• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.61-61
• /
• 2001

The main goal of this study was to produce transgenic piglets by the method of injection of sperm-mediated exogenous DNA. Spermatozoa (1$\times$106 sperm of final concentration) obtained from caudal epididymis were mixed with pBC1-hEPO (20 ng/${mu}ell$) or pcDNA3 LAC Z (20 ng/${mu}ell$), and followed by electroporation (500 V, 25 ㎌). Matured oocytes having the first polar body and dense cytoplasm were selected and centrifuged at 12,000g for 6 min. After sperm injection, the oocytes were activated electrically (1.7 ㎸/cm, 30 $\mu$ sec, single pulse) in 0.3 M mannitol solution. Eggs injected sperm were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air for 192 h. This study were comprised 3 experiments. Experiment 1 compared the developmental efficiencies between the sperm-injected oocytes (Group 1) and further activated electrically (Group 2). Experiment 2 compared the expression of pcDNA3 LAC Z in the embryos produced by Group 1 and Group 2. Finally, experiment 3 carried out transfer of embryos (1-8 cell stage) transfected with pBC1 -hEPO into surrogate recipients synchronized by injection of combination of PG600 with hCG. The rates of cleavage and development into blastocyst stage in Group 2 were significantly higher than those of Group 1 (71.3% and 28.1% vs. 43.3% and 10.3%, respectively, p<0.05). Thirty (24.2%) out of 124 embryos analyzed in Group 2 were positive by X-gal. Similarly, in Group 1, 16.3% (8/49) were positive. After transfer of 789 embryos to 7 recipient gilts, three out of them examined by ultrasound became pregnant. One recipient is in day 50 pregnancy. On day 54 of gestation, two were carried out uterotomy in order to confirm the pregnancy One had 7 and another had 2 fetuses. We conclude that injection of sperm-mediated gene transfer will be used as a valuable tool for the production of transgenic piglets.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.31 no.4
• /
• pp.306-311
• /
• 2005

Despite advances in surgery, radiotherapy, and chemotherapy, the survival of patients with oral squamous cell carcinoma has not significantly improved over the past several decades. Gene therapy is currently under investigation and shows us new possibility of cancer curing method. This experiment was undergone to find out the cell growth inhibition effect and evidence of apoptosis by HCCS-1(human cervical cancer suppressor-1), one of the candidates of tumor suppressor gene, transducted to human oral cancer cell line. To determine the efficiency of the adenovirus as a gene delivery vector cell line was transducted with LacZ gene and analysed with X-gal staining. Northern blot was performed to confirm the transfection with HSCC-1 gene and cell viability was assessed by cell cytotoxicity assay using cell count kit(CCK). To show the evidence of apoptosis, DNA fragmentation assay and flow cytometry(FACS) were performed. We had successfully construct the recombinant HSCC-1 adenovirus(Ad5CMV-HCCS-1), and importation efficiency was 20% at 2 MOI(multiplicity of infection), 80% at 20 MOI. Northern blot analysis showed that a single 0.6kb mRNA transcript was expressed in Ad5CMV-HCCS-1 transducted cell lines. As a result of CCK, when comparing to control subjects, transducted group showed 50% growth inhibition. In DNA fragmentation assay, according to increasing of MOI, DNA volume was diminished. In FACS analysis, DNA distribution showed fragmentation. This results imply that HCCS-1gene has growth inhibition effect in human oral cancer cell lines through apoptosis induction.

• Journal of Life Science
• /
• v.31 no.7
• /
• pp.654-661
• /
• 2021

In this study, in order to isolate excellent whitening agents from fungal cultural broth, various fungi were collected from wild areas in South Korea and then screened for tyrosinase inhibition activity, as tyrosinase is a precursor for the biosynthesis of melanin in regulating skin color. A fungus strain that inhibits tyrosinase activity has been identified and confirmed as Trichoderma viridescens (later renamed T. viridescens SW-1) via ITS sequencing. In T. viridescens SW-1, tyrosinase inhibitory activity was strongest on day three of culture. A 5% culture broth showed a tyrosinase inhibitory activity greater than 90% and exhibited high thermostability on day three. At 10% culture broth, the accumulations of intra- and extracellular melanin were inhibited above 27.1% and 7.5%, respectively. In summary, the physical and functional properties of the tyrosinase inhibitory substances of T. viridescens SW-1 included high levels of inhibition of melanin synthesis and antioxidative activity as well as thermostability. Therefore, we suggest that the whitening substance identified from the cultural broth of T. viridescens SW-1 has potential for application as a functional cosmetic ingredient.

• 한국균학회소식:학술대회논문집
• /
• 2014.05a
• /
• pp.27-28
• /
• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• Development and Reproduction
• /
• v.11 no.3
• /
• pp.235-243
• /
• 2007

Human umbilical cord blood(HUCB) contains a rich source of hematopoietic stem cells, mesenchymal stem cells and endothelial cell precursors. Mesenchymal stem cells(MSCs) in HUCB are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. We studied on transdifferentiation-promoting conditions in neural cells and cholinergic neuron induction of HUCB-derived MSCs. Neural differentiation was induced by addingdimethyl sulphoxide(DMSO) and butylated hydroxyanisole(BHA) in Dulbeco's Modified Essential Medium(DMEM) and fetal bovine serum(FBS). Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor(bFGF), retinoic acid(RA) and sonic hedgehog(Shh). MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including $\beta$-tubulin III, GFAP and MBP, was markedly elevated during this acute differentiation. The differentiation rate was about $32.3{\pm}2.9%$ for $\beta$-tubulin III-positive cells, $11.0{\pm}0.9%$ for GFAP, and $9.4{\pm}1.0%$ for Gal-C. HUCB-MSCs treated combinatorially with bFGF, RA and Shh were differentiated into cholinergic neurons. After cholinergic neuronal differentiation, the $\beta$-tubulin III-positive cell population of total cells was $31.3{\pm}3.2%$ and of differentiated neuronal population, $70.0{\pm}7.8%$ was ChAT-positive showing 3 folds higher in cholinergic population than neural induction. Conclusively, HUCB-derived MSCs can be differentiated into neural and cholinergic neurons and these findings suggest that HUCB are alternative cell source of treatment for neurodegenerative diseases such as Alzheimer's disease.

• The Korean Journal of Physiology and Pharmacology
• /
• v.14 no.3
• /
• pp.139-144
• /
• 2010

In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-$\alpha$ (TNF-$\alpha$) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-$\alpha$ induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-$\alpha$ dose-dependent (5~100 ng/ml). TNF-$\alpha$-stimulated A549 cells evidenced increased Ref-1 expression in a dose-dependent manner. The adenoviral transfection of cells with AdRef-1 inhibited TNF-$\alpha$-induced COX-2 expression relative to that seen in the control cells ($Ad{\beta}gal$). Pretreatment with $10\;{\mu}M$ of SB203580 suppressed TNF-$\alpha$-induced COX-2 expression, thereby suggesting that p38 MAPK might be involved in COX-2 expression in A549 cells. The phosphorylation of p38 MAPK was increased significantly after 5 minutes of treatment with TNF-$\alpha$, reaching a maximum level at 10 min which persisted for up to 60 min. However, p38MAPK phosphorylation was markedly suppressed in the Ref-1-overexpressed A549 cells. Taken together, our results appear to indicate that Ref-1 negatively regulates COX-2 expression in response to cytokine stimulation via the inhibition of p38 MAPK phosphorylation. In the lung cancer cell lines, Ref-1 may be involved as an important negative regulator of inflammatory gene expression.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
• /
• v.22 no.2
• /
• pp.45-55
• /
• 2017

Long chain alkyl diols (LCDs) have been reported in sediments from various marine environments. Rampen et al. (2012) introduced the paleo-sea surface temperature (SST) proxy, Long chain Diol Index (LDI) based on the relative abundance of $C_{30}$ 1,15-diol, $C_{28}$ 1,13-diol, and $C_{30}$ 1,13-diol. In general, CP-Sil5CB and DB-5ms columns have been used for the quantitative and qualitative analysis of LCDs with a GC-MS. In this study, we examined the effect of three different columns (CP-Sil5CB, HP-5ms and DB-5) on the quantitative analysis of LCDs using marine sediments from the East Sea of Korea and the western Arctic Ocean. In general, our study showed that the results of CP-Sil5CB differed significantly from those of HP-5ms and DB-5. However, the differences of the LDI-derived SSTs among three columns were $0.1-0.2^{\circ}C$ for the East Sea and $0.2-0.7^{\circ}C$ for the western Arctic Ocean, which were well within the calibration error range (${\pm}1{\sigma}$). Accordingly, our study showed that the use of different columns resulted in significant differences of LCDs concentrations, but its effect on the LDI was relatively insignificant. Therefore, it appears that the different columns can be used for the paleo-SST reconstruction in the East Sea and the western Arctic Ocean using the LDI proxy.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.48 no.2
• /
• pp.113-121
• /
• 2022

In this study, fermented Lycium chinense (L. chinense) extract was used to analyze the efficacy of nitric oxide (NO) and anti-aging inhibition to verify its usefulness as a functional cosmetic material. L. chinense was extracted with hydrothermal and 70% ethanol as a solvent, fermented, concentrated, and freeze-dried to prepare a sample, followed by MTT assay, NO inhibition assay, western blot assay, and UPLC analysis.As a result of the analysis of NO inhibitory efficacy, fermented L. chinense water extract (FLW) and fermented L. chinense 70% ethanol extract (FLE) showed excellent NO inhibitory efficacy of 47.96% and 56.71%, respectively. As a result of measuring the expression patterns of the wrinkle-related proteins MMP-1, and TRPV-1 through Western blot, both FLW and FLE confirmed the inhibitory efficacy of MMP-1 and TRPV-1. Based on the results of the experiment, the fermented L. chinense extract is expected to have a high application value as a functional cosmetic material related anti-inflammatory, and anti-aging.

• Journal of Life Science
• /
• v.16 no.2 s.75
• /
• pp.274-281
• /
• 2006

A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

• Proceedings of the Korea Water Resources Association Conference
• /
• 2008.05a
• /
• pp.1972-1975
• /
• 2008

우리 인간이 수원으로 가장 많이 이용하는 지표수의 이동 및 저장 역할을 하는 호소는 자연환경과 인간을 이어주는 중요한 매개체임은 분명하다. 그러나 최근에 경제 성장만을 위주로 발전을 추구해온 결과, 급격한 산업화에 따른 도시지역으로의 인구 집중 현상은 호소에 자연적 혹은 인위적으로 큰 영향을 주고 있다. 하천의 자정능력을 초과하여 발생하는 오염물질이 신갈 저수지로 유입되어 생태계를 파괴하고, 하천의 최종 유입지인 호소의 오염을 가중시켰다. 경기도 용인시, 화성군, 오산시 지역의 농 공업용수의 공급원으로 이용되고 있는 신갈 저수지로 유입하는 하천 역시 도시로의 인구 집중으로 인한 막대한 생활하수와 주변 지역에 조성된 공장에서 발생하는 사업장 배수의 유입으로 인해 심각하게 오염되어 신갈 저수지의 오염을 가중시키고 있다. 현재 용인시에서는 기흥 호수공원을 2010년까지 신갈 저수지 일대 118만평에 여가와 문화, 휴양시설이 연계된 대규모 유원지로 조성한다는 계획을 가지고 있다. 이에 본 연구에서는 신갈 저수지의 퇴적물 오염도를 조사하여 호수공원 조성 시 기초 데이터의 확충 및 현황파악을 위하여 신갈 저수지의 퇴적물에 대한 모니터링을 실시하였다. 측정 시기는 1차(2007년 7월)와 2차(2007년 10월)로 총 2회 실시하였으며, 조사 지점선정은 수역 전체의 특성을 가장 대표할 수 있는 지점(호심 또는 가장 깊은 곳 등), 주요 유입하천수가 유입된 후 충분히 혼합되는 지점, 호수가 유출되는 지점, 폐수나 하수의 유입으로 항상 오염이 인정되는 지점을 고려하여 신갈저수지의 6지점을 선정하여 조사하였다. 퇴적물 분석항목 중 VS, COD, T-N, T-P, 중금속 농도를 중심으로 신갈 저수지의 퇴적물 오염도를 조사하였다. 각 지점별 1차 퇴적물 조사 결과, VS $23.1{\sim}46.6\;g/kg$, COD $8.5{\sim}26.4\;mg/g$, T-N $0.35{\sim}1.52\;mg/g$, T-P $0.60{\sim}1.05\;mg/g$ 등의 농도로 조사되었으며, 중금속은 수은, 시안, 카드뮴은 검출되지 않았고 구리의 농도는 $0.1{\sim}0.6\;mg/kg$, 납의 농도는 $2.1{\sim}3.5\;mg/kg$로 조사되었다. 또한 각 지점별 2차 퇴적물 조사 결과, VS $22.8{\sim}41.4\;g/kg$, COD $7.9{\sim}21.5\;mg/g$, T-N $0.31{\sim}1.44\;mg/g$, T-P $0.82{\sim}1.01\;mg/g$ 등의 농도로 조사되었으며, 중금속은 수은, 시안, 카드뮴은 검출되지 않았고 구리의 농도는 $0.2{\sim}0.7\;mg/kg$, 납의 농도는 $2.4{\sim}3.8\;mg/kg$로 조사되었다. 본 연구의 조사 결과는 앞으로 호수공원이 조성되는 신갈 저수지를 효율적으로 관리함에 있어 중요한 근거자료가 될 수 있을 것으로 판단된다.