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Expression and Purification of Recombinant Human Interferon-gamma Produced by Escherichia coli (대장균이 생산한 재조합 인체 감마인터페론의 발현과 정제)

  • Park, Jung-Ryeol;Kim, Sung-Woo;Kim, Jae-Bum;Jung, Woo-Hyuk;Han, Myung-Wan;Jo, Young-Bae;Jung, Joon-Ki
    • KSBB Journal
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    • v.21 no.3
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    • pp.204-211
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    • 2006
  • For the production of the recombinant human interferon-gamma(rhIFN-${\gamma}$) in Escherichia coli, human glucagon and ferritin heavy chain were used as fusion partners. Even though rhIFN-${\gamma}$ is expressed as an inclusion body form in E. coli because of strong hydrophobicity of itself, over 50% of fused rhIFN-${\gamma}$ was expressed as soluble form in E. coli $Origami^{TM}$(DE3) harboring pT7FH(HE)-IFN-${\gamma}$ which encodes ferritin heavy chain-fused rhIFN-${\gamma}$. In the case of using glucagon-ferritin heavy chain hybrid mutant as a fusion partner, 6X His-tag was additionally introduced to N-terminus of GFHM(HE)-IFN-${\gamma}$ for enhancing purification yields of rhIFN-${\gamma}$. Fusion protein HGFHM(HE)-IFN-${\gamma}$ with two 6X His-tag was more effectively bound to Ni-NTA agarose bead than GFHM(HE)-IFN-${\gamma}$ with a 6X His-tag. rhIFN-${\gamma}$ was completely purified from enterokinase-treated HGFHM(HE)-IFN-${\gamma}$ by Ni-NTA affinity column. For high-level production of rhIFN-${\gamma}$, glucose was used as the sole carbon source with simple exponential feeding rate($2.4{\sim}7.2g/h$) in fed-batch process. The effective lactose concentration for the expression of the rhIFN-${\gamma}$ was $10{\sim}20mM$. Under the fed-batch culture conditions, rhIFN-${\gamma}$ production yield reached 11 g DCW/L for 6 hours after lactose induction.

The Diversity of Reproductive and Foraging Behaviors on Breeding Season of Eurasian Oystercatcher (Haematopus ostralegus) (검은머리물떼새의 번식행동 및 번식기 섭식행동의 다양성)

  • Yoon, Moo-Boo
    • The Korean Journal of Ecology
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    • v.27 no.6 s.122
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    • pp.383-390
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    • 2004
  • This study was conducted in three sites, Si-Hwa Lake, Dongman and Seoman island and Janguyeop island, from march, 1999 to september, 2002. The behaviors of pre-breeding season, territorial behaviors, reproductive ecology, foraging sites and behaviors, and the competition of reproduction and foods between intraspecific or interspecific of Eurasian Oystercatcher (Haematopus ostralegus) were observed in each studying sites. The breeding of Eurasian Oystercatcher started on the middle of April in Si-Hwa Lake and on the middle of May in Dongman and Seoman island and Janguyeop island. For intension of pair bond on pre-breeding season, Eurasian Oystercatcher foraged with pair and behaved male-female chasing flight behavior. The pair foraged with male and female before copulation. If other pairs and individuals approached in feeding site of pair, this pair attacked them with piping calling and intruder chasing flight. If continuos serial behaviors were not observed, the discrimination of male-female chasing flight and intruder chasing flight was difficult. Territorial behaviors classified four types; butterfly flight, calling behavior, chasing behavior, fight behavior. The important foraging sites in Si-Hwa Lake are the land place in Daeboo island, tidal flat of Bangameori, tidal flat a front of a stationary net for catching fishes and tidal flat a front of a view station for bird watching. Eurasian Oystercatcher foraged at tidal flat on low water of the tide and foraged at feeding sites near island on flood tide in Dongman and Seoman island. Eurasian Oystercater in Janguyeop island usually foraged feeding sites near island, because water level was not different between low water of the tide and flood tide. Eurasian Oystercatcher competed on foods of intraspecific and interspecific. They chased for taking foods by force in feeding sites and drove out intruders in feeding sites. The foods interspecific competition happened with Black-tailed Gull (Larus crassirostris). Eurasian Oystercatcher was robbed of foods and attacked by Black-tailed Gull. The individual of food competition with Black-tailed Gull was low foods intake rate comparison with other feeding sites and this individual flied out other feeding sites.

Bird Diversity on Area around the Ulsan Mosaic Landscape (울산지역 모자익 경관에서의 조류 다양성)

  • Lee, Won-Ho;Jang, Ji-Doek;Choi, Byung-In;Kang, Sung-Ryong;Kwon, Ki-Chung
    • The Korean Journal of Ecology
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    • v.27 no.6 s.122
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    • pp.325-333
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    • 2004
  • Birds were censused to investigate the composition of landscape structure for bird diversity around Ulsan between May and November 2002. Associations with three main categories of habitat variables were evaluated: 1) amount and type of forest; 2) residual habitats not classified as forest or crops; 3) land-use variables. Cluster analysis of bird community shows the highest forest variables of $79.06\%$, and the others are residual habitat variables ($17.98\%$), land-use variables ($2.94\%$) in spring, and forest variables of $57.77\%$, land-use variables ($23.16\%$), residual habitat variables ($18.47\%$) in autumn, respectively. In Principal Component Analysis of a total of 196 sites, the populations are strongly correlated to Component I ($54.8\%$) based forest habitats and to Component II based on land-use. Species preferring sites were clearly separated with heterogenous forest along the first axis. In autumn, the populations are moderately correlated to Component I based land-use and to component II based forest habitats. Species preferring local habitats were also clearly separated. Fifty three species of 1,700 birds were recorded: Brown-eared Bulbul, Vinous-throated Parrotbill, Great Tit, Tree Sparrow and Black-billed Magpie accounted for over $60\%$ of the observed birds in spring and autumn. The important species were Brown-eared Bulbul, Vinous-throated Parrotbill, Great Tit and Tree Sparrow in spring and autumn. Four habitats in terms of their species richness were computed as follows: Wonhyosan has the highest an expected species number, $E[S_{59}]=19$. Moonsusan has the lowest expected species number, $E[S_{59}]=17$ in spring. In autumn, Kuenamsan has the highest expected species number, $E[S_{63}]=16$. Moonsusan has the lowest expected species number, $E[S_{63}]=12$. Pairwise similarity declined with increasing distance between recording site and recording site from Moonsusan-Wonhyosan (0.62), the same geographical regions clustered separately in a UPGMA cluster tree in spring, and in autumn from Moonsusan-ChungJoksan (0.53).

Effect of Leaf Aqueous Extracts from Some Gymnosperm Plant on the Seed Germination, Seedling Growth and Transplant of Hibiscus syriacus Varieties (수종 나자식물의 잎 수용 추출액이 무궁화의 품종별 종자발아와 유식물 및 초기생장에 미치는 영향)

  • 배병호;김용옥
    • The Korean Journal of Ecology
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    • v.26 no.1
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    • pp.39-47
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    • 2003
  • The leaf aqueous extracts from five gymnosperms plant were investigated for allelopathy with five Hibiscus syriacus varieties. The leaf aqueous extract of Pinus rigida had the highest total phenolic compound of 2.21mg/L, whereas the soil under Pinus koraiensis canopy had the highest total phenolic compound of 1.38mg/L. Fourteen phenolic compounds were isolated from five gymnosperm plants by HPLC. Among them, phenolic compounds were the highest in P. rigida (320.56 g/mg) with the primary compound 5-sulfosalicylic acid (312.55 g/mg). The correlation between leaf total phenolic compound and pH was not significant, while the total phenolic compound of the leaf extract changed soil pH. The relative seed germination of H. syriacus varieties showed 25% was threshold concentration. The germination rates of varieties were similar to the control group or showed slight stimulation to treatment of P. koraiensis extract. H. syriacus Cambanha was similar to the control group or showed stimulation in all treated groups. H. syriacus Seohohyang showed stimulation in both root and shoot growth compared to the control group. In other varieties except Seohohyang, shoot growth was similar to the control group, while root growth was stimulated in all treated groups. The extracts of tested gymnosperms showed significantly more stimulation to transplanted Seohohyang seedlings, whereas others were similar to control or inhibited in the greenhouse. The dry weight of Seohohyang was greater in all treated groups than the control group, while other varieties were inhibited. All gymnosperm extracts stimulated the chlorophyll contents of Seohohyang and H. syriacus Koyoro but other varieties were not significantly affected. Accordingly, it is suggested that Seohohyang seems the most desirable when planted within these five gymnosperms.

Analysis on Distribution Characteristics of Mammals in Relation to Altitude in Juwangsan National Park (주왕산국립공원에 서식하는 포유동물의 고도에 따른 분포 특징의 분석)

  • Chung, Chul-Un
    • Journal of Ecology and Environment
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    • v.29 no.6
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    • pp.511-520
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    • 2006
  • The study analyzed the distribution of mammalia per altitude in Gamebong, the highest trail of Juwangsan National Park and confirmed the inhabitation of 22 different species inhabiting in the park. Starting from 200 m to 800 m above the sea level was segmented into 100 m unit. The result of analysis has shown that the Tamias sibiricus is the dominant specie throughout in all segments. Species confirmed to be inhabiting in all segments of research areas were Mustela sibirica, Caproelus pygargus and squirrel. By analyzing the confirmed Field Sign basing from the observation point, it has shown close relations between types of species and population following the increase in altitude (y=-0.0234 + 25.552, $R^2$=0.9296, $F_{0.05}$(1,4)=7.71, p<0.05). However, the result of analysis targeting nine species inhabiting in at least five out of six 100 m segmented units has shown that only two species, Apodemus sgrarius (y=-0.034X+25.2, $R^2$=0.905, $F_{0.05}$(1, 4)=38.169, p<.0.01) and Sciurus vulgaris (y=-0.0154X+ 12.819, $R^2$=0.781, $F_{0.05}$(1, 4)=14.270, p<0.05) were affected by the altitude. Therefore, it is judged that the distribution of species does not follow the altitude variation but follows the food conditions and the structure of habitat. The result of analysis of Box and Whisker Diagram had shown lowest quartile deviation($Q_U-Q_L$) in Nature Restoration Area that is located in $200{\sim}300m$ above the sea level, where hikers are prohibited from entering. In $500{\sim}600m$ above the sea level segment that has similar food environment and trail, it has shown the widest distribution. Further research is required on this segment for the excretion drops of Prionailurus bengalensis, a Grade II class of endangered species, were found on the trails.

Environmental Adaptability of Eupatorium rugosum : Relationship between Accumulation of Heavy Metals and Phenolic Compounds (서양등골나물의 환경적응력 : 중금속 축적과 Phenolic Compounds의 관계)

  • 김용옥;박종야;이호준
    • The Korean Journal of Ecology
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    • v.26 no.1
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    • pp.5-12
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    • 2003
  • Seed germination rate and seedling growth were measured on 6 different species(Phytolacca americana, Eupatorium rugosum, Rumex acetocella, Echinochloa crusgalli, Cassia mimosoides var. nomame, Setaria viridis) treated with leaf extract of E. rugosum. Total phenolic compound and heavy metal were analyzed on leaf and soil with and without E. rugosum. The growth of P. americana seedlings were stimulated by 10% and 25% of E. rugosum water extract treatment. The content of total phenolic compounds in soil was lower than that of leaf extract, and 25% was confirmed as threshold concentration in natural systems because the total phenolic compounds were not significantly different between the control soils and the soil treated with 10%, and 25% extract. Total phenolic compound concentrations of the leaf extracts were highest (1.66 mg/l) with E. rugosum grown under the Quercus forest canopy and lowest (1.09 mg/l) for the plant grown in the mixed forest edge. Leaf extracts of plants selected in different sampling sites (Forest interior, Forest edge, under Pinus Canopy and Quercus Canopy) were significant, while soil extracts were not. Seed germination of R. acetocella and S. viridis were significantly inhibited at over 50% concentrations of E. rugosum, but C. mimosoides var. nomame was not affected at any concentration. The radicle and shoot growth of the native species group were reduced two times more than those of the exotic species group by the treatment of extracts. Especially, the seed germination percentage and dry weight of E. rugosum were greater than those of the control group by treatments with extracts of 10% and 25%. Analysis of aqueous extracts from E. rugosum by HPLC identified 6 phenolic compounds: caffeic acid (460.9 mg/l), benzoic acid (109.7 mg/l), protocatechuic acid (7.3 mg/l), ρ-hydroquinone (6.0 mg/l), cinnamic acid (2.7 mg/l) and hydroquinone (0.23 mg/l). The seed germination of P. americana was also inhibited dramatically by protocatechuic acid and cinnamic acid even though the content of caffeic acid (460.9 mg/l) was the highest among analyzed phenolic compounds. The heavy metal content of soil without A. altissima was higher than that of soil with E. rugosum. Particularly, Al, Fe and Mn was considerably high and most of the heavy metal were accumulated in leaves where a high level of total phenolic compounds was found.

Effects of Pb and CO2 on Soil Microbial Community Associated with Pinus densiflora-Lab (소나무(Pinus densiflora) 생육토양의 미생물 군집에 미치는 납과 CO2의 영향)

  • Hong, Sun-Hwa;Kim, Sung-Hyun;Kang, Ho-Jeong;Ryu, Hee-Wook;Lee, Sang-Don;Lee, In-Sook;Cho, Kyung-Suk
    • Journal of Ecology and Environment
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    • v.29 no.6
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    • pp.551-558
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    • 2006
  • Effects of Pb and $CO_2$ on soil microbial community associated with Pinus densiflora were investigated using community level physiological profiles (CLPP) and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) methods. Two-years pine trees were planted in Pb-contaminated soils and uncontaminated soils, and cultivated for 3 months in the growth chamber where $CO_2$ concentration was controlled at 380 or 760 ppmv. The structure of microbial community was analyzed in 6 kinds of soil samples (CA-0M : $CO_2$ 380 ppmv + Pb 0 mg/kg + initial, CB-0M : $CO_2$ 380 ppmv + Pb 500 mg/kg + initial, CA-3M : $CO_2$ 380 ppmv + Pb 0 mg/kg + after 3 months, CB-3M : $CO_2$ 380 ppmv + Pb 500 mglkg + after 3 months, EA-3M : $CO_2$ 760 ppmv + Pb 0 mg/kg + after 3 months, EB-3M : $CO_2$ 760 ppmv + Pb 500 mg/kg + after 3 months). After 3 months, the substrate utilization in the uncontaminated soil samples (CA-3M vs EA-3M) was not significantly influenced by $CO_2$ concentrations. However, the substrate utilization in the Pb-contaminated soil samples (CB-3M vs EB-3M) was enhanced by the elevated $CO_2$ concentrations. The results of principal component analysis based on substrate utilization activities showed that the structure of microbial community structure in each soil sample was grouped by Pb-contamination. The similarities of DGGE fingerprints were 56.3 % between the uncontaminated soil samples (CA-3M vs EA-3M), and 71.4% between the Pb-contaminated soil samples (CB-3M vs. EB-3M). The similarities between the soil samples under $CO_2$ 380 ppmv (CA-3M vs CB-3M) and $CO_2$, 760 ppmv (EA-3M vs EB-3M) were 53.3% and 35.8%, respectively. These results suggested that the structure of microbial community associated with Pinus densiflora were sensitively specialized by Pb-contamination rather than $CO_2$ concentration.

Production Medium Optimization for Monascus Biomass Containing High Content of Monacolin-K by Using Soybean Flour Substrates (기능성 원료를 기질로 이용하는 Monacolin-K 고함유 모나스커스 균주의 생산배지 최적화)

  • Lee, Sun-Kyu;Chun, Gie-Taek;Jeong, Yong-Seob
    • KSBB Journal
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    • v.23 no.6
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    • pp.463-469
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    • 2008
  • During the last decade, monacolin-K biosynthesized by fermentation of red yeast rice (Monascus strains) was proved to have an efficient cholesterol lowering capability, leading to rapid increase in the market demand for the functional red yeast rice. In this study, the production medium composition and components were optimized on a shake flask scale for monacolin-K production by Monascus pilosus (KCCM 60160). The effect of three different soybean flours on the monacolin-K production were studied in order to replace the nitrogen sources of basic production medium (yeast extract, malt extract and beef extract). Among the several experiments, the production medium with dietary soybean flour to replace a half of yeast extract was very good for monacolin-K production. Plackett-Burman experimental design was used to determine the key factors which are critical to produce the biological products in the fermentation. According to the result of Plackett-Burman experimental design, a second order response surface design was applied using yeast extract, beef extract and $(NH_4)_2SO_4$ as factors. Applying this model, the optimum concentration of the three variables was obtained. The maximum monacolin-K production (369.6 mg/L) predicted by model agrees well with the experimental value (418 mg/L) obtained from the experimental verification at the optimal medium. The yield of monacolin-K was increased by 67% as compared to that obtained with basic production medium in shake flasks.

Evaluation of Cat Brain infarction Model Using MicroPET (마이크로 PET을 이용한 고양이 뇌 경색 모델의 평가)

  • Lee, Jong-Jin;Lee, Dong-Soo;Kim, Yun-Hui;Hwang, Do-Won;Kim, Jin-Su;Lim, Sang-Moo;Chung, June-Key;Lee, Myung-Chul
    • The Korean Journal of Nuclear Medicine
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    • v.38 no.6
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    • pp.528-531
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    • 2004
  • Purpose: PET has some disadvantage in the imaging of small animal due to poor resolution. With the advent of microPET scanner, it is possible to image small animals. However, the image quality was not good enough as human image. Due to larger brain, cat brain imaging was superior to mouse or rat. In this study, we established the cat brain infarction model and evaluate it and its temporal charge using microPET scanner. Materials and Methods: Two adult male cats were used. Anesthesia was done with xylazine and ketamine HCl. A burr hole was made at 1cm right lateral to the bregma. Collagenase type IV 10 ${\mu}l$ was injected using 30 G needle for 5 minutes to establish the infarction model. $^{18}F$-FDG microPET (Concorde Microsystems Inc., Knoxville, TN) scans were performed 1, 11 and 32 days after the infarction. In addition, $^{18}F$-FDG PET scans were performed using human PET scanner (Gemini, Philips medical systems, CA, USA) 13 and 47 days after the infarction. Results: Two cat brain infarction models were established. The glucose metabolism of an infarction lesion improved with time. An infarction lesion was also distinguishable in the human PET scan. Conclusion: We successfully established the cat brain infarction model and evaluated the infarcted lesion and its temporal change using $^{18}F$-FDG microPET scanner.

Effects of Fermented Rice Wine by Using Mycelium of Phellinus linteus on the Expression of Inflammation-Related Proteins in Human Hepatoma Cells and Rat Liver (상황버섯 균사체를 이용한 발효주가 인체간암세포와 흰쥐 간의 염증관련 단백질 발현에 미치는 영향)

  • Ahn Seung-Min;Lee Jun-Hyuk;Choi Yung-Hyun;Lee Yong-Tae;Chung Kyung-Tae;Jeong Young-Kee;Jo Un-Bock;Choi Byung-Tae
    • Journal of Life Science
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    • v.16 no.1
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    • pp.101-107
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    • 2006
  • We have recently discovered that mycelium of Phellinus linteus, popular medical mushrooms in Korea, possess alcohol dehydrogenase and produce alcohol. In the present study, it was examined that the effect of fermented rice wine made by using mycelium of P. linteus (FLMP) on the expression of in-flammation-related proteins in both $HepG_2$ cells and rats. To examine the effect of FLMP on the morphology and expression of inflammatory proteins in $HepG_2$ cells, the cells were incubated with ethanol, and FLMP for 24 hours, and then analyzed by microscopic observation and Western blot and reverse transcription polymerase chain reaction (RT-PCR). While ethanol induced the morphological change accompanied with cell debris formation and scattering on $HepG_2$ cells, FLMP had no effect. The results of Western blot and RT-PCR analyses showed that the level of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1 and COX-2 was induced by ethanol, however, FLMP inhibited the expression of these proteins and its mRNAs. In the animal model, the value of flutamate oxaloacetate transaminase and glutamate pyruvate transaminase was significantly increased by administration with ethanol. But the group administrated with FLMP showed lower levels on the changes of these markers compared with ethanol-administrated group. Besides, the results of Western blot and RT-PCR analyses showed that the expression of inflammatory proteins such as iNOS, COX-1 and COX-2 was not affected by FLMP administration in rat liver. About histopathological and immunohistochemical observations, inflammatory loci were markedly decreased in the FLMP-administrated rat compared to ethanol-administrated rats and showed weaker COX-2 and iNOS jmmunoreactions. These results suggested that FLMP showed slight changes on the inflammatory proteins expression compared to ethanol and FLMP may be used as a functional alcoholic beverage.