• Journal of Ginseng Research
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• 제29권4호
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• pp.167-175
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• 2005

The $Ca^{2+}-activated$ chloride channel (CLCA) was activated by ginseng total saponin (GTS) in Xenopus oocytes. The reverse transcription PCR (RT-PCR) method was performed with gene specific primers on oocytes. The gene specific primers were deduced from spleen cDNA in expressed sequence tags (EST) database showing high homology to the mouse CLCA. Full length of cDNA sequence was completed by linkage of several 5' and 3'-half cDNA fragments have been sequenced. We named the full cDNA to oCLCA transiently. The oCLCA gene encodes a protein of 911 amino acids with $48.9\%$ identity overall to that of mouse CLCA (mCLCA4). A predicted oCLCA amino acids sequence shows the molecular weight of 108 kDa and has four or more transmembrane domains, and also the one hydrophobic C­terminal domain. oCLCA gene was expressed ubiquitously in various tissues included oocytes, also interfered in oocytes by siRNA for oCLCA. Here, we suggest that oCLCA is a endogenous chloride channel gene in oocytes. We are studying for the identification of oCLCA gene and further physiological research.

• Journal of Gastric Cancer
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• 제15권1호
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• pp.39-45
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• 2015

Purpose: Recent studies have revealed recurrent alterations in the cell adhesion gene FAT4, a candidate tumor suppressor gene, in cancer. FAT atypical cadherin 4 (FAT4) is a transmembrane receptor involved in the Hippo signaling pathway, which is involved in the control of organ size. Here, we investigated the loss of FAT4 expression and its association with clinicopathological risk factors in gastric cancer. Materials and Methods: We assessed the expression of FAT4 by using immunohistochemistry on three tissue microarrays containing samples from 136 gastric cancer cases, radically resected in the Soonchunhyang University Cheonan Hospital between July 2006 and June 2008. Cytoplasmic immunoexpression of FAT4 was semi-quantitatively scored using the H-score system. An H-score of ${\geq}10$ was considered positive for FAT4 expression. Results: Variable cytoplasmic expressions of FAT4 were observed in gastric cancers, with 33 cases (24.3%) showing loss of expression (H-score <10). Loss of FAT4 expression was associated with an increased rate of perineural invasion (H-score <10 vs. ${\geq}10$, 36.4% vs. 16.5%, P=0.015), high pathologic T stage (P=0.015), high tumor-node-metastasis stage (P=0.017), and reduced disease-free survival time (H-score <10 vs. ${\geq}10$, mean survival $62.7{\pm}7.3$ months vs. $79.1{\pm}3.1$ months, P=0.025). However, no association was found between the loss of FAT4 expression and tumor size, gross type, histologic subtype, Lauren classification, lymphovascular invasion, or overall survival. Conclusions: Loss of FAT4 expression appears to be associated with invasiveness in gastric cancer.

• 한국수정란이식학회지
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• 제29권4호
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• pp.375-383
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• 2014

Klotho (KL) is a single transmembrane protein composed of KL1 and KL2 repeats possessing ${\beta}$-glucuronidase activity and maintains calcium homeostasis in physiological state. It has been implicated in pigs that calcium is important for the establishment and maintenance of pregnancy, and our previous study has shown that transient receptor potential vanilloid type 6 (TRPV6), a calcium ion transporter, is predominantly expressed in the uterine endometrium during pregnancy in pigs. However, expression and function of KL in the uterine endometrium has not been determined in pigs. Thus, the present study determined expression and regulation of KL in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of KL mRNA decreased between Days 12 to 15 of the estrous cycle, and its expression showed a biphasic manner during pregnancy. KL mRNA was expressed in conceptuses and in chorioallantoic tissues during pregnancy. Explant culture study showed that expression levels of KL were not affected by treatment of steroid hormones or interleukin-1beta during the implantation period. Furthermore, levels of KL mRNA in the uterine endometrium from gilts carrying somatic cell nuclear transfer (SCNT)-derived embryos were significantly lower than those from gilts carrying natural mating-derived embryos on Day 12 of pregnancy. These results exhibited that KL was expressed at the maternal-conceptus interface in a pregnancy status- and stage-specific manner, and its expression was affected by SCNT procedure, suggesting that KL may play an important role in the establishment and maintenance of pregnancy in pigs.

• 대한수의학회지
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• 제42권1호
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• pp.55-64
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• 2002

HIV-1의 envelope glycoprotein은 중화항체에 의한 체액성 면역반응의 중요한 target으로 surface glycoprotein인 gp120과 transmembrane glycoprotein인 gp41로 이루어져 있다. gp120과 gp41의 ectodomain으로 이루어진 gp140 유전자를 PCR의 방법으로 증폭하고 Semliki Forest virus(SFV) 유래 expression system을 이용하여 mammalian 세포에서 발현하였다. 발현된 gp140은 natural HIV-1에서와 같이 oligomer를 형성하였다. 발현된 gp140을 정제하여 BALB/c 마우스에 접종하여 항체가 형성되었음을 확인하였다.

• Molecules and Cells
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• 제38권1호
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• pp.75-80
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• 2015

Osteoclasts are unique cells responsible for the resorption of bone matrix. MicroRNAs (miRNAs) are involved in the regulation of a wide range of physiological processes. Here, we examined the role of miR-26a in RANKL-induced osteoclastogenesis. The expression of miR-26a was upregulated by RANKL at the late stage of osteoclastogenesis. Ectopic expression of an miR-26a mimic in osteoclast precursor cells attenuated osteoclast formation, actin-ring formation, and bone resorption by suppressing the expression of connective tissue growth factor/CCN family 2 (CTGF/CCN2), which can promote osteoclast formation via upregulation of dendritic cell-specific transmembrane protein (DC-STAMP). On the other hand, overexpression of miR-26a inhibitor enhanced RANKL-induced osteoclast formation and function as well as CTGF expression. In addition, the inhibitory effect of miR-26a on osteoclast formation and function was prevented by treatment with recombinant CTGF. Collectively, our results suggest that miR-26a modulates osteoclast formation and function through the regulation of CTGF.

• Molecules and Cells
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• 제44권10호
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• pp.758-769
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• 2021

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I_CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I_CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I_CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I_CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.

• Journal of Interdisciplinary Genomics
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• 제3권1호
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• pp.21-24
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• 2021

Purpose: Activating mutations of the calcium-sensing receptor (CASR) are a rare genetic disorder, and result in autosomal dominant hypocalcemia with hypercalciuria (ADHH). ADHH exhibited varying degrees of hypocalcemia. In this study, we report the clinical and molecular characteristics of activating variants in CASR patients diagnosed in Korea. Methods: This study included three patients with activating variants of CASR confirmed by biochemical and molecular analysis of CASR. Clinical and biochemical findings were reviewed chart retrospectively. Mutation analysis of CASR was performed by Sanger sequencing. Results: Subject 1 showed severe symptoms from the neonatal period and had difficulty in controlling the medications that were administered. Subject 2 was identified as having a novel variant of CASR with hypocalcemia and a low parathyroid hormone that were found in the neonatal period. During a course without medication, hypocalcemia occurred suddenly around 2 years of age. Subject 3 was diagnosed with hypoparathyroidism with hypocalcemic seizures starting from the neonatal period. About 4 years without taking medication with any symptom. However, at 10 years old revisited by repetitive hypocalcemic seizure events. Subject 1 and 3, were heterozygous for c.2474A>T (p.Y825F), c.2395G>A (p.E799K) located in the transmembrane domain (TMD) of CASR. Subject 2 was heterozygous for c.403A>C (S430L) located in the extracellular domain (ECD) of CASR. Conclusion: We reported 3 patients who have activating CASR variant with different onset and severity of symptoms. In the future, further study is needed to determine how the protein level according to the location of the mutation of CASR affects the degree of symptoms.

• Molecules and Cells
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• 제41권12호
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• pp.1052-1060
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• 2018

Triclosan (TCS) is a phenolic antimicrobial chemical used in consumer products and medical devices. Evidence from in vitro and in vivo animal studies has linked TCS to numerous health problems, including allergic, cardiovascular, and neurodegenerative disease. Using Caenorhabditis elegans as a model system, we here show that short-term TCS treatment ($LC_{50}$: ~0.2 mM) significantly induced mortality in a dose-dependent manner. Notably, TCS-induced mortality was dramatically suppressed by co-treatment with non-ionic surfactants (NISs: e.g., Tween 20, Tween 80, NP-40, and Triton X-100), but not with anionic surfactants (e.g., sodium dodecyl sulfate). To identify the range of compounds susceptible to NIS inhibition, other structurally related chemical compounds were also examined. Of the compounds tested, only the toxicity of phenolic compounds (bisphenol A and benzyl 4-hydroxybenzoic acid) was significantly abrogated by NISs. Mechanistic analyses using TCS revealed that NISs appear to interfere with TCS-mediated mortality by micellar solubilization. Once internalized, the TCS-micelle complex is inefficiently exported in worms lacking PMP-3 (encoding an ATP-binding cassette (ABC) transporter) transmembrane protein, resulting in overt toxicity. Since many EDCs and surfactants are extensively used in commercial products, findings from this study provide valuable insights to devise safer pharmaceutical and nutritional preparations.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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• pp.1288-1298
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• 2019

Bacterial ATP synthases drive ATP synthesis by a rotary mechanism, and play a vital role in physiology and cell metabolism. Corynebacterium glutamicum is well known as an industrial workhorse for amino acid production, and its ATP synthase operon contains eight structural genes and two adjacent genes, cg1360 and cg1361. So far, the physiological functions of Cg1360 (GenBank CAF19908) and Cg1361 (GenBank CAF19909) remain unclear. Here, we showed that Cg1360 was a hydrophobic protein with four transmembrane helices (TMHs), while no TMH was found in Cg1361. Deletion of cg1360, but not cg1361, led to significantly reduced cell growth using glucose and acetic acid as carbon sources, reduced F1 portions in the membrane, reduced ATP-driven proton-pumping activity and ATPase activity, suggesting that Cg1360 plays an important role in ATP synthase function. The intracellular ATP concentration in the ${\Delta}cg1360$ mutant was decreased to 72% of the wild type, while the NADH and NADPH levels in the ${\Delta}cg1360$ mutant were increased by 29% and 26%, respectively. However, the ${\Delta}cg1361$ mutant exhibited comparable intracellular ATP, NADH and NADPH levels with the wild-type strain. Moreover, the effect of cg1360 deletion on L-valine production was examined in the L-valine-producing V-10 strain. The final production of L-valine in the $V-10-{\Delta}cg1360$ mutant reached $9.2{\pm}0.3g/l$ in shake flasks, which was 14% higher than that of the V-10 strain. Thus, Cg1360 can be used as an effective engineering target by altering energy metabolism for the enhancement of amino acid production in C. glutamicum.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.