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Enzymatic Characterization of Salmonella typhimurium Mannitol Dehydrogenase Expressed in Escherichia coli (Salmonella typhimurium에서 유래한 Mannitol Dehydrogenase 유전자의 대장균 내 발현 및 효소특성 규명)

  • Jang, Myoung-Uoon;Park, Jung-Mi;Kim, Min-Jeong;Kang, Jung-Hyun;Lee, So-Won;Kim, Tae-Jip
    • Korean Journal of Microbiology
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    • v.48 no.2
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    • pp.156-162
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    • 2012
  • A mannitol dehydrogenase (StMDH) gene was cloned from Salmonella typhimurium LT2 (KCTC 2421) and overexpressed in Escherichia coli. It has a 1,467 bp open reading frame encoding 488 amino acids with deduced molecular mass of 54 kDa, which shares approximately 36% of amino acid identity with known long-chain dehydrogenase/reductatse (LDR) family enzymes. The recombinant StMDH showed the highest activity at $30^{\circ}C$, and pH 5.0 and 10.0 for D-fructose reduction and D-mannitol oxidation, respectively. On the contrary, it has no activity on glucose, galactose, xylose, and arabinose. StMDH can catalyze the oxidative/reductive reactions between D-fructose and D-mannitol only in the presence of $NAD^+$/NADH as coenzymes. These results indicate that StMDH is a typical $NAD^+$/NADH-dependent mannitol dehydrogenase (E.C. 1.1.1.67).

Inhibitory Effects of a Recombinant Viral Cystatin Protein on Insect Immune and Development (바이러스 유래 시스타틴 재조합 단백질의 곤충 면역 및 발육 억제효과)

  • Kim, Yeongtae;Eom, Seonghyun;Park, Jiyeong;Kim, Yonggyun
    • Korean journal of applied entomology
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    • v.53 no.4
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    • pp.331-338
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    • 2014
  • Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus (CpBV). Our previous study indicated that a transient expression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. To directly demonstrate the protein function, this study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to determine its inhibitory activity against cysteine protease and to assess its physiological alteration in insect immune and development. The open reading frame of CpBV-CST1 encodes a polypeptide of 138 amino acids (${\approx}15kDa$). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was over-expressed by 0.5 mM IPTG for 4 h. In biological activity assay, the purified rCpBV-CST1 showed a significant inhibition against papain activity. It inhibited a cellular immune response of hemocyte nodule formation in the beet armyworm, Spodoptera exigua. Moreover, its oral administration retarded larval development of the diamondback moth, Plutella xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 may be applied to control insect pest populations.

Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40

  • Kim, Sung Chan;Kang, Seung Ha;Choi, Eun Young;Hong, Yeon Hee;Bok, Jin Duck;Kim, Jae Yeong;Lee, Sang Suk;Choi, Yun Jaie;Choi, In Soon;Cho, Kwang Keun
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.1
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    • pp.126-133
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    • 2016
  • A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

Cloning and Characterization of Monofunctional Catalase from Photosynthetic Bacterium Rhodospirillum rubrum S1

  • Lee, Dong-Heon;Oh, Duck-Chul;Oh, You-Sung;Malinverni, Juliana C.;Kukor, Jerome J.;Kahng, Hyung-Yeel
    • Journal of Microbiology and Biotechnology
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    • v.17 no.9
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    • pp.1460-1468
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    • 2007
  • In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from $20^{\circ}C$ to $60^{\circ}C$C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's $K_m$ value and $V_{max}$ of the catalase for $H_2O_2$ were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of $A_{406}$ to $A_{280}$ for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1 in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.

A Study on the Illustration Content Used in Secondary School Textbooks : Focusing on the 'Society.Culture' Textbooks (중등학교 교과서에서 삽화 콘텐츠 활용 연구 : 고등학교 '사회.문화' 교과서를 중심으로)

  • Min, Il-Hong
    • Cartoon and Animation Studies
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    • s.18
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    • pp.57-72
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    • 2010
  • Textbooks used in Information Society need to use many illustrations and pictures which have positive effect in motivating and triggering students to study. "Pictures mean more than thousands of the word" says that it is significant to use media in class through visual materials. To meet the needs of the times, I examined one of the units, "IV Understanding Humans and Cultural Phenomena", among 7 authorized 'Society Culture' textbooks, so that there are 123 illustrations and 342 pictures that the percentage of them was 35.65% totally in the unit. On the examination of the frequency of using contents in each category, the illustrations are used 59 times (47.96%) in the research activity and the pictures 145 times (42.4%) in the context, which are most frequently used. Also on the examination into the actual states using contents among 'Society Culture' teachers by in-depth interviewing, they often use them when their class starts. And they require more increase in the illustrations than the pictures for easy and clear understanding and need more contents offering in the research activity to help students to study more interesting. Finally, on the result of the analysis of contents used in textbooks, exemplary cases were available to convey enough information without reading the context in the textbook because the proposed illustrations expressed the research activity's subject and the context's subject effectively. Even more, one illustration was able to indicate the sub-unit's subject while also presenting the content to be learned in the unit. However, improper cases included illustrations which are somewhat unrealistic or difficult to understand. Further, there are also some illustrations which are not related to the context. If these points are revised in the future, textbooks would be better.

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A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens

  • Lee, Su-Jin;Lee, Youngdeuk;Park, Gun-Hoo;Umasuthan, Navaneethaiyer;Heo, Soo-Jin;Zoysa, Mahanama De;Jung, Won-Kyo;Lee, Dae-Won;Kim, Hanjun;Kang, Do-Hyung;Oh, Chulhong
    • Journal of Microbiology and Biotechnology
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    • v.26 no.6
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    • pp.1115-1123
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    • 2016
  • L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the L-asparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40℃, pH 9. Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-L-ASPG86 was 687.1 units/mg under optimum conditions (37℃, pH 9, and 5 mM MnSO4).

Assessing the Prevalence of Recurrent Neck and Shoulder Pain in Korean High School Male Students: A Cross-sectional Observational Study

  • Koh, Min-Jung;Park, Sun-Young;Woo, Young-Sun;Kang, Sung-Hyun;Park, Sang-Hoon;Chun, Hye-Jung;Park, Eun-Jung
    • The Korean Journal of Pain
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    • v.25 no.3
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    • pp.161-167
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    • 2012
  • Background: Neck and shoulder pain (NSP) is fairly common in adolescents, which is associated with a high prevalence of NSP found during adulthood as well; therefore, its significance during adolescence should not be underestimated. We surveyed the prevalence of recurrent NSP, lifestyle, and risk factors in Korean high school students, and examined the influence of recurrent NSP on the quality of life. Methods: Nine hundred thirty one male students (16-19 years old) from two academic high schools in Seoul were included in this study. The survey consisted of a questionnaire to assess the prevalence of recurrent NSP, with questions regarding having an occurrence more than once a week, characteristics of NSP, activity and lifestyle of the students, and the risk factors for recurrent NSP. A 36-item Short Form questionnaire was also examined. Results: We found that 44.3% of the high school students surveyed had recurrent NSP (more than once a week) and the overall prevalence of NSP was 79.1%. The average sitting time was $10.2{\pm}2.7$ h/day. 59.0% did not sit straight, 14.7% used assisting devices during reading, and 11.9% answered that they stretched regularly. Found from their self assessed health, frequent fatigue and frequent depressed mood presented significant associations with the higher prevalence of recurrent NSP. Conclusions: Korean high school students had a high prevalence of recurrent NSP. Clinical attention is needed for the prevention and resolution of recurrent NSP found in high school students.

An Analysis of Spactial Practice of Morden People appeared in the early 20th century film (20세기 초 영화에 나타난 근대인의 공간적 실천 분석 연구)

  • Lee, Young-Soo;Roh, Eun-Joo
    • Korean Institute of Interior Design Journal
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    • v.20 no.6
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    • pp.124-134
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    • 2011
  • The space has been interpreted from various perspectives, such as hierarchical, cultural, economic, political factors, etc. So we can see the space as a social existence. Space is now being formed through the dialectical relations of these elements. From this point of view, this study started to research the spatial practice of morden people through the case in the early 20th century film. With the discourse of Henri Lefebvre and David Harvey, and Michel de Certeau's theory, this research tried to find the mechanisms of spatial practice. Also Benjamin is a philosopher who intervenes the relationship between modernity and cultural production and his way of reading cultural phenomena seems to serve as the useful methodology of cultural studies. Modern people were individual unawared of the era, awakened to the ego. They were wandering the room and the street, private and public places. They were city dwellers walking around, collecting goods, and living of everyday life. Spatial practice is a fixed activity and have continuity. spatial practice appeared in the early 20th century film is at the intersection of social practices and the practice of everyday life. Social practices are a fixed practice and continuous practice. The practices of everyday life are nomadic practice and amusable practice. Modern people accommodate and adapt to a given space of the city through fixed practice. They realizes the access and the distance from spaces through continuous practice. They select and approved the spaces through nomadic practice. And they possess exclusively and utilize the spaces through amusable practice. Through These research spatial practices, it could easily found similarities and differences between modern space on the early 20th century and contemporary space of 21st century. True modern is not the past but the present.

Molecular Cloning of a Gene Encoding a Putative Antibacterial Peptide from Bombyx mori (누에에서 새로운 항세균성 펩타이드 유사 유전자의 분리와 염기서열 결정)

  • 김상현;제연호;윤은영;강석우;김근영;강석권
    • Korean journal of applied entomology
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    • v.35 no.4
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    • pp.321-325
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    • 1996
  • To isolate a novel gene for antibacterial peptide, an inducible clone(BmInc8) was selected by differential screening strategy from Bombyx mori cDNA library prepared from lavae injected with Escherichia coli. This clone contained a cDNA insert of 564 nucleotides and encoded 59 amino acids with an apparent molecular mass of 6.3 kDa. The cDNA sequence of BmInc8 had 61.2% identity compared to that of the bactericidin from Manduca sexta and also the deduced amino acids sequences from this insert had 65% identity compared to that of the cecropin D peptide Hyalophora cecropia. The transient expression assay of this insert using prokaryotic expression vector system revealed that the expressed peptide displayed the antibacterial activity. The cDNA sequence was deposited in GenBank under the accession number U30289.

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Effects of Safflower Seed Extracts and Bovine Bone on Regeneration of Bone Defects in Mongrel Dogs (홍화씨 추출물 및 우골유도합성골이 성견골 결손부 재생에 미치는 영향)

  • Seo, Jae-Jin;Kim, Tak;Pi, Sung-Hee;Yun, Gi-Yun;Yu, Hyung-Keun;Shin, Hyung-Shik
    • Journal of Periodontal and Implant Science
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    • v.30 no.3
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    • pp.553-569
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    • 2000
  • Many natural medicines have been studied for their capacity and effects of antibacterial, anti-inflammatory and regenerative potential in periodontal tissues. Safflower seed has been traditionally used as a drug for treatment of bone fracture in oriental medicine. The purpose of the present study was to compare the effects of safflower seed extract and bone substitute on bone formation and regeneration in artificial defects in mongrel dogs. The bony defects were made with round bur at mandible and tibia. Extracts of safflower seed and bovine bone were placed directly at each defect for experimental group, and the defect of control group was sutured without any other treatment. Experimental animals were sacrificed at 8 weeks. And then histopathologic reading and histomorphometric study was done. There was not significant differences between control and experimental groups in osteoclastic activity and infiltration of inflammatory cells. However, new capillary proliferation, fibrosis and new bone formation were prominent in safflower seed extract group. The mandibular defects of safflower seed extract group were healed with dense connective and bony tissues, and endochondral bone formation was observed in tibial defect of safflower seed extract group only. New bone area of safflower seed extract group was more significantly increased than that of control and that of bone substitute group. These results indicate that direct local application of safflower seed extracts on bony defects seems to reduces the early inflammatory response and to promotes the bone regeneration.

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