• Journal of Microbiology and Biotechnology
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• 제28권3호
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• pp.473-481
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• 2018

Owing to its high protein secretion capacity, simple nutritional requirements, and GRAS (generally regarded as safe) status, Bacillus licheniformis is widely used as a host for the industrial production of enzymes, antibiotics, and peptides. However, as compared with its close relative Bacillus subtilis, little is known about the physiology and stress responses of B. licheniformis. To explore its temperature-stress metabolome, B. licheniformis strains ATCC 14580 and B186, with respective optimal growth temperatures of $42^{\circ}C$ and $50^{\circ}C$, were cultured at $42^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$ and their corresponding metabolic profiles were determined by gas chromatography/mass spectrometry and multivariate statistical analyses. It was found that with increased growth temperatures, the two B. licheniformis strains displayed elevated cellular levels of proline, glutamate, lysine, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, and octadecanoic acid, and decreased levels of glutamine and octadecenoic acid. Regulation of amino acid and fatty acid metabolism is likely to be associated with the evolution of protective biochemical mechanisms of B. licheniformis. Our results will help to optimize the industrial use of B. licheniformis and other important Bacillus species.

• Journal of Microbiology and Biotechnology
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• 제27권1호
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• pp.179-188
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• 2017

White-rot basidiomycetes are the organisms that decompose lignin most efficiently, and Trametes villosa is a promising species for ligninolytic enzyme production. There are several publications on T. villosa applications for lignin degradation regarding the expression and secretion of laccase and manganese peroxidase (MnP) but no reports on the identification and characterization of lignin peroxidase (LiP), a relevant enzyme for the efficient breakdown of lignin. The object of this study was to identify and partially characterize, for the first time, gDNA, mRNA, and the corresponding lignin peroxidase (TvLiP) protein from T. villosa strain CCMB561 from the Brazilian semiarid region. The presence of ligninolytic enzymes produced by this strain grown in inducer media was qualitatively and quantitatively analyzed by spectrophotometry, qPCR, and dye fading using Remazol Brilliant Blue R. The spectrophotometric analysis showed that LiP activity was higher than that of MnP. The greatest LiP expression as measured by qPCR occurred on the $7^{th}$ day, and the ABSA medium (agar, sugarcane bagasse, and ammonium sulfate) was the best that favored LiP expression. The amplification of the TvLiP gene median region covering approximately 50% of the T. versicolor LPGIV gene (87% identity); the presence of Trp199, Leu115, Asp193, Trp199, and Ala203 in the translated amplicon of the T. villosa mRNA; and the close phylogenetic relationship between TvLiP and T. versicolor LiP all indicate that the target enzyme is a lignin peroxidase. Therefore, T. villosa CCMB561 has great potential for use as a LiP, MnP, and Lac producer for industrial applications.

• 셀메드
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• 제3권2호
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• pp.18.1-18.7
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• 2013

Inflammation in rheumatoid arthritis is characterized by immune cell infiltration and cytokine secretion. In particular, mast cells and their cytokines play an important role in the pathogenesis of rheumatoid arthritis. Korean medicine, BaekJeol-Tang (BT) was designed by traditional Korean medicine theory. We already reported therapeutic effect of BT in rheumatoid arthritis. Here, we report the specific underlying mechanism of BT in activated human mast cells, HMC-1 cells. In addition, we report for the first time that BT significantly inhibited the production and mRNA expression of proinflammatory cytokines including thymic stromal lymphopoietin, interleukin (IL)-$1{\beta}$, IL-6, IL-8, and tumor necrosis factor-${\alpha}$ in activated HMC-1 cells. BT also decreased the activation of mitogen-activated protein kinases, nuclear factor-${\kappa}B$, and caspapase-1. Taken together, these results indicate that BT has potential as a regulator of inflammatory reactions for the treatment of arthritis such as osteoarthritis and rheumatoid arthritis.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.689-698
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• 2013

The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.

• 치위생과학회지
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• 제14권2호
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• pp.223-229
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• 2014

본 연구에서는 체내 염증지표 변화를 모니터링 함으로써 새로운 천연추출물인 polycan 및 calcium gluconate 복합제제의 치주질환 개선 효과를 평가하고자 하였으며, 다음과 같은 결론을 얻었다. 1. 치은열구액 내 $IL-1{\beta}$는 대조군과 시험군 모두 시간에 따른 유의한 차이는 없었으나(p>0.05), 4주째 시험군이 대조군보다 낮았다(p<0.05). 2. 치은열구액 내 MMP-8과 $TNF-{\alpha}$는 4주째 시험군에서 초기에 비해 감소하였으며, 시험군이 대조군보다 낮았다. 3. 혈액 내 MMP-8은 4주째 시험군에서 유의하게 감소하였고(p<0.05), 대조군에 비해 통계적으로 유의하게 낮았다(p<0.05). 그러나 혈액 내 CRP는 유의한 변화가 관찰되지 않았다(p>0.05). 이상의 결과들을 종합해 보았을 때, polycan을 함유한 calcium gluconate 복합제 복용이 전신적인 염증성 생체지표에 영향을 미쳐 치은열구액 내의 염증매개물질들을 감소시킴으로써 치은건강에 긍정적인 효과를 나타내는 것으로 사료된다.

• 한국식품영양과학회지
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• 제42권10호
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• pp.1576-1584
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• 2013

이상의 결과를 종합해 볼 때, 높은 수준의 포도당 섭취는 공복혈당과 공복혈당면적을 높이고 혈중 인슐린 농도와 아티포넥틴의 수준을 낮추어 혈당조절 능력을 억제시켰다. 반면, 높은 수준의 과당 섭취는 인슐린을 요구하지 않는 과당대사의 특이성으로 인해 혈당조절에는 효과적으로 보인다. 그러나 고과당 섭취는 간 조직 및 혈중 중성지방의 농도를 높이고 염증조절복합체 구성단백질의 발현을 조절하여 전염증인자의 발현을 증가시켰다. 이는 간조직에 있어 과당이 포도당보다 높은 수준의 염증반응을 유도하여 NAFLD의 발병과 진행에 보다 유의적인 영향을 준다는 것을 보여준다. 본 연구의 제한점은 일상에서 과당이나 포도당을 단독으로 섭취하는 경우가 드물다는 점과 3주라는 짧은 중재기간에 의한 실험 결과라는 것이다. 앞으로의 연구는 단순당을 장기간 섭취했을 때 혈청과 간 조직을 포함한 다른 대사 관련조직에서 나타나는 변화에 초점을 맞출 필요가 있으며, 염증조절복합체가 염증인자의 발현을 증가시키는 기전을 구체화하는 것이 요구된다.

• 한국가금학회지
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• 제33권1호
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• pp.49-55
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• 2006

이 연구는 시호의 첨가가 육용종계의 생산성, 혈액의 성상, 호르몬 분비에 미치는 영향을 구명하기 위해서 8주간 실시하였다. 총 800수의 20주령 육용종계를 4개 처리구에 각각 200수씩 배치하였고, 시호는 사료에 0, 0.5, 0.1, 0.2% 수준으로 첨가하여 급여하였다. 기초사료의 영양소 함량은 육성기와 산란기로 구분하여 각각 CP 15.0%, ME 2,900 kcal/kg 수준과 CP 17.5%, ME 2,800 kcal/kg 수준으로 하였다. 시호를 육성후기에($20{\sim}24$주) 급여하여 육용종계의 증체량을 조사한 결과, 처리구간에 차이는 없었으나 산란초기($24{\sim}28$주)의 산란율과 산란량은 시호 급여구에서 대조구에 비하여 유의적으로 증가하였다(p<0.05). 시호의 첨가에 따른 육용종계의 혈중 변화를 관찰한 결과, 혈중 콜레스테롤 농도의 경우 대조구에 비하여 시호 2.0% 급여구에서 가장 낮은 수치를 나타내었다(p<0.05). 이와는 다르게 혈중 중성지방 농도에서는 시호의 급여 효과는 관찰되지 않았다. 반면, 혈중 헤마토크릿, 포도당, 칼슘, 인 및 마그네슘 농도에서 시호의 첨가 효과는 관찰할 수 없었다. 혈중 총 단백질과 알부민 함량 또한 시호 급여에 따른 변동은 관찰할 수 없었으나 알부민/글로불린 비율은 시호 급여구가 유의하게 감소하였다(p< 0.05). 혈중 GGT, AST, ALT 및 BUN 농도에서도 시호 급여구와 대조구간의 차이는 관찰되지 않았다. 28주령 육용종계의 혈중 에스트로겐 농도는 시호 급여구가 전체적으로 증가하는 양상을 보였으며, 그 중 시호 0.5% 급여구의 에스트로겐 농도가 현저하게 증가하였다(p<0.05). 또한 IGF-I 농도는 시호 급여구가 대조구와 비교하여 유의하게 증가하였다(p<0.05). 이 연구의 결과 육용종계 사료에 시호의 첨가는 생산능력을 개선하고, 혈중 콜레스테롤 농도와 A/G 비율을 감소시켰으며, 에스트로겐과 IGF-I 분비를 증가시켰다.

• Asian-Australasian Journal of Animal Sciences
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• 제30권5호
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• pp.653-659
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• 2017

Objective: This trial was performed to examine the effects of ruminally degradable starch (RDS) levels in total mixed ration (TMR) with low corn-based starch on the milk production, whole-tract nutrient digestibility and nitrogen balance in dairy cows. Methods: Eight multiparous Holstein cows (body weight [BW]: $717{\pm}63kg$; days in milk [DIM]: $169{\pm}29$) were assigned to a crossover design with two dietary treatments: a diet containing 62.3% ruminally degradable starch (% of total starch, low RDS) or 72.1% ruminally degradable starch (% of total starch, high RDS). Changes to the ruminally degradable levels were conducted by using either finely ground corn or steam-flaked corn as the starch component. Results: The results showed that dry matter intake, milk yield and composition in dairy cows were not affected by dietary treatments. The concentration of milk urea nitrogen was lower for cows fed high RDS TMR than low RDS TMR. The whole-tract apparent digestibility of neutral detergent fiber, acid detergent fiber and crude protein decreased, and that of starch increased for cows fed high RDS TMR over those fed low RDS TMR, with no dietary effect on the whole-tract apparent digestibility of dry matter and organic matter. The proportion of urinary N excretion in N intake was lower and that of fecal N excretion in N intake was higher for cows fed high RDS TMR than those fed low RDS TMR. The N secretion in milk and the retention of N were not influenced by the dietary treatments. Total purine derivative was similar in cows fed high RDS TMR and low RDS TMR. Consequently, estimated microbial N flow to the duodenum was similar in cows fed high RDS TMR and low RDS TMR. Conclusion: Results of this study show that ruminally degradable starch levels can influence whole-tract nutrient digestibility and nitrogen balance in dairy cows fed low corn-based starch diets, with no influence on performance.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.5825-5831
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• 2013

Background/Objectives: Maintenance of cellular function in culture is vital for transfer and development following adoptive immunotherapy. Dual properties of IL-21 in activating T cells and reducing activation induced cell death led us to explore the mechanism of action of IL-21 enhanced proliferation and cytotoxic potential of CIK cells. Method: CIK cells cultured from PBMCs of healthy subjects were stimulated with IL-21 and cellular viability and cytotoxicity to K562 cells were measured. To elucidate the mechanism of action of IL-21, mRNA expression of cytotoxic factors was assessed by RT-PCR and protein expression of significantly important cytotoxic factors and cytokine secretion were determined through flow cytometry and ELISA. Western blotting was performed to check the involvement of the JAK/STAT pathway following stimulation. Results: We found that IL-21 did not enhance in vitro proliferation of CIK cells, but did increase the number of cells expressing the CD3+/CD56+ phenotype. Cytotoxic potential was increased with corresponding increase in perforin ($0.9831{\pm}0.1265$ to $0.7592{\pm}0.1457$), granzyme B ($0.4084{\pm}0.1589$ to $0.7319{\pm}0.1639$) and FasL ($0.4015{\pm}0.2842$ to $0.7381{\pm}0.2568$). Interferon gamma and TNF-alpha were noted to increase ($25.8{\pm}6.1ng/L$ to $56.0{\pm}2.3ng/L$; and $5.64{\pm}0.61{\mu}g/L$ to $15.14{\pm}0.93{\mu}g/L$, respectively) while no significant differences were observed in the expression of granzyme A, TNF-alpha and NKG2D, and NKG2D. We further affirmed that IL-21 signals through the STAT-3 and STAT-5b signaling pathway in the CIK cell pool. Conclusion: IL-21 enhances cytotoxic potential of CIK cells through increasing expression of perforin, granzyme B, IFN-gamma and TNF-alpha. The effect is brought about by the activation of STAT-3 and STAT-5b proteins.

• 한국미생물·생명공학회지
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• 제32권2호
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• pp.135-141
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• 2004

인간 과립구 성장인자(hG-CSF)는 골수에서 생산되는 단백질로 호중구의 분화 및 생성을 촉진시키는 역할을 한다. 현재 재조합 hG-CSF는 암화학요법에 의한 호중구감소증, 골수이식시 호중구 감소증, 재생불량성 빈혈에 수반되는 호중구 감소증 등으로 적응증이 확대되고 있다. 본 연구에서는 OmpA signal sequence를 삽입하여 인간 과립구 성장인자(hG-CSF)가 분비발현되도록 고안된 T7 promoter 에 의하여 발현되는 pYRCl 발현백터를 제조하였다. E. coli BL2l (pYRCl) 발현시 $37^{\circ}C$에서 배양하는 경우 많은 양의 봉입체(aggregates)를 형성한다. 이에 비하여 $10\mu$M ucose를 포함하는 변형된 MBL배지에서 10 g/$\ell$IPTG를 유도물질로 7시간동안 $25^{\circ}C$에서 배양하였을 때 전체 periplasm단백질의 15%가 soluble rhG-CSF이었다. 또한, 유가식 배양방법을 사용하여 E. coli BL2l(pYRCl)에서 soluble rhG-CSF의 생산조건을 조사하였다. 유가식 배양에서 rhG-CSF의 발현량이 비증식속도를 $0.43 h^{-1}$ 에서 0.14 $h^{-1}$ 으로, 유도 배양시간을 최적화함으로써 rhG-CSF의 발현량이 4.4mg/$\ell$에서 24mg/$\ell$ 로 증가하였다.