• Korean Journal of Agricultural Science
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• v.38 no.1
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• pp.65-70
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• 2011

Development of mouse fetus brains can be defined morphologically and functionally by three developmental stages, embryo day (ED) 16, postnatal stage one week and eight weeks. These defined stages of brain development may be closely associated with differential gene expression rates due to limited cellular resources such as energy, space, and free water. Complex patterns of expressed genes and proteins during brain development suggests the changes in relative concentrations of proteins rather than the increase in numbers of new gene products. This study was designed to evaluate early protein expression pattern in mouse fetus brain. The mouse brain proteome of fetus at ED 15.5, and 19.5 was obtained using 2-dimensional gel electrophoresis (DE). Analysis of the 2-DE gels in pH 3-10 range revealed the presence of 15 differentially expressed spots, of which 11 spots were identified to be known proteins following MALDI-TOF analysis; 3 spots were up-regulated and 8 spots were down-regulated in the mouse fetus brain at ED 15.5. UP-regulated proteins were identified as MCG18238, isoform M2 of pyruvate kinase isozymes M1/M2, isoform 2 of heterogeneous nuclear ribonucleoprotein K, heterogeneous nuclear ribonucleoprotein H2, creatine kinase B-type, 40S ribosomal protein SA and hemoglobin subunit beta-H1. Down-regulated proteins were putative uncharacterized protein, lactoylglutathione lyase and secreted acidic cysteine rich glycoprotein. Our results revealed composite profiles of mouse fetus brain proteins related to mouse fetus development by 2-DE analysis implying possible roles of these proteins in neural differentiation.

• Fisheries and Aquatic Sciences
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• v.18 no.3
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• pp.273-281
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• 2015

The follistatin (FST) gene encodes a monomeric glycoprotein that plays a role in binding and inhibiting the functions of members of the transforming growth factor (TGF)-${\beta}$ superfamily. Thus, FST facilitates a wide variety of functions, ranging from muscle growth, to inflammation and immunity. In this study, we sought to characterize an FST counterpart, RbFST, which was identified from rock bream Oplegnathus fasciatus. The RbFST cDNA sequence (2,419 bp) contains a 933-bp open reading frame (ORF) that encodes a putative amino acid sequence for RbFST (35 kDa). The putative amino acid sequence contains a Kazal-type serine protease inhibitor domain (51-98 residues) and an EF-hand, calcium-binding domain (191-226 residues). Additionally, this sequence shares a high identity (98.7%) with the Siniperca chuatsi FST sequence, with which it also has the closest evolutionary relationship according to a phylogenetic study. Omnipresent distribution of RbFST transcripts were detected in the gill, liver, spleen, head kidney, kidney, skin, muscle, heart, brain, and intestine of healthy animals, with significantly higher expression levels in the heart, followed by the liver tissue. Under pathogenic stress caused by two bacterial pathogens, Streptococcus iniae and Edwardsiella tarda, RbFST transcription was found to be significantly up-regulated. Altogether, our findings suggest the putative role of RbFST in immune related responses against pathogenic infections, further prefiguring its significance in rock bream physiology.

• Proceedings of the Plant Resources Society of Korea Conference
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• v.18 no.1
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• pp.41-51
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• 2005

We examined whether extracts from 14 local citrus spp. on Jeju Island (Korea) contained chemosensitizing activity that would increase the cytotoxic effect of vincristine(VCR) in drug-resistant cancer cells. We report that methanol extracts from fruits and flowers of some species had a chemosensitizing effect that reversed P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). Using drug-sensitive AML-2/WT and drug-resistant AML-2/D100 in the absence of VCR in human acute myelogenous leukemia cells we found that fruit or flower extracts alone generally had low cytotoxicity $(IC_{50}>200\;{\mu}g/ml)$. In studies examining the effect of extracts on 120 ng/ml VCR cytotoxicity in drug-resistant AML-2/D100 cells, we found that immature fruit extracts had greater chemosensitizing activity than either extracts from mature fruit or flower. Of the 14 species examined, the immature fruit extract from Inchangkyool (Citrus ichangiensis) showed the hishest chemosensitizing index(CI) valus. Immature fruit extracts of Hongkyool(C. tachibana), Byungkyool(C. platymamma), Cheongkyool(C. nippokoreana) and Jinkyool (C. sunki) also strongly potentiated VCR cytotoxicity in AML-2/D100 cells. The chemosensitizing effect of peel extracts was 2-10-fold that of whole fruit extracts from Hongkyool (C. tachibana), Byungkyool (C. platymamma) and Inchangkyool (C. inchangiensis). The CI values for flower extracts were higher than those for mature fruit extracts, but lower than those for immature fruit extracts. These results indicate that immature citrus fruits contain compounds that do not exert their activity solely through cytotoxicity. In particular, Incahngkyool (C. inchangiensis), Byungkyool(C.platymamma), Cheongkyool(C. nippokoreana) and Hongkyool (C. tahibana) may be useful sources of chemosensitizing compounds.

• IMMUNE NETWORK
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• v.14 no.4
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• pp.207-218
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• 2014

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, ${\alpha}$-galactosylceramide (${\alpha}GC$) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific $CD8^+$ T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by ${\alpha}GC$ can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naïve mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after ${\alpha}GC$-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naïve and chronically infected mice. Similar to naïve B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 $CD8^+$ T cells in vivo, thereby allowing the proliferation of functional $CD8^+$ T cells. Importantly, when ${\alpha}GC$ and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific $CD8^+$ T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between ${\alpha}GC$-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.

• BMB Reports
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• v.43 no.8
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• pp.547-553
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• 2010

Biochemical tests such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are useful for diagnosing patients with liver disease. In this study, we tested the association between copy number variation and the hepatic biomarkers AST and ALT based on 8,842 samples from population-based cohorts in Korea. We used Affymetrix Genome-Wide Human 5.0 arrays and identified 10,534 CNVs using HelixTree software. Of the CNVs tested using univariate linear regression, 100 CNVs were significant for AST and 16 were significant for ALT (P < 0.05). We identified 39 genes located within the CNV regions. DKK1 and HS3ST3B1 were shown to play roles in heparan sulfate biosynthesis and the Wnt signaling pathway, respectively. NAF1 and NPY1R were associated with glycoprotein processes and neuropeptide Y receptor activity based on GO categories. PTER, SOX14 and TM7SF4 were expressed in liver. DPYS and CTSC were found to be associated with dihydropyrimidinuria and Papillon-Lefevre syndrome phenotypes using OMIM. NPY5R was found to be associated with dyslipidemia using the Genetic Association Database.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.278-281
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• 2004

The $\alpha$1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 plays a key role for the outer chain initiation of the N-linked oligosaccharides. A search for Hansenula polymorpha genes homologous to S. cerevisiae OCHI (ScOCH1) has revealed seven open reading frames (ORF100, ORF142, ORF168, ORF288, ORF379, ORF576, ORF580). All of the seven ORFs are predicted to be a type II integral membrane protein containing a transmembrane domain near the amino-terminal region and has a DXD motif, which has been found in the active site of many glycosyltransferases. Among this seven-membered OCH1 gene family of H. polymorpha, we have carried out a functional analysis of H. polymorpha ORF168 (HpOCH2) showing the highest identity to ScOCH1. Inactivation of this protein by disruption of corresponding gene resulted in several phenotypes suggestive of cell wall defects, including hypersensitivity to hygromycin B and sodium deoxycholate. The structural analysis of N-glycans synthesized in HpOCH2-disrupted strain (Hpoch2Δ) and the in vitro $\alpha$1,6-mannosyltransferase activity assay strongly indicate that HpOch2p is a key enzyme adding the first $\alpha$1,6-mannose residue on the core glycan Man$_{8}$GlcNAc$_2$. The Hpoch2Δ was further genetically engineered to synthesize a recombinant glycoprotein with the human compatible N-linked oligosaccharide, Man$_{5}$GlcNAc$_2$, by overexpression of the Aspergillus saitoi $\alpha$1,2-mannosidase with the 'HDEL” ER retention signal.gnal.

• BMB Reports
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• v.28 no.3
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• pp.243-248
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• 1995

Cholesteryl ester transfer protein (CETP), a hydrophobic glycoprotein promoting transfer of cholesteryl esters (CE) from high-density lipoproteins (HDL) to lower-density lipoproteins in the plasma, has been recognized a potent atherogenic factor during the development of coronary artery diseases. This study demonstrated a possible utilization of antisense RNA to inhibit expression of the CETP gene using vaccinia virus as an expression system. The CETP cDNA was inserted into a transfer vector (pSC11) in sense and antisense orientations and used to generate recombinant viruses. Recombinants containing sense or antisense orientations of the CETP cDNA were isolated by $TK^-$ selection and X-gal test. The inserted CETP cDNAs in the recombinants were identified by Southern blot analysis and allowed to transcribe in host cells (CV-1). Expressions of the exogenous CETP mRNA, extracted from the CV-1 cells coinfected with viruses containing sense and antisense DNAs, were monitored by Northern blot analysis using the CETP cDNA probe, by Western blot analysis using monoclonal antibody against the C-terminal active region of the CETP and by the CETP assay. Decreased expressions of the exogenous CETP cDNA were clearly evident in the Northern and Western blot analyses as the dose of antisense expression increased. In the CETP assay, the CETP activities decreased compared to the activity obtained from the cell extracts infected with sense construct only.

• Korean Journal of Pharmacognosy
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• v.32 no.1 s.124
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• pp.31-38
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• 2001

A lectin was purified from Allomyrina dichotoma (ADL) by physiological saline extraction, ammonium sulfate fractionation, anion exchange column chromatography on DEAE Sephadex A-50 and gel filtration column chromatography on Sephadex G-200. Several biochemical properties of ADL were characterized as follows: ADL from gel filtration column chromatography showed single band on SDS-PAGE. ADL agglutinated the erythrocytes of rabbit and human A, B, O, AB. Agglutinability was relatively stable at basic pH, and was stable at temperature below $40^{\circ}C$. Agglutinability was not affected by metal ions and EDTA. This lectin was proved to be a glycoprotein which contains 0.47% of sugars. The molecular weight of ADL was estimated to be 97,000 dalton by SDS-PAGE. By amino acid analysis, ADL exhibited high amounts of aspartic acid. The lectin's immunomodulating effect was measured as cytokine production. The productions of 5 cytokines $(IL-1{\alpha},\;IL-2,\;IL-6,\;IFN{\gamma}\;and\;TNF{\alpha})$ from peripheral blood mononuclear cells were measured by ELISA. The lectin induced the highest secretion of IL-2 at 8 hr, $TNF{\alpha}$ at 4 hr, and $IFN{\gamma}$ at 24hr, respectively. These results suggest that ADL can elicit the production of detectable cytokines from PBMC.

• The Journal of Korean Society of Virology
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• v.28 no.4
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• pp.383-391
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• 1998

A murine monoclonal antibody (mAb) specific for the envelope glycoprotein gp120 of human immunodeficiency virus type-I (HIV -1) was chemically coupled to pokeweed antiviral protein (PAP) from Phytolacca americana. The immunotoxin was purified by FPLC using S200 colum. The purified immunotoxin efficiently bound to HIV-infected T cells as evidenced by fluorescenceactivated cell sorter analysis. The immunotoxin selectively killed human T lymphoid lines infected with $HIV-1_{IIIB}$ at less than 250 pM of the immunotoxin cells, while PAP or mAb alone did not have any significant effect on infected cells. The uninfected control T cell lines were not affected. Human cells infected with HIV-2 or other HIV-1 strains were not killed, suggesting that the killing depends completely on the antibody used for coupling. These in vitro results suggest that the PAP-mAb conjugate may be used to selectively remove cells expressing viral antigens from individuals infected with HIV.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.722-727
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• 2005

Cytosolic sialidase (Neu2), a member of the sialidase family that is responsible for hydrolysis of sialic acid from the terminal position of sialoglycoconjugates, is poorly expressed in skeletal muscle and not detected in any other adult tissues. Thus, we isolated Neu2 cDNA using splicing by overlap extension (SOEing). In order to further characterize this enzyme, a His-tagged derivative was expressed in the bacterial expression system and purified by $Ni^{2+}$-affinity chromatography. A recombinant product of approximately 42 kDa had sialidase activity toward 4-methyl-umbelliferyl-$\alpha$-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH and temperature of the recombinant Neu2 for 4MU-NeuAc was 6.0 and $37.5^{\circ}C$, respectively. The metal ions, such as $Cu^{2+}\;and\;Cd^{2+}$, showed strong inhibitory effect on the activity of the enzyme. The enzyme efficiently hydrolyzed the gangliosides GM3 and GD3 and had relatively low activities on ganglioside GD1a and GD1b, $\alpha$2-3 sialyllactose, and sialylated glycoproteins such as fetuin, transferrin, and orsomucoid, but had hardly any activities on $\alpha$2-6 sialyllactose and ganglioside GM1 and GM2. We concluded that the recombinant Neu2 has a sialidase activity toward glycoproteins as well as gangliosides.