• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.188-191
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• 2002

A glucan and a fructan producing enzymes from Leuconostoc mesenteroides NRRL B-1149 were prepared and concentrated from the cu1ture of 1.5% sucrose using polysulfone ultrafiltration hollow fiber in the presence of 0.1% (w/v) Tween 80, 1 mM $CaCl_2$, and 0.02% $NaN_3$. The molecular masses of the enzymes were estimated to be about 213.6 kDa and 180 kDa, respectively, based on the PAS staining for the glucosyltransferase and Mukasa method for fructosyltransferase. Polymers produced by the enzymes showed different solubility; an insoluble glucan and a soluble fructan. The linkages of polymers were determined by methylation using Hakomori reagent and following acid hydrolysis. The glucan was composed of ${\alpha}$-1,6 and 1,3 linkages and the fructan showed similar linkage data of levan.

• Archives of Pharmacal Research
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• v.21 no.6
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• pp.703-706
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• 1998

The anticomplementary activity of stilbenes from medicinal plants in Korea was investigated in vitro. 3,5-Dihydroxy-4'-methoxystilbene (3) was most potent with $IC_{50}$ value of $1.5{\times}10^{-4}M$ followed by rhapontigenin (4), oxyresverastrol (2), 2,3,4',5-tetrahydroxystilbene-2-O-beta-glucoside (9), rhaponticin (8), resverastrol (1), and piceid (7). The activity was found to be increased by a methylation on a hydroxy group of C-4' of 1, but decreased by further methylation on hydroxy groups of C-3 and C-5 and glucosylation on any hydroxy group of 1. Addition of hydroxy group on C-2' of 1 or C-3' of 3 was little affected on the anticomplementary activity but the activity was increased by O-glucosylation on C-2 of 1.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.364-369
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• 2005

This study was performed to elucidate substrate specificity to the locust bean gum galactomannan by Trichoderma harzianum ${\beta}-mannanase$. The medium composition for enzyme production were determined 3% cellulose, 3% corn steep liquor, 1% $KH_2PO_4$, 0.2% $(NH_4){_2}SO_4$, and incubated for 115 hr at $28^{\circ}C$. The ${\beta}-mannanase$ exhibited maximum activity at pH 4.5 and $60^{\circ}C$. Locust bean gum galactomannan was hydrolyzed by the ${\beta}-mannanase$, and then hydrolysates separated by activated carbon column chromatography. The main hydrolysates were composed of D.P 4 and 7 galactosyl mannooligosaccharides by TLC. For the elucidate the structure of D.P 4 and 7 oligosaccharides, methylation analysis was performed. D.P 4 and 7 were identified as M-M-M-M and M-M-M-M-M (G- and M-represent ${\alpha-1,6-D-galactosidic\;and\;{\beta}-1,4-mannosidic$ linkages, respectively). //G-G To investigate the effects of locust bean gum galactosyl mannooligosaccharides on the in vitro growth of B. longum, B. bifidum, B. infantis, and B. breve, Bifidobacterium spp. were cultivated individually on the modified-MRS medium containing carbon source such as D.P 4 and 7 galactosyl mannooligosaccharides, respectively. B. longum grew up 3.4-fold and 4.3-fold more effectively by the replacement of D.P 4 and 7 galactosyl mannooligosaccharides as the carbon source in a comparasion of standard MRS.

• Korean Journal of Microbiology
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• v.32 no.3
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• pp.208-214
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• 1994

We screened many species from a wide variety of bacterial genera for a new type II restriction endonuclease. The purification and characterization of SolI from a soil isolate, Streptoverticillium olivoverticillatum are described here. The enzyme turned out to be an isoschizomer of BamHI. It recognized the hexanucleotide sequence of 5'-G$\downarrow$GATCC-3' and cleaved as in dicated by the arrow, generating a 4 base 5' extension. Unlike its isoschizomer, BamHI, the activity was sensitive to dam methylation within the recognition sequence. Following ammonium sulfate fractionation of the crude extract, heparin-agarose and Affi-gel Blue column chromatography were employed to purify the enzyme. SolI required at least 0.2 mM of $MgCl_2$ for the cleavage to occur. The enzyme exhibited its maximal activity in the absence of NaCl, but was inhibited completely in the presence of 120 mM NaCl. The pH and temperature optima for activity were pH 8.6 and $40^{\circ}C$, respectively. The molecular weight of SolI was estimated to be 43,000 Da by Superose-12 gel filtraion chromatography.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.505.1-505
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• 1986

The gene for iso-1-cytochrome c for Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP6 promoter. The iso-1-cytochrome c gene was cloned as an 856 bp Xho 1-Hind III fragment. When the resulting plasmid was digested at the Hind 111 site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system and the protein products analyzed on SDS polyacrylamide gels. One major band was detected by autofluorography. This band was found to have a molecular weight of 12,000 Da and coincided with the Coomassie staining band of apocytochrome c from S. cerebisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectric focusing gel. The in vitro synthesized iso-a-cytochrome c was methylated by adding partially purified S-adenosyl-L-methionine . protein-lysine N-methyltransferase (Protein methylase III; EC 2.1.1.43) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor pu omycin. The methyl derivatives in the protein were identified as $\varepsilon$-N-mono, di and trimethyllysine by amino acid analysis. The molar ratio of methyl groups incorporated to that of cytochrome c molecules synthesized showed that 23% of the translated cytochrome c molecules were methylated by protein methylase III.

• IMMUNE NETWORK
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• v.24 no.2
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• pp.3.1-3.23
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• 2024

Cigarette smoke extract (CSE)-treated mouse airway epithelial cells (MAECs)-derived exosomes accelerate the progression of chronic obstructive pulmonary disease (COPD) by upregulating triggering receptor expressed on myeloid cells 1 (TREM-1); however, the specific mechanism remains unclear. We aimed to explore the potential mechanisms of CSE-treated MAECs-derived exosomes on M1 macrophage polarization and pyroptosis in COPD. In vitro, exosomes were extracted from CSE-treated MAECs, followed by co-culture with macrophages. In vivo, mice exposed to cigarette smoke (CS) to induce COPD, followed by injection or/and intranasal instillation with oe-TREM-1 lentivirus. Lung function and pathological changes were evaluated. CD68⁺ cell number and the levels of iNOS, TNF-α, IL-1β (M1 macrophage marker), and pyroptosis-related proteins (NOD-like receptor family pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain, caspase-1, cleaved-caspase-1, gasdermin D [GSDMD], and GSDMD-N) were examined. The expression of maternally expressed gene 3 (MEG3), spleen focus forming virus proviral integration oncogene (SPI1), methyltransferase 3 (METTL3), and TREM-1 was detected and the binding relationships among them were verified. MEG3 increased N6-methyladenosine methylation of TREM-1 by recruiting SPI1 to activate METTL3. Overexpression of TREM-1 or METTL3 negated the alleviative effects of MEG3 inhibition on M1 polarization and pyroptosis. In mice exposed to CS, EXO^-CSE further aggravated lung injury, M1 polarization, and pyroptosis, which were reversed by MEG3 inhibition. TREM-1 overexpression negated the palliative effects of MEG3 inhibition on COPD mouse lung injury. Collectively, CSE-treated MAECs-derived exosomal long non-coding RNA MEG3 may expedite M1 macrophage polarization and pyroptosis in COPD via the SPI1/METTL3/TREM-1 axis.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.121-129
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• 2022

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an N6-methyladenosine (m⁶A) RNA modification regulator and a key determinant of prem-RNA processing, mRNA metabolism and transportation in cells. Currently, m⁶A reader proteins such as hnRNPA2/B1 and YTHDF2 has functional roles in mice embryo. However, the role of hnRNPA2/B1 in porcine embryogenic development are unclear. Here, we investigated the developmental competence and mRNA expression levels in porcine parthenogenetic embryos after hnRNPA2/B1 knock-down. HhnRNPA2/B1 was localized in the nucleus during subsequent embryonic development since zygote stage. After hnRNPA2/B1 knock-down using double stranded RNA injection, blastocyst formation rate decreased than that in the control group. Moreover, hnRNPA2/B1 knock-down embryos show developmental delay after compaction. In blastocyste stage, total cell number was decreased. Interestingly, gene expression patterns revealed that transcription of Pou5f1, Sox2, TRFP2C, Cdx2 and PARD6B decreased without changing the junction protein, ZO1, OCLN, and CDH1. Thus, hnRNPA2/B1 is necessary for porcine early embryo development by regulating gene expression through epigenetic RNA modification.

• Archives of Pharmacal Research
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• v.22 no.3
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• pp.237-242
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• 1999

A proteinacious inhibitor with a molecular weight of 1,600 Da which inhibits S-adenosyl-L-methionine-dependent transmethylation reactions was purified from porcine liver to homogeneity by procedures including boiling, Sephadex G-25 column chromatography and repeated HPLC. Employing both Nuclear Magnetic Resonance (NMR) and Fast Atom Bombardment-Mass (FAB-Mass) spectroscopy, S-adenosylhomocysteine was conclusively identified as an integral part of the inhibitor. The purified S-adenosylhomocysteine was competitive with S-adenosyl-L-methionine with Ki value of $6.3{\times}10^{-6}$ M towards protein methylase II.

• Bulletin of the Korean Chemical Society
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• v.18 no.10
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• pp.1050-1052
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• 1997

The complexes M(CO)4-1,2-(PPh2)2-1,2-C2B10H10 (M=Cr 2a, Mo 2b, W 2c) have been prepared in good yields from readily available bis-diphenylphosphino-o-carboranyl ligand, closo-1,2-(PPh2)2-1,2-C2B10H10 (1), by direct reaction with Group Ⅵ metal carbonyls. The infrared spectra of the complexes indicate that there is an octahedral disposition of chelate bis-diphenylphosphino-o-carboranyl ligand around the metal atom. The crystal structure of 2a was determined by X-ray diffraction. Complex 2a crystallizes in the monoclinic space group P21/n with cell parameters a = 12.2360(7), b = 17.156(1), c = 16.2040(6) Å, V = 3354.1(3) Å3, and Z =4. Of the reflections measured a total of 2514 unique reflections with F2 > 3σ(F2) was used during subsequent structure refinement. Refinement converged to R1 = 0.066 and R2 = 0.071. Structural studies showed that the chromium atom had a slightly distorted pseudo-octahedral configuration about the metal center with two phosphine groups of o-carborane occupying the equatorial plane cis-orientation to each other. These metal carbonyl complexes are rapidly converted to the corresponding metal carbene complexes, [(CO)3M=C(OCH3)(CH3)]-1,2-(PPh2)2-1,2-C2B10H10 (M= Cr 3a, Mo 3b, W 3c), via alkylation with methyllithium followed by O-methylation with CF3SO3CH3.

• IMMUNE NETWORK
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• v.12 no.2
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• pp.58-65
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• 2012

Background: A cell line with transfected Wilms' tumor protein 1 (WT1) is has been used for the preclinical evaluation of novel treatment strategies of WT1 immunotherapy for leukemia due to the lack of appropriate murine leukemia cell line with endogenous WT1. However, silencing of the transgene occurs. Regarding the effects of hypomethylating agents (HMAs) on reactivation of silenced genes, HMAs are considered to be immune enhancers. Methods: We treated murine WT1- transfected C1498 (mWT1-C1498) with increasing doses of decitabine (DAC) and azacitidine (AZA) to analyze their effects on transgene reactivation. Results: DAC and AZA decreased the number of viable cells in a dose- or time-dependent manner. Quantification of WT1 mRNA level was analyzed by real-time polymerase chain reaction after mWT1-C1498 treated with increasing dose of HMA. DAC treatment for 48 h induced 1.4-, 14.6-, and 15.5-fold increment of WT1 mRNA level, compared to untreated sample, at 0.1, 1, and $10{\mu}M$, respectively. Further increment of WT1 expression in the presence of 1 and $10{\mu}M$ DAC was evident at 72 h. AZA treatment also induced up-regulation of mRNA, but not to the same degree as with DAC treatment. The correlation between the incremental increases in WT1 mRNA by DAC was confirmed by Western blot and concomitant down-regulation of WT1 promoter methylation was revealed. Conclusion: The in vitro data show that HMA can induce reactivation of WT1 transgene and that DAC is more effective, at least in mWT1-C1498 cells, which suggests that the combination of DAC and mWT1-C1498 can be used for the development of the experimental model of HMA-combined WT1 immunotherapy targeting leukemia.