• Molecules and Cells
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• 제25권4호
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• pp.566-571
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• 2008

Calcineurin (Cn) is a calcium/calmodulin-dependent serine/threonine protein phosphatase that has diverse functions in different cell types and organisms. We screened proteins interacting with the C. elegans CnA homolog, TAX-6, by the yeast two-hybrid system. CNP-3 (Calcineurin interacting protein-3) is a novel protein that physically interacts with the catalytic domain of TAX-6. It is strongly expressed in the nuclei of intestine, hypodermis, dorsal uterine regions and spermatheca. Expression begins around the 60-cell stage and proceeds during all larval stages and the adult. To elucidate the biological function of cnp-3 we isolated a cnp-3 deletion mutant. Since CNP-3 binds CnA, we looked at factors associated with calcineurin loss-of-function mutants, such as brood size, body size, serotonin- and levamisole-mediated egg-laying behavior. The cnp-3(jh145) single mutant had no gross defects compared to wild-type animal. However, the phenotypes of the double mutants, tax-6(p675);cnp-3(jh145) and cnb-1(jh103);cnp-3(jh145), were more severe in terms of brood size, body size and serotonin-mediated egg-laying defects than tax-6(p675) and cnb-1(jh103), respectively. These results suggest that dysfunction of cnp-3 enhances certain calcineurin loss-of-function phenotypes in C. elegans.

• The Plant Pathology Journal
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• 제35권6호
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• pp.684-691
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• 2019

Evolution of adaptive mechanisms to abiotic stress is essential for plant growth and development. Plants adapt to stress conditions by activating the abscisic acid (ABA) signaling pathway. It has been suggested that the ABA receptor, clade A protein phosphatase, SnRK2 type kinase, and SLAC1 anion channel are important components of the ABA signaling pathway. In this study, we report that the shaker type potassium (K⁺) channel, GORK, modulates plant responses to ABA and abiotic stresses. Our results indicate that the full length of PP2CA is needed to interact with the GORK C-terminal region. We identified a loss of function allele in gork that displayed ABA-hyposensitive phenotype. gork and pp2ca mutants showed opposite responses to ABA in seed germination and seedling growth. Additionally, gork mutant was tolerant to the NaCl and mannitol treatments, whereas pp2ca mutant was sensitive to the NaCl and mannitol treatments. Thus, our results indicate that GORK enhances the sensitivity to ABA and negatively regulates the mechanisms involved in high salinity and osmotic stresses via PP2CA-mediated signals.

• 생명과학회지
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• 제33권11호
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• pp.859-867
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• 2023

루페올은 5환성 트리테르펜의 일종으로 많은 질병에 치료 효과가 있는 것으로 보고되었으나, 인슐린 저항성에 미치는 영향은 명확하지 않다. 본 연구에서는 3T3-L1 지방세포에서 루페올의 IRS-1 인산화 억제능을 통해 인슐린 저항성 개선효과를 조사하였다. 3T3-L1 세포를 배양하고 TNF-α를 24시간 동안 처리하여 인슐린 저항성을 유도하였다. 서로 다른 농도의 루페올(15, 30 μM) 또는 100 nM의 rosiglitazone을 처리한 세포를 배양한 후, 용해된 세포를 이용하여 western blotting을 시행하였다. 실험결과 루페올은 지방세포에서 TNF-α에 의해 유발되는 인슐린 신호전달의 음성 조절자와 염증 활성화 단백질 kinase에 대한 개선 효과를 나타냈다. 인슐린 신호전달의 음성 조절자인 PTP-1B와 JNK의 활성 및 IKK와 염증활성화 단백질키나아제의 활성을 억제하였다. 또한, 루페올은 IRS-1의 serine 인산화는 하향 조절하고 tyrosine 인산화는 상향 조절하였다. 그 후, 하향 조절된 PI3K/AKT 경로가 활성화되고, GLUT 4의 세포막 전위가 자극되어, 결과적으로 인슐린 저항성이 유도된 3T3-L1 지방세포에서에서 세포내 포도당 흡수가 증가하였다. 본 연구결과, 루페올은 3T3-L1 지방세포에서 인슐린 신호전달 및 염증 활성화 단백질 kinsase들의 음성 조절인자를 억제하여, IRS-1의 serine 인산화를 하향 조절함으로써 TNF-α 유발 인슐린 저항성을 개선할 수 있을 것으로 사료된다.

• Molecules and Cells
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• 제39권4호
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• pp.322-329
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• 2016

Voltage-gated $Ca^{2+}$ ($Ca_V$) channels are dynamically modulated by Gprotein-coupled receptors (GPCR). The $M_1$ muscarinic receptor stimulation is known to enhance $Ca_V2.3$ channel gating through the activation of protein kinase C (PKC). Here, we found that $M_1$ receptors also inhibit $Ca_V2.3$ currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated $Ca_V2.3$ currents by ~two-fold. After the PMA-induced potentiation, stimulation of $M_1$ receptors decreased the $Ca_V2.3$ currents by $52{\pm}8%$. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) is responsible for the muscarinic suppression of $Ca_V2.3$ currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of $PI(4,5)P_2$ to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed $Ca_V2.3$ currents, by $53{\pm}3%$. Next, dephosphorylation of both PI(4)P and $PI(4,5)P_2$ to PI by PJ translocation further decreased the current by up to $66{\pm}3%$. The results suggest that $Ca_V2.3$ currents are modulated by the $M_1$ receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane $PI(4,5)P_2$. Our results also suggest that there is rapid turnover between PI(4)P and $PI(4,5)P_2$ in the plasma membrane.

• 대한소아치과학회지
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• 제27권2호
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• pp.300-308
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• 2000

Fibroblast growth factor(FGF)는 세포의 성장과 이동, 분화와 생존과 관련된 여러가지 과정을 조절하는 것으로 알려져 있다. 이들의 prototype인 FGF-1과 FGF-2는 FGF receptors (FGFRs)를 통해서 세포내로 신호를 전달하는데, 두개봉합부의 조기융합을 보이는 craniosynostosis는 FGFRs중, 특히 FGFR-2의 point mutation에 의해서 야기된다. 최근 여러 보고에서 FGF/FGFR 신호전달은 골아세포의 분화에 있어 필수적인 역할을 한다고 하였으며, bFGF soaked bead를 쥐 두개골의 시상봉합부의 mid-mesenchyme과 osteogenic front부위에 적용하였을 때 osteopontin(OPN) 유전자의 발현을 유도한다고 하였다. 이에 본 연구에서는 ST-2 cell line를 이용한 in vitro실험에서 bFGF가 OPN 유전자 발현에 미치는 영향과 그 기전을 Northern blot analysis를 통해서 연구하고자 하였다. 1 ng/ml bFGF의 투여는 OPN, fibronectin 유전자 발현을 증가시켰으며, type I collagen 유전자 발현은 감소시켰으나 alkaline phosphatase 유전자 발현에는 영향을 미치지 않았다. OPN은 그 발현양상이 bFGF의 농도 증가에 따라 증가하는 양상으로 나타났으며, 시간결과에 따른 발현양상도 bFGF 투여 3시간째부터 bFGF를 투여한 군에서 대조군에 비해 높게 나타났으며 이는 24시간까지 시간의 경과에 따라 증가하는 양상을 보였다. 단백질 합성 억제제인 cycloheximide를 처리한 군에서는 OPN의 증가 양상을 보이지 않았는데 이는 bFGF에 의한 OPN유전자 발현이 새로운 전사조절 단백질 합성 등의 여러 단계를 거쳐서 일어남을 의미한다. 결론적으로 bFGF는 새로운 전사조절 단백질의 합성을 통해서 OPN 유전자 발현을 농도 및 시간 의존적으로 증가시킨다.

• 대한치과교정학회지
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• 제23권2호
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• pp.229-248
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• 1993

결합 조직의 대사를 억제하고, 골조직이나 골세포의 골기질 대사 역시 억제하는 것으로 알려져 있는 $Interferon-\gamma(IFN-\gamma)$의 교정치료에의 이용 가능성을 평가하기 위하여, 교정력에 의한 골 개조 과정에서 중심적 역할을 하는 것으로 알려진 치주인대 세포를 primary culture하여 배양된 세포에 $IFN-\gamma$를 투여함으로써 이것이 골조직 기질의 합성능에 미치는 영향에 대하여 관찰하였다. $IFN-\gamma$는 세포의 DNA합성능을 약하게 증가시켰으며, 세포내 DNA 총량에는 영향을 미치지 않았다. 따라서 이 실험에서 사용한 용량의 $IFN-\gamma$는 세포에 독성이 없다고 할 수 있으며, 다른 결합조직 세포에서 나타나는 항증식 효과와는 반대되는 결과였다. $IFN-\gamma$는 비교원성 단백질(NCP)의 합성을 증가시키는 양상을 나타내었으나, 교원질(CDP)의 합성은 감소시키는 경향을 보여, 총단백질에 대한 교원질의 합성비율은 $IFN-\gamma$에 의해 용량 의존적으로 감소하였다. 또한 교원질의 mRNA양은 단백질 합성과 유관하게 $IFN-\gamma$에 의해 억제되었다. 따라서 $IFN-\gamma$는 교원질 합성의 전사과정 혹은 그 이후의 과정에 영향을 미칠 것으로 예상할 수 있다. 한편 Indomethacin의 투여로 $IFN-\gamma$에 의해 억제된 교원질의 합성이 영향을 받지 않았기 때문에 $IFN-\gamma$에 의한 교원질 합성 과정에 prostaglandin이 관련되지 않았음을 알 수 있었다. 반면 fibronectin의 합성은 10 및 100 U/ml의 $IFN-\gamma$ 투여시에는 영향을 받지 않았으나, 1000U/ml의 $IFN-\gamma$투여시에는 유의한 증가를 나타내어, 교원질에서와는 다른 영향을 나타내었다. 또한 mRNA steady state level에서도 $IFN-\gamma$는 교원질의에서와는 달리 fibronectin mRNA 양에는 영향을 미치지 않았다. 즉 $IFN-\gamma$ fibronectin 유전자 발현에 영향을 미치는 부위는 전사나, 전사후 변형단계가 아닌 단백질 합성단계에서 영향을 미침을 보여주었다. 따라서 $IFN-\gamma$ 교원질과 fibronectin합성 조절 영향을 미치는 부위 서로 다름을 알 수 있겠다. Alkaline phospatase의 활성은 10-1000 U/ml의 $IFN-\gamma$를 투여시 약하게 증가시키는 경향을 보였으나 석회화가 일어날 정도로 높게 증가시키지는 못했다. 따라서 $IFN-\gamma$는 골기질의 주성분인 type I 교원질의 합성을 선택적으로 억제하는 기능과 alkaline phosphatase의 활성을 크게 증가시키지 못한 점 등으로 미루어 볼 때, 골개조를 억제하는 방향으로 작용한다고 볼 수 있으며, 교정치료 과정중 골개조를 억제하는 부위에서 사용을 시도해 볼 수 있겠다.

• The Korean Journal of Physiology and Pharmacology
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• 제27권4호
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• pp.417-426
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• 2023

The TRPM4 gene encodes a Ca²⁺-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.

• Anatomy and Cell Biology
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• 제56권4호
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• pp.552-561
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• 2023

The endocrinology of type 2 diabetes (T2D) and its predisposing factors have been studied extensively while its skeletal effects have received negligible research despite this being a global disease. The cellular and molecular association between proximal humeral fractures and T2D has not been fully elucidated. We aimed to study bone cell quantities and immunolabel osteogenic and antiosteogenic cytokines. The study used 12-week-old rats (23 males) consisting of 8 Sprague Dawley (SD) and 15 Zucker Diabetic Sprague Dawley (ZDSD). Weekly mass measurements were taken while fasting blood glucose levels were recorded every 2 weeks with oral glucose tolerance tests conducted once every 4 weeks. Upon termination at the age of 28 weeks, humeri were fixed in 10% buffered formalin, prior to decalcification in ethylenediaminetetraacetic acid. The bone samples were then processed in ascending grades of alcohol using an automatic processor before embedding in paraffin wax. Sections were cut at 5 ㎛ thickness in a series for Haematoxylin and Eosin stain, and immunohistochemistry was performed with the anti-tartrate-resistant acid phosphatase (TRAP), anti-alkaline phosphatase (ALP), anti-bone morphogenetic protein 3 (BMP3), anti-transforming growth factor beta 1 (TGFβ1), anti-aged glycation end product (AGE) antibodies in the sequence. ZDSD rats had more adipocytes, BMP3 and AGEs expression with higher numbers of TRAP positive osteocytes and fewer ALP cells although no differences were found in TGFβ1 immunopositivity. We also found that T2D increases the number of AGEs immuno-positive cells, as well as its extracellular expression, thus providing a conducive environment for the interaction of the osteogenic cytokine and its antagonist to suppress osteoblastogenesis. ZDSD groups had higher adipocyte numbers therefore increased marrow adiposity in T2D.

• Journal of Microbiology and Biotechnology
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• 제23권9호
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• pp.1206-1211
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• 2013

The selective inhibition of PTP1B has been widely recognized as a potential drug target for the treatment of type 2 diabetes and obesity. In the course of screening for PTP1B inhibitory fungal metabolites, the organic extracts of several fungal species isolated from marine environments were found to exhibit significant inhibitory effects, and the bioassay-guided investigation of these extracts resulted in the isolation of fructigenine A (1), cyclopenol (2), echinulin (3), flavoglaucin (4), and viridicatol (5). The structures of these compounds were determined mainly by analysis of NMR and MS data. These compounds inhibited PTP1B activity with 50% inhibitory concentration values of 10.7, 30.0, 29.4, 13.4, and 64.0 ${\mu}M$, respectively. Furthermore, the kinetic analysis of PTP1B inhibition by compounds 1 and 5 suggested that compound 1 inhibited PTP1B activity in a noncompetitive manner, whereas compound 5 inhibited PTP1B activity in a competitive manner.

• BMB Reports
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• 제40권3호
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• pp.302-314
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• 2007

N type calcium channels (CaV2.2) play a key role in the gating of transmitter release at presynaptic nerve terminals. These channels are generally regarded as parts of a multimolecular complex that can modulate their open probability and ensure their location near the vesicle docking and fusion sites. However, the proteins that comprise this component remain poorly characterized. We have carried out the first open screen of presynaptic CaV2.2 complex members by an antibody-mediated capture of the channel from purified rat brain synaptosome lysate followed by mass spectroscopy. 589 unique peptides resulted in a high confidence match of 104 total proteins and 40 synaptosome proteome proteins. This screen identified several known CaV2.2 interacting proteins including syntaxin 1, VAMP, protein phosphatase 2A, $G_{o\alpha}$, G$\beta$ and spectrin and also a number of novel proteins, including clathrin, adaptin, dynamin, dynein, NSF and actin. The unexpected proteins were classified within a number of functional classes that include exocytosis, endocytosis, cytoplasmic matrix, modulators, chaperones, and cell-signaling molecules and this list was contrasted to previous reports that catalogue the synaptosome proteome. The failure to detect any postsynaptic density proteins suggests that the channel itself does not exhibit stable trans-synaptic attachments. Our results suggest that the channel is anchored to a cytoplasmic matrix related to the previously described particle web.