• Laboraroty Animal Research
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• 제34권4호
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• pp.264-269
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• 2018

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors $p16^{Ink4a}$ (Cdkn2a, cyclin-dependent kinase inhibitor 2a), $p19^{Arf}$ (an alternative reading frame product of Cdkn2a,), and $p27^{Kip1}$ (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The $p16^{Ink4a}$ and $p19^{Arf}$ knockout mice were generated via transcription activator-like effector nucleases (TALENs), and $p27^{Kip1}$ knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.650-655
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• 1998

The chromosomal DNA fragment from Phaffia rhodozyma CBS 6938 which is able to autonomously replicate in the yeast Saccharomyces cerevisiae was cloned on an integrative URA3 plasmid. Its minimal fragment exhibiting autonomously replicating activiy in the S. cerevisiae gave a higher frequency transformation efficiency than that found for centromere-based plasmid, and enabled extrachromosoma1ly stable transmission of the plasmids in one copy per yeast cell under non-selective culture condition. The 836-bp DNA element lacked an ORF and did not contain any acceptable match to an ARS core consensus. Sequence analysis, however, displayed a cluster of three hairpin-Ioop-sequences with individual $\triangle {G_{25}}^{\circ}C$ free energy value of -10.0, -17.5, and -17.0 kcal. $mor^{-l}$as well as a 9-bp sequence with two base pair mismatches to the S. cerevisiae/E. coli gyrase-binding site. This 836-bp sequence also included one 7-bp sequence analogous to the core consensus of centromeric DNA element III (CDEIII) of S. cerevisiae, but CDEIII-like 7 bp sequence alone did not give a replicative function in this yeast.

• 대한치과교정학회지
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• 제21권1호
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• pp.97-111
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• 1991

It is the aim of this study to investigate the effects of sodium fluoride and sodium orthovanadate upon the proliferation and activity of the osteoblast (MC3T3-E1 cells). MC3T3-E1 cells were cultured in $\alpha-MEM$ containing $10\%$ FBS and various concentration of sodium fluoride and sodium orthovanadate was appended to serum free media. DNA synthesis was examined through the $[^3H]$ thymidine incorporation into DNA. Collagen synthesis was examined through the $[^3H]$ proline incorporation into collagenase digestible protein and noncollagen protein. The following results were drawn; 1. Sodium fluoride stimulated the DNA synthesis of osteoblast significantly in dose-dependent manner within the concentration from $2{\mu}M$ to $10{\mu}M$ (P < 0.005). 2. Sodium orthovanadate stimulated the DNA synthesis of osteoblast significantly in dose-dependent manner within the concentration from $2{\mu}M\;to\;8{\mu}M$, however showed diminution at $10{\mu}M$ (P < 0.001). 3. Sodium fluoride and sodium orthovanadate stimulated the percent collagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from $5{\mu}M$ to $10{\mu}M$ (P < 0.001). 4. Sodium fluoride and sodium orthovanadate stimulated the noncollagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from $5{\mu}M\;to\;10{\mu}M$ (P < 0.001). In conclusion, sodium fluoride and sodium orthovanadate stimulate the proliferation and activity of osteoblast by stimulation of DNA synthesis and collagen and noncollagen synthesis in osteoblast.

• Biomolecules & Therapeutics
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• 제21권5호
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• pp.349-357
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• 2013

6'-O-galloylpaeoniflorin (GPF) is a galloylated derivate of paeoniflorin and a key chemical constituent of the peony root, a perennial flowering plant that is widely used as an herbal medicine in East Asia. This study is the first investigation of the cytoprotective effects of GPF against hydrogen peroxide ($H_2O_2$)-induced cell injury and death in human HaCaT keratinocytes. GPF demonstrated a significant scavenging capacity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, $H_2O_2$-generated intracellular reactive oxygen species (ROS), the superoxide anion radical ($O_2^-$), and the hydroxyl radical (${\cdot}$OH). GPF also safeguarded HaCaT keratinocytes against $H_2O_2$-provoked apoptotic cell death and attenuated oxidative macromolecular damage to DNA, lipids, and proteins. The compound exerted its cytoprotective actions in keratinocytes at least in part by decreasing the number of DNA strand breaks, the levels of 8-isoprostane (a stable end-product of lipid peroxidation), and the formation of carbonylated protein species. Taken together, these results indicate that GPF may be developed as a cytoprotector against ROS-mediated oxidative stress.

• 생명과학회지
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• 제21권9호
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• pp.1266-1273
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• 2011

사포닌은 동양의 전통적인 약재로 널리 알려진 인삼의 주요한 성분이다. 사포닌의 다양한 생물학적 효능이 밝혀져 있으나, 피부재생과 관련된 효능은 현재까지도 명백하지 않다. 본 연구에서는 세포외 시스템에서 사포닌의 항산화 효과 뿐만 아니라 사람 진피 섬유아세포에서 기질금속단백질 분해효소(MMP)에 대한 효능이 조사되었다. 먼저 MTT assay를 이용한 세포생존력에 대한 사포닌의 효능을 조사한 결과, 10 ${\mu}g$/ml 이하의 농도에서 사포닌은 세포생존력을 증가시켰으나 25 ${\mu}g$/ml 이상의 농도에서는 세포독성을 나타내었다. 항산화에 대한 사포닌 효능을 조사한 결과, 1 ${\mu}g$/ml 이상의 농도에서 사포닌은 환원력 뿐만 $H_2O_2$에 대한 억제 효능을 보여주었다. 특히, DNA 산화에 대한 보호 효과도 나타내었다. 더욱이 gelatin 및 casein zymography 시험결과, 사포닌은 MMP-2를 활성화 시키고 MMP-1의 활성을 증가시키는 것으로 보아, 항노화 및 피부재생 약효제로 잠재적인 가능성이 기대된다.

• 한국수산과학회지
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• 제34권1호
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• pp.21-26
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• 2001

Alginate를 가열에 의해 저분자화하여 저분자화에 따른 소화생리 특성을 검토하기 위해, 랫드에 저분자 alginate인 HAG-10,HAG-50, HAG-100 및 alginate를 장기간 섭취시켰을 때, 체중, 각종 장기의 무게와 길이 및 췌장과 소장의 소화효소활성과 단백질, DNA 및 RNA 함량을 측정하였고 소장응모의 미세구조의 변화를 관찰하였다. 랫드의 체중은 $5\%$와 $10\%$ HAG-50 및 $10\%$ alginate에서 체중의 증가가 유의적으로 현저히 억제되었다. 간장의 무게는 유의적인 차이가 없었고, 위의 무게와 길이는 $10\%$ HAG-10, $1\%$, $5\%$ 및$10\%$ HAG-50, $1\%$와 $5\%$ HAG-100 그리고 $5\%$ alginate에서 증가하는 경향을 보였으며, 췌장의 무게와 길이는 모두 약간 증가하였다. 소장과 맹장의 무게와 길이는 $1\%$와 $5\%$ HAG-10을 제외하고 모두 증가하였으나 대장의 무게와 길이는 전반적으로 감소하였다. 췌장의 amylase 활성은 유의적인 차이는 보이지 않았고, lipase 활성은 HAG-50에서 약간 저하하였으며, protease 활성은 $1\%$ HAG-10만 제외하고 유의적으로 저하하였다. 췌장의 단백질 함량은 모두 증가하였으나, DNA와 RNA 함량은 유의 적 인 차이가 없었다. 소장의 단백질 함량은 $5\%$와 $10\%$ HAG-50에서 그리고DNA는 $5\%$ HAG-50에서 가장 높은 함량을 보였으며, RNA는 전반적으로 증가하였다 소장융모의 미세구조는 HAG-50에서 주름이 많고 표면적이 넓은 잎사귀 모양의 응모세포와 돌림주름 및 Boblet cell이 현저히 발달되어 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.206-209
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• 2001

Recombinant human GM -CSF was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, pMYN44. containing the hGM -CSF cDNA. Regulated expression and secretion of hGM -CSF from this vector achieved using the promoter, signal peptide, and terminator from a rice alfa-amylase gene Amy3D. The Amy3D gene is expressed in response to sugar deprivation. The recombinant hGM -CSF was expressed from the transgenic rice cell culture on the sugar-free medium as a yield of about 110 mg/L in the culture filtrate, which was determined by ELISA. Biological activity of hGM-CSF was confirmed by measuring the proliferation of the hGM -CSF dependent TF -1 cells.(This work was supported by a grant from the NRL program of the Korean Ministry of Science and Technology. Shin, Y.- J.. Lee. J.-H and Kwon, T.-H. have been supported by BK21 program from the Korean Ministry of Education)

• Biomolecules & Therapeutics
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• 제10권2호
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• pp.78-84
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• 2002

Taurine (2-ethaneaminosulfonic acid, $^+{NH}_3{CH_2}{CH_2}{SO_3^{-}}$) is endogenous amino acid with functions as modulator of osmoregulation, antioxidation, detoxification, transmembrane calcium transport, and a free radical scavenger in mammalian tissues. Taurine transporter(TAUT) contains 12 transmembrane helices, which are typical of the $Na^+$- and $Cl^-$-dependent transporter gene family, and has been cloned recently from several species and tissues. To analyze the expression of TAUT mRNA, one step RT-PCR was performed from human and mouse cultured cell lines and from various mouse tissues. The primers were designed to encode highly conserved amino acid sequences at the second transmembrane domain and at the fourth and fifth intracellular domains. RT-PCR analysis showed both of the human intestine HT-29 and mouse macrophage RAW264.7 cell lines expressed mRNA of TAUT. To define the expression patterns of the TAUT mRNA in the murine organs, RT-PCR was performed to detect cDNA representing TAUT mRNA from seven different mouse tissues. The TAUT was detected in all of the mouse tissues analyzed such as heart, lung, thymus, kidney, liver, spleen and brain. A large amount of transcript was fecund from heart, liver, spleen, kidney, and brain, while lung contained a very small amount of transcript.

• Environmental Analysis Health and Toxicology
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• 제13권1_2호
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• pp.1-9
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• 1998

The ROS-producing potency of chromium compounds of several oxidation states were determined in the H4 cells. $K_2Cr_2O_7$ as Cr (VI), synthetic Cr (V) compounds and Cr (III) as TPP produced high level of ROS. However, ROS values of Cr-picolinate as Cr (III), CrCl$_2$, CrCI$_2$, were almost equal to the control. The effects of physiological antioxidants compounds which react with free radicals were examined for their effects on chromate-induced production of reactive oxygen species (ROS) in A549 cells after the addition of $K_2Cr_2O_7$. The compounds used were vitamin C (ascorbate), vitamin E ($\alpha$-tocopherol), superoxide dismutase (SOD) and catalase. The preincubation of ascorbate (200uM) with A549 cells for 20hr resulted in a significant reduction of hexavalent chromate(100uM) induced ROS. However, there is no effects of preincubation of the cells with vitamin E succinate (10 and 20uM, 20hr) on the ROS production. Also, the effects of Cr (VI) on the cell cycle of A549 cells was measured by adding the DNA intercalating agent, propidium iodide. S phase of the cell cycle was increased by the chromium (VI) compounds up to 20uM indicating toxicity or possible mitogenic action of the cell. The shoulder in Go/G1 phase at 20uM Cr (VI) with 24 hr treatment indicates apoptosis.

• 미생물학회지
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• 제29권4호
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• pp.250-257
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• 1991

An obligate methanol-oxidizing bacterium, Methylobacillus sp. strain SK1, which grows only on methanol was isolated from soil. The isolate was nonmotile Gram-negtive rod. It does not have internal membrane system. The colonies were small, whitish-yellow, and smooth. The guanine plus cytosine content of the DNA was 48 mol%. Cellular fatty acids consisted predominantly of large amounts of straight-chain saturated $C_{16:0}$ acid and unsaturated $C_{16:1}$ acid. The major ubiquinone was Q-8, and Q-10 was present as minor component. The cell was obligately aerobic and exhibited catalase, but no oxidase, activity. Poly-.betha.-hydroxybutyrate, endospores, or cysts were not observed. the isolate could grow only on methanol in mineral medium. Growth factors were not required. The isolate was unable to use methane, formaldehyde, formate, methylamine, and several other organic compounds tested as a sole source of carbon and energy. Growth was optimal at 35.deg.C and pH 7.5. It could not grow at 42.deg.C. The doubling time was 1.2h at 30.deg.C when grown with 1.0%(v/v) methanol. The growth was not affected by antibiotics inhibiting cell wall synthesis and carbon monoxide but was completely suppressed by those inhibiting protein synthesis. Methanol was found to be assimilated through the ribulose monophosphate pathway. Cytochromes of b-, c-, and o- types were found. Cell-free extracts contained a phenazine methosulfate-linked methanol dehydrogenase activity, which required ammonium ions as an activator. Cells harvested after the late exponential phase seemed to contain blue protein.ein.