• 한국식품과학회지
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• 제22권6호
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• pp.632-639
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• 1990

곰취, 참취 그리고 참나물로부터 polyphenoloxidase를 추출하여 polyphenol 화합물인 pyrogallol, hydroxyhyd-roquinone, catechol 그리고 3,4-dihydroxytoluene과 반응시켜 얻어진 12종류의 효소갈변반응 생성물의 생리작용을 검토한 결과 시료 모두 rec-assay, 돌연변이원성 실험 그리고 DNA 절단실험에서 돌연병원성을 나타내지 않았다. 그러나 12종류의 시료 모두 S-9mix를 첨가한 Salmonella/microsomal 실험에서 $benzo({\alpha})pyrene(B({\alpha})P)$, 3-amino-1,4-dimethyl-5H-pyrido(4,3-b) indole(Trp-P-1) 그리고 2-aminofluorene(2-AF) 등 변이원 물질들을 강하게 억제시키는 활성을 나타내었다. DNA repair 실험에서 효소갈변반응 생성물의 E. coli HB101에 plasmid pGA658의 transformation 빈도수는 곰취 효소갈변반응 생성물의 첨가에 의해 높은 수치를 나타내었으며 transformation 전에 DNA와 효소갈변반응 생성물을 혼합하여 자외선을 조사하였을 때 곰취의 pyrogallol, catechol, hydroxyhydroquinone의 효소갈변반응 생성물은 높은 수치를 나타내었다.

• Archives of Pharmacal Research
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• 제18권4호
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• pp.243-248
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• 1995

Seventeen transformation-deficient mutants of streptococcus pneumoniae, which are defective in competence induction (com), DNA uptake(ent) of recombination(rec), were investigated to determine sensitivity to ethylmethane sulfonate(EMS), methylmethane sulfonate(MMS), UV and mitomycin C. In ethylmethane sulfonate assay, the viability of most $com^-, \; rec^-\; and ent^-$ mutants was decreased about 2-10 times and the viability of ent-9 and ent-13 mutant was decreased about 33 and 25 times, respectively. On the other hand only half of the transformation-deficient mutants tested was sensitive to methylmethane sulfonate about 2 times and ent-12 mutant was sensitive to 2.0% MMS about 8 times. After UV and mitomycin C treatment, most of the mutants are not sensitive to UV and mitomycin C, although the viability of some transformation-deficient mutants was decreased slightly. Especially none of the com mutants were sensitive to DNA damage suggesting that competence is not involved in DNA repair. Also DNA uptake and recombination gane might be related to DNA repair function.

• Reproductive and Developmental Biology
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• 제29권2호
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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.

• Journal of Microbiology and Biotechnology
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• 제28권10호
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• pp.1749-1759
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• 2018

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with >95% purity. The purified recPrPs predominantly adopted an ${\alpha}$-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.

• 미생물학회지
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• 제50권2호
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• pp.95-100
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• 2014

Hrq1은 곰팡이 유전체에서 생물정보분석에 의해 발견된 새로운 RecQ helicase이다. 이 단백질은 인간의 RECQL4와 가장 상동성이 높으며 최근의 유전학적 생화학적 연구를 통해서 유전체 안정성을 유지하는데 어떤 역할을 할 것으로 예상되었다. 본 연구에서는 RECQL4와 상호작용하는 것으로 알려진 인간 유전자들과 상동성이 있는 효모 유전자들이 Hrq1과 상호작용하는지를 yeast two-hybrid assay를 이용하여 조사하였다. 총 11개의 유전자를 조사한 결과, nucleotide excision repair (NER) 인자 중의 하나인 Rad14이 Hrq1과 상호작용하는 것을 발견하였다. 또한 정제한 단백질을 이용한 pull-down assay로 Hrq1과 Rad14 사이의 직접적인 상호작용을 확인하였다. Hrq1과 Rad14 사이의 yeast two-hybrid 상호작용은 4-nitroquinoline-1-oxide에 의한 DNA 손상으로 더욱 증가하였으며, 이러한 상호작용의 증가는 또 다른 NER 인자인 Rad4에 의존적이었다. 이러한 결과들은 Hrq1이 Rad14과의 상호작용을 통하여 NER 과정에 어떤 역할을 할 가능성을 제시하고 있다.

• 한국약용작물학회지
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• 제10권5호
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• pp.403-408
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• 2002

마가목 잎, 수피, 열매 추출물의 Spore-rec assay와 Ames test에 의한 항돌연변이 활성탐색 결과, 추출물 모두 돌연변이원성을 나타내지 않았으며 열매와 수피의 에탄올 추출물에서 MNNC에 대하여 높은 항돌연변이원성을 나타내었다. 인간 암세포주를 이용한 항암효과 실험은 먼저 정상간세포인 WRL68에 대한 세포독성효과를 검토한 결과, 0.75mg/ml 이하의 농도에서 수피의 물 추출물을 제외하고는 모두 20% 이하의 낮은 세포독성을 나타내어 추출물 자체의 세포 독성은 낮은 것으로 확인되었다. 인간 폐암세포(A549), 유방암세포(MCF7), 간암세포(HepG2)에 대한 세포독성 결과, 수피의 에탄올 추출물이 폐암세포주인 A549에 대하여 1.0 mg/ml의 농도에서 94%의 가장 높은 암세포 생육억제능을 나타내었으며, 0.75mg/ml 이상의 농도에서 65%이상의 생육억제 활성을 나타내었다. 유방암세포주인 MCF7에 대하여서는 0.75mg/ml 이상의 농토에서 58%이상의 생육억제능을 나타내었으며, 열매의 에탄올 추출물이 1.0mg/ml의 농도에서 91%의 생육을 억제하였다. 간암세포주인 HepG2에 대하여서는 수피와 열매의 추출물들이 0.75mg/ml 이상의 농도에서 56%이상의 생육억제활성을 나타내었다. 이와 같은 결과로부터 마가목 잎, 열매, 수피 추출물들은 시료자체의 독성은 낮은 반면 항돌연변이원성 및 암 세포주에 대한 생육억제활성이 높아 앞으로 이들 추출물을 이용한 기능성 식품소재로의 이용 가능성이 높을 것으로 사료된다.

• 생약학회지
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• 제36권2호통권141호
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• pp.93-96
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• 2005

Mutagenicity of Gamgung-tang (GGT) was tested using in vitro S-9 mixture in vitro host-mediated assay with Salmonella typhimurium. In the previous reports, GGT was tested for the safety using Ames(-S-9), Bacillus subtilis Rec, and umu gene expression mutagenicity tests. Mutagenic activity in any assays we tested was not found. In this report, we further investigated safety of GGT after metabolic activation in vivo. Ames test with S-9 mixture and host-mediated assay with Salmonella typhimurium TA98 were used to identify metagenic property of GGT. GGT was administered 3 times with i.m. to Balb/c mice did not induced mutagenic effect in Salmonella typhimurium TA98 recovered from the liver after 3.5h with i.p. treatment. Over the entire dose range $(3{\sim}150mg/mouse)$ tested no toxicity was detected to the bacterial cells. These results suggest that there was no DNA damage and mutagenicity by GGT.

• 한국식품영양과학회지
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• 제23권4호
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• pp.698-703
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• 1994

In spore rec-assay using B. subtillus H17(rec) and M 45(rec) , the ethanol extract of buckwheat leaves showed antimutagenicity in condition of low concentrations, but its did comutagenicity in condition of high concentrations. In Ames test, the ethanol extract of buckwheat leaves reduced the mtabenicity of N-methyl-N' -nitro-N-nitrosoguaidine (MNNG), benzo (a) pyrene(B($\alpha$)P), 2-amino-fluorene(2AF), and 3-amino -1, 4-dime-thyl-5-H-pyrido(4, 3-b) indol(Trp-P-1) in Salmonella typhimurium TA98 and TA100. The ethanol extract was fractionated by hexane, ethylacetate, butanol and water. Among Them hexane fraction showed the highest inhibition rate on the mutagenicity of B($\alpha$)P, and so did chloroform fraction on the mutagenicity of MNNG in S. typhimurium Ta98 and TA100. To elucidate the antimutagenic mechanism of the ethanol extract, it was mixed and co-incubated with various metagens, S9 mix, and the bacteria with different experimental orders and different reaction times. The ethanol extract did not affect reversion rate of pre-mutated. S.typhimurium. However, when the ethanol extract was added to the mutagens before their interaction with S.typhimurium , it reduced the mutation rate to 152$\pm$12-273$\pm$18 colonies/plates in case of MNNG, and 135$\pm$13-195$\pm$10 colonies/ plates in case of B($\alpha$)P), showing strong desmutagenic activity.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.77-83
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• 2009

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.