• Journal of Animal Science and Technology
• /
• v.46 no.6
• /
• pp.909-916
• /
• 2004

CCAAT/enhancer binding proteins(C/EBP) are a group of transcription factors expressed during preadipocyte differentiation. In the C/EBPs, C/EBPa plays an important role in lipid deposition and adipocyte differentiation. In this studies, we report the identification, characterization, and expression of a Hanwoo CIEBP$\alpha$ The Hanwoo C/EBP$\alpha$DNA includes a 1059 bp open reading frame encoding a protein of 353 amino acids. The CIEBPa amino acid sequences of the Hanwoo show strong conservation with the corresponding sequences reported in other species. The distribution of C/EBP$\alpha$ mRNA in various tissues of Hanwoo aged 12 months were investigated using Northern blotting analysis. The highest expression was detected in adipose tissue and more lower expression was detected in colon and lung. We also identified expression of C/EBPa mRNA in Hanwoo sirloin and adipose tissue aged 12, 26, and 30 months by real-time RT-PCR. The higest expression were detected at 26 months in the sirloin and at 12 and 26 months in the adipose tissue.

• Journal of Periodontal and Implant Science
• /
• v.34 no.1
• /
• pp.205-221
• /
• 2004

Periodontal ligament(PDL) cells have been known as playing an important roles in periodontal regeneration and gingival fibroblasts are also important to periodontal regeneration by forming connective tissue attachment. There were rare studies about the gene expression patterns of PDL cells and gingival fibroblasts, therefore in this study, we tried cDNA microarray-based gene expression monitoring to explain the functional differences of PDL cells and gingival fibroblasts in vivo and to confirm the characteristics of PDL cells. Total RNA were extracted from PDL cells and gingival fibroblasts of same person and same passages, and mRNA were isolated from the total RNA using Oligotex mRNA midi kit(Qiagen) and then fluorescent cDNA probe were prepared. And microarray hybridization were performed. The gene expression patterns of PDL cells and gingival fibroblasts were quite different. About 400 genes were expressed more highly in the PDL cells than gingival fibroblasts and about 300 genes were more highly expressed in the gingival fibroblasts than PDL cells. Compared growth factor- and growth factor receptor-related gene expression patterns of PDL cells with gingival fibroblasts, IGF-2, IGF-2 associated protein, nerve growth factor, placental bone morphogenic protein, neuron-specific growth- associated protein, FGF receptor, EGF receptor-related gene and PDGF receptor were more highly expressed in the PDL cells than gingival fibroblasts. The results of collagen gene expression patterns showed that collagen type I, type III, type VI and type VII were more highly expressed in the PDL cells than gingival fibroblasts, and in the gingival fibroblasts collagen type V, XII were more highly expressed than PDL cells. The results of osteoblast-related gene expression patterns showed that osteoblast specific cysteine-rich protein were more highly expressed in the PDL cells than gingival fibroblasts. The results of cytoskeletal proteins gene expression patterns showed that a-smooth muscle actin, actin binding protein, smooth muscle myosin heavy chain homolog and myosin light chain were more highly expressed in the PDL cells than gingival fibrobalsts, and ${\beta}-actin$, actin-capping protein(${\beta}$ subunit), actin- related protein Arp3(ARP) and myosin class I(myh-1c) were more highly expressed in the gingival fibroblasts than PDL cells. Osteoprotegerin/osteoclastogenesis inhibitory factor(OPG/OCIF) was more highly expressed in the PDL cells than gingival fibroblasts. According to the results of this study, PDL cells and gingival fibroblasts were quite different gene expression patterns though they are the fibroblast which have similar shape. Therefore PDL cells & gingival fibroblasts are heterogeneous populations which represent distinct characteristics. If more studies about genes that were differently expressed in each PDL cells & gingival fibroblasts would be performed in the future, it would be expected that the characteristics of PDL cells would be more clear.

• BMB Reports
• /
• v.39 no.6
• /
• pp.677-685
• /
• 2006

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be targeted for refolding or degradation to maintain the homeostasis of the ER. Derlin-1 was reportedly implicated in the retro-translocation of misfolded proteins from the ER to the cytosol for degradation. In this report, we showed that Derlin-1 was down-regulated in the endothelial cells derived from human hepatic cavernous hemangioma (CHEC) compared with other tested cells. Electron microscopy analysis showed that ER was aberrantly enlarged in CHEC cells, but not in other tested cells. When overexpressed, Derlin-1 induced the dilated ER to return normal size. This ER dynamic was associated with the activation of unfolded protein response (UPR). In CHEC cells where Derlin-1 was down-regulated, increased expression of the immunoglobulin heavy chain-binding protein (Bip) and UPR-specific splicing of X-box DNA-binding protein 1 (XBP1) mRNA were detected, as compared with that in other tested cells, indicating that UPR was activated. After Derlin-1 overexpression, the extent of UPR activation diminished, as evidenced by decreased expression of Bip, reduced amount of the spliced form of XBP1 ($XBP1_S$), and elevated expression of the unspliced form of XBP1 ($XBP1_U$). Taken together, these findings provide another example of a single protein being able to affect ER dynamic in mammalian cells, and an insight into the possible molecular mechanism(s).

• KSBB Journal
• /
• v.24 no.4
• /
• pp.403-409
• /
• 2009

H2AX, a crucial component of chromatin, is implicated in DNA repair, cell cycle check point and tumor suppression. The aim of this study was to identify direct binding partners of H2AX to regulate cellular responses to above mechanisms. Literature reviews and bioinformatical tools were attempted intensively to find binding partners of H2AX, which resulted in identifying two potential proteins, breast cancer-1 (BRCA1) and BRCA1-associated RING domain 1 (BARD1). Although it has been reported in vivo that BRCA1 co-localizes with H2AX at the site of DNA damage, their biochemical mechanism for H2AX were however only known that the complex monoubiquitinates histone monomers, including unphosphorylated H2AX in vitro. Therefore, it is important to know whether the complex directly interacts with H2AX, and also which regions of these are specifically mediated for the interaction. Using in vitro GST pull-down assay, we present here that BRCA1 and BARD1 directly bind to H2AX. Moreover, through combinational approaches of domain analysis, fragment clonings and in vitro binding assay, we revealed molecular details of the BRCA1-H2AX and BARD1-H2AX complex. These data provide the potential evidence that each of the BRCA1 nuclear localization signal (NLS) and BARD1 BRCA1 C-terminal (BRCT) repeat domain is the novel mediator of H2AX recognition.

• Biomedical Science Letters
• /
• v.9 no.2
• /
• pp.59-65
• /
• 2003

When cells are exposed to proteotoxic stresses such as heat shock, amino acid analogs, and heavy metals, they increase the synthesis of the heat shock proteins (HSPs) by activating the heat shock transcription factor 1 (HSF1), whose activity is controlled via multiple steps including homotrimerization, nuclear translocation, DNA binding, and hyperphosphorylation. Under unstressed conditions, the HSF1 activity is repressed through its constitutive phosphorylation by glycogen synthase kinase 3$\beta$ (GSK3$\beta$), extracellular regulated kinase 1/2 (ERK1/2), and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). However, the protein kinase (s) responsible for HSF1 hyperphosphorylation and activation is not yet identified. In the present study, we observed that profile of p38 mitogen-activated protein kinase (p38MAPK) activation in response to heat shock was very similar to those of HSF1 hyperphosphorylation and nuclear translocation. Therefore, we investigated whether p38MAPK is involved in the heat shock-induced HSF1 activation and HSP expression. Here we show that the p38MAPK inhibitors, SB202190 and SB203580, but not other inhibitors including the MEK1/2 inhibitor PD98059 and the PI3-K inhibitor LY294002 and wortmannin, suppress HSF1 hyperphosphorylation in response to heat shock and L-azetidine 2-carboxylic acid (Azc), but not to heavy metals. Furthermore, heat shock-induced HSF1-DNA binding and HSP72 expression was specifically prevented by the p38MAPK inhibitors, but not by the MEK1/2 inhibitor and the PI3-K inhibitors. These results suggest that SB202190- and SB203580-sensitive p38MAPK may positively regulate HSP gene regulation in response to heat shock and amino acid analogs.

• Journal of Life Science
• /
• v.28 no.1
• /
• pp.99-104
• /
• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• Korean Journal of Environmental Agriculture
• /
• v.25 no.1
• /
• pp.7-13
• /
• 2006

Plastichouse cultivation for crops and vegetables in the winter has been widely popularized in Korea. In the vinylhouse Ultraviolet B penetration is lower than in the field, and so some problems, as plant overgrowth and outbreak of disease, occurred frequently. The effect of artificial supplement ultraviolet B $(UV-B:280{\sim}320nm)$ radiation on the physiological responses and yield of perilla (perilla frutescens) was investigated UV-B ray was radiated on perilla with the 10th leaf stage at the distance of 90, 120 and 150 cm from the plant canopy for 30 days after planting in the vinylhouse. The production of fresh perilla leaves was high in the order of plastic house, ambient+50% of supplemental UV-B, ambient ambient+100% of supplemental UV-B. Enhanced UV-B radiation affected the intensity of thirty-three proteins in 2-dimensional electrophoretic analysis of proteins and ten proteins out of them seemed to be responsive to UV-B : a protein was, ATP synthase CF1 alpha chain, down regulated and nine proteins (Chlorophyll a/b bindng protein type I, Chlorophyll a/b binding protein type II precursor, Photosystem I P700 chlorophyll a apoprotein A2, DNA recombination and repair protein recF, Galactinol synthase, S-adenosyl-L-methionine, Heat shock protein 21, Calcium-dependent protein kinase(CDPK)-like, Catalase) were up-regulated.

• Journal of Life Science
• /
• v.13 no.5
• /
• pp.642-650
• /
• 2003

To develop a new approach to the treatment of neuroblastoma cells we evaluated the effect of cAMP on the Ewing's sarcoma cell line CHP-100. We observed that the proliferation-inhibitory effect of cAMP analogs was due to cell cycle arrest and induction of apoptosis, which was confirmed by observing the morphological changes and DNA fragmentation. DNA flow cytometric analysis revealed that cAMP arrested the cell cycle progression at the G1 phase, which effects were associated with inhibition of phosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB and the transcription factor E2F-1. cAMP also suppressed the cyclin-dependent kinase (Cdk) 2 and cyclin E-associated kinase activity without changes of their expressions. Furthermore, cAMP induced the levels of Cdk inhibitor $p21^{WAF1/CIP1$ expression and p21 proteins induced by cAMP were associated with Cdk2. Overall, our results identify a combined mechanism involving the inhibition of pRB phosphorylation and induction of p21 as targets for cAMP, and this may explain some of its anti-cancer effects.

• Journal of Ginseng Research
• /
• v.41 no.3
• /
• pp.330-338
• /
• 2017

Background: Ginsenoside Rh2 (G-Rh2) is a ginseng saponin that is widely investigated because of its remarkable antitumor activity. However, the molecular mechanism by which (20S) G-Rh2 triggers its functions and how target animals avoid its cytotoxic action remains largely unknown. Methods: Phage display was used to screen the human targets of (20S) G-Rh2. Fluorescence spectroscopy and UV-visible absorption spectroscopy were used to confirm the interaction of candidate target proteins and (20S) G-Rh2. Molecular docking was utilized to calculate the estimated free energy of binding and to structurally visualize their interactions. MTT assay and immunoblotting were used to assess whether human serum albumin (HSA), bovine serum albumin (BSA), and bovine serum can reduce the cytotoxic activity of (20S) G-Rh2 in HepG2 cells. Results: In phage display, (20S) G-Rh2-beads and (20R) G-Rh2-beads were combined with numerous kinds of phages, and a total of 111 different human complementary DNAs (cDNA) were identified, including HSA which had the highest rate. The binding constant and number of binding site in the interaction between (20S)-Rh2 and HSA were $3.5{\times}10^5M^{-1}$ and 1, and those in the interaction between (20S) G-Rh2 and BSA were $1.4{\times}10^5M^{-1}$ and 1. The quenching mechanism is static quenching. HSA, BSA and bovine serum significantly reduced the proapoptotic effect of (20S) G-Rh2. Conclusion: HSA and BSA interact with (20S) G-Rh2. Serum inhibited the activity of (20S) G-Rh2 mainly due to the interaction between (20S) G-Rh2 and serum albumin (SA). This study proposes that HSA may enhance (20S) G-Rh2 water solubility, and thus might be used as nanoparticles in the (20S) G-Rh2 delivery process.

• IMMUNE NETWORK
• /
• v.16 no.5
• /
• pp.296-304
• /
• 2016

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation.