• 한국임상수의학회지
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• 제12권1호
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• pp.877-886
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• 1995

The recycling nuclear transplantation(NT) technique has the powerful potential of producing a large number of genetically identical embryos and offsprings from one embryo. Multiple generational cloning by this technique utilizes the NT embryo itself as the donor for the next generation of cloning. In this experiment, we have produced the third generational cloned embryos by recycling NT. Further we examined comparatively the electrofusion rate and in vitro developmental potential in the cloned embryos of the first second and third generations. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulberco's phosphate buffered saline containing 10 % fetal calf serum(FCS) at 47 hours after hCG injection. In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gl/S transition of 32-cell stage. The first and second generation NT embryos developed to 16-cell were used as donor nuclei for second and third generation. The recipient cytoplasms were utilized the oocytes collected at 14 hours after hCG injection, following revoming the nucleus and the first polar body by micromanipulation. The separated blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were fused by electrical stimulation. The electrofusion rate was seen to be 78.0, 88.0 and 90.3 % in the first second and third generation NT rabbit embryos, respectively. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10 % FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The in vitro developmental potential to blastocyst stage was significantly(P<0.05) decreased in the third(7.2 %) generation NT embryos compared to the first(53.1 %) and second(16.1 %) generation NT embryos. Following in vitro development to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The mean blastomere numbers and cell cycle numbers of NT embryos during the culture period were significantly(p<0.05) decreased in the second(93.9 cells and 6.55 cylces) and third(81.5 cells and 1.35 cylces) generation, compared to the first(189.9 cells and 7.55 cylces) generation.

• Applied Biological Chemistry
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• 제37권4호
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• pp.221-225
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• 1994

농산폐섬유소 자원을 이용하여 세균 단세포단백질(single cell protein)을 생산하기 위한 연구로서 부엽토에서 분리된 Cellulomonas sp. KL-6의 증식에 미치는 열처리 및 항생물질의 효과에 대해 조사하였다. KL-6 균주는 $65^{\circ}C$에서 5분, $75^{\circ}C$에서 1분 및 $85^{\circ}C$에서 15초까지 열처리시에는 증식하였고 그 이상의 온도에서는 처리시간에 관계없이 자라지 못하였다. $48^{\circ}C$에서 10분간의 예비 열처리는 본 균주의 열저항성을 증가시키지 못하였다. 기질 중 casamino acid(1%), yeast extract(1%), xylose(5%)는 KL-6 균주의 열저항성을 증가시켰으나 cysteine(0.03 M), sodium citrate(1%), casein(1%)은 반대로 열저항성을 감소시켰다. KL-6 균주는 $penicillin-G(1.536\;{\mu}g/ml)$와 $ampicillin(3.125\;{\mu}g/ml)$에 감수성인 반면, kanamycin에 대해서는 $200\;{\mu}g/ml$ 이상에서도 강한 내성을 보였다. SDS 처리에 의해 kanamycin의 내성이 제거되었고 제거율은 $9.2{\sim}31.2%$로 각각 나타났다.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.537-543
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• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.576-581
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• 2005

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast. Saccharomyces cerevisiae, by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) of Aspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast ${\alpha}-agglutinin$, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced into S. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.226-226
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• 2017

This study was to evaluate methods to high quality food ramie rice cake, thereby increasing farm income. This study investigated the effects of different covering material on stable winter survival management with edible leaf in Ramie(Boehmeria nivea L.). The method of winter survival with covering material were conducted under three condition compose to Non covering, Rice straw cutting covered with 500kg.10a-1, Rice husks covered with 1,000kg.10a-1(covered 4~5 cm thickness in the soil surface). Method of application were standard application(N-P-K-Compost applied at 27-9-27-600kg.10a-1. Compost and fused phosphate applied at 100% of basal fertilizer in March 25. 20% of top dressing were four times application in March 25 - October 5. Planting year were March 15, 2011. Plants were spaced 60 cm apart in rows 25 cm apart with open cultivation. According to non covering < Rice husks covered with 1,000kg.10a-1 < Rice straw cutting covered with 500kg.10a-1 cultivation this order, aerial part as a result were plenty amount of growth. Sprout time and winter survival rates was uncovering control plot compared to 2 - 5 days quickly, 45-57% highly by rice husks and rice straw covering. Green leaf yields is untreated control plot (12,44 kg.10a-1) compared to rice husks covering 7% higher, and rice straw covering increased to 18% of the most.

• 한국수정란이식학회지
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• 제9권2호
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• pp.153-160
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• 1994

This study evaluated the influence of cell stage of donor nucleus on nuclear injection, electrofusion and in vitro development in the rabbit to improve the efficiency of nuclear transplantation in the rabbit. The embryos of 8-, 16- and 32-cell stage were collected from the mated does by flushing viducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FGS) at 44, 54 and 60 hours after hCG injection. The blastorneres separated from these embryos were used as donor nucleus. The ovulated oocytes collected at 14 hours after hCG injection were used as recipient cytoplasm following removing the nucleus and the first polar body. The separated blastomeres were injected into the enucleated oocytes by micromanipulation and were electrofused in 0.28 M mannitol solution at 1.5 kV /cm, 60 $\mu$sec for three times. The fused oocytes were cocultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FGS for 72~120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The cultured nuclear transplant embryos were stained with Hoechst 33342 solution and the number of cells were counted by fluorescence microscopy. The successful injection rate of 8-, 16- and 32-cell-stageblastomeres into enucleated oocytes was 86.7, 91.0 and 93.9%, respectively. The electrofusion rate of 8-, 16- and 32-cell-stage blastomeres with enucleated oocytes was 93.3,89.3 and 79.0%, respectively. Development of blastomeres to blastocyst was similar with 8-,16- and 32-cell-stage donor nuclei(26.2, 25.8 and 26.6%, respectively, P<0.05). The mean number of cell cycle per day during in vitro culture in nuclear transplant embryos which received 8-, 16- and 32-cell- stage nuclei was 1.87, 1.81 and 1.43, respectively.

• 한국초지조사료학회지
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• 제21권4호
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• pp.253-258
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• 2001

This pot experiment was conducted to find out the forage productivity and quality in a grasslclover sward as affected by the application of three different fertilizers; double superphosphate(DS), fused Mg-phosphate (FP), and complex fertilizer(CF) on newly reclaimed hilly soil. This part was concerned with the mutual balances of mineral nutrients in the soils and mixed grass/clover sward in relation to grass tetany hazard. The results obtained are summarized as follows : 1. Concentration of exchangeable Mg and relative proportion of Mg to CEC in the soils before experiment were considerably below the critical level for good forage growth and prevention of grass tetany. It seems that these properties would be able to handicap by liming and NPK applications. 2. Comparing with the critical level for likelihood of tetany(Mg <0.2%, K >2.5%, and W(Ca+Mg) >2.2 in forages), mean concentration of Mg ranged from 0.14 in DS plot and 0.18 in FP plot to 0.24% in CF plot. Meanwhile, hazards of grass tetany in relation to the %K and ratio of K/(Ca+Mg) were not recognized. 3. Comparing with the optimum level of Carp(% ratio)=2.0 in forages for animal health, these ranged from 6.1 to 7.1. (Key words : Grass tetany, Fertilizer. Soil. Mineral nutrients)

• Bulletin of the Korean Chemical Society
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• 제31권6호
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• pp.1496-1500
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• 2010

Sibutramine enantiomers were separated successfully by capillary zone electrophoresis using substituted cyclodextrins as chiral selectors. The effects of cyclodextrin concentration, pH, voltage, buffer type, and electrolyte concentration on the migration time and resolution of enantiomers were examined. Separation of sibutramine enantiomers on an unmodified fused silica capillary (total length, 54 cm; effective length, 45 cm) was achieved using a mixed buffer of 20 mM phosphate/10 mM citrate containing either 5 mM methyl-${\beta}$-cyclodextrin (pH 4.3) or 5 mM carboxymethyl-${\beta}$-cyclodextrin (pH 6.5). Samples were injected with a pressure of 50 mbar for 5 s and were detected at a wavelength of 223 nm. The established method showed good precision and accuracy, with intra- and inter-day variations of less than 2.9 and 4.7%, respectively, and recoveries of 95.7 - 103.8%. The stability constants of (R)- and (S)-sibutramine demonstrated that the resolution of sibutramine enantiomers was attributable primarily to the difference in stability constants. When this optimized method was applied to the determination of sibutramine enantiomers in commercial drug formulations, it proved to be economical and convenient, affording sufficient accuracy, precision, and reproducibility as well as sensitivity and selectivity.

• Journal of Microbiology and Biotechnology
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• 제31권8호
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• pp.1088-1097
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• 2021

Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate-buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of Escherichia coli BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV removal efficacy of the bacteria and viral cytotoxicity after bacterial adsorption treatment. Although both constructs were successfully expressed, only BL21/lpp-Mx exhibited GNNV binding activity; BL21/lpp-Mx cells removed GNNV and protected GF-1 cells from GNNV infection more efficiently. Moreover, salinity affected the GNNV removal efficacy of BL21/lpp-Mx. Thus, our GNNV-binding bacterium is an efficient microparticle for removing GNNV from 10‰ brackish water and for preventing GNNV infection in groupers.

• 한국토양비료학회지
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• 제10권4호
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• pp.211-217
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• 1978

수종토양(數種土壤)에 2종(種)의 인산질비료(燐酸質肥料)를 시용(施用)한 후(後) 토양중(土壤中) 무기태인산(無機態燐酸)의 형태별(形態別) 함량변화(含量變化)와 수종침출액(數種浸出液)에 의한 유효인산침출량(有效燐酸浸出量)을 실내실험(室內實驗)으로 조사(調査)하여 비교(比較) 검토(檢討)한바 얻어진 결과(結果)는 다음과 같다. 1. Al-P 함량(含量)은 미경지(未耕地)에 비(比)해 기경전(旣耕田) 토양(土壤)에서 많고 Fe-P 함량(含量)은 사질(砂質)에 비(比)해 식질토양(埴質土壤)에서 많았다. 2. 시용(施用)된 중과석(重過石)의 인산(燐酸)은 토양중(土壤中)에서 주로 Al-P로, 용성인비(熔成燐肥)의 인산(燐酸)은 Al-P와 Ca-P로 변화(變化) 되었으며 Fe-P의 증가(增加)는 모두 아주 낮았다. 3. 유효 인산(燐酸)의 용출량(溶出量)은 침출액간(浸出液間)에 차이가 컸으며 대체(大體)로 중과석(重過石)을 시용(施用)한 토양(土壤)에서는 Bray No. 2>Lancaster${\fallingdotseq}$Bray No. 1>Truog>Olsen 침출액(浸出液)의 순(順)이었고 용린(熔燐)을 시용(施用)한 토양(土壤)에서는 Lancaster> Bray No. 2>Truog>Bray No. 1>Olsen 침출액(浸出液)의 순(順)이었다. 4. 모든 유효린산침출액(有效燐酸浸出液)의 무기린산형태별(無機燐酸形態別)에 대(對)한 침출액(浸出液)은 Al-P>Ca-P>Fe-P의 순(順)이었으나 이중 Bray No. 1과 Bray No. 2는 Fe-P에 대(對)한 침출능(浸出能)이 가장 낮았다. 5. Bray No. 2와 Bray No. 1 및 Olsen법(法)은 Ca-P의 침출능(浸出能)에 비(比)해 상대적(相對的)으로 Al-P에 대(對)한 용출능(溶出能)이 강(强)하니 Lancaster와 Truog법(法)은 Al-P에 비(比)해 Ca-P에 대(對)한 용출능(溶出能)이 더욱 큰 것으로 나타났다.