• 한국식품영양과학회지
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• 제20권6호
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• pp.519-526
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• 1991

평균 체중이 $137.51{\pm}4.84g$인 Sprague-Dawley종 수컷 22마리를 세 군으로 나누어 각각 68% cornstarch 식이(cornstarch군)와 20% sucrose+48% cornstarch 식이(sucrose군), 그리고 20% glycosylsucrose+48% cornstarch 식이(glycosyl-sucrose군)로 6주간 사육하여 glycosyl-sucrose가 혈당 및 혈청 지질 농도, 당 가수분해 효소의 활성에 미치는 영향을 검토하였다. 사료 섭취량, 체중 증가량 및 사료 효율에 있어서는 세 군간에 유의적인 차이를 볼 수 없었다. Glycosyl-sucrose군에서 공복시의 혈당 농도는 sucrose군과 비교해 차이가 없었으나 포도당 투여 30분 후, 60분 후의 혈당치는 낮은 경향을 보였다. 또한 충혈당바응면적(total integrated blood glucose response area)도 sucrose군에 비해 낮은 경향을 보였다. 혈청 지질 함량은 분석한 지질(중성 지방, 인지질, 총콜레스테롤, HDL-콜레스테롤) 모두에 있어 세 군간에 통계적 유의성은 없었다. 췌장-amylase 활성은 glycosyl-sucrose 군에서 증가하는 경향을 보였다. 소장 점막, 특히 공장과 회장에서의 서당 가수분해 효소 활성은 glycostyl-surcose군이 다른 두 군에 비해 유의적으로 감소되었다. 맥아당 가수분해 효소의 활성은 cornstarch군에 비해 glycostyl-surcose군의 공장과 회장 부위에서 낮게 나타났으며 특히 공장에서는 sucrose군보다도 유의적으로 낮은 수치를 보였다.

• 한국식품영양과학회지
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• 제32권6호
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• pp.887-892
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• 2003

신령버섯의 균사체 배양액이 성장기 흰쥐에 미치는 영향을 관찰하고자 생후 7주령의 흰쥐 수컷을 대상으로 음료수에 20% 및 30% 수준으로 신령버섯 균사체 배양액을 혼합하여 5주간 급여하고 성장률, 식이섭취량, 식이효율, 장기의 무게, 간과 혈청의 지질 농도, 혈청의 단백질 농도 및 효소 활성을 조사한 결과는 다음과 같다. 체중증가량, 식이섭취랑, 식이효율 및 장기의 무게는 대조군과 신령버섯 음료군들이 유의한 차이를 보이 지 않았다. 간과 혈청 의 중성 지질과 콜레스테롤 농도 및 혈청의 HDL-콜레스테를 농도는 대조군과 신령버섯 음료군들이 비슷한 수준으로 나타났다. 혈청의 LDL-콜레스테를 농도 및 동맥경화지수는 대조군에 비해 신령버섯 음료군들이 유의하게 감소되었다. 혈청의 총 콜레스테롤에 대한 HDL-콜레스테롤의 비율은 대조군에 비해 신령버섯 음료군들이 유의하게 증가되었다. 혈청의 총 단백질, 알부민, 혈당, 혈색소, 마그네슘 등의 농도는 각 수준별 신령버섯 음료군이 대조군과 비슷한 농도로 신령버섯 음료 섭취에 따른 영향은 나타나지 않았다. 혈청의 GPT, GOT, LDH및 ALP 등의 활성도 신령버섯 음료 섭취에 따른 영향은 나타나지 않았다. 혈청의 칼슘과 인의 농도는 대조군에 비해 신령버섯 음료군들이 유의하게 감소되었다. 이상의 결과로보아 성장기 흰쥐에 신령버섯 균사체 배양액을 20% 및 30% 수준으로 급여시 성장률, 간과 혈청의 콜레스테를 및 중성지질 농도, 단백질 농도 및 효소 활성은 정상수준을 유지하였고, 혈청의 LDL 콜레스테를 농도 및 동맥경화지수를 낮추는 효과가 있고, 혈청의 총 콜레스테롤에 대한 HDL-콜레스테롤의 비율을 높여주는 효과가 있는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.373-381
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• 2019

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency ($k_{cat}/K_M$) of the ${\underline{M151A}}$/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using ${\underline{V115A}}$/C295A/I86A mutant in asymmetric reduction. The ${\underline{A85G}}$/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'-methoxyacetophenone, with improved yield. In terms of thermal stability, the ${\underline{M151A}}$/C295A/I86A and ${\underline{V115A}}$/C295A/I86A mutants significantly increased ${\Delta}T_{1/2}$ by $+6.8^{\circ}C$ and $+2.4^{\circ}C$, respectively, with thermal deactivation constant ($k_d$) close to the wild-type enzyme. The ${\underline{M151A}}$/C295A/I86A mutant reacts optimally at $70^{\circ}C$ with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'-methylacetophenone by ${\underline{V115A}}$/C295A/I86A, and (R)-1-phenylethanol from acetophenone by ${\underline{M151A}}$/C295A/I86A mutant, in large-scale bioreduction processes.

• 대한한방내과학회지
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• 제18권1호
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• pp.191-206
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• 1997

In order to investingate the effects of Jukyeopseokgotanggagambang Extract on antitumor effects after human cell lines(A549, hep3B, Caki-1, Ehrlich) transplantation into the peritoneal cavity or right groin in mice induced by RPMI 1640 and GIBCO etc., the extracts of its herbal medicines were orally administered for 10 or 12days. Experimental studies were performed for measurance of antitumor effect of MMC(Mitomycin C) and lysosomal enzyme's activities using colony forming efficiency, SRB assay which were regarded as a valuable method for antitumor effects of unknown compound on tumor cell lines. The results obtained in this studies were as follows: 1. According to the change of colony-forming efficiency and SRB assay of Caki-1 cell, hep3B and A549 cells after exposure to the extract of Jukyeopseokgotanggagambang extract, that extract depressed the growth of tumor cells depending on its concentration. 2. Antitumor activities of the ethanol extract from Jukyeopseokgotanggagambang extract and MMC on ascites form of Ehrlich carcinoma in mice is a little improved. Especially mean survival times of the group of Jukyeopseokgotanggagambang extract 200mg/kg and MMC 0.1mg/kg is improved over 30%. 3. When Jukyeopseokgotanggagambang extract and MMC are administerated together, the weight of tumor is more decreased than MMC alone. 4. The effects of the Jukyeopseokgotanggagambang extract and MMC on the lysosomal enzymes in Ehrlich ascites carcinoma cell are more significantly improved than MMC alone. 5. Jukyeopseokgotanggagambang extract also increased the uptake of MMC into Ehrlich carcinoma cells. According to the above results, it could be suggested that Jukyeopseokgotanggagambang extract has indirect autitumor effects by strengthening the effects of MMC on tumor cells.

• 대한한방종양학회지
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• 제3권1호
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• pp.149-167
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• 1997

In order to investingate the effects of Jukyeopseokgotanggagambang Extract on antitumor effects after human cell lines(A549, hep3B, Caki-1, Ehrlich) transplantation into the peritoneal cavity or right groin in mice induced by RPMI 1640 and GIBCO etc., the extracts of its herbal medicines were orally administered for 10 or 12days. Experimental studies were performed for measurance of antitumor effect of MMC(Mitomycin C) and lysosomal enzyme's activities using colony forming efficiency, SRB assay which were regarded as a valuable method for antitumor effects of unknown compound on tumor cell lines. The results obtained in this studies were as follows: 1. According to the change of colony-forming efficiency and SRB assay of Caki-1 cell, hep3B and A549 cells after exposure to the extract of Jukyeopseokgotanggagambang extract, that extract depressed the growth of tumor cells depending on its concentration. 2. Antitumor activities of the ethanol extract from Jukyeopseokgotanggagambang extract and MMC on ascites form of Ehrlich carcinoma in mice is a little improved. Especially mean survival times of the group of Jukyeopseokgotang-gagambang extract 200mg／kg and MMC 0.1mg／kg is improved over 30%. 3. When Jukyeopseokgotanggagambang extract and MMC are administerated together, the weight of tumor is more decreased than MMC alone. 4. The effects of the Jukyeopseokgotanggagambang extract and MMC on the lysosomal enzymes in Ehrlich ascites carcinoma cell are more significantly improved than MMC alone. 5. Jukyeopseokgotanggagambang extract also increased the uptake of MMC into Ehrlich carcinoma cells. According to the above results. it could be suggested that Jukyeopseokgotanggagambang extract has indirect autitumor effects by strengthening the effects of MMC on tumor cells.

• Asian-Australasian Journal of Animal Sciences
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• 제17권6호
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• pp.836-842
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• 2004

An experiment was conducted to study the performance of broilers chicks (2 to 42 d of age) fed diets containing pearl millet (PM, Pennisetum typhoides), foxtail millet (FOM, Setaria italica) or finger millet (FIM, Elusine coracana) totally replacing (w/w) yellow maize (YM) with and with out supplementing non-starch polysaccharide (NSP) hydrolysing enzymes at the rate of 0.5 g/kg diet. Enzyme preparation contained amylase 2,400 units, hemi-cellulase 5,400 units, cellulase 12,000 units, protease 2,400 units and beta-glucanase 106 units/g. Each diet was fed to eight replicates (five female Vencob broilers/replicate) housed in stainless steel battery brooders. The estimated metabolizable energy (ME) contents of YM, PM, FOM and FIM were FM (PM) were about 3,389, 2,736, 3,303 and 2,846 kcal/kg, respectively. Total replacement of YM with FOM did not influence the body weight gain, ready to cook yield, relative weights of giblet, liver, intestine, lymphoid organs (bursa and spleen) and length of intestine, antibody titers and livability at 42 d of age. But the food efficiency decreased significantly in FOM fed broilers compared those fed YM. Further, the fat content in thigh muscle reduced with FOM fed groups compared to those fed YM. The performance of broilers decreased significantly in PM and FIM fed broilers compared to those fed YM. The relative weights of giblet, gizzard and liver increased in FIM fed groups compared to those fed YM as the principal source of energy in broilers. Incorporation of NSP hydrolysing enzymes in commercial broiler diets improved the efficiency of feed utilization during starter phase but not at 42 d of age. The results thus indicate that yellow maize can be replaced in toto on weight basis in commercial broiler diets without affecting the performance. Supplementation of NSP hydrolysing enzymes was beneficial in enhancing feed utilization during the starter phase.

• Asian Pacific Journal of Cancer Prevention
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• 제16권2호
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• pp.571-578
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• 2015

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-${\gamma}$, TNF-${\beta}$ and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-${\gamma}$, TNF-${\beta}$ and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

• International Journal of Oral Biology
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• 제37권4호
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• pp.181-188
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• 2012

As the demand for large-scale analysis of gene expression using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We compared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM) of mercaptohexanol or hepthanethiol, which create hydrophilic or hydrophobic surface properties, respectively. A hydrophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA molecules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The optimal DNA concentration for immobilization was found to be 0.5 ${\mu}M$. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized conditions, molecular biology techniques including DNA hybridization, ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We demonstrated from our present analysis the importance of surface polarity in solid-phase molecular biological applications. A hydrophilic SAM generated a far more favorable environment than hydrophobic SAM for solid-state molecular techniques. Our findings suggest that the conditions and methods identified here could be used for DNA-DNA hybridization applications such as DNA chips and for the further development of solid-phase genetic engineering applications that involve DNA-enzyme interactions.

• The Plant Pathology Journal
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• 제35권6호
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• pp.575-584
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• 2019

Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH₄Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 ㎍/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.

• Biomolecules & Therapeutics
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• 제21권6호
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• pp.487-492
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• 2013

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.