• Reproductive and Developmental Biology
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• v.39 no.4
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• pp.127-132
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• 2015

To overcome the hyperacute immune rejection during pig-to-non-human primates xenotranasplantation, we have produced and bred ${\alpha}$-1,3-galactosyltransferase knock-out ($GalT^{-/-}$) pigs. In this study, the somatic cells and tissues from the $GalT^{-/-}$ pigs were characterized by an analysis of the expression of Gal${\alpha}$-1,3-Gal (${\alpha}-Gal$) epitope. Briefly, ear fibroblast cell lines of 19 homozygous $GalT^{-/-}$ pigs were established and cryopreserved. The expression of ${\alpha}-Gal$ epitope in the cells was measured by fluorescence activated cell sorter (FACS) analysis using BS-I-B4 lectin. Also, the homozygous ($GalT^{-/-}$) cells and tissues samples were immunostained with BS-I-B4 lectin for analysis of ${\alpha}-Gal$ epitope expression. The results showed that the expression of ${\alpha}-Gal$ epitope in $GalT^{-/-}$ cells (0.2 %) were significantly (p<0.05) down-regulated to the range of cynomolgus monkey fibroblast (0.2 %) cells compared to heterozygous ($GalT^{-/+}$) (9.3 %) and wild type ($GalT^{+/+}$) (93.7 %) fibroblast cells. In the immunostaining results, while the expression of ${\alpha}-Gal$ epitope was detected a partly in $GalT^{-/+}$ cells and mostly in $GalT^{+/+}$ cells, it was almost not detected in the $GalT^{-/-}$ cells. Also, immunostaining results from various tissues of the $GalT^{-/-}$ pig showed that the expression of ${\alpha}-Gal$ epitope was not detectable, whereas various tissues from $GalT^{+/+}$ pig showed a strong expression of ${\alpha}-Gal$ epitope. Our results demonstrated that ${\alpha}-Gal$ epitope expressions from $GalT^{-/-}$ pigs were successfully knocked out to prevent hyperacute immune rejection for further study of xenotransplantation.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.43 no.4
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• pp.373-379
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• 2017

We carried out the enzymatic synthesis of 1, 2-hexanediol galactoside (HD-gal) by transgalactosylation reaction using recombinant Escherichia coli ${\beta}-galactosidase$ (${\beta}-gal$). The amounts of ${\beta}-gal$ and 1, 2-hexanediol (HD), pH, and temperature, respectively, were first optimized (${\beta}-Gal$, 4.8 U/mL; HD, 75 mM; pH, 7.0; temperature, $37^{\circ}C$). Under these optimal conditions, about 96% HD was converted to HD-gal. When we investigated the water holding capacities (WHCs) of HD and HD-gal using pig epidermis in the concentrations of 84.4, 126.6, 168.8, 211.0 mM, WHC of HD-gal was superior to HD. In particular, at 168.8 mM HD and HD-gal, WHC of HD-gal showed about 20% greater than that of HD. However, it was observed that MIC values against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus of HD-gal were about three to ten times greater than those of HD, although MIC value of HD-gal against Enterococcus faecalis was almost the same as that of HD. Finally, it was concluded that the covalent bonding of a galactose molecule to HD (transgalactosylation) resulted in an increase in WHC of HD-gal and a decrease in anti-bacterial activity.

• Journal of the Korean Applied Science and Technology
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• v.38 no.3
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• pp.629-637
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• 2021

To develop a safer cosmetic preservative, we carried out a comparative study on characteristics of OD and OD-gal, where OD-gal was synthesized from OD using E. coli β-gal. OD-gal synthesis was confirmed by mass spectrometry analysis as sodium adduct ion (m/z=331.1731) and protonated ion (m/z=309.1926) of OD-gal. To compare the antimicrobial activities of OD and newly synthesized OD-gal, MIC values were investigated using E. coli, S. aureus, C. albicans, and A. niger. As a result, it was observed that there was no remarkable difference between MIC values of OD and OD-gal. In addition, to compare the cytotoxicity of OD-gal and OD, HaCaT cells were treated with OD or OD-gal, and then cell viability was quantified using EZ-Cytox assay. In the case of 1.5% OD, the cell viability was 64% at 24 h and 42% at 48 h compared to the control, and cell viability of 1.5% OD-gal-treated cells showed no significant change at 24 h and was 85% at 48 h. Consequently, the cytotoxicity of OD-gal-treated cells was reduced by more than 40% when compared with that of OD-treated cells. Thus, the newly synthesized OD-gal in this study is safer than the existing OD used as a cosmetic additive. In the future, OD-gal will be applicable as a substitute for OD as a less toxic preservative for the cosmetic industry.

• 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.

• Journal of the Korean Applied Science and Technology
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• v.36 no.2
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• pp.572-580
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• 2019

Recently, it has been reported that benzyl alcohol (BzOH) as an additive in cosmetics, food, and medicine lead to toxicity and allergy problem. Then, to circumvent this hurdle, we carried out the synthesis of benzyl alcohol galactoside (BzO-gal). Previously, it was confirmed that BzO-gal was synthesized by transgalactosylation reaction using Escherichia coli (E. coli) ${\beta}$-galactosidase (${\beta}-gal$). Meanwhile, in this study, two peaks of BzO-gal as sodium adduct ion (m/z=293.1004) and protonated ion (m/z=271.1180) were detected in the reaction mixture by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). In addition, the amount of ${\beta}-gal$ and BzOH concentration, temperature, pH, and lactose concentration, respectively, were optimized (${\beta}-gal$, 0.75 U/mL; BzOH, 185 mM; temperature, $40^{\circ}C$, pH, 7.5; lactose, 350 g/l). Under these optimal conditions, 185 mM BzOH was converted into about 131 mM BzO-gal, in which the conversion yield was about 72%. In the future, BzO-gal will be applicable as a substitute for BzOH as a less toxic preservative for the cosmetic, pharmaceutical, and food industries, and we are planning to investigate the characteristics of BzO-gal as a preservative.

• Journal of the Korean Applied Science and Technology
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• v.38 no.1
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• pp.99-106
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• 2021

To circumvent the skin problem from phenylethanol (PhE), we have studied on the enzymatic synthesis of phenylethanol galactoside (PhE-gal) as an alternative to PhE. Base on the previous study, we optimized the reaction conditions for PhE-gal synthesis from PhE using E. coli β-galactosidase (β-gal). The optimal amount of β-gal, PhE concentration, pH, and temperature for PhE-gal synthesis were 0.45 U/ml, 1%, 8.0, 40℃, respectively. Under these conditions, about 81.9 mM PhE was converted into about 47.4 mM PhE-gal, in which the conversion yield was about 57.9%. Meanwhile, when the reaction mixture containing PhE and PhE-gal was mixed and fractionated with water-immiscible solvent (EA or MC), it was observed that PhE-gal was distributed in water phase, and PhE was distributed in solvent phase. Additionally, PhE-gal was clearly distributed into water phase when MC was used, but PE-gal was not when EA was used. In the future, we are planning to carried out the continuing study on developing an alternative cosmetic preservative using PhE-gal.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.393-397
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• 1999

The gene (galE) encoding UDP-galactose-4-epimerase, operative in the galactose metabolic pathway, was cloned together with the $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis ATCC7962 (L. lactis 7962). galE was found to have a length of 981 bps and encoded a protein with a molecular mass of 36,209 Da. The deduced amino acid sequence showed a homology with GalE proteins from several other microorganisms. A Northern analysis demonstrated that galE was constitutively expressed by its own promoter. When galactose or lactose was added into medium, the galE transcription was induced by several upstream promoters. The structure of the gal/lac operon of L. lactis 7962 was partially characterized and the gene order around galE was galT-lacA-lacZ-galE-orfX.

• The Korean Journal of Zoology
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• v.39 no.4
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• pp.393-399
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• 1996

A zero-Iength croessinking procedure for studying protein-protein interactlons in preinitiation complex has heen developed. Preinltiadon complexes were assembled with immobilized DNA templates coupled to metal beads. Pudfied complexes were dIrectly crosslinked by 1-ethyl-3.(3-dimethylaminopropyl)carbodlimide (EDC). The reaction was stopped by addition of $\beta$-mercaptoethanol, and the complexes were isolated from EDC immedIately. An appllcatlion of this method with a preinltlation complex assembled with TBP, TFIIB, and GAL4-AH demonstrated that ThP dIrectly interacts with GAL4-AH and TFIIB in the prelnitlatlon complex. However, croeslinked produd between GAL4-AH and ThilB was not observed. These resutts lndlcate that GAL4-AH does not stably Interact with TFIIB In the GAL4-AH-TFIIB-TBP-DNA prelnitlatlon complex.

• Journal of Life Science
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• v.25 no.10
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• pp.1164-1168
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• 2015

Previous research showed that chlorphenesin galactoside (CPN-Gal), a preservative in cosmetics, was safer than CPN against human skin cells [9]. To establish a stable and long-term process for CPN-Gal production, we investigated the repeated-batch process. In this process, β-gal-producing recombinant Escherichia coli cells were immobilized in calcium alginate beads, and CPN was converted to CPN-Gal by the transgalactosylation reaction. The process was conducted in a 300 ml flask, which contained E. coli cell-immobilized alginate beads, 33.8 mM of CPN, and 400 g/l of lactose. The pH and temperature were 7.0 and 40℃, respectively. During the repeated-batch operation, four consecutive batch operations were conducted successfully until 192 hr. The conversion yield of CPN to CPN-Gal was 64% during 192 hr, which was higher than the values in previous reports [3, 13]. Thereafter, however, the conversion yield gradually decreased until the operation was finished at 336 hr. Western blotting of immobilized E. coli cells revealed that β-gal gradually decreased after 192 hr. In addition, alginate beads were cracked when the operation was finished. It is probable that, including this loss of E. coli cells by cracks, deactivation, and product inhibition of E. coli β-gal might lead to a gradual decrease in the production of CPN-Gal after 192 hr. However, as the purification of β-gal is not necessary with β-gal-producing recombinant E. coli cells, β-gal-producing E. coli cells might be a practical and cost-effective approach for enzymatically synthesizing CPN-Gal. It is expected that this process will be extended to long-term production process of CPN-Gal for commercialization.

• Journal of the Korean Applied Science and Technology
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• v.34 no.4
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• pp.954-961
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• 2017

We investigated the purifications of PE-gal and CPN-gal, synthesized by transgalactosylation reaction using recombinant ${\beta}$-gal. The reaction mixture containing PE and PE-gal was first mixed with EA, and thereafter PE and PE-gal were distributed in two-phase (EA/water) system. In this system, PE and PE-gal was selectively moved into EA and water phase, respectively. Then, the water phase was collected, and silica gel chromatography was carried out using the collected water phase. Finally, we compared two purified PE-gal samples using HPLC and TLC analysis, in which the one was purified only by silica gel chromatography and the other was purified by EA extraction followed by silica gel chromatography. In the latter case, the residual PE was almost completely removed, whereas, in the former case, the residual PE was remained remarkably. Additionally, the purification yield of PE-gal was about 21% on the basis of mole. In the same purification protocol, CPN-gal was able to be purified using EA extraction followed by silica gel chromatography, in which the residual CPN was almost removed when CPN-gal was purified by EA extraction followed by silica gel chromatography.