• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.68-72
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• 2002

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin is present in the two closely related bacterial species, Streptomyces lividans and Streptomyces coelicolor, it normally is expressed only in S. coelicolor---generating the deep blue colonies responsible for the S. coelicolor name. However, multiple copies of the afsR2 gene, which activates actinorhodin synthesis, result in the ability of S. lividansto also synthesize large amounts of actinorhodin. Here we report that the phenotypic property that historicially distinguishes these two Streptomycesspecies is determined conditionally by the carbon source used for culture. Whereas growth on glucose repressed actinorhodin production in S. lividans, culture on solid media containing glycerol as the sole carbon source dramatically increased the expression of afsR2 mRNA---leading to extensive actinorhodin synthesis by S. lividansand obliterating its phenotypic distinction from S. coelicolor. afsR2 transcription under these conditions was developmentally regulated, rising sharply at the time of aerial mycelium formation and coinciding temporally with the onset of actinorhodin production. Our results, which identify media-dependent parallel pathways that regulate actinorhodin synthesis in S. lividans, demonstrate carbon source control of actinorhodin production through the regulation of afsR2 mRNA synthesis. The nucleotide sequences of afsR2 revealed two putative important domains; the domain containing direct repeats in the middle and the domain homologous to sigma factor sequence in the C-terminal end. In this work, we constructed various sized afsR2-derivatives and compared the actinorhodin stimulating effects in S. lividans TK21. The experimental data indicate that the domain homologous to sigma factor sequence in the C-terminal end of afsR2 plays a critical role as an antibiotic stimulating function. In addition, we also observed that the single copy integration of afsR2 regulatory gene into S. lividans TK21 chromosome significantly activates antibiotic overproduction.

• Journal of Microbiology and Biotechnology
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• 제1권2호
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• pp.116-120
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• 1991

The transformant Bacillus subtilis ED213 carrying the pSO100 which cloned the cdd gene encoding cytidine deaminase (cytidine /2'-deoxycytidine aminohydrolase, EC 3.5.4.5, CDase) originated from wild type B. subtilis was cultivated in Spizizen minimal medium (SMM). To overcome poor expression of the cdd gene in SMM medium, the medium compositions and growth conditions were optimized. The optimized medium compositions and growth conditions were cytidine concentration of 80 mg/l, glycerol of 25 g/l, and $(NH_4)_2SO_4$ of 10 g/l, along with $37^{\circ}C$ and pH 7.0. The intracellular CDase production was increased 3 times from 1,000 unit/ml to 3,200 unit/ml, and extracellular CDase also increased from nearly undetectable amounts to 1,500 unit/ml. The cytidine concentration was signified as the most critical compositional factor for overproduction of CDase by increasing the cell density mainly in culture broth. The plasmids were more stable in cells that were grown in original SMM medium with stability of 90% compared to those grown in optimized SMM medium with stability of 80% after 48 hours cultivation. The most active amplification of plasmid was occurred in the logarithmic phase, which showed a value around four times higher than the initial copy number. In the exponential phase, the CDase production was closely related to the plasmid copy number along with the cell density. However, it was not accorded with cell density at the stationary phase.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1538-1545
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• 2007

Clavulanic acid (CA) is an inhibitor of ${\beta}$-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHLl, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHLl3, and a 23.8-fold increase with pNQ1. The integrative pNQl strain has been successfully applied to enhance production.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.183-187
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• 2008

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

• 한국가축번식학회지
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• 제24권4호
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• pp.321-333
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• 2000

High production of milk and its components are necessary to allow maximal growth of developing piglets. In this study, transgenic pigs were produced containing the $\alpha$ -lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein $\alpha$ -lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine $\alpha$ -lactalbumin gene. The gene construct contained 2.0 kb of 5'flanking region, the 2.0 kb coding region and 329 bp of 3'flanking region. Sows hemizygous for the trans gene produced as much as 0.9 g of bovine $\alpha$-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50% increase in the total $\alpha$ -lactalbumin concentration in pig milk throughout lactation. The concentration of bovine $\alpha$ -lactalbumin was highest on day 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine $\alpha$ -lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on day 0 of lactation, but by day 20 of lactation the ratio was 0.43 to 1. This suggested that the bovine transgene and the endogenous porcine gene were under slightly different control mechanisms. The higher level of total $\alpha$-lactalbumin present on day 0 of lactation was correlated with higher lactose percentage on day 0 in transgenic sows (3.8%) as compared to controls (2.6%) (P＜0.01). Although there was also a trend for higher lactose percentage in transgenic sows on day 5 and 10 of lactation, no significant differences were observed. These data suggest that $\alpha$ -lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of $\alpha$ -lactalbumin early in lactation may boost milk output.

• 대한한방내과학회지
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• 제29권1호
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• pp.177-188
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• 2008

Objectives : This study was performed to investigate the anti-fibrogenic effect of Artemisiae Capillaris Herba on cultured rat hepatic stellate cells. Materials and Methods : Hepatic stellate cells(HSC-T6) were treated with various concentrations of Artemisiae Capillaris Herba extract for 24 hours. The extraction was done either with distilled water or 50% EtOH. After the treatment, cell viability, proliferation, procollagen levels and the mRNA of the collagen type 1a2 and ASMA were measured by using MTT assay, BrdU assay, RT-PCR, and Procollagen Type I C-peptide EIA Kit. Results : The viability and proliferation of the hepatic stellate cells were decreased as the concentration increased. The mRNA expression decreased consistently with the volume of the secreted procollagen with the extraction made with distilled water, which indicates the herb has inhibitory effect on fibrogenesis of the liver by regulating one of the fibrosis associated genes in transcription. However, it increased in 50% EtOH extraction, which shows that a more stable reaction is expected of the extraction made with distilled water than the extraction made with 50% EtOH. The production of procollagen was decreased by a low-concentration treatment with Artemisiae Capillaris Herba, but increased by a high concentration. It seemed that the cells were responding to Artemisiae Capillaris Herba in low- concentrations, thus producing small amounts of collagen. When the drug was administered at high enough concentration to give direct toxicity to cells, the ability of cells to produce collagen was activated, and the overproduction of collagen was observed as an undesirable results. Conclusion : These results suggest that Artemisiae Capillaris Herba is beneficial in the treatment of cirrhotic patients as well as for the patients with chronic hepatitis when extracted with water in the proper concentrations.

• Biomolecules & Therapeutics
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• 제22권1호
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• pp.10-16
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• 2014

Derivatives of caffeic acid have been reported to possess diverse pharmacological properties such as anti-inflammatory, anti-tumor, and neuroprotective effects. However, the biological activity of methyl p-hydroxycinnamate, an ester derivative of caffeic acid, has not been clearly demonstrated. This study aimed to elucidate the anti-inflammatory mechanism of methyl p-hydroxycinnamate in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Methyl p-hydroxycinnamate significantly inhibited LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and $PGE_2$ and the protein expression of iNOS and COX-2. Methyl p-hydroxycinnamate also suppressed LPS-induced overproduction of pro-inflammatory cytokines such as IL-$1{\beta}$ and TNF-${\alpha}$. In addition, methyl p-hydroxycinnamate significantly suppressed LPS-induced degradation of $I{\kappa}B$, which retains NF-${\kappa}B$ in the cytoplasm, consequently inhibiting the transcription of pro-inflammatory genes by NF-${\kappa}B$ in the nucleus. Methyl p-hydroxycinnamate exhibited significantly increased Akt phosphorylation in a concentration-dependent manner. Furthermore, inhibition of Akt signaling pathway with wortmaninn abolished methyl p-hydroxycinnamate-induced Akt phosphorylation. Taken together, the present study clearly demonstrates that methyl p-hydroxycinnamate exhibits anti-inflammatory activity through the activation of Akt signaling pathway in LPS-stimulated RAW264.7 macrophage cells.

• Journal of Plant Biotechnology
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• 제5권1호
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• pp.43-49
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• 2003

Resistant cell lines to azetidine-2-carboxylic acid (AZCA) were selected through rice embryo culture after mutagenic treatment of callus irradiated with 30,50,70,90 and 120 Gy. The optimum AZCA concentration for the selection of resistant cell lines was 3 or 4 mM AZCA considering $LD_{50}$ and the fresh weight of callus. Survival rate of the AZCA resistant callus showed remarkable increase in the callus irradiated with 50 and 70 Gy. Regeneration rate of the AZCA resistant callus was much lower on the whole. Ninety and 120 Gy increased the regeneration rate for calli selected from 3 and 4 mM AZCA, respectively. Based on fresh weight, survival rate and regeneration for selection of the AZCA resistant cell line, 50-90 Gy was considered as the optimum range of gamma irradiation. Irradiated calli selected from AZCA were more tolerant to NaCl than those from non-irradiated calli. It suggests that elevated resistance to osmotic stress resulted from mutagenic treatment. The level of free proline content in the AZCA resistant cell line was increased up to 3.5 times compared with that in the control. Proline content in the regenerant derived from the AZCA resistant cell line also increased to 1.7 times that from the control plants regenerated from callus grown in AZCA free medium. Selection of proline overproducing cell lines by in vitro mutagenesis was successful and seems to be useful for improvement of stress tolerance in this crop.

• 한국식품과학회지
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• 제50권2호
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• pp.209-215
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• 2018

치자 추출물의 극성에 따른 분획-헥세인, 에틸아세테이트와 뷰탄올 분획의 폴리페놀 함량을 평가한 결과, 에틸아세테이트 분획이 다량의 폴리페놀을 함유함을 하였고, DPPH와 ABTS 라디칼 제거와 환원력 실험에서 강력한 라디칼 제거와 환원능력을 나타냈다. 또한 치차 에틸아세테이트 분획(GJ-EA)의 라디칼 제거 활성을 세포수준에서 평가한 결과, LPS로 활성화된 BV-2 미세아교세포에서 발생하는 과량의 산화질소 생성을 억제하였고, 과산화수소를 처리한 췌장암 세포주의 증식과 이동성에 대해 억제효과를 나타내었다. 따라서 치자 에틸아세테이트 분획(GJ-EA)이 라디칼 제거효과, 과산화수소 유도형 암세포의 증식과 이동성에 대한 억제효과를 가진다는 것을 확인하여 치자 에틸아세테이트 분획(GJ-EA)을 이용한 기능성 식품소재 개발을 위한 기초자료로 활용될 수 있음을 제시하였다.

• Biomolecules & Therapeutics
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• 제20권6호
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• pp.532-537
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• 2012

Avicularin, quercetin-3-${\alpha}$-L-arabinofuranoside, has been reported to possess diverse pharmacological properties such as anti-inflammatory and anti-infectious effects. However, the underlying mechanism by which avicularin exerts its anti-inflammatory activity has not been clearly demonstrated. This study aimed to elucidate the anti-inflammatory mechanism of avicularin in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Avicularin significantly inhibited LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and $PGE_2$ and the protein levels of iNOS and COX-2, which are responsible for the production of NO and $PGE_2$, respectively. Avicularin also suppressed LPS-induced overproduction of pro-inflammatory cytokine IL-$1{\beta}$. Furthermore, avicularin significantly suppressed LPS-induced degradation of $I{\kappa}B$, which retains NF-${\kappa}B$ in the cytoplasm, consequently inhibiting the transcription of pro-inflammatory genes by NF-${\kappa}B$ in the nucleus. To understand the underlying signaling mechanism of anti-inflammatory activity of avicularin, involvement of multiple kinases was examined. Avicularin significantly attenuated LPS-induced activation of ERK signaling pathway in a concentration-dependent manner. Taken together, the present study clearly demonstrates that avicularin exhibits anti-inflammatory activity through the suppression of ERK signaling pathway in LPS-stimulated RAW 264.7 macrophage cells.