• BMB Reports
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• v.44 no.7
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• pp.490-495
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• 2011

Transcription factor AP-$2{\alpha}$ involves in the process of mammalian embryonic development and tumorigenesis. Many studies have shown that AP-$2{\alpha}$ functions in association with other interacting proteins. In a two-hybrid screening, the regulatory subunit ${\beta}$ of protein casein kinase 2 ($CK2{\beta}$) was identified as an interacting protein of AP-$2{\alpha}$; we confirmed this interaction using in-vitro GST pull-down and in-vivo co-immunoprecipitation assays; in an endogenous co-immunoprecipitation experiment, we further found the catalytic subunit ${\alpha}$ of protein casein kinase 2 ($CK2{\alpha}$) also exists in the complex. Phosphorylation analysis revealed that AP-$2{\alpha}$ was phosphorylated by CK2 kinase majorly at the site of Ser429, and such phosphorylation could be blocked by CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in a dose-dependent manner. Luciferase assays demonstrated that both $CK2{\alpha}$ and $CK2{\beta}$ enhanced the transcription activity of AP-$2{\alpha}$; moreover, $CK2{\beta}$ increased the stability of AP-$2{\alpha}$. Our data suggest a novel cellular function of CK-2 as a transcriptional co-activator of AP-$2{\alpha}$.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• Yonsei Medical Journal
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• v.59 no.9
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• pp.1064-1071
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• 2018

Purpose: To explore the influence of S100 calcium binding protein A4 (S100A4) knockout (KO) on methionine-choline-deficient (MCD) diet-induced non-alcoholic fatty liver disease (NAFLD) in mice. Materials and Methods: S100A4 KO mice (n=20) and their wild-type (WT) counterparts (n=20) were randomly divided into KO/MCD, Ko/methionine-choline-sufficient (MCS), WT/MCD, and WT/MCS groups. After 8 weeks of feeding, blood lipid and liver function-related indexes were measured. HE, Oil Red O, and Masson stainings were used to observe the changes of liver histopathology. Additionally, expressions of S100A4 and proinflammatory and profibrogenic cytokines were detected by qRT-PCR and Western blot, while hepatocyte apoptosis was revealed by TUNEL staining. Results: Serum levels of aminotransferase, aspartate aminotransferase, triglyceride, and total cholesterol in mice were increased after 8-week MCD feeding, and hepatocytes performed varying balloon-like changes with increased inflammatory cell infiltration and collagen fibers; however, these effects were improved in mice of KO/MCD group. Meanwhile, total NAFLD activity scores and fibrosis were lower compared to WT+MCD group. Compared to WT/MCS group, S100A4 expression in liver tissue of WT/MCD group was enhanced. The expression of proinflammatory ($TGF-{\alpha}$, $IL-1{\beta}$, IL-6) and profibrogenic cytokines ($TGF-{\beta}1$, COL1A1, ${\alpha}-SMA$) in MCD-induced NAFLD mice were increased, as well as apoptotic index (AI). For MCD group, the expressions of proinflammatory and profibrogenic cytokines and AI in KO mice were lower than those of WT mice. Conclusion: S100A4 was detected to be upregulated in NAFLD, while S100A4 KO alleviated liver fibrosis and inflammation, in addition to inhibiting hepatocyte apoptosis.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.2012-2018
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• 2016

Coxsackievirus B3 (CVB3) is the main cause of acute myocarditis and dilated cardiomyopathy. Plant extracts are considered as useful materials to develop new antiviral drugs. We had previously selected candidate plant extracts, which showed anti-inflammatory effects. We examined the antiviral effects by using a HeLa cell survival assay. Among these extracts, we chose the Amomi Cardamomi (Amomi) extract, which showed strong antiviral effect and preserved cell survival in CVB3 infection. We investigated the mechanisms underlying the ability of Amomi extract to inhibit CVB3 infection and replication. HeLa cells were infected by CVB3 with or without Amomi extract. Erk and Akt activities, and their correlation with virus replication were observed. Live virus titers in cell supernatants and viral positive- and negative-strand RNA amplification were measured. Amomi extract significantly increased HeLa cell survival in different concentrations ($100-10{\mu}g/ml$). CVB3 capsid protein VP1 expression (76%) and viral protease 2A-induced eIF4G1 cleavage (70%) were significantly decreased in Amomi extract ($100{\mu}g/ml$) treated cells. The levels of positive- (20%) and negative-strand (80%) RNA were dramatically decreased compared with the control, as revealed by reverse transcription-PCR. In addition, Amomi extract improved mice survival (51% vs 26%) and dramatically reduced heart inflammation in a CVB3-induced myocarditis mouse model. These results suggested that Amomi extract significantly inhibited Enterovirus replication and myocarditis damage. Amomi may be developed as a therapeutic drug for Enterovirus.

• Asian Journal of Turfgrass Science
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• v.3 no.2
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• pp.77-82
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• 1989

This experiment was carried out to determine the value of turfgrass clippings as a source of protein for animal feed. Levels of crude protein(CP) and in vitro dry matter digestibility(JVDMD) were determined for Korean lawngrass (Zoysia Japonica Steud.) under three levels of nitrogen fertilizer ( 0 , 350 and 700kg ha year and three levels of mowing interval between cuts (lO, 20, and 30 days). Sampling was done on the three-year old turffield on which nitrogen and mowing treatments were Imposed. Clippings were collected after mowing, and CP levels were calculated from a measurement of total N(CP = N >< 6.25) l)y Auto-analyser system-2 procedure and IVDMD by Maeng's method which was partly modified with Tilley and Terry's procedure. With the increased nitrogen legime the CP content of leaf clippings varied from 6.6 to 13.6% and that Of stem clippings from 4 to 10.3%. The CP levels of clippings were directly proportional to the N fertilization rate. The highest CP content was recorded in early July when it was 14.1% at 35OkgN ha -and I 18.l%kgN ha-1 with 10-wcek mowing interval. Mowing interval had little effect on the CP content in both leaf and stem clippings. The level of IVDMD in the Korean lawngrass clippings varied with mowing interval. The highest level measured was 47%, hut this varied during the growing season and occurred in the following order : early summer > summer > fall. This experiment has shown that Korean lawngrass clippings can he helpful source of protein for use in some animal feeds and that a high concentration of CP can occur depending upon N fertllizer and that of I VDM I) upon mowing practices.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2383-2386
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• 2013

Senescence marker protein 30 (SMP30), a hepatocellular carcinoma (HCC) associated antigen, was earlier shown by our research group to be highly expressed in HCC paracancerous tissues, but have low levels in HCC tissues. In order to detect anti-SMP30 antibody in serum of HCC patients, we established pET30a-SMP30 and pColdIII-SMP30 expression systems in Escherichia coli. However, the expression product was mainly in the form of inclusion bodies. In this research, we used several combinations of chaperones, four molecular chaperone plasmids with pET30a-SMP30 and five molecular chaperone plasmids with pColdIII-SMP30 to increase the amount of soluble protein. Results showed that co-expression of HIS-SMP30 with pTf16, combined with the addition of osmosis-regulator, and a two-step expression resulted in the highest enhancement of solubility. A total of 175 cases of HCC serum were studied by ELISA to detect anti-SMP30 antibody with recombinant SMP30 protein. Some 22 were positive and x2 two-sided tests all showed P>0.05, although it remained unclear whether there was a relationship between positive cases and clinical diagnostic data.

• Korean Journal of Poultry Science
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• v.46 no.1
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• pp.17-24
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• 2019

Chickens have been considered as well-defined animal bioreactor. The optimized ovalbumin promoter is essential for recombinant protein production in transgenic chicken. Here we try to compare the activity and identify the effect of estrogen on ovalbumin promoter according to each promoter length with estrogen response element (ERE) existence. We cloned two (2.8 and 5.5 kb) ovalbumin promoters that the 5.5 kb contained the ERE but the 2.8 kb did not, and these two promoters were cloned to pGL4.11 vector. Additionally, we constructed another pGL4.11 vector containing of the 4.4 kb (with ERE) ovalbumin promoter deleted with 1 kb between ERE region and the 2.8 kb promoter. For reporter assay, HeLa, MES-SA, LMH/2A, and cEF cells were transfected with all the pGL4.11 vectors. The comparative analysis showed that the mutated 4.4 kb promoter has more potent activity than the 2.8 and 5.5 kb promoters in HeLa, MES-SA, and LMH/2A cells. However, there is no significant difference in cEFs. Also, these cells transfected with the mutated 4.4 kb promoter were treated with the $17{\beta}$-estradiol (0~3,000 nM) and HeLa, MES-SA, and LMH/2A cells showed estrogen responsibilities, but cEFs did not. Besides, the mutated 4.4 kb promoter has still higher activity than the 2.8 and 5.5 kb promoter, and there is no transcriptional induction effect in 2.8 kb promoter at 500 nM estrogen that is blood concentration of laying hens. Hence our study strongly suggested that the mutated 4.4 kb promoter is considered as one of the most efficient length for generating transgenic chicken.

• Animal Bioscience
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• v.36 no.12
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• pp.1842-1852
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• 2023

Objective: Hairy vetch is considered to improve the nutritional value of corn because of its high protein and mineral levels. To better understand the mechanism underlying hairy vetch regulated whole-plant corn silage fermentation, this experiment investigated the fermentation quality and bacterial community of whole-plant corn and hairy vetch mixture. Methods: Whole-plant corn and hairy vetch were mixed at ratios of 10:0 (Mix 10:0), 8:2 (Mix 8:2), 6:4 (Mix 6:4), 4:6 (Mix 4:6), 2:8 (Mix 2:8), and 0:10 (Mix 0:10) on a fresh weight basis. After ensiling 60 days, samples were collected to examine the fermentation dynamics, ensiling characteristics, and bacterial communities. Results: Mix 0:10, Mix 2:8, and Mix 4:6 showed poor fermentation characteristics. Mix 8:2 and Mix 6:4 silages showed high quality, based on the low pH, acetic acid, and ammonia nitrogen levels and the high lactic acid, crude protein, and crude fat contents. The bacterial diversity was affected by the mixing ratio of the two forage species. The genus Lactobacillus dominated the bacterial community in Mix 10:0 silage, whereas with the addition of hairy vetch, the relative abundance of unclassified-Enterobacter increased from 7.67% to 41.84%, and the abundance of Lactobacillus decreased from 50.66% to 13.76%. Conclusion: The silage quality of whole-plant corn can be improved with inclusion levels of hairy vetch from 20% to 40%.

• Clinical and Experimental Pediatrics
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• v.61 no.12
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• pp.403-406
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• 2018

Floating-Harbor syndrome is a rare autosomal dominant genetic disorder associated with SRCAP mutation. To date, approximately 50 cases of Floating-Harbor syndrome have been reported, but none have been reported in Korea yet. Floating-Harbor syndrome is characterized by delayed bony maturation, unique facial features, and language impairment. Here, we present a 6-year-old boy with a triangular face, deep-set protruding eyes, low-set ears, wide nose with narrow nasal bridge, short philtrum, long thin lips, clinodactyly, and developmental delay that was transferred to our pediatric clinic for genetic evaluation. He showed progressive delay in the area of language and cognition-adaption as he grew. He had previously undergone chromosomal analysis at another hospital due to his language delay, but his karyotype was normal. We performed targeted exome sequencing, considering several syndromes with similar phenotypes. Library preparation was performed with the TruSight One sequencing panel, which enriches the sample for about 4,800 genes of clinical relevance. Massively parallel sequencing was conducted with NextSeq. An identified variant was confirmed by Sanger sequencing of the patient and his parents. Finally, the patient was confirmed as the first Korean case of Floating-Harbor syndrome with a novel SRCAP (Snf2 related CREBBP activator protein) mutation (c.7732dupT, p.Ser2578Phefs*6), resulting in early termination of the protein; it was not found in either of his healthy parents or a control population. To our knowledge, this is the first study to describe a boy with Floating-Harbor syndrome with a novel SRCAP mutation diagnosed by targeted exome sequencing in Korea.

• Environmental Mutagens and Carcinogens
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• v.21 no.2
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• pp.82-88
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• 2001

Recently, there has been significant progress in understanding the control process of the cell division cycle. To investigate the influence of toxic substances on the cell cycle, the effect of benzo(a)pyrene (BAP) and mitomycine C (MMC) on synchronized HeLa cells was analyzed during the cell cycle. To synchronize the HeLa cells, 10$^{6}$ cells were grown for 1 day and then treated with 1 mM hydroxyurea for 14 h. The arrested cells were then allowed to proceed through their cell cycle by removing the hydroxyurea and resupplying a fresh medium. The arrested cells in the G1/S transition then proceeded to the S phase after 4 h, the G2/M phase after 8h, and the G1 phase after 12 h, subsequent to the resupply of a fresh medium. In the untreated HeLa cells, the p34$^{cdc2}$ kinase activity, measured using a p34$^{cdc2}$ specific peptide, peaked after 8h (G2/M) and then declined after 12 h (G1). However, treatment with 30 $\mu$M BAP delayed the peak of the p34$^{cdc2}$ kinase activity. The amount of p34$^{cdc2}$ remained unchanged in the untreated, BAP-, and MMC-treated cells throughout the cell cycle. The cyclin B level peaked after 8 h in the untreated cells, yet peaked after 10-12 h in the BAP-treated cells. There was no significant change in the cyclin B level in the MMC-treated cells.