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GENETIC AND BIOCHEMICAL ANALYSIS OF A THERMOSTABLE CHITOSANASE FROM Bacillus sp. CK4

  • Yoon, Ho-Geun;Cho, Hong-Yon
    • Proceedings of the Korean Society for Applied Microbiology Conference
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    • 2000.04a
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    • pp.157-167
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    • 2000
  • A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

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Antimicrobial Efficacy of Penicillium amestolkiae elv609 Extract Treated Cotton Fabric for Diabetic Wound Care

  • Rozman, Nur Amiera Syuhada Binti;Hamin, Nurhanis Syafiqah Binti Mohd Nor;Ring, Leong Chean;Nee, Tan Wen;Mustapha, Mahfuzah Binti;Yenn, Tong Woei
    • Mycobiology
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    • v.45 no.3
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    • pp.178-183
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    • 2017
  • Diabetes mellitus is a chronic disorder which affects millions of population worldwide. Global estimates published in 2010 reported the world diabetic prevalence as 6.4%, affecting 285 million adults. Foot ulceration and wound infection are major forms of disabilities arising from diabetic diseases. This study was aimed to develop a natural antimicrobial finishing on medical grade textile that meets American Association of Textiles Chemists and Colorists (AATCC) standard. The textile samples were finished with the ethanolic extract of Penicillium amestolkiae elv609, an endophytic fungus isolated from Orthosiphon stamineus Benth (common name: cat's whiskers). Endophyte is defined as microorganism that reside in the living plant tissue, without causing apparent disease symptom to the host. The antimicrobial efficacy of the ethanolic extract of P. minioluteum was tested on clinical pathogens isolated from diabetic wound. The extract exhibited significant inhibitory activity against 4 bacteria and 1 yeast with the minimal inhibitory concentration ranged from 6.25 to 12.5 mg/mL. The results indicate different susceptibility levels of the test microorganism to the ethanolic extract. However, the killing activity of the extract was concentration-dependent. The finished medical textile showed excellent antimicrobial efficacy on AATCC test assays. All the microbial cultures treated with the textile sample displayed a growth reduction of 99.9% on Hoheinstein Challenge Test. The wash durability of the finished textile was found good even after 50 washes with commercial detergent. Besides, the gas chromatography mass spectrometry analysis showed that 6-octadecenoic acid and diethyl phthalate were the main bioactive constituents of the extract. In conclusion, the developed medical textile showed good antimicrobial efficacy on laboratory tests. This work can be extended to in vivo trials for developing healthcare textile products for antimicrobial applications.

The Digital Mock-Up Information System for New Car Development

  • Min, Sung-Ki;Lee, Chul-Woo
    • Proceedings of the CALSEC Conference
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    • 1999.07a
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    • pp.277-299
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    • 1999
  • Since Chrysler Motor Co. had experienced the digital development system in the beginning of 1990's, most of leading automobile companies are trying to apply a digital information system for their own business process reengineering based upon concurrent engineering system from product planning phase. This is called as virtual DMU(Digital Mock-Up) system instead of the traditional PMU(Physical Mock-Up) system. By using the virtual prototype, all of the design requirements and system specifications can be checked, changed and optimized more quickly and more efficiently. This paper consists of five chapters for the DMU information system. In the 1$^{st}$ chapter, the principle of digital design system is suggested by using four basic modules such as product design module, process design module, manufacturing system design module and central control module. The basic scheme of DMU is introduced with the benefits of application in the chapter 2. In the chapter 3, a digital design process of new car development is explained with the detailed DMU design and design review processes. In the chapter 4, the practical DMU manufacturing techniques and applications are introduced as CAD/CAM analyses, DPA(Digital Pre-Assembly)reviews for development, production, operation and maintenance phases, digital tolerance analyses and digital factory analyses for assembling line simulation, automated robot welding processes, production jig & fixtures and painting process simulation. Finally, the activities of digital design support; CAS-styling, CAE-engineering and CAT-testing are summarized for design optimization in the chapter 5. As today's automobile manufactures and related business organizations are struggling to compete in the global marketplace, they are concentrating on efficient use of DMU information system to reduce the new car development cost, to have shorten the delivery schedule and to improve product design quality. To meet the demand of those automobile industries on digital information systems, the CALS(Computer aided Acquisition and Logistics Support) and EC(Electronic Commerce)initiative has been focused as a dominant philosophy in defense & commercial industries, specially automobile industries.s.

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Nitric oxide modulates antioxidant defense and the methylglyoxal detoxification system and reduces salinity-induced damage of wheat seedlings

  • Hasanuzzaman, Mirza;Hossain, Mohammad Anwar;Fujita, Masayuki
    • Plant Biotechnology Reports
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    • v.5 no.4
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    • pp.353-365
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    • 2011
  • The present study investigates the possible regulatory role of exogenous nitric oxide (NO) in antioxidant defense and methylglyoxal (MG) detoxification systems of wheat seedlings exposed to salt stress (150 and 300 mM NaCl, 4 days). Seedlings were pre-treated for 24 h with 1 mM sodium nitroprusside, a NO donor, and then subjected to salt stress. The ascorbate (AsA) content decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) and the GSH/GSSG ratio increased with an increase in the level of salt stress. The glutathione S-transferase (GST) activity increased significantly with severe salt stress (300 mM). The ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glutathione peroxidase (GPX) activities did not show significant changes in response to salt stress. The glutathione reductase (GR), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, especially at 300 mM NaCl, with a concomitant increase in the $H_2O_2$ and lipid peroxidation levels. Exogenous NO pretreatment of the seedlings had little influence on the nonenzymatic and enzymatic components compared to the seedlings of the untreated control. Further investigation revealed that NO pre-treatment had a synergistic effect; that is, the pre-treatment increased the AsA and GSH content and the GSH/GSSG ratio, as well as the activities of MDHAR, DHAR, GR, GST, GPX, Gly I, and Gly II in most of the seedlings subjected to salt stress. These results suggest that the exogenous application of NO rendered the plants more tolerant to salinity-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.

Study on the Changes in Evapotranspiration according to the Decentralized Rainwater Management (분산식 빗물관리시설 적용에 따른 증발산 변화 연구)

  • Han, Young-Hae;Lee, Tae-Goo
    • KIEAE Journal
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    • v.12 no.5
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    • pp.3-10
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    • 2012
  • In this study, the influence of decentralized rainwater management over the changes in evapotranspiration was analyzed. The analysis method was obtained by establishing the decentralized rainwater management plan according to different scenarios, and subsequently examined evapotranspiration in the plan. Scenario 1 refers to the analysis of the existing situation, in which was 100% of a parking lot is asphalt pavement. In Scenario 2, the pavement of the parking surface in the parking lot is replaced with lawn blocks. In Scenario 3, some asphalt pavement was removed to establish a flower-bed type infiltration system to allow rainwater to permeate. In Scenario 4, infiltration and storage of rain water would be achieved by transforming the parking surface into lawn blocks, keeping the asphalt for the parking road while establishing a vegetation strip. The amount of evapotranspiration of the target site was analyzed with a water budget analysis program (CAT) using the 2001 meteorological data for each scenario According to the analysis values of S2 and S3, it was found that evapotranspiration is critically affected by the amount of area replaced with pervious area in the total target site. An energy equivalent to 680kWh is required for 1 ton of water to evaporate. Hence, it can be seen that the active inducement of evapotranspiration in urban area makes a positive contribution not only to heat island mitigation, but also to the small-scale water circulation process in a city.

An Analysis of Posthuman's Body Type and Fashion in SF Movies (SF 영화에 나타난 포스트휴먼의 신체 유형 및 패션 분석)

  • Choi, Jung-Hwa
    • The Research Journal of the Costume Culture
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    • v.19 no.3
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    • pp.473-487
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    • 2011
  • The purpose of this study is to analyze the body type and fashion of posthuman in SF movies. The method of this study was to analyze documentaries, internet web site, fashion books and so forth. The results were as follows: The body types of posthuman were expressed as mutation type, prosthetic type, clone type by biological hybrid or renovation and digital type by computer simulation. The mutation type was expressed as reinforcement of masculinity or feminity and reinforcement of body functions. The fashion item was expressed as a black tailored suit, leather jacket, cat suit, whip, black sunglass, garter belt, high heel shoes, short pants, black one piece dress and functional body suit. The prosthetic type was expressed as reinforcement of body functions and reinforcement of masculinity or feminity. The fashion item was expressed as a military item, high-tech power suit and ergonomic armor suit. The clone type was expressed as the plural ego with reinforcement of body functions. The fashion item was expressed as a power shoulder jacket, fake fur coat, vinyl, black see-through look and functional suit. The digital type was expressed as reinforcement of masculinity or feminity and the plural ego with reinforcement of body functions. The fashion item was expressed as a data suit, leather jacket, black over coat, boots, black sun glass, ethnic items and military items. The meanings of posthuman fashion in SF movies were impurity of posthuman, display of superhuman's power by sexuality, metaphor of power and fantasy of superhero in opposition futuristic dystopia. As mentioned above, posthuman body type and fashion in SF movies become the conversational topic in the real world. The fact that we think about utopia and identity of posthuman in the future is of great significance.

IN VIVO EFFECT OF WET BONDING AND COLLAGEN DISSOLUTION IN ACID CONDITIONED DENTIN ON MARGINAL LEAKAGE (In vivo에서 습윤접착과 상아질 교원섬유용해가 변연누출에 미치는 영향)

  • Hwang, Su-Jin;Kim, Myung-Su;Lee, Kwang-Won;Son, Ho-Hyun
    • Restorative Dentistry and Endodontics
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    • v.21 no.1
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    • pp.254-266
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    • 1996
  • The purpose of this study was to evaluate the degree of marginal leakage that is made by wet bonding to dentin and dissolution of dentinal collagen layer in vivo and in vitro. Class V cavities were prepared to 32 cat's canine teeth and they were randomly assigned into the following 4 groups : Group 1 (not acid conditioned) ; Group 2 (acid-conditioned and blot-dried) ; Group 3 (acid-conditioned and air-dried for 30 seconds) ; Group 4 (acid-conditioned and retreated with 10% NaOCl solution). The acetone-based primer and bonding agent of All Bond 2(Bisco Inc., U.S.A.) and composite resin(Z-100, 3M. Dent. Prod., U.S.A.) were then applied. The resin collar and cap attached to each specimen and 50% silver nitrate solution was placed inside the resin cap for 3 hours. After the canine tooth was resected in level of cervical third, specimens were highly polished and observed the degree of the dye penetration on incisal and gingival margins. The results were as follows ; 1. There was no significant difference of marginal leakage in all the groups between in vivo and in vitro. 2. In vivo, the degree of leakage on the gingival margin showed the lowerest in group 4 and followed by group 2, 3 and group 1 with ascending order. However, there was no statistical difference among all the groups(p>0.05). 3. In vitro, the degree of leakage on the gingival margin showed the lowerest in group 2 and followed by group 4, 3 and group 1 with ascending order. However, there was no significant difference among the experimental groups(p>0.05). 4. The marginal leakage of the incisal margin in vitro was significantly higher in group 1 than the remaining groups(p<0.05).

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Pharmacological Studies of Zizyphus Seed Extract on Central Nervous System and Blood Pressure (산조인의 중추신경 및 심혈관계에 대한 약리작용)

  • Ahn, Y.S.;Kim, K.H.;Cho, T.S.;Kim, W.J.;Hong, S.S.
    • The Korean Journal of Pharmacology
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    • v.18 no.1
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    • pp.17-22
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    • 1982
  • Zizyphus seed(Zizyphus vulgaris Lamark var. Spinosus Bunge) has long been used as hypnotics and sedatives in oriental medicine, and it is reported that the Zizyphus seed elicited a variety of pharmacologic actions besides CNS depression. Present study was undertaken to investigate the effects of Zizyphus seed on the central nervous system and on the blood pressure. The effect of Zizyphus seed on the central nervous system was measured by the influence of thiopental sleeping time and by inhibition of chemical convulsion (strychnine and pentylenetetrazol induced). Blood pressure changes by Zizyphus extract and its mode of action were investigated. The ground Zizyphus seed was extracted with hexane and methanol, consecutively and the supernatants were discarded. The precipitate was re-extracted with distilled water and the supernatant was evaporated to a dark-brownish sticky liquid, which was used as Zizyphus seed extract in this study after dissolving in saline prior to experiment. The results are as follows. 1) Zizyphus seed extract caused marked prolongation of the thiopental sleeping time in mice. 2) The chemical convulsion by strychnine and pentylenetetrazol, and the mortality by them in chicks were not affected by pretreatment of Zizyphus seed extract. 3) Zizyphus seed extract produced transient fall of blood pressure in the cat, and this hypotentive effect was blocked partially by atropine but not affected by bilateral vagotomy and/or hexamethonium, nor propranolol and, chlorpheniramine and/or cimetidine. With the above results, it may be suggested that the water extract of Zizyphus seeds contains components producing CNS depression and hypotension. Furthermore it is felt that the cholinergic effect, but not the adrenergic or histaminergic, is partly responsible for the hypotensive effect of Zizyphus seed extract.

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Effect of Mutagenesis of V111 and L112 on the Substrate Specificity of Zymomonas mobilis Pyruvate Decarboxylase

  • Huang, Chang-Yi;Nixon, Peter F.;Duggleby, Ronald G.
    • BMB Reports
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    • v.32 no.1
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    • pp.39-44
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    • 1999
  • Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and $Mg^{2+}$, for activity. Zymomonas mobilis PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L112I, all mutants retain activity towards pyruvate with $k_{cat}$ values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for $Mg^{2+}$. Two of the mutants, V111G and V111A, show an increase in the $K_m$ for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

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In vitro Polymerization and Copolymerization of Poly-3-hydroxypropionyl-CoA with the PHB Synthase from Ralstonia eutropha

  • Song, Jae-Jun;Goodwin, Steve;Lenz, Robert W.
    • 한국생물공학회:학술대회논문집
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    • 2000.11a
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    • pp.48-51
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    • 2000
  • The poly(3-hydroxybutyrate) (PHB) synthase of Ralstonia. eutropha, which was produced by a recombinant strain E. coli and purified in one-step with a methyl-HIC column to a purity of more than 90%, was used to polymerize 3-hydroxypropionyl-CoA (3HPCoA) and to copolymerize 3HPCoA with 3-hydroxybutyryl-CoA (3HBCoA) in vitro. A $K_m$ of $189\;{\mu}M$ and a $k_{cat}$ of $10\;sec^{-1}$ were determined for the activity of the enzyme in the polymerization reaction of 3HPCoA based on the assumption that the dimer form of PHB synthase was the active form. Free coenzyme A was found to be a very effective competitive inhibitor for the polymerization of 3HPCoA with a $K_i$ of $85\;{\mu}M$. The maximum degree of conversion of 3HPCoA to polymer was less than 40 %. In the simultaneous copolymerization reactions of these two monomers, both the turnover number for the copolymerization reaction and the maximum degree of conversion of 3HPCoA and 3HBCoA to copolymers increased with an increase in the amount of 3HBCoA in the monomer mixture. However, the maximum conversion of 3HPCoA to a copolymer was less than 35 % regardless of the ratio of 3HPCoA to 3HBCoA. Block copolymers were obtained by the sequential copolymerization of the two monomers and these copolymers had a much narrower molecular weight distribution than those obtained by the simultaneous copolymerization of the same molar ratio of 3HPCoA and 3HBCoA.

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