• Journal of Embryo Transfer
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• v.19 no.3
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• pp.201-208
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• 2004

This study was performed to examine the effects of catalase and $\beta$-mercaptoethanol ($\beta$ME) on the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were passaged two times before use. A single cell was seeded in 96-well plates and cultured in medium supplemented with or without catalase or $\beta$ ME. Cell colonies were passaged two times into 4-well dish. Cell lines with proliferating potential were classified as an established clonal cell line. In experience 1, the establishment efficiencies were examined by addition of catalase (100ng/$m\ell$) or $\beta$ME (100 uM) in culture medium. The establishment efficiency of $\beta$ME-added group (8.3％) was significantly higher than that of control group (3.2％, P<0.05). However, catalase did not have a positive efffct on the establishment efficiency. In experience 2, the establishment efficiencies were examined by addition of different concentrations of catalase (0-1,000 ng/$m\ell$) in culture medium. However, establishment efficiencies were not different among the different concentrations of catalase (0-2.6％). In experience 3. the establishment efficiencies were examined by addition of different concentrations of $\beta$ME(0-1,000 uM) in culture medium. The establishment efficiency was significantly higher in 100 uM $\beta$ME-added group (9.4％) compare to others (0-1.6％). The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 100uM $\beta$ME. However, catalase did not have a positive effect on the establishment efficiency.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.97-97
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• 2002

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2${\times}$10$\^$6//${\mu}\ell$. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6${\times}$10$\^$6//${\mu}\ell$. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.

• Korean Chemical Engineering Research
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• v.54 no.5
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• pp.665-670
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• 2016

This study deals with the possible degradation of toluene and acetic acid when subjected to cell-free enzyme system from the toluene degrading bacteria Pseudomonas putida and acetic acid degrading bacteria Cupriavidus necator. P. putida produces toluene dioxygenase only under the existence of toluene in culture medium and toluene is degraded to cis-toluene dihydrodiol by this enzyme. C. necator produces acetyl coenzyme A synthetase-1 and converts acetic acid to acetyl CoA in order to synthesize ATP to need for growth or PHA which is biodegradable polymer. In case of toluene degradation, the experiment was conducted before and after production of toluene dioxygenase as this enzyme, produced by P. putida, is an inducible enzyme. Toluene was detected using gas chromatography (GC). Similar amount of toluene was found in control group and before production of toluene dioxygenase (experimental group 1). However, reduction in toluene was detected after the production of toluene dioxygenase (experimental group 2). Acetic acid was detected through application of gas chromatography-mass spectrometer (GC-MS). The results showed the acetic acid peak was not detected in the experimental group to apply cell-free enzyme system. These results show that the cell-free enzyme system obtained from P. putida and C. necator retained the ability to degrade toluene and acetic acid. However, P. putida needs to produce the inducible enzyme before preparation of the cell-free enzyme system.

• Journal of Mushroom
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• v.4 no.2
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• pp.48-52
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• 2006

Oyster mushroom, Pleurotus ostreatus is bifactorial heterothallism. Single basidiospore isolates from fruiting bodies are homokaryotic and self-sterile. However, we found that homokaryons derived from some strains of P. ostreatus could develop fruiting bodies with two different types. One hundred and two isolates out of 155 monospore isolates formed fruiting bodies (65.8%). First group did not have only mature or sporulating fruiting bodies but also clamp connections, which initial isolate also did not present clamp connections (Abortive homokaryotic fruiting, AHF). Second group had developed fruiting bodies with clamp connections even though initial homokaryotic colony did not form clamp connections (Pseudo- homokaryotic fruiting, PHF). The mycelial colonies derived from PHF by tissue culture formed clamp connections, while mycelial colonies of AHF lacked them. We obtained 535 PHF and 79 AHF inter-strain hybrids among 8 strains of P. ostreatus by hyphal anastomosis. The fruiting body yield of PHF group is higher than that of AHF group in bottle cultivation. A preselection of single spore isolates for fertility would save labour in strain improvement of P. ostreatus.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.262-270
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• 2007

Of the twelve lytic bacteriophages recovered from five different fermenting cucumber tanks that were inoculated with Pediococcus sp. LA0281, a lytic phage, ${\phi}ps05$, was characterized in the present study. The plaques were mostly clear and round-shaped on the lawn of starter strain, indicating lytic phage. Overall appearance indicated that it belongs to the Siphoviridae family or Bradley's group B1, with a small isometric head and a flexible noncontractile tail with swollen base plate. The average size was found to be 51.2 nm in head diameter and 11.6 nm wide ${\times}$ 129.6 nm long for the tail. The single-step growth kinetics curve showed that the eclipse and the latent period were 29 min and 34 min, respectively, and an average burst size was calculated to be 12 particles per infective center. The optimum proliferating temperature ($35^{\circ}C$) was slightly lower than that of cell growth ($35\;to\;40^{\circ}C$). The structural proteins revealed by SDS-PAGE consisted of one main protein of 33 kDa and three minor proteins of 85, 58, and 52 kDa. The phage genome was a linear double-stranded DNA without cohesive ends. Based on the single and double digestion patterns obtained by EcoRI, HindIII, and SalI, the physical map was constructed. The overall size of the phage genome was estimated to be 24.1 kb. The present report describes the presence of a lytic phage active against a commercial starter culture Pediococcus sp. LA0281 in cucumber fermentation, and a preliminary study characterizes the phage on bacterial successions in the process of starter-added cucumber fermentation.

• Journal of the korean academy of Pediatric Dentistry
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• v.24 no.1
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• pp.1-17
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• 1997

The purpose of this study was to investigate the aspects of proliferation and bone nodule formation of osteogenic precursor cells. To determine the effects of ascorbic acid and dexamethasone upon capacity of osteoblast proliferation and bone nodule formation, cells were maintained in the presence of one or some of these additives for up to 30 days. Group I culture was maintained in standard medium(DMEM plus 10% plus antibiotics), group II was maintained in supplemented medium containing dexamethasone, group III was maintained in supplemented medium containing ascorbic acid and sodium-${\beta}$-glycerophosphate, and group IV was maintained in supplemented containing ascorbic acid, sodium-${\beta}$-glycerophosphate and dexamethasone. Morphology of bone nodules was observed with light microscope and electron microscope. The results were as follows: ${\bullet}$ Proliferation capacity of osteoblasts was not affected by single use of dexamethasone, but it was chiefly affected by ascorbic acid. ${\bullet}$ Cellular morphology was fibroblastic appearance initially, but, it was gradually changed to polygonal shape accompanied by confluency stage. ${\bullet}$ Pluripotent mesenchymal cells existed during primary culture, they were differentiated to adipocyte, chondrocyte, osteocyte according to culture condition. ${\bullet}$ Dexamethasone increased bone nodule formation under the condition that the culture was maintained with supplemented medium ascorbic acid and sodium-${\beta}$-glycerophosphate. ${\bullet}$ when the cultures were stained with alizarin red, the group supplemented with dexamethasone, ascorbic acid and sodium-${\beta}$-glycerophosphate showed the marked increase of bone nodule formation, but the group supplemented with ascorbic acid and sodium-${\beta}$-glycerophosphate revealed only small amounts of bone nodules. And the groups cultured without ascorbic acid showed no observed any of bone-like mass independent of dexamethasone addition.

• Childhood Kidney Diseases
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• v.21 no.2
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• pp.75-80
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• 2017

Purpose: To investigate the frequency, presentation, management, and outcome of cytomegalovirus (CMV) infection in pediatric patients who underwent renal transplantation. Methods: We performed a retrospective chart review of 70 patients under the age of 18, who underwent renal transplantation between January 1990 and November 2014. A diagnosis of CMV infection was based on serology, molecular assays, antigenemia assays, and culture. CMV infection was defined as detection of virus and CMV disease was diagnosed when clinical signs and symptoms were present. Results: The number of patients with CMV infection was 18 (25.7% of renal transplant recipients). Twelve were male (66.7%), and the $mean{\pm}standard$ deviation (SD) age at infection was $13.3{\pm}3.9$ years. Median time of infection after renal transplantation was 4 months (range 1.0-31.0 months). Pretransplantation CMV status in the infected group was as follows: donor (D)+/recipient (R)+, 11 (61.1%); D+/R-, 7 (38.9%); D-/R+, 0; and D-/R- 0. Nine patients had CMV disease with fever, leukopenia, thrombocytopenia, or organ involvement such as enteritis, hepatitis, and pneumonitis. The age of disease occurrence was $13.1{\pm}3.9$ years and the median time to disease onset after renal transplantation was 8 months (range 1.0-31.0). Immunosuppressive agents were reduced or discontinued in 14 patients (77.8%), antiviral agents were used in 11 patients (61.1%), and all patients with CMV infection were controlled. Conclusions: A quarter of the patients had CMV infection about 4 months after renal transplantation. CMV infection was successfully treated with reduction of immunosuppressants or with antiviral agents.

• Pediatric Infection and Vaccine
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• v.13 no.2
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• pp.124-129
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• 2006

Purpose : We investigated the causative organisms of bacterial meningitis by age distribution from 1996 to 2005. Methods : Retrospective data were obtained from the medical records with diagnosis of bacterial meningitis or neonatal meningitis from 1996 through 2005. A case was defined by isolation of organism or detection of its antigen by latex agglutination from cerebrospinal fluid. Results : A total of 46 cases(27 neonates and 19 children) were identified. 15 of 27 episodes(55.6%) of neonatal meningitis had a concomitant-positive blood culture. Group B streptococci were the most common bacterial causes of neonatal meningitis(44.4%). Nine of 12 episodes(75.0%) were late-onset infections in neonatal meningitis caused by group B streptococci. 16 of 19 children(84.2%) with bacterial meningitis beyond the neonatal period were younger than 5 years of age(median age, 23 months). Of 19 cases, 8 infections were with Streptococcus pneumoniae, 8 were with Haemophilus influenzae and 3 were with Neisseria meningitidis. Since 2001 there was no case of meningococcal meningitis in this study. Conclusion : In neonates group B streptococci are the most common causative organisms of bacterial meningitis, especially late-onset infections. In infants and young children, the predominant causes of bacterial meningitis are H. influenzae and S. pneumoniae; meningitis caused by the former are likely to decrease after the introduction of the conjugate vaccine for H. influenzae type b.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.334-339
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• 2007

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of $CaCl_2$ and 0.1 mM of $MgCl_2$ for $30{\mu}s$ using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of $H_2O_2$ in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and $100{\mu}M$ L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and $100{\mu}M$ L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and $200{\mu}M$ L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the $H_2O_2$ level in developing embryos in a dose-dependant manner. The $H_2O_2$ level (pixels/ embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and $200{\mu}M$ L-ascorbic acid, respectively. So, we recommend to supplement 50 or $100{\mu}M$ L-ascorbic acid in porcine in vitro culture medium.

• Journal of Microbiology
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• v.40 no.2
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• pp.109-118
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• 2002

The present work is aimed at providing additional new pure cultures of oxalate utilizing bacteria and its preliminary characterization for further work in the field of oxalate-metabolism and taxonomic studies. The taxonomy of 14 mesophilic, aerobic oxalotrophic bacteria isolated by an enrichment culture technique from soils rhizosphers, and the juice of the petiole/stem tissue of plants was investigated. Isolates were characterized with 95 morphological, biochemical and physiological tests. Cellular lipid components and carotenoids of isolates were also studied as an aid to taxonomic characterization. All isolates were Gram-negative, oxidase and catalase positive and no growth factors were required. In addition to oxalates, some of the strains grow on methanol and/or formate. The taxonomic similarities among isolates, reference strains or previously reported oxalotrophic bacteria were analysed by using the Simple Matching (S/ sub SM/) and Jaccard (S$\_$J/) Coefficients. Clustering was performed by using the unweighted pair group method with arithmetic averages (UPGMA) algorithm. The oxalotrophic strains formed five major and two single-member clusters at the 70-86% similarity level. Based on the numerical taxonomy, isolates were separated into three phenotypic groups. Pink-pigmented strains belonged to Methylobacterium extorquens, yellow-pigmented strains were most similar to Pseudomonas sp. YOx and Xanthobacter autorophicus, and heterogeneous non-pigmented strains were closely related to genera Azospirillum, Ancylobacter, Burkholderia and Pseudomonas. New strains belonged to the genera Pseudomonas, Azospirillum and Ancylobacter that differ taxonomically from other known oxalate oxidizers were obtained. Numerical analysis indicated that some strains of the yellow-pigmented and nonpigmented clusters might represent new species.