Establishment of a Human Embryonic Germ Cell Line and Comparison with Mouse and Human Embryonic Stem Cells

Hyun Soo Yoon,김철근,Jong Hyuk Park,Sun Jong Kim,Jung Bok Lee,Ji Min Song,Sung Il Roh 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly estab-lished, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differ-entiation over a year of continuous culture. They are karyotypically normal and express high levels of alka-line phosphatase, Oct-4, and several cell-surface mark-ers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.

Graphene Films Show Stable Cell Attachment and Biocompatibility with Electrogenic Primary Cardiac Cells

김태용,김도한,강영호,이탁희,이광희 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.6

Graphene has attracted substantial attention due to its advantageous materialistic applicability. In the present study, we tested the biocompatibility of graphene films synthesized by chemical vapor deposition with electrogenic primary adult cardiac cells (cardiomyocytes) by measuring the cell properties such as cell attachment, survival, contractility and calcium transients. The results show that the graphene films showed stable cell attachment and excellent biocompatibility with the electrogenic cardiomyocytes, suggesting their useful applications for future cell biology studies.

Cytoplasmic Localization and Redox Cysteine Residue of APE1/Ref-1 Are Associated with Its Anti-Inflammatory Activity in Cultured Endothelial Cells

박명수,전병화,김척승,주희경,이유란,강건,김수진,최성아,이상도,박진봉,김국성 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

Apurinic/apyrimidinic endonuclease1/redox factor-1 (APE1/ Ref-1) is a multifunctional protein involved in base excision DNA repair and transcriptional regulation of gene expression. APE1/Ref-1 is mainly localized in the nucleus, but cytoplasmic localization has also been reported. However, the functional role of cytoplasmic APE1/Ref-1 and its redox cysteine residue are still un-known. We investigated the role of cytoplasmic APE1/Ref-1 on tumor necrosis factor-alpha (TNF-alpha)-induced vascular cell adhesion molecule-1 (VCAM-1) expressions in endothelial cells. Endogenous APE1/Ref-1 was mainly observed in the nucleus, however, cytoplasmic APE1/Ref-1 was increased by TNF-alpha. Cytoplasmic APE1/ Ref-1 expression was not blunted by cycloheximide, a protein synthesis inhibitor, suggesting cytoplasmic trans-location of APE1/Ref-1. Transfection of an N-terminus deletion mutant APE1/Ref-1(29-318) inhibited TNF-alpha-induced VCAM-1 expression, indicating an anti-inflam- matory role for APE1/Ref-1 in the cytoplasm. In contrast, redox mutant of APE1/Ref-1 (C65A/C93A) transfection led to increased TNF-alpha-induced VCAM-1 expression. Our findings suggest cytoplasmic APE1/Ref-1 localization and redox cysteine residues of APE1/Ref-1 are associated with its anti-inflam-matory activity in endothelial cells.

New Tools and New Biology: Recent Miniaturized Systems for Molecular and Cellular Biology

Morgan Hamon,Jong Wook Hong 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.6

Recent advances in applied physics and chemistry have led to the development of novel microfluidic systems. Microfluidic systems allow minute amounts of reagents to be processed using m-scale channels and offer several advantages over conventional analytical devices for use in biological sciences: faster, more accurate and more reproducible analytical performance, reduced cell and reagent consumption, portability, and integration of functional components in a single chip. In this review, we introduce how microfluidics has been applied to biological sciences. We first present an overview of the fabrication of microfluidic systems and describe the distinct technologies available for biological research. We then present examples of microsystems used in biological sciences, focusing on applications in molecular and cellular biology.

Cholesterol-Responsive Metabolic Proteins Are Required for Larval Development in Caenorhabditis elegans

가와사키이찌로,심용희,정명환,윤유준,신윤경 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

Caenorhabditis elegans, a cholesterol auxotroph, showed defects in larval development upon cholesterol starvation (CS) in a previous study. To identify cholesterol-respon-sive proteins likely responsible for the larval arrest upon CS, a comparative proteomic analysis was performed between C. elegans grown in normal medium supplemented with cholesterol (CN) and those grown in medium not supplemented with cholesterol (cholesterol starvation, CS). Our analysis revealed significant change (more than 2.2-fold, p < 0.05) in nine proteins upon CS. Six proteins were down-regulated [CE01270 (EEF-1A.1), CE08852 (SAMS-1), CE11068 (PMT-2), CE09015 (ACDH-1), CE12564 (R07H5.8), and CE09655 (RLA-0)], and three proteins were up-regu-lated [CE29645 (LEC-1), CE16576 (LEC-5), and CE01431 (NEX-1)]. RNAi phenotypes of two of the down-regulated genes, R07H5.8 (adenosine kinase) and rla-0 (ribosomal protein), in CN were similar to that of larval arrest in CS, and RNAi of a down-regulated gene, R07H5.8, in CS further enhanced the effects of CS, suggesting that down-regulation of these genes is likely responsible for the larval arrest in CS. All three up-regulated genes con-tain putative DAF-16 binding sites and mRNA levels of these three genes were all decreased in daf-16 mutants in CN, suggesting that DAF-16 activates expression of these genes.

Human Cytomegalovirus (HCMV) US2 Protein Interacts with Human CD1d (hCD1d) and Down-Regulates Invariant NKT (iNKT) Cell Activity

한지혜,전태훈,노승배,이재연,배준범,박세호,이석준,이상열,안규리,김재영 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-mem-brane glycoprotein US2, which interferes with antigen presenting mechanism of major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and 3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells.

Anticancer Effects of the Engineered Stem Cells Transduced with Therapeutic Genes via a Selective Tumor Tropism Caused by Vascular Endothelial Growth Factor Toward HeLa Cervical Cancer Cells

김혜선,최경철,이보림,황경아,김승우 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.4

The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTECs) expressing bacterial cytosine deaminase (CD) and/ or human interferon-beta (IFN-) gene against HeLa cervical cancer and the migration factors of the GESTECs toward the cancer cells. Anticancer effect of GESTECs was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed so as to assess the migration capability of the stem cells to cervical cancer cells. Next, several che-moattractant ligands and their receptors related to a selective migration of the stem cells toward HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by CD gene and it caused the cell death in a co-culture system. When IFN-β was additionally expressed with CD gene by these GESTECs, the anticancer activity was significantly increased. In the migration assay, the GESTECs selectively migrated to HeLa cervical cancer cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GES-TECs. These GESTECs transduced with CD gene and IFN- may provide a potential of a novel gene therapy for anti-cervical cancer treatments via their selective tumor tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTECs.

The Protein Kinase 2 Inhibitor CX-4945 Regulates Osteoclast and Osteoblast Differentiation In Vitro

손유화,김지연,문성희 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

Drug repositioning can identify new therapeutic applica-tions for existing drugs, thus mitigating high R&D costs. The Protein kinase 2 (CK2) inhibitor CX-4945 regulates human cancer cell survival and angiogenesis. Here we found that CX-4945 significantly inhibited the RANKL-indu-ced osteoclast differentiation, but enhanced the BMP2-induced osteoblast differentiation in a cell culture model. CX-4945 inhibited the RANKL-induced activation of TRAP and NFATc1 expression accompanied with sup-pression of Akt phosphorylation, but, in contrast, it enhanced the BMP2-mediated ALP induction and MAPK ERK1/2 phosphorylation. CX-4945 is thus a novel drug candidate for bone-related disorders such as osteoporosis.

Enzymatic Synthesis of Apigenin Glucosides by Glucosyltransferase (YjiC) from Bacillus licheniformis DSM 13

Rit Bahadur Gurung,송재경,김은희,오태진 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.4

Apigenin, a member of the flavone subclass of flavono-ids, has long been considered to have various biological activities. Its glucosides, in particular, have been reported to have higher water solubility, increased chemical stability, and enhanced biological activities. Here, the synthesis of apigenin glucosides by the in vitro glucosylation reaction was successfully performed using a UDP-glucosyltrans-ferase YjiC, from Bacillus licheniformis DSM 13. The glucosylation has been confirmed at the phenolic groups of C-4′ and C-7 positions ensuing apigenin 4′-O-glucoside, apigenin 7-O-glucoside and apigenin 4′,7-O-diglucoside as the products leaving the C-5 position unglucosylated. The position of glucosylation and the chemical structures of glucosides were elucidated by liquid chromatography/ mass spectroscopy and nuclear magnetic resonance spec- troscopy. The parameters such as pH, UDP glucose concentration and time of incubation were also analyzed during this study.

Identification of Loop Nucleotide Polymorphisms Affecting MicroRNA Processing and Function

Xiaoxing Xiong,Shengmei Zhu,Xianhui Kang,Yueying Zheng,Sibiao Yue 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.6

MicroRNAs are short 21-22 nucleotide single strand RNAs that are involved in post-transcriptional regulation of gene expression. Most microRNAs are first transcribed as long primary microRNAs and then undergo a two step-wise sequential processing to yield single-stranded mature microRNAs. It has been suggested that the loop region of primary microRNAs plays an important role in regulating microRNA biogenesis and target recognition. However, despite the fact that several single nucleotide polymorphisms have been identified in mature microRNA sequences and are related to human diseases, it remains unclear whether and how the single nucleotide polymorphisms in the loop regions of primary microRNAs would affect the biogenesis and function of microRNAs. Herein, we provide evidence that primary microRNAs loop nucleo-tides control the accuracy and efficiency of microRNA processing. Accordingly, we identified 32 single nucleotide polymorphisms in the loop regions of human primary microRNAs using bioinformatics, and further validated three loss-of-function and one gain-of-function single nucleotide polymorphisms using dual-luciferase assays. Thus, these results reveal a critical regulatory role encoded in the loop nucleotides of primary microRNAs for microRNA processing and function.