루는지에 대하여 실험을 통해 확인하였다. 그 결과 S1 도메인의 N 말단 부분이 pAPN과의 결합에서 중요한 부위임을 확인할 수 있었다. 본 연구에서 도출된 결과는 향후 PEDV의 S 단백질과 pAPN간의 분자적 상호작용을 이해하는 데에 도움을 줄 것으로 판단된다.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1247-1255
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• 2016

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, $40{\mu}g/mLs$ semen were standardized to find out the optimum dose and $20{\mu}g/mLs$ was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with $20{\mu}g/mL$ SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/$32.7{\mu}M$/$10^9$ spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species.

• Bulletin of the Korean Chemical Society
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• 제7권2호
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• pp.137-142
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• 1986

The luminescence spectra of $Ru(bpy)_3^{2+}$ in poly(methacrylic acid) (PMA) solutions varied sensitively with pH. At pH < 5.5, the emission intensity increased with pH up to 4 times, while it decreased with pH beyond the pH. The enhanced emission intensity was accompanied by blue-shift of the emission maxima as much as 15 nm. The enhancement of emission intensity was attributed to the restricted rotational mobility of ligand of the cation bound to densely coiled PMA molecules at pH < 5.5. The sharp decrease in emission intensity with increasing pH near pH 5.6 was accounted for conformational transition of the polymer to more extended structure, which was also revealed in viscosity measurement. The enhancement of emission intensity became higher as NaCl concentration of the solution increased. The binding constant of $Ru(bpy)_3^{2+}$ with two carboxylate groups of PMA was calculated as $2{\times}10^5\;M^{-1}$ in 0.1 M NaCl at pH 5.2. The pH dependence of luminescence quenching rate of $Ru(bpy)_3^{2+}$ by $Cu^{++}$ also showed maximum near pH 5, and the rate was more than $10^3$ times higher than that in water, whereas the maximum enhancement of quenching rate (about 20 times) in poly(acrylic acid) (PAA) solution occurred at pH 4.5. On the other hand, the pH dependence for neutral water soluble nitrobenzene (NB) exhibited opposite trend to that of $Cu^{++}$. The quenching constant vs pH curve for $MV^{++}$ was composite of those for $Cu^{++}$ and NB. The anomalous high quenching rate for $Cu^{++}$ in PMA solution at pH < 5.5 was attributed to the binding of $Ru(bpy)_3^{2+}$ and $Cu^{++}$ to the same region of PMA, when it conforms densely coiled structure in the pH range. The observation of mininium quenching rate for NB near pH 5.5 indicated that the $Ru(bpy)_3^{2+}$ bound to the densely coiled PMA is not accessible by NB, which is in bulk water phase. The composite nature of the pH dependence of quenching rate for $MV^{++}$ in PMA solution was attributed to the smaller binding affinity of the cation to PMA, compared to that of $Cu^{++}$. The sharp, cooperative conformational transition with pH observed in PMA was not revealed in PAA. But, the pH dependence of quenching rates in this polymer reflected increased charge density and, thus, binding of cations to the polymer, and expansion of the polymer chain with pH.

• Journal of Ginseng Research
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• 제48권2호
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• pp.202-210
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• 2024

Background: Panax ginseng Meyer polysaccharides exhibit various biological functions, like antagonizing galectin-3-mediated cell adhesion and migration. Galectin-8 (Gal-8), with its linker-joined N- and C-terminal carbohydrate recognition domains (CRDs), is also crucial to these biological processes, and thus plays a role in various pathological disorders. Yet the effect of ginseng-derived polysaccharides in modulating Gal-8 function has remained unclear. Methods: P. ginseng-derived pectin was chromatographically isolated and enzymatically digested to obtain a series of polysaccharides. Biolayer Interferometry (BLI) quantified their binding affinity to Gal-8, and their inhibitory effects on Gal-8 was assessed by hemagglutination, cell migration and T-cell apoptosis. Results: Our ginseng-derived pectin polysaccharides consist mostly of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG). BLI shows that Gal-8 binding rests primarily in RG-I and its β-1,4-galactan side chains, with sub-micromolar K_D values. Both N- and C-terminal Gal-8 CRDs bind RG-I, with binding correlated with Gal-8-mediated function. Conclusion: P. ginseng RG-I pectin β-1,4-galactan side chains are crucial to binding Gal-8 and antagonizing its function. This study enhances our understanding of galectin-sugar interactions, information that may be used in the development of pharmaceutical agents targeting Gal-8.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제36권5호
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• pp.192-200
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• 2014

Purpose: The objective of this study was to evaluate the interaction between 4-hexylresorcinol (4HR) and antibody as that affects the performance of a silk-4HR combination graft for soft tissue augmentation in an animal model. Methods: The silk graft materials consisted of four types: silk+10% tricalcium phosphate (TCP) (ST0), silk+10% TCP+1% 4HR (ST1), silk+10% TCP+3% 4HR (ST3), and silk+10% TCP+6% 4-HR (ST6). The antibody binding assay tested the 4HR effect and scanning electron microscopic (SEM) exam was done for silk grafts. The animal experiment used a subcutaneous pocket mouse model. The graft - SH0 or SH1 or SH3 or SH6 - was placed in a subcutaneous pocket. The animals were killed at one, two, and four weeks, postoperatively. The specimens were subjected to histological analysis and lysozyme assay. Results: Groups with 4HR applied showed lower antibody binding affinity to antigen compared to groups without 4HR. In the SEM examination, there was no significant difference among groups. Histological examinations revealed many foreign body giant cells in ST0 and ST1 group at four weeks postoperatively. Both ST3 and ST6 groups developed significantly lower levels of giant cell values compared to ST0 and ST1 groups (P < 0.001) at four weeks postoperatively. In the lysozyme assay, the ST1 and ST3 groups showed denser signals than the other groups. Conclusion: 4HR combined silk implants resulted in high levels of vascular and connective tissue regeneration.

• 대한의생명과학회지
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• 제23권1호
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• pp.8-16
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• 2017

p-Coumaric acid is an organic compound that is a hydroxyl derivative of cinnamic acid. Due to its multiple biological activities p-coumaric acid has been widely studied in biochemical and cellular systems and is also considered as a useful therapeutic candidate for various neuronal diseases. However, the efficacy of p-coumaric acid on zebrafish developmental regulation has not been fully explored. In this study, therefore, we first investigated the action mechanism of the p-coumaric acid on the zebrafish development in a whole-organism model. p-Coumaric acid treated group significantly inhibited the pigmentation of the developing zebrafish embryos compared with control embryos without any severe side effects. In addition, p-coumaric acid down-regulated more effectively in a lower concentration than the well-known zebrafish's melanogenic inhibitor, phenylthiourea. We also compared the molecular docking property of p-coumaric acid with phenylthiourea on the tyrosinase's kojic acid binding site, which is the key enzyme of zebrafish embryo pigmentation. Interestingly, p-coumaric acid interacted with higher numbers of the amino acid residues and exhibited a tight binding affinity to the enzyme than phenylthiourea. Taken all together, these results strongly suggest that p-coumaric acid inhibits the activity of tyrosinase, consequently down-regulating zebrafish embryo pigmentation, and might play an important role in the reduction of dermal pigmentation. Thus, p-coumaric acid can be an effective and non-toxic ingredient for anti-melanogenesis functional materials.