• 대한의생명과학회지
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• 제13권4호
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• pp.263-272
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• 2007

Nebulin is a giant actin binding protein (600-900 kDa) which is specific to skeletal muscle. This protein is known to regulate thin filaments length in sarcomere as a molecular template. The C-terminus of nebulin is located in the Z-disc of muscle sarcomere and is bound to other proteins such like myopalladin, titin, archvillin, and desmin. The N-terminus of nebulin binds to tropomodulin at the pointed ends of the thin filaments. In recent research, nebulin not only found in brain but also expressed in heart, stomach, and liver. So, the roles of nebulin in non-muscle tissue have been studied. However, lack of information or studies on nebulin binding proteins and nebulin function in brain are available so far. Therefore, the current study have investigated a novel binding partner of Nebulin C-terminus by using yeast two-hybrid screening with human brain cDNA library. Nebulin C-terminus, containing simple repeats, serine rich and SH3 domain, interacts with osteonectin C-terminal region. The specific interaction of nebulin and osteonectin were confirmed in vitro by using GST pull-down assay and reconfirmed in vivo by using transfected COS-7 cells with EGFP-tagged nebulin and DsRed-tagged osteonectin. Consequently, this study identified SH3 domain in nebulin C-terminus specifically binds to extracellular Ca-binding (EeC domain in osteonectin. Also, nebulin C-terminus fusion protein colocalized with osteonectin EC domain fusion protein in transfected COS-7 cells. The current study found the interaction between nebulin and osteonectin in human brain for the first time and suggested the nebulin in brain may be associated with osteonectin, as a regulator of cell cycle progression and mitosis.

• BMB Reports
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• 제39권6호
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• pp.696-702
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• 2006

Recently three proteins, playing central roles in the bidirectional transport of urate in renal proximal tubules, were identified: two members of the organic anion transporter (OAT) family, OAT1 and OAT3, and a protein that designated renal urate-anion exchanger (URAT1). Antibodies against these transporters are very important for investigating their expressions and functions. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages including high specificity and high recognition to the native protein compared with current methods. We fused high antigenicity fragments of the three transporters to the plasmids pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin, respectively. Gene gun immunization with these recombinant plasmids and two other adjuvant plasmids, which express granulocyte/macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, induced high level immunoglobulin G antibodies, respectively. The native corresponding proteins of URAT1, OAT1 and OAT3, in human kidney can be recognized by their specific antibodies, respectively, with Western blot analysis and immunohistochemistry. Besides, URAT1 expression in Xenopus oocytes can also be recognized by its corresponding antibody with immuno-fluorescence. The successful production of the antibodies has provided an important tool for the study of UA transporters.

• BMB Reports
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• 제46권8호
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• pp.422-427
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• 2013

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs), the molecular mechanism involved remains to be fully elucidated. Here, we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover, BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together, our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy, however, is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs, implying that activation of JNKs is essential for BMP9 osteoinductive activity.

• 대한한의학방제학회지
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• 제16권2호
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• pp.1-9
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• 2008

Objective : The purpose of this report was to provide the information about activity and safety of Ojeok-san by analyzing domestic/international papers about Ojeok-san. Methods : Domestic/international papers related to Ojeok-san were reviewed and analyzed. These papers were then classified by year, experimental method and subject. Results : The following results were obtained in this study. 1. The studies of Ojeok-san started from 1984 and has continuously increased. The studies were mainly focused on experimental models rather than clinical studies. 2. By subject, papers related to safety were most common with 5 papers among 20 papers. Besides there were papers related to efficacy of analgesic, anti-hyperlipidemic, anti-blood stasis and treatment for uterine myoma. 3. The papers related to safety were mainly focused on the effect of Okeok-san on liver function, renal function or metal concentration of organs such as blood, brain, liver, kidney and bone. Ojeok-san proved to be safe, but more clinical studies regarding the safety are needed hereafter. 4. Papers related to analgesic, anti-pyretic, anti-phlogistic activities of Ojeok-san were in vivo studies, and other papers were about anti-hyperlipidemic activity, apoptosis inducing activity on uterine myeloma cell line and anti blood static activity on hydrocortisone acetate induced blood statis model. 5. Case reports were about anti-lipidemia, analgesic effect for mastalgia/back pain and anxiety disorder due to climacteric changes. Conclusion : Ojeok-san is being used in various ways with analgesic, anti-pyretic, anti-phlogistic, anti-hyperlipidemic, anti-tumor or anti-blood statis activity. However, mechanism study should be conducted at the molecular biology level and more clinical studies on the efficacy of Ojeok-san are needed.

• Asian-Australasian Journal of Animal Sciences
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• 제15권2호
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• pp.172-178
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• 2002

The present study examined whether treatment of in vitro matured pig oocytes with calcium ionophore (A23187) could prevent polyspermic penetration in vitro. When oocytes cultured for maturation for 33, 36 or 44 h were subsequently treated with $50{\mu}M$ A23187 in medium with fetal calf serum (FCS) for 1, 2 and 3 h and then cultured for 12 h without spermatozoa, virtually no activation occurred. In the absence of FCS, however, 31-42, 45-49 and 56-64% of oocytes were activated, respectively. When oocytes treated with $50 {\mu}M$ A23187 in medium with FCS for 3 h were inseminated in vitro, the penetration rates (14-57%) were lower (p<0.01) with a higher (p<0.01) incidence (35-67%) of monospermy compared with untreated oocytes (69-80% penetration and 15-17% monospermy). However, sperm penetration was completely blocked in all oocytes treated with A23187 in the absence of FCS. When oocytes matured for 33 h were treated with different concentrations of A23187 for 3 h and inseminated in vitro, the penetration rate did not change but there was an increased incidence (p<0.05) of monospermy at $10-20{\mu}M$ and $2.5-5{\mu}M$ A23187 in the presence and absence of FCS, respectively, compared with at $0{\mu}M$ A23187. With these lower concentrations of A23187, treatment of oocytes for at least 60 and 30 min in the presence and absence of FCS, respectively, was required to increase the incidence of monospermy without reducing penetration rate. These results indicate that a high concentration ($50{\mu}M$) of A23187 in medium without FCS, but not in medium with FCS, stimulated in vitro matured pig oocytes to induce parthenogenetic activation and a complete block to sperm penetration in vitro. However, treatment of oocytes with lower concentrations of A23187 ( $10-20{\mu}M$ and $2.5-5{\mu}M$) both in the presence and absence of FCS maintained sperm penetration in vitro and increased the incidence of monospermy.

• 한국발생생물학회지:발생과생식
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• 제15권1호
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• pp.61-69
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• 2011

Previously, we have shown that Bcl2l10 as a member of Bcl-2 family, key regulators of the apoptotic process, is dominantly expressed in oocytes of ovary but several member of the Bcl-2 family are not expressed in oocytes. Recent our studies had been processed about roles and regulatory mechanisms of Bcl2l10 in oocytes. Microinjection of Bcl2l10 RNAi into the cytoplasm of germinal vesicle oocytes resulted in metaphase I (MI) arrest and exhibited abnormalities in their spindles and chromosome configurations (Yoon et al., 2009). The present study was conducted to elucidate the downstream genes regulated by Bcl2l10 and signaling networks in Bcl2l10 RNAi microinjected oocytes by using microarray analysis. Surprisingly, we found that a large proportion of genes regulated by Bcl2l10 RNAi were involved in the cell cycle and actin skeletal system regulation as important upstream genes of Bcl2l10. Among the transcripts with highly significant fold changes more than 2-fold, Tpx2 and Cep192 are 16.1- and 8.2-fold down regulated respectively by Bcl2l10 RNAi. Tpx2 and Cep192 are known as cofactors that control Aurora A kinase activity and localization. Therefore, we concluded that Bcl2l10 may have important roles during oocyte meiosis as functional upstream regulator of Tpx2 and Cep192.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.305-312
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• 2009

단분화성 정원줄기세포의 장기간 체외배양 중에 확립되는 다분화능 정원줄기세포는 배아줄기세포와 유사한 특성을 가져 3배엽성 세포로 체외분화가 가능하며 기형종을 형성할 수 있다. 본 연구에서는 선행 연구를 통해 outbred 생쥐(ICR strain)로부터 확립된 다분화능 정원줄기세포의 형질전환 가능성을 확인하며, 배아 내로 주입하여 유전적 키메라를 형성하는 효율을 배아줄기세포와의 비교를 통하여 검증하고자 하였다. 다분화능 정원줄기세포를 넣은 배아로부터 태어난 산자는 총 47마리(4.8%)가 태어나, 67마리(11.7%)가 태어난 배아줄기세포군에 비해 그 효율이 낮았다(P<0.05). 그러나 산자들 중의 키메라 생쥐의 비율은 다분화능 정원줄기세포 군으로 부터 3마리(6.4%)가 태어나 배아줄기세포 군으로부터 태어난 5마리(7.5%)와 유사하였다(P>0.05). 태어난 유전자변형 생쥐의 장기를 확인한 결과, 췌장, 심장, 뇌, 근육, 위, 피부, 정소에 GFP가 발현되는 것을 확인하였다. 또한 배아의 근육, 위, 뼈 등에서 anti-GFP 항체의 발현을 확인하였다. 이상의 결과를 종합하면 outbred 생쥐로부터 확립된 다분화능 정원줄기세포가 inbred 생쥐로부터 확립된 배아줄기세포와 마찬가지로 키메라 생쥐를 생산할 수 있는 전분화능을 가짐을 확인하였고, 새로운 유전자변형 동물의 생산을 위한 매개체로서의 가능성을 가진 것으로 여겨진다.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.249-256
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• 2009

구조적으로 estrogen 수용체(estrogen receptor, ER)와 유사한 estrogen receptor-related receptor(ERR)는 포유동물에서 배발생 후기에 외배엽 형성과 관련되어 있다고 알려진 고아핵수용체(orphan nuclear receptor)이다. ERR은 ER과 DNA binding domain의 보존성은 유사하지만, ligand 결합 및 전사 활성은 다르다. 포유동물의 ERR에 관한 연구에 비하여 해양 무척추동물의 ERR에 대한 기능 연구는 매우 부족하다. 본 연구에서는 우리나라 동해안에 주로 서식하는 둥근성게(Strongylocentrotus nudus) ERR의 초기 발생기 유전자 발현 변화와 전사 활성 기능을 조사하기 위해 먼저 polymerase chain reaction(PCR)을 이용하여 cDNA를 동정하였다. 둥근성게 ERR은 S. purpuratus와 91%의 높은 상동성을 보였으며, 계통수 분석을 통해 무척추동물 ERR의 clade에 포함되는 것을 알았다. 둥근성게 배발생 시기에 ERR 유전자 발현을 알아보기 위하여 real-time PCR을 실시한 결과, 4~64세포기와 유생기에 mRNA level이 높은 경향을 나타내었다. 또한 호르몬 및 co-regulator에 의한 둥근성게 ERR의 전사 활성을 조사한 결과, 호르몬에 의한 특이성은 확인되지 않았지만, peroxisome proliferator activated receptor-(PPAR) $\gamma$ coactivator $1\alpha$(PGC-$1\alpha$)가 둥근성게 ERR의 coactivator임을 입증할 수 있었다. 이 연구 결과는 향후 새로운 ligand 발굴과 coregulator와 의 상호작용 연구에 도움이 될 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제20권4호
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• pp.289-296
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• 2016

This report describes the sex differentiation of the Korean rose bitterling, Rhodeus uyekii, from hatching to 170 days post-hatch (DPH) in relation to total length (TL), body weight (BW), and integral water temperature (IWT). The growth curve of TL from just hatching to 83 DPH was $5.144e^{0.045t}$ ($R^2=0.961$; t, time), and that of BW was $2.398e^{0.086t}$ ($R^2=0.725$). Primordial germ cells (PGCs) were observed at 17 DPH (7.9 mm TL, 3.74 mg BW, $374^{\circ}C$ IWT), and thereafter began to protrude into the peritoneal cavity. At 21 DPH ($9.2{\pm}0.14mm$ TL, $4.8{\pm}0.07mg$ BW, $462^{\circ}C$ IWT), some PGCs contained condensed chromatin and oocyte were observed in meiotic prophase. In contrast to the ovaries, which grew gradually after sexual differentiation, testes began multiplying at 25 DPH (10.1 mm TL, 5.42 mg BW, $550^{\circ}C$ IWT), when testicular differentiation was first identified, and multiplied continuously thereafter. At 33 DPH (11.2 mm TL, 10.5 mg BW, $726^{\circ}C$ IWT), the developing testes contained spermatogonia that exhibited mitotic activity. No spermatocyte or sperm cell was observed until 83 DPH (18.9 TL, 48.2 mg BW, $1,826^{\circ}C$ IWT). At 170 DPH (32.5 mm TL, 270.1 mg BW, $3,740^{\circ}C$ IWT), which was the end point of this study, the mature ovaries showed germinal vesicle breakdown, while the mature testes contained observable spermatocytes and sperm cells. These results allow us to identify the sex differentiation type of the Korean rose bitterling as differentiated gonochoristic.

• Reproductive and Developmental Biology
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• 제39권3호
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• pp.83-88
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• 2015

이 연구에서는 정소가 제거된 흰쥐에 비타민 E와 셀레늄(Selevit)을 5주간 투여 후 체중, 장기 무게, 혈액학적 그리고 생화학적 변화를 관찰하였다. 체중의 변화에서는 모든 실험군에서 증가가 나타냈다. Orch+Selevit군의 체중 증가는 $11.2{\pm}10.25g$으로 가장 낮았으며, Intact군, Sham군, Orch군과 비교 시 유의적으로 감소되었다. 장기 무게의 변화에서는 Orch+Selevit군의 심장과 간장 무게는 Intact군, Sham군과 비교 시 유의적으로 감소되었다. Intact군, Sham군, Orch군의 신장 무게는 Orch+Selevit군 비교 시 유의적인 증가를 확인하였다. 백혈구 수의 혈액학적 변화에서는 Orch+Selevit군은 다른 모든 군과 비교해 유의적인 증가를 확인하였으며, 적혈구수, 평균적혈구용적, 평균적혈구혈색소량, 평균적혈구색소농도 등과 같은 혈액학적 측정치에서는 유의성은 인정되지 않았다. 생화학적 변화에서는 Orch+Selevit군의 혈청총단백질, 알부민은 Orch군과 비교 시 유의적으로 증가하였으며, 알부민은 Intact군, Sham군 그리고 Orch군과 비교 시 유의적으로 감소했다. AST와 ALT는 모든 실험군에서 유의성이 없었다.