• 한국식품영양학회지
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• 제6권1호
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• pp.37-46
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• 1993

Brain, heart, liver, lung, kidney and thymus etc. 12 organs were removed and homogenized from Dawley-Sprague rats after suffocation. After fractionation of the tissue cytosols, enzymatic activities of the key enzymes in metabolic inositol phosphates cycle, PLC, IPSK and Ins(1,4,5) P35-phosphatase, were measured respectively. Hybridoma monoclones producing anti-lP3K murine monoclonal antibodies were obtained by the fusion of SP2/Ag 0-14 and spleen cells of mouse immunized with purified 53KDa IPSK, screening and cloning procedures. 18 cloned hybridoma cells were obtained, background due to nonspecific binding was very low with 10 clones. These Abs were purified from ascitic fluids by using affi-gel 15, and determined subtype of Abs. When immunoreactivities for rat tissues IP3K were exercised by adding the mixed Abs of 19Gl and 19G2b, they showed an overall similarity with noncompetitive inhibition. Brain tissue has high sensitivity for anti-lP3K Ab, whereas heart tissue has very low activity. In kinetic parameters Km value was 1.58 mM and Vmx value was 5.41umol/min/ml, respectively Only one form of 40 KDa IPSK was detected in heart tissues, however rat brain contains at least three immunologically distinct IP3K (53, 51 and 40 KDa) in western blot analysis. Of them 53 KDa protein was major enzyme in enzymatic activity. Northern blot analysis with 32P-labeled CDNA probe which encodes 1.8 Kb IPSK gene was performed. These results suggest that IPSK are regulated at transcriptional level during rat tissue development.

• 한국동물생명공학회지
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• 제35권3호
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• pp.215-222
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• 2020

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

• 자연과학논문집
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• 제16권1호
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• pp.1-13
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• 2005

안지오텐신(ACE) 저해제는 항고혈 효과를 갖고 있으므로 오랫동안 고혈압의 예방이나 치료에 이용되어 왔다. 본 연구는 재조합 대장균으로부터 새로운 ACE 저해제를 생산하고 정제하며 나아가 이들이 구조-기능 관P를 규명하기 위해 수행되었다. Saccharomyces cerevisiae의 ACE 저해 펩타이드 유전자를 함유하고 있는 재조합 pGEX-4T-3을 대장균 BL21(DE3)로 형질전환 시켰다. 재조합 pGEX-4T-3을 갖고 있는 대장균 BL21(DE3)로부터 생산된 Glutathione-s 전이효소(GST) 융합 단백질을 얻어서 그중 ACE저해 펩타이드를 Sephadex G-25 컬럼 크로마토그래피로 정제하였다. 정제된 ACE 저해 펩타이드는 타이로신-아스파틱엑시드-그리신-글리신-발린-페닐알라린-아르기닌-발린-타이로신-트레오닌의 서열을 가진 새로운 decapeptide이었고 ACE에 대하여 경쟁적으로 저해하였다.

• 대한화장품학회지
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• 제30권4호
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• pp.479-483
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• 2004

MITF는 미백관련 유전자의 대표적인 조절 인자 단백질로서 미백관련 유전자의 E-box와의 결합정도를 단백질 칩을 이용하여 측정하였다. 융합 단백질 형태의 MITF를 유리 칩에 고정시켰고 E-box를 포함하는 DNA oligomer가 결합하는 것을 확인하였다. 형광법, SPR (surface plasmon resonance), SPRi (surface plasmon resonance imaging)방법 중 형광법이 가장 효과적이었으며, DNA 저해제를 사용시 결합이 감소하는 것을 확인하였다. 이 결과 MITF를 이용한 미백원료의 고속스크리닝(HTS)의 가능성을 보여주었다.

• BMB Reports
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• 제40권6호
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• pp.952-958
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• 2007

We isolated many genes induced from pepper cDNA microarray data following their infection with the soybean pustule pathogen Xanthomonas axonopodis pv. glycines 8ra. A full-length cDNA clone of the Capsicum annuum ankyrin-repeat domain $C_3H_1$ zinc finger protein (CaKR1) was identified in a chili pepper using the expressed sequence tag (EST) database. The deduced amino acid sequence of CaKR1 showed a significant sequence similarity (46%) to the ankyrin-repeat protein in very diverse family of proteins of Arabidopsis. The gene was induced in response to various biotic and abiotic stresses in the pepper leaves, as well as by an incompatible pathogen, such as salicylic acid (SA) and ethephon. CaKR1 expression was highest in the root and flower, and its expression was induced by treatment with agents such as NaCl and methyl viologen, as well as by cold stresses. These results showed that CaKR1 fusion with soluble, modified green fluorescent protein (smGFP) was localized to the cytosol in Arabidopsis protoplasts, suggesting that CaKR1 might be involved in responses to both biotic and abiotic stresses in pepper plants.

• Parasites, Hosts and Diseases
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• 제51권2호
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• pp.247-253
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• 2013

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was $10^9TCID_{50}/ml$. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-${\gamma}$ and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.

• Asian-Australasian Journal of Animal Sciences
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• 제29권7호
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• pp.1037-1043
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• 2016

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• 미생물학회지
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• 제29권6호
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• pp.392-396
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• 1991

Pseudomonas sp. DJ77의 chromosomal DNA로부터 6.9kb XhoI 절편 상에 존재하는 phenanthrene 분해에 관련된 유전자군을 vector pBLUESCRIPT SK(+)에 클로닝하였다. 이렇게 얻은 재조합 plasmid인 pHENX7을 가지고 있는 JM101 균주는 3-methylcarechol을 노란색의 meta-cleavage 화합물로 전환할 수 있었다. 그러나 삽입된 절편의 방향이 반대가 되도록 제조한 pHENX7은 extradiol dioxygenase 활성을 나타내지 않기 때문에 전사방향을 알 수 있었다. pHENX7과 이의 듀도체들을 지니는 JH101균주에서 PhnC(24kDa), PhnD(31KDa), PhnE(34kDa), PhnF(KDa)의 4 polypeptide를 확인 할 수 있었고 개개의 유전자의 위치와 범위를 알 수 있었다. 유전자 순서는 phnC-phnD-phnE-phnF-phnG이었으며, phnC, phnD, phnE, phnF, phnG는 각기 glutathione S-transferase, meta-cleavage compound hydrolase extradiol dioxygenase, meta-cleavage compound dehydrogenase의 유전자이었다.

• 미생물학회지
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• 제44권2호
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• pp.105-109
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• 2008

발아효모 Saccharomyces cerevisiae의 핵공단백질인 Nic96 단백질과 유사성을 보이는 분열효모 Schizosaccharomyces pombe의 Nup97의 기능과 세포 내 위치를 조사하였다. nup97을 과 발현시켰을 때는 생장과 $poly(A)^{+}$ RNA의 분포에 별다른 이상을 보이지 않았다. 하지만, $kan^{r}$ 유전자를 이용하여 제작한 ${\Delta}nup97$ 결실돌연변이는 nup97의 발현이 억제되면, $poly(A)^{+}$ RNA가 핵 안에 축적되었고 비정상적인 DNA 분포를 보였으며 결국 생장하지 못했다. 한편, Nup97p의 N말단 또는 C말단에 GFP를 붙인 Nup97-GFP 융합단배질의 세포 내 위치를 확인하고자 하였다. 이러한 융합단백질들이 ${\Delta}nup97$ 결실돌연변이의 생장결함을 정상적으로 상보하는 것을 확인하고, nup97-GFP 유전자를 염색체의 nup97 유전자 위치에 삽입한 균주를 제작하였다. 자신의 프로모터에서 발현된 Nup97-GFP 융합단백질은 정상적인 기능을 보였으며, 핵막 주위에 점점 이 위치하였다. 이와 같은 결과들은 Nup97 단백질 역시 핵공단배질로 mRNA의 핵에서 세포질로의 이동에 중요하다는 것을 시사한다.