• Journal of Life Science
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• v.28 no.4
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• pp.389-397
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• 2018

The structure of glycan residues attached to glycoproteins can influence the biological activity, stability, and safety of pharmaceutical proteins delivered from transgenic pig milk. The production of therapeutic glycoprotein in transgenic livestock animals is limited, as the glycosylation of mammary gland cells and the production of glycoproteins with the desired homogeneous glycoform remain a challenge. The ${\beta}$-1,3-N-acetylglucosaminylatransferase1 (B3GNT1) gene is an important enzyme that attaches N-acetylglucosamine (GlcNAc) to galactose (Gal) residues for protein glycosylation; however, there is limited information about pig glycosyltransferases. Therefore, we cloned the pig B3GNT1 (pB3GNT1) and investigated its functional properties that could attach N-acetylglucosamine to galactose residue. Using several different primers, a partial pB3GNT1 mRNA sequence containing the full open reading frame (ORF) was isolated from liver tissue. The ORF of pB3GNT1 contained 1,248 nucleotides and encoded 415 amino acid residues. Organ-dependent expression of the pB3GNT1 gene was confirmed in various organs from adult and juvenile pigs. The pB3GNT1 mRNA expression level was high in the muscles of the heart and small intestine but was lower in the lungs. For functional characterization of pB3GNT1, we established a stable expression of the pB3GNT1 gene in the porcine kidney cell line (PK-15). As a result, it was suggested that the glycosylation pattern of pB3GNT1 expression in PK-15 cells did not affect the total sialic acid level but increased the poly N-acetyllactosamine level. The results of this study can be used to produce glycoproteins with improved properties and therapeutic potential for the generation of desired glycosylation using transgenic pigs as bioreactors.

• Journal of Korean Medicine Rehabilitation
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• v.23 no.4
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• pp.9-21
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• 2013

Objectives The purpose of this study was investigating effects of Jengjengamiyijin-tang (zhengzhuanjiaweierchentang) (JGYT) on lowering lipid, antioxidation and production of inflammatory mediators being used rats fed on high oxidized fat. Methods We divided fat Sprague-Dawley rats fed on high oxidized into 4 groups. Each of 8 rats was divided into a control group and experimental groups. We fed a control group of rats a basal diet and administered normal saline (100 mg/kg, 1 time/1 day) for 4 weeks. And We fed each experimental group of rats basal diet and administered an extract of JGYT extracts (100 mg/kg, 200 mg/kg, 300 mg/kg, 1 time/1 day) for 4 weeks. At the end of the experiment, the rats were sacrificed to determine their chemical composition. We measured lipid of plasma and liver, concentration of proinflammatory cytokines, antioxidative activity and plasma tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), plasma interleukin-6 (IL-6), Apo-B, Apo-E and Leptin gene expression. Results 1. Concentration of plasma FFA, LDL-cholesterol, plasma and liver total cholesterol showed a significant decrement in JGYT groups. However, concentration of plasma HDL-cholesterol showed a significant increment in JGYT groups. 2. Concentration of plasma and liver TG, TBARS showed a significant decrement in JGYT groups. However, concentration of liver GSH-Px, SOD and CAT showed a significant increment in JGYT groups. 3. Plasma GPT activity and concentration of plasma IL-6, TNF-${\alpha}$, NO, Ceruloplasmin, ${\alpha}1$-acid glycoprotein showed a significant decrement in JGYT groups. 4. In the analysis of RT-PCR, gene expression of Apo-B and Apo-E in the JGYT groups showed a low expression than that of control group. However, the gene expression of leptin showed no difference in all the treatment groups. 5. The ratio of leptin expression per ${\beta}$-actin expression showed no significant difference among all treatment groups. However, The ratio of Apo-B and Apo-E expression per ${\beta}$-actin expression showed a significant decrement in JGYT groups. Conclusions According to this study, extract of JGYT showed a positive effect in lowering lipid, antioxidation and control of inflammatory mediators production.

• Archives of Pharmacal Research
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• v.16 no.1
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• pp.36-42
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• 1993

To find biologically active components of the higher fungi of Korea, the carpophores of Auricularia polytricha, a well-known edible mushroom, were extracted with 0.14 M NaCl solution. The extract was successively fractionated by adding ammonium sulfate at various concentrations, and the respective precipitates were separated by centrifugation, then dialyzed and freeze-dried. When a does of 60 mg/kg of each was injected i.p. into ICR mice, the fraction which precipitated at 20% ammonium sulfate showed the highest toxicity, killing seven out of seven mice within two days. The fraction obtained at 40% ammonium sulfate showed the second highest toxicity. The two fractions were named auritoxin I and II after the genus name. However, they Nere shown to have nearly identical composition by physicochemical and 6.8% protein. The polysaccharide moiety was found to have 12.3% $\alpha$-linkage and 87.7% $\beta$-linkage and to be a heteromannoglucan consisting of 45.1% glucose, 435 mannose and 11.0% xylose. The protein moiety contained ten amino adids. The molecular weight of the toxin was $1.5\times10^6$ dalton by Sepharose CL-4B gel filtration. The modian lethal doses of auritoxin in mice were 56.4, 157.2 and 454.6 mg/kg by i.p., s.c. and p.o.administrations, respectively. The signs of intrxication were convulsion during the first 30 minutes after the injection, coma or sleeping within an hour, termor, lacrimation, nasal bleeding congestion, and death in 24 hours. Smong the various organs, the spleen was found to be enlarged remarkably. Human platelet aggregation was inhibited by the addition of auritoxin. The activity of malic dehydrogenase in vitro was inhibited by the toxin.

• 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.

• Journal of Ginseng Research
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• v.39 no.3
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• pp.279-285
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• 2015

Background: Korean Red Ginseng has been used as a traditional oriental medicine to treat illness and to promote health for several thousand years in Eastern Asia. It is widely accepted that ginseng saponins, ginsenosides, are the major active ingredients responsible for Korean Red Ginseng's therapeutic activity against many kinds of illness. Although the crude saponin fraction (CSF) displayed antiplatelet activity, the molecular mechanism of its action remains to be elucidated. Methods: The platelet aggregation was induced by collagen, the ligand of integrin ${\alpha}_{II}{\beta}_I$ and glycoprotein VI. The crude saponin's effects on granule secretion [e.g., calcium ion mobilization and adenosine triphosphate (ATP) release] were determined. The activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 MAPK, and phosphoinositide 3-kinase (PI3K)/Akt was analyzed by immunoblotting. In addition, the activation of integrin ${\alpha}_{II}b{\beta}_{III}$ was examined by fluorocytometry. Results: CSF strongly inhibited collagen-induced platelet aggregation and ATP release in a concentration-dependent manner. It also markedly suppressed $[Ca^{2+}]_i$ mobilization in collagen-stimulated platelets. Immunoblotting assay revealed that CSF significantly suppressed ERK1/2, p38, JNK, PI3K, Akt, and mitogen-activated protein kinase kinase 1/2 phosphorylation. In addition, our fraction strongly inhibited the fibrinogen binding to integrin ${\alpha}_{IIb}{\beta}_3$. Conclusion: Our present data suggest that CSF may have a strong antiplatelet property and it can be considered as a candidate with therapeutic potential for the treatment of cardiovascular disorders involving abnormal platelet function.

• 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 Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.133.1-133
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• 2003

Gummy stem blight, caused by Didymella bryoniae occurs exclusively on cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. Through this study, we optimized cultural conditions for mass-production of pycnidiospore by Metal Halide Lamp irradiation. In brief, the mycelial was cultured at $26^{\circ}C$ on PDA, for 2 days under the darkness, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$, a great number of pycnidia was simultaneously formed. Thus produced pycnidiospores were used as immunogen. From fusions of myeloma cell (v-653) with splenocytes from immunifed mice were car ried out. And, two hybridoma cell lines that recognized the immunogen Didymella bryoniae were obtained. One Monoclonal Antibody, Db1, recognized the supernatant and the other monoclonal antibody, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid two MAb Db1 and Db15, were immunotyped and identified as IgG1 and IgG2b, respectively. Titer of MAb Db1 and MAb Db15 was measured absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with Didymella bryoniae but none reacted with other of fungi and CMV, CGMMV Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{3}$ well by indirect ELISA characterization of the MAb Db1, Db15 antigen by heat and protease treatments show that the epitope recognized by the MAb Bb1, Db15 were a glycoprotein.

• 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.

• 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.