• Journal of Life Science
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• v.19 no.3
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• pp.305-310
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• 2009

Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the $M13{\Delta}lac182/229$ phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.

• Journal of Veterinary Clinics
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• v.12 no.1
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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.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2010.06a
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• pp.15-15
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• 2010

$Na_2Ti_6O_{13}$ (NT)가 도핑된 $BaTiO_3-(Bi_{0.5}Na_{0.5})TiO_3$ BBNT) PTCR 세라믹스를 변형된 세라믹공정을 이용하여 제조하였다. 제조된 BBNT 세라믹의 미세구조와 PTCR 특성에 미치는 NT의 효과를 조사하였다. $1300^{\circ}C$에서 합성된 BBNT 세라믹은 NT의 도핑량이 증가함에 따라 비정상적으로 성장된 입자의 수가 증가하였다. 뿐만 아니라, NT의 도핑량 증가는 상온비저항을 약간 증가시켰지만 큐리온도 (Tc) 부근의 최대비저항/최소비저항으로 정의되는 PTC 점프 특성을 크게 향상시켰다. 특히, 0.01mol%의 NT 도핑 시 상온비저항은 $425\;\Omega{\cdot}cm$, PTC 점프는 ($2.02{\times}^10^5$) 저항온도계수는 69.8% 및 Tc는 $155^{\circ}C$의 우수한 결과를 나타내었다.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.133-139
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• 1997

Large scale production of cloned embryos requires the technology of multiple generational nuclear transfer(NT) by using NT embryos itself as the subsequent donor nuclei. In this work we investigated comparatively the effects of enucleated oocytes treated with ionomycin and 6-DMAP on the electrofusion rate and in vitro developmental potential in the first and second NT embryos. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 15 hours after hCG injection. The enucleated oocytes were pre-activated by 5 min incubation in 5$\mu$M ionomycin and 2 hours incubation in 2 mM 6-DMAP at 19~20 hours post-hCG before microinjection. In the first and second generation NT, the unsynchronized 16-cell stage embryos were used as nuclear donor. The separated donor blastomeres were injected into the enucleated activated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of single pulse for 60 $\mu$sec at 1.25kV/cm in $Ca^2$+, $Mg^2$+ - free 0.28 M mannitol solution. In the non-preactivation group, the electrofusion and electrical stimulation was given 3 pulses for 60 $\mu$sec at 1.25 kV/cm in 100$\mu$M $Ca^2$+, $Mg^2$+ 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in TCM-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The results obtained were summarized as follows: 1. In the first generational NT embryos, the electrofusion rate of preactivated and non-activated oocytes(80.4 and 87.8%) was not significantly different, but in the second generational NT embryos, the electrofusion rate was significantly(P<0.05) higher in the non-activated oocytes(85.7%) than in the preactivated oocytes(70.1%). 2) In the first and second generational NT embryos, the developmental potential to biastocyst stage was significantly(P<0.05) higher in the preactivated oocytes(39.3 and35.7%) than in the non-preactivated oocytes(16.0 and 13.3%). No significant difference in the developmental potential was shown between the first and second generational NT embryos derived from the preactivated oocytes. In conclusion, it may be efficient to use the oocytes preactivated with ionomycin and 6-DMAP for the multiple production of cloned embryos by recycling nuclear transfer.

• Molecules and Cells
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• v.24 no.3
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• pp.323-328
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• 2007

SOX (Sry-related HMG box) family proteins, which have an evolutionarily conserved DNA binding domain, have crucial roles in cell differentiation. However, their target genes remain enigmatic. Some members of the SOX family may have roles in regulation of cell proliferation. We established stable NT2/D1 cell lines overexpressing SOX15 (SOX15-NT2/D1), and a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the SOX15-NT2/D1 cells exhibited significantly slower growth than the controls. Flow cytometry analysis revealed that an increased fraction of the SOX15-NT2/D1 cells were in G1-G0. In addition, a microarray analysis identified 26 genes that were up-regulated in the SOX15-NT2/D1 cells, but none that were down-regulated genes. Among the up-regulated genes, IGFBP5, S100A4, ID2, FABP5, MTSS1, PDCD4 have been shown to be related to cell proliferation and/or the cell cycle.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.168-173
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• 2004

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -20$-20^{\circ}C$, $-30^{\circ}C$ or $-80^{\circ}C$ in the presence of 5% DMSO used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstracted from the somatic cells, 62.3%, 76.6% to 65% showed cleavage 70.5%, 81.9% to 78.5% reached the stage of morula formation and 39.7%, 43.2% or 47.6% reached the blastocyst stage. There was no significant difference in development when the NT embryos were compared with those reconstracted from fresh somatic cell derieved skin tissues (72%, 75.3%, and 45.2%, for cleavage, and development to morula and blastocyst stage, respectively). NT embryos were then placed in a portable $CO_2$ incubator and carried to China from Japan by air. After reaching to farm, two NT embryos were transferred to each of 5 recipients. We obtained 2 NT calves which birth weights is 30kg and 36kg female, and gestation periods is 281 and 284 days, respectively. There were no observation any abnormality from those calves. The results indicated that cell lines derieved from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer using the portable $CO_2$ incubator.

• The Journal of Korean Physical Therapy
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• v.24 no.2
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• pp.134-142
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• 2012

Purpose: This study examined the effect on postural control during the stimulation of haptic touch with fingertip on the stable surface at quiet standing posture, squat flexion stage, 60 degrees squat stage and squat extension stage. Methods: The postural sway was measured on the force platform, while 30 subjects were squatting, under three different haptic touch conditions (No Touch [NT], Light Touch [LT], Heavy Touch [HT]), above the touch pad in front of their body midline. Three different haptic touch conditions were divided into 1) NT condition; squatting as right index fingers held above the touch pad, 2) LT condition (<1N); squatting as the touch pad was in contact with right index fingers pulp with a pressure not exceeding 1N and 3) HT condition; squatting as subjects were allowed to use the touch pad for mechanical support by transmitting onto it with as much force, choosing with their index fingers. Results: There was significant decrease in LT, rather than that of NT (p<0.01), and in HT, rather than that of LT (p<0.01), as the results of the distance and velocity of center of pressure (COP) in mediolateral direction at quiet standing position. In anteroposterior direction, the distance and velocity of COP in LT and HT showed significant decrease, when compared to that of the data of NT (p<0.01). There was no significant difference between the 3 conditions (NT, LT, and HT), with respect to the distance and velocity of COP in mediolateral direction, during dynamic balance (squat flexion stage, squat extension stage) (p>0.05). In anteroposterior direction, the results of the distance and velocity of COP in HT showed significant decrease when compared to that of the data of NT (p<0.05). Conclusion: Light touch, during the task, decreased the postural sway at static balance. The results suggest that haptic touch should be applied, appropriately, because it varies the effects according to different conditions.

• Journal of the Korean Chemical Society
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• v.56 no.1
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• pp.34-46
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• 2012

Dopants and defects can be introduced as well as the intercalation of metals into single wall carbon nanotubes (SWCNTs) to modify their electronic and magnetic properties, thus significantly widening their application areas. Through spinpolarized density functional theory (DFT) calculations, we have systemically studied the following: (i) (10,0) and (5,5) SWCNT doped with nitrogen ($CN_xNT$), (ii) (10,0) and (5,5) SWCNT with pyridine-like defects (3NV-$CN_xNT$), and (iii) chemical functionalization of (10,0) and (5,5) 3NV-$CN_xNT$ with 12 different transition metals (TMs) (Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Pd, and Pt). Attention was done in searching for the most stable configurations, deformation, calculating the formation energies, and exploring the effects of the doping concentration of nitrogen and pyridine-like nitrogenated defects on the electronic properties of the nanotubes. Also, calculating the corresponding binding energies and effects of chemical functionalization of TMs on the electronic and magnetic properties of the nanotubes has been made. We found out that the electronic properties of SWCNT can be effectively modified in various ways, which are strongly dependent not only on the concentration of the adsorbed nitrogen but also to the configuration of the adsorbed nitrogen impurities, the pyridine-like nitrogenated defects, and the TMs absorbed; due to the strong interaction between the d orbitals of TMs and the p orbitals of N atoms, the binding strengths of TMs with the two 3NV-$CN_xNT$ are significantly enhanced when compared to the pure SWCNTs.

• Proceedings of the KSME Conference
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• 2008.11a
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• pp.1585-1587
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• 2008

Fibroblast is constantly subjected to mechanical loads in connective tissues where mechanical signals are converted to intercellular biochemical events. The aim of this study is to understand the effects of tensile stress on the neurotrophin (NT) and transforming growth factor (TGF) expression of fibroblast in vitro. Nerve growth factor (NGF) stimulates fibroblast migration, and TGF is related to tissue repair. In this study, at the uniaxial stretch of 10% strain and frequency of 0.5 Hz, different resting times of 0, 20, and 60 min are placed in between 10 min stimulations periods. Results show increase in NGF mRNA levels and a substantial decrease in NT3 mRNA after 1 hr of stimulation, indicating that the tensile stress may regulate NGF and NT3, key factors for the neurocosmetic applications. The mRNA level for TGF-${\alpha}$ and TGF-${\beta}2$ had increased up to two-folds after 1 hr of stimulation, showing that the tensile stress may control TGF, an important part of wound healing.

• Journal of Radiation Industry
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• v.11 no.3
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• pp.145-149
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• 2017

The cel7A sequence variation was analyzed between the wild type (Lentinula edodes KACC42378) and its cellulase activity enhanced mutant LER277. LER277 was induced by using gamma ray radiation ($^{60}Co$) at the $LD_{99}$ dose (0.94 kGy). Cloning and sequencing results showed that the cel7A coding DNA sequence (CDS) of LER277 had five nucleotide substitutions ($T{\rightarrow}C$, 201, 285 and 744 nt; $A{\rightarrow}G$, 525 nt; $C{\rightarrow}T$, 540 nt) and one hexanucleotide repeat insertion (GGCACC, within 1375-1392 nt) compared to that of the wild type. The Five nucleotide substitutions did not change the deduced amino acids and the hexanucleotide insertion elongated the GT repeat in a serine/threonine/glycine-rich linker. These results suggest that the enhancement of the cellulase activity in LER277 partly stemmed from cel7A changes by which the GT repeat of the linker is elongated.