• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1665-1672
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a useful method to preserve endangered species and to study the reprogramming event of a nuclear donor cell by the oocyte. Although several studies of iSCNT using murine cells and bovine oocytes have been reported, the development of murine-bovine iSCNT embryos beyond the 8-cell stage has not been successful. In this paper, we examined the developmental potential of embryos reconstructed with a murine embryonic fibroblast as the nuclear donor and a bovine oocyte as the cytoplasm recipient. The reconstructed embryos were cultured in CZB (murine medium) or CR1aa (bovine medium). In addition, for the development of a murine-bovine iSCNT blastocyst, the antioxidant ${\beta}$-mercaptoethanol (${\beta}ME$) was supplemented to CR1aa medium. Furthermore, to verify the mouse genome activation in murine-bovine iSCNT embryos, RT-PCR analysis of murine Xist was performed. The development of the murine-bovine iSCNT embryos cultured in CR1aa was significantly higher than that in CZB (p<0.05). With respect to the effect of BME on the development of the murine-bovine iSCNT blastocyst, addition of BME produced a significant increase in blastocyst development (p<0.05). Karyotype analysis confirmed that the reconstructed embryos were derived from murine cells (40XX). The Xist gene was gradually increased from the 8-cell stage to the blastocyst stage. This is the first report of blastocyst development of iSCNT embryos derived from murine somatic cells and bovine oocytes. These results demonstrate that bovine cytoplasm can support the development of later stages of a preimplantation embryo from murine-bovine iSCNT.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.7-12
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• 2004

This study was performed to establish the effective culture condition for the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were cultured and passaged two times before use. A single cell was seeded in 96-well plates, cultured in medium supplemented with different concentrations of FBS, catalase or $\beta$-mercaptoethanol ($\beta$ME), and classified by cell size and morphology. Cells were passaged two times into 4-well dish before freezing. The establishment efficiencies were not different among different concentrations of FBS (0.3 to 5.1%). However, population doubling time (PDT) was significantly decreased by increasing the FBS concentration (P＜0.05). The establishment efficiency of $\beta$ME-added group (10.4%) was significantly higher than those of catalase-added and control groups (3.5%, and 3.5%, respectively, p＜0.05), and PDT was significantly decreased (23.6 vs 28.1, and 25.5 h, respectively, p＜0.05). However, catalase did not show a positive effect on the establishment efficiency. Cell size and morphology did not affect the establishment efficiency and PDT of clonal lines. The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 30% FBS and $\beta$ME.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.938-943
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• 2004

A gene responsible for the chlorothalonil-biotransformation was cloned from the chromosomal DNA of Ochrobactrum anthropi SH35B, an isolated bacterium strain from soil. We determined the nucleotide sequences and found an open reading frame for glutathione S-transferase (GST). The drug-hypersensitive Escherichia coli KAM3 cells transformed with a plasmid carrying the GST gene can grow in the presence of chlorothalonil. The GST of O. anthropi SH35B was expressed in E. coli and purified by affinity chromatography. The fungicide chlorothalonil was rapidly transformed by the purified GST in the presence of glutathione. No significant difference in the chlorothalonil-biotransformation effect was observed among the thiol compounds (cysteine, reduced glutathione, and $\beta$-mercaptoethanol). Thus, the result reported here is the first evidence on the chlorothalonil-biotransformation by conjugation with the cellular free thiol groups, especially glutathione, catalyzed by the bacterial GST.

• Korean Journal of Microbiology
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• v.36 no.1
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• pp.26-32
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• 2000

Catechol 2,3-dioxygenase was purified from recombinant strain E. coli CNU312 carrying the tomB gene which was cloned from toluene-degrading Burkholderia cepacia G4. The purification of this enzyme was performed by acetone precipitation, Sephadex G-75 chromatography, electrophoresis and electro-elution. The molecular weight of native enzyme was about 140.4 kDa and its subunit was estimated to be 35 kDa by SDS-PAGE. It means that this enzyme consists of four identical subunits. This enzyme was specifically active to catechol, and$K_(m)$ value and $V_(max)$value of this enzyme were 372.6 $\mu$M and 39.27 U/mg. This enzyme was weakly active to 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol, but rarely active to 2,3-DHBP. The optimal pH and temperature of the enzyme were pH 8.0 and $40^{\circ}C$. The enzyme was inhibited by $Co^(2+)$, $Mn^(2+)$, $Zn^(2+)$, $Fe^(2+)$, $Fe^(3+)$, and $Cu^(2+)$ ions, and was inactivated by adding the reagents such as N-bromosuccinimide, and $\rho$-diazobenzene sulfonic acid. The activity of catechol 2,3-dioxygenase was not stabilized by 10% concentration of organic solvents such as acetone, ethanol, isopropyl alcohol, ethyl acetate, and acetic acid, and by reducing agents such as 2-mercaptoethanol, dithiothreitol, and ascorbic acid. The enzyme was inactivated by the oxidizing agent $H_(2)$$O_(2)$, and by chelators such as EDTA, and ο-phenanthroline.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.153-156
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• 2002

The characterization of a partially purified, cellulase-free, thermostable alkaline xylanase from thermoalkalophilic Bacillus sp. JB-99 was investigated. The xylanase production was the highest when birchwood xylan was added to a medium containing finely powdered rice bran, showing 4,826 IU$ml^-1$ of activity for 15 h of incubation. The partially purified xylanase exhibited an optimum temperature and pH at $70^C{\circ}$ and 10, respectively. The enzyme was stable at pH 5-11 at $50^C{\circ}$. The xylanase activity was strongly inhibited by $Hg^2+$, while dithiothreitol, cysteine, and ${\beta}$-mercaptoethanol enhanced the activity.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Korean Journal of Food Science and Technology
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• v.22 no.4
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• pp.426-433
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• 1990

The conditions for immobilization of the partially purified ${\beta}-galactosidase$ form Bacillus subtilis HP4 and the properties of the immobilized enzyme have been investigated. The crude enzyme precipitated with cold acetone was purified about 68-fold through DEAE-cellulose and sephadex G-100 chromatography and its recovery was 19.9% The optimal conditions for Immobilization of enzyme were obtained in 2%(w/v) sodium alginate, 15%(v/v) enzyme solution and 2%(w/v) calcium chloride, and also the optimal stirring thme was 2 hours on the above conditions. The optimum temperature and pH values for immobilized enzyme were $55^{\circ}C$ and 6.5, respectively. Its residual activity was show 25% after heat treatment for an hour at $65^{\circ}C$, and found its high stability in pH 6.0 to 8.0. The enzyme activity was not affected b)· EDTA, 2-mercaptoethanol, KCN, protective agents, and other methal ions except Hg ion and Cu ion. The $K_m\;and\;V_{max}$ values of the immobilized enzyme on ONPG were $1.82{\times}10^{-2}M\;and\;3.57{\times}10^{-8}mole/min$, whereas those on lactose were $2.94{\times}10^{-2}M\;and\;1.68{\times}10^{-7} mole/min$, respectively. The remained enzyme activity for the immobilized enzyme was 95%t of original activity after storage of 40 days at $4^{\circ}C$, and when reused for 5 times was 81%. When skim milk(4.8% lactose) and 5% lactose solution were reacted with the immobilized enzyme(250 units/g) of lactose were 51% and 43%, respectively.

• Elastomers and Composites
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• v.48 no.1
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• pp.10-17
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• 2013

Polyolefin-g-poly(t-butylstyrene) as one of the high-temperature polyolefin-based thermoplastic elastomers was synthesized by the graft-from anionic living polymerization from the styrene moieties of the linear poly(ethylene-ter-1-hexene-ter-divinylbenzene) as a soft block to form the hard end blocks, poly(t-butylstyrene). The chemistry of the anionic graft-from polymerization involved complete lithiation of the pendant styrene unit of the soft polyolefin elastomer with sec-BuLi/TMEDA followed by the subsequent graft anionic polymerization of 4-tert-butylstyrene with Mn=10,000~30,000 g/mol. The graft-from living anionic polymerization were very effective and the grafting size increased proportionally with increasing monomer concentration and the reaction times. The synthetic methodology for the multi-hydroxyl chain-end modified polyolefin-g-poly(t-butylstyrene) was proposed by using the thiol-ene click reaction between 2-mercaptoethanol and the polyolefin-g-[poly(t-butylstyrene)-b-high vinyl polyisoprene], which was obtained from the subsequent living block copolymerization using polyolefin-g-Poly(t-butylstyrene) with isoprene. The result indicated that this process produced a new well-defined functionalized graft-type polyolefin-based TPE with high $T_g$ hard block(> $145^{\circ}C$).

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.201-208
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• 2004

This study was performed to examine the effects of catalase and $\beta$-mercaptoethanol ($\beta$ME) on the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were passaged two times before use. A single cell was seeded in 96-well plates and cultured in medium supplemented with or without catalase or $\beta$ ME. Cell colonies were passaged two times into 4-well dish. Cell lines with proliferating potential were classified as an established clonal cell line. In experience 1, the establishment efficiencies were examined by addition of catalase (100ng/$m\ell$) or $\beta$ME (100 uM) in culture medium. The establishment efficiency of $\beta$ME-added group (8.3％) was significantly higher than that of control group (3.2％, P<0.05). However, catalase did not have a positive efffct on the establishment efficiency. In experience 2, the establishment efficiencies were examined by addition of different concentrations of catalase (0-1,000 ng/$m\ell$) in culture medium. However, establishment efficiencies were not different among the different concentrations of catalase (0-2.6％). In experience 3. the establishment efficiencies were examined by addition of different concentrations of $\beta$ME(0-1,000 uM) in culture medium. The establishment efficiency was significantly higher in 100 uM $\beta$ME-added group (9.4％) compare to others (0-1.6％). The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 100uM $\beta$ME. However, catalase did not have a positive effect on the establishment efficiency.

• Development and Reproduction
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• v.2 no.1
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• pp.29-37
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• 1998

To investigate the role of oviductal environment in early mammalian development, we examined the effects of bovine oviductal fluid (bOF) on the development of mouse 2-cell embryos in vitro. All of the embryos cultured in medium containing 5% or more of bOF underwent degeneration after 48 hr, whereas only 5% of embryos cultured in the absence of bOF degenerated. When bOF was heated at 65 \circ C for 30 min and then added to the culture medium, the embryotoxic effect of bOF was not removed at all such that none of the embryos remained alive after 48 hr. However, when bOF heated at 90 \circ C for 30 min was added to the culture, nearly most (95%) of embryos was alive. Similarly, pretreatment of bOF with 0.1% chymotrypsin for 1 hr or overnight following heating at 65 \circ C resulted in the development of 95.5% of mouse 2-cell embryos to early blastula after 48 hr culture in the presence of treated bOF. Interestingly addition of an anti-oxidant removed the evbryotoxic effect of bOF so that 91.0% of 2-cell embryos developed to morulae or blastulae in the presence of both 5% bOF and 10 mM of glutathione (GSH) after 48 hr culture. Neither oxidized form of GSH (GSSG) nor other antioxidants, however, could support the embryonic development in the presence of bOF. From these results, it is suggested that bOF contains a protein-like factor(s) which becomes embryotoxic by exposing in vitro, probably via oxidation reaction.