• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1384-1391
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• 2006

The signal transduction system is one of the most important devices involved in maintaining life, and many protein kinases are included in the cellular signal transduction system. Finding a protein kinase inhibitor is very valuable, as it can be used to study cell biology and applied to pharmaceuticals. For the efficient and rapid screening of protein kinase inhibitors, two assay systems were combined; the nonradioactive protein kinase assay system that uses an FITC-labeled IRS-2 peptide and the cell-based paper disc assay system that uses Streptomyces griseus as the indicator strain. Among 330 kinds of herb extracts tested, the extract of Dalbergia odorifera exhibited the strongest inhibitory activity in the two assay systems and was selected for further isolation. Based on solvent extraction and many steps of chromatography, seven compounds were finally separated to homogeneity and their structures determined by $^{1}H$ and $^{13}C$ NMR spectroscopies. Four were to be flavonoids and identified as butin ($C_{15}H_{12}O_5$, Mw=272.07), 3'-hydroxymelanetin ($C_{16}H_{12}O_6$, Mw=300.06), liquiritigenin ($C_{15}H_{12}O_4$, Mw=256.07), and 2'-hydroxyformononetin ($C_{16}H_{12}O_{5}$, Mw=284.07). 3'-Hydroxymelanetin inhibited the phosphorylation of the GSK3 protein by Akt to 37% at a concentration of $10{\mu}g/ml$ and showed the strongest cytotoxicity ($ED_{50}<50{\mu}g/ml$) against the human cancer cell line HCT116. Under the same conditions, liquiritigenin also inhibited the phosphorylation of GSK3 by Akt to 26%, and its cytotoxicity against the HCT116 cell line was lower than $100{\mu}g/ml$.

• 유기물자원화
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• 제13권4호
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• pp.100-106
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• 2005

사탕무즙을 이용한 알콜생산에서 배출되는 증류폐액(sugar beet stillage)을 기질로 고온성 효모인 Candida rugosa, Kl. marxianus, C. utilis를 이용해 단세포단백질을 생산 할 때 균체 생산량 및 COD 감소를 연구하였다. pH가 높아짐에 따라 균체생산량은 증가했으나 단백질 함량은 오히려 감소하였다. 조단백질생산량은 3.68g/l로 C. rugosa 가 가장 높았고 이어서 C. utilis가 2.90g/l, Kl. marxianus가 2.30g/l로 나타났다. 감소한 증류폐액기질의 COD값에 대한 조단백질생산량비율도 C. rugosa가 0.35~0.39g/l를 나타내 가장 높은 값을 보여주어 사탕무우알콜증류폐액을 기질로 이용한 단세포 단백질 생산에 있어서 종균으로서 우수성을 보여주었다.

• The Plant Pathology Journal
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• 제31권2호
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• pp.108-114
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• 2015

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

• 생명과학회지
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• 제14권2호
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• pp.309-314
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• 2004

cAMP수용성 단백질(CRP)은 E. coli의 100가지 이상의 유전자 전자조절에 관계된다. CRP는 dimer로 존재하며 cAMP의 결합으로 활성인 형태로 전환된다. 이중체인 CRP 단백질의 열 안정성과 구조 전이의 연구에 효과적인 온도 기울기 전기영동장치를 이용하여 확인하였다. 본 연구에서 야생형과 S83C CRP 단백질의 melting temperature (Tm)는 산성인 완충용액[89.8 mM Glycine, 24mM Boric acid (pH 5.8)]에서 57$\pm$1(야생형 CRP)과 55$\pm$1$^{\circ}C$ (S83G CRP)였다. 그리고 온도에 따른 CRP 단백질의 구조변화도 protease digestion과 CD spectropolarimeter을 이용하여 확인하였다.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.122-128
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• 1992

Runaway replciation plasmid pSY35AT를 이용하여 이 plasmid가 가지고 있는 $cI_{857}$ repressor를 숙주 MC1065를 이용하여 $30^{\circ}C$에서 $37^{\circ}C$로 온도를 올려 shift-up 법으로 생산하였다. $cI_{857}$은 wild type cI repressor 정제법을 수정하여 사용하였으며 정제된 $cI_{857}$ repressor 농도는 0.11mg/ml이었다. 그리고 정제된 $cI_{857}$ repressor와 $^3H-CTP$로 labelinf한 PrOr과의 결합활성은 cell 파쇄액보다 약 23배 높았으며, 온도가 올라갈수록 결합활성은 떨어졌다.

• Journal of Yeungnam Medical Science
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• 제10권2호
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• pp.298-305
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• 1993

임신주령과 진통 및 분만의 진행정도에 따른 정상 임부의 혈청 CRP치의 동태를 파악하기 위해서 1992년 3월 1일 부터 1993년 8월 31일 까지 18개월간 영남대학교 의과대학 부속병원 산부인과에서 임신 20주에서 44주 사이의 건강한 임부 521명을 대상으로 혈청 CRP치를 측정하여 임신 주령과 진통의 유무, 양막의 파열여부 및 분만의 진행정도에 따라 비교 분석한 결과는 다음과 같다. 1. 혈청 CRP치 0.8mg/dl 이상과 2.0mg/dl 이상을 나타낸 임부의 빈도는 각각 12%(61/521)와 4%(22/521)이었다. 2. 임신주령 37주이하의 만삭전 임부군과 38주 이상의 만삭 임부군, 양막 파열 임부군과 비파열 임부군, 자궁경관 개대 3cm이하 임부군과 4cm 이상 임부군의 비교에서 혈청 CRP치 0.8mg/dl 이상과 2.0mg/dl 이상을 나타낸 임부의 빈도는 통계적으로 유의한 차이가 없었다. 3. 진통이 없는 임부군과 진통중인 임부군의 비교에서 혈청 CRP치 0.8mg/dl 이상을 나타낸 임부의 빈도는 각각 5.93%와 13.73%로 통계적으로 유의한 차이(p<0.05)가 있었으나 혈청 CRP치 2.0mg/dl 이상을 나타낸 임부의 빈도는 차이가 없었다. 4. 임신주령 37주 이하의 만삭전 임부군에서 혈청 CRP치 0.8mg/dl 이상을 나타낸 임부의 빈도는 진통중인 임부군이 23.64%로 진통이 없는 임부군의 4.69% 보다 현저히 높았으며(p<0.001), 혈청 CRP치 2.0mg/dl 이상을 나타낸 임부의 빈도도 진통중인 임부군과 진통이 없는 임부군이 각각 12.73%와 3.13%로 통계적으로 유의성 있는 차이(p<0.05)가 있었다. 임신주령 38주 이상의 만삭 임부군에서 진통이 없는 임부군과 진통중인 임부군을 비교하여 혈청 CRP치 0.8mg/dl 이상과 2.0mg/dl 이상을 나타낸 임부의 빈도는 통계적으로 유의성 있는 차이가 없었다. 이상 본 연구의 결과를 종합하면 임부의 감염을 조기 예측하는데 있어 기왕에 조기진통 임부에서 임상적 감염의 지표로 이용되고 있는 혈청 CRP치 2.0mg/dl은 진통중인 만삭임부에서도 양막파열 여부와 분만의 진행정도와 무관하게 유용한 것으로 추정되며, 조기진통 임부의 처치시 혈청 CRP치가 0.8mg/bl 이상인 경우 임부의 불현성 감염을 의심하여야 할 것으로 생각된다.

• 생명과학회지
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• 제26권4호
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• pp.387-397
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• 2016

본 연구는 17-DMAG이 골격근에서 autophagy에 관여하는 가를 조사하기 위해, C2C12세포와 마우스 골격근에서 17-DMAG (Hsp90 억제제/Hsp72 활성제)을 처치하는 그룹과 autophagy 억제제(Bafilomycin 또는 colchicine)를 처치하는 그룹과 처치하지 않는 그룹을 동시에 두고 autophagy flux를 측정하였다. C2C12 배양세포에서 17-DMAG이 Hsp90 억제/hsp72 활성화시켰으며 Akt-mTOR 신호체계를 유의하게 감소시켰지만(p<0.05) autophagy marker 단백질인 LC3 II와 p62를 증가시키지 않았다. in vivo 모델의 경우 17-DMAG 처치가 배양세포에서 발견된 것처럼 Hsp90억제/hsp72를 활성화시켰고 Akt-mTOR 신호체계를 유의하게 감소시켰다(p<0.05). 반면 LC3 II와 p62 단백질 수준은 autophagy 억제제(colchicine) 처치 수준보다 더 높게 증가되었다. 이는 17-DMAG이 골격근에서 autophagy를 증가시키지만 C2C12 배양세포에서는 autophagy의 활성화가 제한적임을 암시한다. 현재 이러한 in vitro와 in vivo 모델에서의 차이는 불분명하다.

• 대한의생명과학회지
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• 제12권4호
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• pp.419-425
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• 2006

Phospholipase $C-{\gamma}1\;(PLC-{\gamma}1)$ is an important signaling molecule for cell proliferation and differentiation. $PLC-{\gamma}1$ contains two pleckstrin homology (PH) domains, which are responsible for protein-protein interaction and protein-lipid interaction. $PLC-{\gamma}1$ also has two Src homology (SH)2 domains and a SH3 domain, which are responsible for protein- protein interaction. To identity proteins that specifically binds to PH domain of $PLC-{\gamma}1$, we prepared and incubated the glutathione S-transferase(GST)-fused PH domains of $PLC-{\gamma}1$ with COS7 cell lysate. We found that 90 kDa protein specifically binds to PH domain of $PLC-{\gamma}1$. By matrix-assisted laser desorption ionization time of flight-mass spectrometry, the 90 kDa protein revealed to be heat shock protein (Hsp) $90{\beta}$. Hsp $90{\beta}$ is a molecular chaperone that stabilizes and facilitates the folding of proteins that are involved in cell signaling, including receptors for steroids hormones and a variety of protein kinases. To know whether Hsp $90{\beta}$ affects on $PLC-{\gamma}1$ activity, we performed $PIP_2$ hydrolyzing activity of $PLC-{\gamma}1$ in the presence of purified Hsp $90{\beta}$ in vitro. Our results show that the Hsp $90{\beta}$ dose-dependently inhibits the enzymatic activity of $PLC-{\gamma}1$ and further suggest that Hsp $90{\beta}$ regulates cell growth and differentiation via regulation of $PLC-{\gamma}1$ activity.

• 한국가금학회지
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• 제44권2호
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• pp.67-73
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• 2017

The purpose of our study was to determine the effects of varying levels of energy, protein, and amino acids on the performances of laying hens. A total of 240 Hy-Line Brown laying hens at 36 weeks of age were used in this 4-week feeding trial. The hens were randomly allocated to five treatment diets, with eight replications of six hens in each replicate cage. The treatment diets were as follows: A- basal diet + 18% crude protein, metabolizable energy 2,800 kcal, total (methionine + cysteine) 0.65%; B- basal diet + 17% crude protein, metabolizable energy 2,700 kcal, total (methionine + cysteine) 0.59%; C- basal diet + 16.5% crude protein, metabolizable energy 2,700 kcal, total (methionine + cysteine) 0.59%; D- basal diet + 16.5% crude protein, metabolizable energy 2,700 kcal, total (methionine + cysteine) 0.54%; and E- basal diet + 16% crude protein, metabolizable energy 2,680 kcal, total (methionine + cysteine) 0.54%. The study results revealed that the hen-day egg production of hens that were fed with low-energy diets (B, C, and D) was comparable with that of hens fed with high-energy diet A, whereas average daily feed intake in hens fed treatment diet D and E was significantly higher (P<0.05) than that in hens fed treatment diet A. Overall, the eggshell thickness was unaffected by any of the treatment diets. Egg weight was comparable among the treatment diets, except for treatment diet E. Haugh unit improved with decreasing levels of dietary energy, protein, and methionine + cysteine in the diet. We can summarize that laying hens fed with low dietary energy and low crude protein treatment diets B, C, and D had satisfactory performance compared with those fed with high-energy treatment diet A. This indicates that there is the potential to reduce feed costs by formulating diets with lower energy and low protein levels.

• 농업과학연구
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• 제44권3호
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• pp.392-399
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• 2017

The present study was conducted to investigate the effects of different dietary proteins as fraction-enriched protein, defined by Cornell net carbohydrates and protein system (CNCPS), on in vivo ruminal fermentation pattern and blood metabolites in Holstein steers fed total mixed ration (TMR) containing 17.2% crude protein. Four ruminally cannulated Holstein steers in a $4{\times}4$ Latin square design consumed TMR only (control) and TMR with rapeseed meal (AB1), soybean meal (B2), and perilla meal (B3C). Each protein was substituted for 23.0% of crude protein in TMR. Rumen digesta were taken through ruminal cannula at 1 h interval during the feeding cycle in order to analyze ruminal pH, ammonia-N, and volatile fatty acids (VFA). Plasma metabolites in blood taken via the jugular vein after the rumen digesta sampling were analyzed. Feeding perilla meal significantly (p < 0.05) decreased mean ruminal pH compared with control and the other protein feeding groups. Compared with control, feeding protein significantly (p < 0.05) increased ruminal ammonia-N concentration except for AB1. Statistically (p > 0.05) similar total VFA appeared among control and the supplemented groups. However, control, AB1, and B2 showed higher (p < 0.05) acetate concentrations than B3C, and propionate was vice versa. CNCPS fractionated protein significantly (p < 0.05) affected concentrations of albumin and total protein in blood; i.e. plasma albumin was lower for control and B2 groups than AB1 and B3C groups. Despite lack of significances (p > 0.05) in creatinine and blood urea nitrogen, AB1 and B2 groups were numerically higher than the others.