• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.69-73
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• 2005

The hematopoietic growth factor erythropoietin (EPO) is required for the maintenance, proliferation, and differentiation of the stem cells that produce erythrocytes. To analyse the biological activity of the recombinant human EPO (rec-hEPO), we have cloned the EPO cDNA and genomic DNA and produced rec-hEPO in the CHO cell lines. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of rec-hEPO. MIT assay values were increased by survival of F36E cells at 24h or 72h. The hematocrit and RBC values were increased by subcutaneous injection of 20 IU (in mice) and 100IU(in rats) rec-hEPO. Hematocrit values remarkably increased at $13.2\%$ (in mice) and $12.2\%$ (in rats). The pharmacokinetic behavior with injection of 6 IU of rec-hEPO remained detectable after 24 h in all mice tested. The highest peat appeared at 2h after injection. The long half-life of rec-hEPO is likely to confer clinical advantages by allowing less frequent dosing in patients treated for anemia. These data demonstratethat ree-hEPO produced in this study has a potent activity in vivo and in vitro. The results also suggest that biological activity of ree-hEPO could be remarkably enhanced by genetic engineering that affects the potential activity, including mutants with added oligosaccharide chain and designed to produce EPO-EPO fusion protein.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Journal of Applied Biological Chemistry
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• v.54 no.1
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• pp.41-46
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• 2011

A galactose-fermenting yeasts, Saccharomyces cerevisiae No. 9, was selected by screening their abilities to produce carbon dioxide gas when grown on galactose. The selected strain, No. 9 and the reference strains NRRL Y-1528 which was exceptionally resistant to high concentration of substrate, were acclimated on sugars such as glucose, mannose, and galactose, and then their ethanol productivities were investigated during fermentation on these three carbon sources. Ethanol productivity of the strain No. 9 reached to the maximum levels after 18 h of fermentation and the ethanol yield was from 36 to 38% when presented as $[EtOH]_{max}/[Sugar]_{ini}(g/g)$, regardless of the conditions of acclimation. From the results obtained by acclimation and fermentation, it was concluded that the ethanol yields from galactose were not affected by the sugars acclimated. Improvements of the strain S. cerevisiae No. 9 were attempted to increase the fermentation efficiency and/or ethanol yields on high concentration of substrate by the conventional mutation methods employing methanesulfonic acid, ethyl ester (EMS). Mutants, Mut-5 (SJ1-40), -17 (LK4-25) and -24 (LK2-48) fermented galactose at the concentration of 20% in the levels of higher 39.9~51.6% than the mother strain, No. 9, however, their ethanol yields never exceeded those of the reference strain.

• Microbiology and Biotechnology Letters
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• v.34 no.2
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• pp.136-142
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• 2006

Haematococcus pluvialis is a great producer of astaxanthin (3,3'-dihydroxy-$\beta$,$\beta$-carotene-4,4'-dione). The activities of astaxanthin include potential cancer prevention, immune response enhancement, antioxidant activity, and so on. Nevertheless, it tried to manipulate by mutation for overcoming low growth rate of wild type and limited production of astaxanthin. Mutated colony that is lager and more reddish one than wild type was selected by attempting to expose strains to UV irradiation and to treat chemical such as EMS and colchicines as mutagen. Selected mutants were further screened using inhibitors of the carotenoid biosynthetic pathway. Inhibitors used were nicotine and diphenylamine and both had decreased the survival rate by 40-50%. Among over 50,000 mutant colonies screened, two strains were selected. One selected mutant strain (U15-5) from UV treatment showed 1.68-fold higher total carotenoid contents per cell than that of the wild type strain. On the other hand, the other selected mutant strains (DS, M4-3) from colchicine treatment showed 20$\sim$30% faster cell growth than the wild type strain.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.771-781
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• 2015

To examine if the molecular chaperone DnaK operon proteins of Streptococcus suis type 2 (SS2) are involved in adhesion to host cells, the abundance values of these proteins from the surface of two SS2 strains of different adhesion capability were compared. Their roles in growth and adhesion to human laryngeal epithelial cell line HEp-2 cells were investigated on SS2 strain HA9801 and its mutants with DnaK operon genes partially knocked-out (PKO mutant) under heat stress. The major difference was that DnaJ was more abundant in strain HA9801 than in strain JX0811. Pretreatment of the bacteria with hyperimmune sera to DnaJ, but not with those to other proteins, could significantly reduce SS2 adhesion to HEp-2 cells. PKO of dnaJ g ene resulted in decreased SS2 growth at 37℃ and 42℃, and reduced its adhesion to HEp-2 cells. The wild-type strain stressed at 42℃ had increased expression of DnaJ on its surface and elevated adhesion to HEp-2 cells, which was also inhibitable by DnaJ specific antiserum. These results indicate that the DnaJ of S. suis type 2 is important not only for thermotolerance but also for adhesion to host cells. Because DnaJ expression is increased upon temperature upshift with increased exposure on the bacterial surface, the febrile conditions of the cases with systemic infections might help facilitate bacterial adhesion to host cells. DnaJ could be one of the potential candidates as a subunit vaccine because of its good immunogenicity.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2125-2134
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• 2015

IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad-IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.

• Microbiology and Biotechnology Letters
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• v.36 no.2
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• pp.158-164
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• 2008

The fusants which contain starch utilizing ability and alcohol fermenting ability were developed by protoplast fusion of Saccharomyces cerevisiae KOY-1 and Saccharomyces diastaticus KCTC 1804. Sacharomyces cerevisiae KH-12 was obtained by haploid induction from Saccharomyces cerevisiae KOY-1. The auxotropic mutants of yeast were obtained by using an ethylmethane sulfonate (EMS). The frequency of protoplast formation in Saccharomyces cerevisiae KOY-1 $(Met^-)$ and Saccharomyces diastaticus KCTC 1804 $(Trp^-)$ were 90.5% and 97.7%, respectively. The frequency of fusant formation was $1.79{\times}10^{-4 }$ for the regenerated protoplast and the 1,000 fusants were obtained. Fusant FA 776 was selected as a potential yeast which contain an alcohol fermenting ability in the starch medium. The genetic stability was 4.64% for 10 passages of generation. Fusant FA 776 produced 13mg/ml of alcohol in 24% starch medium and showed 1.86-fold higher alcohol fermenting ability than Saccharomyces diastaticus KCTC 1804.

• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.1023-1031
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• 2017

The conformational change of cellular prion protein ($PrP^C$) to its misfolded counterpart, termed $PrP^{Sc}$, is mediated by a hypothesized cellular cofactor. This cofactor is believed to interact directly with certain amino acid residues of $PrP^C$. When these are mutated into cationic amino acid residues, $PrP^{Sc}$ formation and prion replication halt in a dominant negative (DN) manner, presumably due to strong binding of the cofactor to mutated $PrP^C$, designated as DN PrP mutants. Previous studies demonstrated that plasminogen and its kringle domains bind to PrP and accelerate $PrP^{Sc}$ generation. In this study, in vitro binding analysis of kringle domains of plasminogen to Q167R DN mutant PrP (PrPQ167R) was performed in parallel with the wild type (WT) and Q218K DN mutant PrP (PrPQ218K). The binding affinity of PrPQ167R was higher than that of WT PrP, but lower than that of PrPQ218K. Scatchard analysis further indicated that, like PrPQ218K and WT PrP, PrPQ167R interaction with plasminogen occurred at multiple sites, suggesting cooperativity in this interaction. Competitive binding analysis using $\small{L}$-lysine or $\small{L}$-arginine confirmed the increase of the specificity and binding affinity of the interaction as PrP acquired DN mutations. Circular dichroism spectroscopy demonstrated that the recombinant PrPs used in this study retained the ${\alpha}$-helix-rich structure. The ${\alpha}$-helix unfolding study revealed similar conformational stability for WT and DN-mutated PrPs. This study provides an additional piece of biochemical evidence concerning the interaction of plasminogen with DN mutant PrPs.

• The Korean Journal of Mycology
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• v.17 no.3
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• pp.137-144
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• 1989

Effects of incompatibility existing between intra-and interspecies in Pleurotus spp. on protoplast fusion, clamp formation of their fusants and fruitbody production were investigated. Protoplast fusion between intra-and interspecies of the fungus was achieved by Poly ethylene glycol treatment. The fusion frequency between intraspecies was a little higher than that of interspecies. Fusion frequency between interspecies was not correlated with their similarities based on isozyme patterns. In case of protoplast fusion between intra-and interspecies, the fusants from the compatible isolates produced normal fruit bodies, while those from the incompatible isolates did not produce clamp connections and fruit bodies except those of a few isolates presumed mutants.

• The Korean Journal of Mycology
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• v.17 no.3
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• pp.119-123
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• 1989

Interorder heterokaryons were obtained by polyethylene glycol induced fusion of protoplasts from auxotrophic mutants of Pleurotus ostreatus in agaricales and Ganoderma applanaturm in aphyllophorales. When transferred to MMM plates, all fusion colonies exhibited an extremely growth rate. During three times subcultivation on MCM the growth rate of fusants showed faster little by little. Seventy-five % fusion products of potoplasts showed mixed morphologies between those of P. ostreatus and G. applannatum in the first subcultivation on MCM and MGM. The phenotype of these fusants changed similar those of P. osteatus type after three times subcultivation on MCM. However, phenotype of 25% stable strains did not change on subcultivation. Hyphae of all fusion products did not form true clamp connection. All these types did not produce primordia. A comparrison of interorder somatic hybrids was made using isozyme analysis of esterase, malate dehydrogenase and peroxidase. In most cases the enzyme patterns of G. applanatum were not distinct, however, fusant showed non-parental bands.