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Growth Temperature-Dependent Conversion of De novo-Synthesized Unsaturated Fatty Acids into Polyhydroxyalkanoic Acid and Membrane Cyclopropane Fatty Acids in the Psychrotrophic Bacterium Pseudomonas fluorescens BM07

  • LEE , HO-JOO;RHO, JONG-KOOK;YOON, SUNG-CHUL
    • Journal of Microbiology and Biotechnology
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    • v.14 no.6
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    • pp.1217-1226
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    • 2004
  • A psychrotrophic bacterial strain, Pseudomonas fluorescens BM07, synthesized unsaturated fatty acids (UFA) from fructose in response to lowering of growth temperature, and incorporated them into both polyhydroxyalkanoic acid (PHA) and membrane lipid. The blocking of PHA synthesis by adding 5 mM 2-bromooctanoic acid to the growth medium, containing 70 mM fructose, was found to be a useful means to profile the composition of membrane lipid by gas chromatography. As the growth temperature changed from 35 to $50^{\circ}C$, the total content of two UFA, 3-hydroxy-cis-5­dodecenoic acid ($C_{12:1}$) and 3-hydroxy-cis-7-tetradecenoic acid ($C_{14:1}$), in PHA increased from 31 to 44 $mol\%$. The growth at lower temperatures also led to an increase in the level of two major UFA, palmitoleic acid (C16:1 cis9) and cis-vaccenic acid (C18:1 cis11), in membrane lipid. A fraction of these membrane-lipid UFA was converted to their corresponding cyclopropane fatty acids (CFA). The CFA conversion was a function of culture time, exhibiting biphasic increase before and after entering the stationary phase. However, pH changes in growth media had no effect on the CFA conversion, which is contrary to the case of E. coli reported. The cells grown at $30^{\circ}C$ responded to a cold shock (lowering the medium temperature down to $10^{\circ}C$) by increasing the level of C16:1 cis9 and C 18: I cis II up to that of $10^{\circ}C$-grown control cells and concomitantly decreasing the relative level of cis-9,10­methylenehexadecanoic acid (the CFA converted from C16:1 cis9) from 14 to 8 $mol\%$, whereas the 10-grown cells exhibited little change in the lipid composition when exposed to a warmer environment of $30^{\circ}C$ for 12 h. Based on this one- way response, we suggest that this psychrotrophic strain responds more efficiently and sensitively to a cold shock than to a hot shock. It is also suggested that BM07 strain is a good producer of two unsaturated 3-hydroxyacids, $C_{12:1}\;and\;C_{141:1}$.

Effects of Microbacterium laevaniformans Levans Molecular Weight on Cytotoxicity

  • Oh, Im-Kyung;Yoo, Sang-Ho;Bae, In-Young;Cha, Jae-Ho;Lee, Hyeon-Gyu
    • Journal of Microbiology and Biotechnology
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    • v.14 no.5
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    • pp.985-990
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    • 2004
  • Levans produced from Microbacterium laevaniformans were isolated, characterized, and fractionated by molecular weight. TLC, HPLC, and GC-MS analyses of the exopolysaccharide showed that it was a fructan-type polymer and was composed of (2,6)- and (2,1)-glycosidic linkages. $^{13}C$-NMR analysis proved that the polysaccharide was mainly a $\beta$-(2,6)-linked levan-type polysaccharide. To investigate the cytotoxicity of the acetone-precipitated levan fractions such as M1, M2, and M3, HepG2, P388D1, U937, SNU-1, and SNUC2A cell lines were screened. Among the cell lines tested, the cytotoxicity of M1- M3 fractions were detected from only SNU-1 and HepG2 cells at the dosage level of $100-800\mu\textrm{g}ml$. The M2 fraction M_r$, 80,000) at 400 $mu{g/ml}$ had the greatest cell growth inhibition (84.6%) on SNU-1, while the M1 $(M_r$, 50,000) at $800\mu\textrm{g}ml$ showed the greatest (46.32%) on HepG2. To obtain more uniform M_r$ fractions of levan, the levan was further fractionated from S1 $(M_r$ 1,000,000) to S5 $(M_r$ 10,000) using gel permeation chromatography. Again, the S1-S5 fractions had strong cytotoxicity on SNU-1 and HepG2 cell lines. The greatest inhibition effects of S4 $(M_r$ 80,000) on SNU-1 and S5 $(M_r$ 10,000) on HepG2 were shown to be 49.5% and 73.0%, respectively. The cytotoxicity of the levan fractions was more effective on SNU-1 than on HepG2. Although the relationship between the Mw and the cytotoxicity was not clear, smaller $M_r$, fractions of levan showed greater growth inhibition effect on the cancer cell lines in general. Therefore, it was indicated that a specific Mw class of levan is responsible for the effective cytotoxicity.

Evaluation of Novel Constitutive Expression Vectors Equipped with Mined Promoters from Metagenome (메타게놈에서 발굴한 프로모터를 장착한 새로운 항시발현 벡터의 가치평가)

  • Han, Sang-Soo;Kim, Geun-Joong
    • Microbiology and Biotechnology Letters
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    • v.36 no.4
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    • pp.260-267
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    • 2008
  • The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system (pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and $\beta$-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of $2{\sim}3%$ of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.

Hydrogenation and Electrochemical Characteristics of Amorphous-nanostructured Mg-based Alloys

  • Gebert, A.;Khorkounov, B.;Schultz, L.
    • Journal of Powder Materials
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    • v.13 no.5 s.58
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    • pp.327-335
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    • 2006
  • In the development of new hydrogen absorbing materials for a next generation of metal hydride electrodes for rechargeable batteries, metastable Mg-Ni-based compounds find currently special attention. Amor phous-nanocrystalline $Mg_{63}Ni_{30}Y_7$ and $Mg_{50}Ni_{30}Y_{20}$ alloys were produced by mechanical alloying and melt-spinning and characterized by means of XRD, TEM and DSC. On basis of mechanically alloyed Mg-Ni-Y powders, complex hydride electrodes were fabricated and their electrochemical behaviour in 6M KOH (pH=14,8) was investigated. The electrodes made from $Mg_{63}Ni_{30}Y_7$ powders, which were prepared under use of a SPEX shaker mill, with a major fraction of nanocrystalline phase reveal a higher electrochemical activity far hydrogen reduction and a higher maximum discharge capacity (247 mAh/g) than the electrodes from alloy powder with predominantly amorphous microstructure (216 mAh/g) obtained when using a Retsch planetary ball mill at low temperatures. Those discharge capacities are higher that those fur nanocrystalline $Mg_2Ni$ electrodes. However, the cyclic stability of those alloy powder electrodes was low. Therefore, fundamental stability studies were performed on $Mg_{63}Ni_{30}Y_7$ and $Mg_{50}Ni_{30}Y_{20}$ ribbon samples in the as-quenched state and after cathodic hydrogen charging by means of anodic and cathodic polarisation measurements. Gradual oxidation and dissolution of nickel governs the anodic behaviour before a passive state is attained. A stabilizing effect of higher fractions of yttrium in the alloy on the passivation was detected. During the cathodic hydrogen charging process the alloys exhibit a change in the surface state chemistry, i.e. an enrichment of nickel-species, causing preferential oxidation and dissolution during subsequent anodization. The effect of chemical pre-treatments in 1% HF and in $10\;mg/l\;YCl_3/1%\;H_2O_2$ solution on the surface degradation processes was investigated. A HF treatment can improve their anodic passivation behavior by inhibiting a preferential nickel oxidation-dissolution at low polarisation, whereas a $YCl_3/H_2O_2$ treatment has the opposite effect. Both pre-treatment methods lead to an enhancement of cathodically induced surface degradation processes.

Effect of Benzoyl Peroxide on the Activity of Drug-metabolizing Enzyme System and Lipid Peroxidation in Rats (Benzoyl peroxide가 흰쥐의 지질과산화현상에 미치는 영향)

  • Lee, H.W.;Rhee, K.S.;Hong, S.U.
    • The Korean Journal of Pharmacology
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    • v.18 no.1
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    • pp.55-63
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    • 1982
  • Lipid peroxidation is the reaction of oxidative deterioration of polyunsaturated lipids and this peroxidation involves the direct reaction of oxygen and lipid to form free radical intermediates, which can lead to autocatalysis. As results of the extensive studies on the lipid peroxidation by many authors, the relationship between lipid peroxidation and the drug metabolizing system as well as the actions of free radicals on the peroxidation was reasonably well known. For a long time, the mechanism of hepatotoxicity of $CCl_4$ was not clearly understood. However, it is now quite well established that $CCl_4$ is activated in vivo to a free radical which is a highly reactive molecule. Therefore, lipid peroxidation which induces the reduction of cytochrome P-450 and aminopyrine demethylase activity is known as decisive event of $CCl_4$ hepatotoxicity. On the other hand, it was also reported that singlet molecular oxygen produces lipid peroxidation in liver microsomes. In this study the effects of benzoyl peroxide on the lipid peroxidation and drug-metabolizing enzyme were examined. Benzoyl peroxide mixed with starch and phosphates etc. is usually used as a food additive for flour bleaching and maturing purpose because of its oxidative property. Albino rats were used for the experimental animals. Benzoyl peroxide was suspended in soybean oil and sesame oil and administered intraperitoneally or orally. TBA value and aminopyrine demethylase activity were determined in liver microsomal fraction and serum. The results were summerized as following. 1) Body weights of animals administered benzoyl peroxide suspension were decreased while that of oil administered group were increased. 2) The activity of aminopyrine demethylase was generally decreased in animals administered oil suspension of benzoyl peroxide. Furthermore, the marked reduction of the enzyme activity was observed in animals administered benzoyl peroxide intraperitoneally. 3) Generally, microsomal TBA values as well as serum TBA were significantly elevated in benzoyl peroxide group in comparison with the control group. However, the more remarkable increase of serum TBA than microsomal TBA was observed in animals administered orally for 6 days. 4) Specifically, the changing pattern of TBA value was notable in serum rather than in liver microsome by intraperitoneal administration of benzoyl peroxide.

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Atmospheric Distribution Characteristics of Airborne Bacteria in Part of Seoul Area (일부 서울 지역 대기 부유세균 분포 현황 조사)

  • Kim, Ki-Youn;Kim, Yoon-Shin;Lee, Cheol-Min;Cho, Man-Su;Byeon, Sang-Hoon
    • Journal of Korean Society for Atmospheric Environment
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    • v.25 no.6
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    • pp.493-502
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    • 2009
  • The concentrations of outdoor airborne bacteria measured in Seoul, Korea for one year (Jan. 2008~Dec. 2008) ranged from $500CFU/m^3$ to $7,500CFU/m^3$. In monthly concentration distribution, the level of outdoor airborne bacteria was highest in September and October and lowest in March. In seasonal concentration distribution, the order of level of outdoor airborne bacteria was autumn>winter>spring>summer. In regional concentration distribution, the highest level of outdoor airborne bacteria was generally found in the forest, followed by general area and traffic site. In distribution characteristics according to particle size, outdoor airborne bacteria showed 31% for >$7.0{\mu}m$ (stage 1), 21% for $4.7{\sim}7.0{\mu}m$(stage 2), 15% for $3.3{\sim}4.7{\mu}m$(stage 3), 19% for $2.1{\sim}3.3{\mu}m$(stage 4), 10% for $1.1{\sim}2.1{\mu}m$(stage 5), and 4% for $0.65{\sim}1.1{\mu}m$(stage 6) and its mean respirable fraction was 48%. In concentration distribution by yellow dust, the mean levels of outdoor airborne bacteria were 803 $(\pm479)CFU/m^3$ for period of yellow dust and 691 $(\pm1,134)CFU/m^3$ for period of non-yellow dust. Although the level of outdoor airborne bacteria was higher in period of yellow dust than period of non-yellow dust, there was no significant difference between period of yellow dust and non-yellow dust (p>0.05). In correlation analysis between outdoor airborne bacteria and atmospheric factors (temperature and relative humidity), there was no significant correlation between outdoor airborne bacteria and atmospheric factors. The predominant airborne bacteria were identified to the Bacillus-derived species.

Pollution Characteristics of Rainwater at Jeju Island during 2009~2010 (2009~2010년 제주지역 강우의 오염 특성 연구)

  • Kim, Ki-Ju;Bu, Jun-Oh;Kim, Won-Hyung;Lee, Yoon-Sang;Hyeon, Dong-Rim;Kang, Chang-Hee
    • Journal of Korean Society for Atmospheric Environment
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    • v.29 no.6
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    • pp.818-829
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    • 2013
  • The collection of rainwater samples was made at Jeju area during 2009~2010, and the major ionic species were analyzed. In the comparison of ion balance, conductivity, and acid fraction for the validation of analytical data, the correlation coefficients showed a good linear relationship within the range of 0.966~0.990. The volume-weighted mean pH and electric conductivity were 4.9 and $17.8{\mu}S/cm$, respectively, at the Jeju area. The volume-weighted mean concentrations of ionic species in rainwater were in the order of $Cl^-$ > $Na^+$ > $nss-SO_4{^{2-}}$ > $NH_4{^+}$ > $NO_3{^-}$ > $Mg^{2+}$ > $H^+$ > $nss-Ca^{2+}$ > $HCOO^-$ > $K^+$ > $PO_4{^{3-}}$ > $CH_3COO^-$ > $NO_2{^-}$ > $F^-$ > $HCO_3{^-}$ > $CH_3SO_3{^-}$. The ionic strength of rainwater was $0.26{\pm}0.21$ mM during the study period. The composition ratios of ionic species were such as 50.1% for the marine sources ($Na^+$, $Mg^{2+}$, $Cl^-$), 30.9% for the anthropogenic sources ($NH_4{^+}$, $nss-SO_4{^{2-}}$, $NO_3{^-}$), and 4.7% for the soil source ($nss-Ca^{2+}$), and 3.1% for organic acids ($HCOO^-$, $CH_3COO^-$). From the seasonal comparison, the concentrations of $NO_3{^-}$, $nss-Ca^{2+}$, and $nss-SO_4{^{2-}}$ increased in winter and spring seasons, indicating a reasonable possibility of long range transport from Asia continent. Especially, the acidifying contributions by major inorganic acids ($nss-SO_4{^{2-}}$ and $NO_3{^-}$) and organic acids ($HCOO^-$ and $CH_3COO^-$) were 87.6% and 12.4%, respectively. In comparison by sectional inflow pathway of air mass during the rainy sampling days, the concentrations of $nss-SO_4{^{2-}}$ and $NO_3{^-}$ were relatively high when the air mass was moved from the China continent into Jeju area.

Characterization and Purification of a Microsomal 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Rice Seedling (벼 HMG-CoA 환원효소의 특성연구)

  • Kim, Jai-Hyun;Paik, Young-Ki;Kim, Jong-Bum;Kim, Jong-Guk;Hwang, Young-Soo;Ha, Sun-Hwa
    • Applied Biological Chemistry
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    • v.41 no.1
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    • pp.47-52
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    • 1998
  • 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonic acid, the first intermediate of isoprenoid biosynthetic pathway in plants. The enzyme was solubilized with 0.4% Brij (polyoxyethylene ether) W-1 from a microsomal fraction of etiolated rice seedlings (Oryza sativa L.) in which its maximal activity was observed on the fourth day after germination. HMGR was purified to near homogeneity by employing $(NH_4)_2SO_4$ fractionation plus chromatographic procedures including DEAE-Sephadex A-50 and HMG-CoA-hexane-agarose affinity column. The size of the purified enzyme was estimated to be 55 kDa when judged by SDS-PAGE analysis with silver staining method. The apparent $K_m$ and $V_{max}$ values for HMG-CoA were determined to be $180\;{\mu}M$ and 107 pmol/min/mg, and those for NADPH were $810\;{\mu}M$ and 32.1 pmol/min/mg, respectively.

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Angiotensin I Converting Enzyme Inhibitory Effects of Gelatin Hydrolysates Prepared from Tilapia mossambica Scales by Hot Water and Enzymatic Extraction (열수 및 효소적 가수분해로 제조된 틸라피아 비늘 젤라틴 가수분해물의 ACE 저해 활성)

  • Ahn, Yong-Seok;Lee, Won-Woo;Lee, Seung-Hong;Ahn, Gin-Nae;Ko, Chang-Ik;Oh, Chang-Kyung;Oh, Myung-Cheol;Kim, Dong-Woo;Jeon, You-Jin;Kim, Soo-Hyun
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.42 no.5
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    • pp.426-433
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    • 2009
  • Fish scales have potential in functional food preparation due to their antioxidant and antihypertensive properties. We investigated the angiotensin I converting enzyme (ACE) inhibitory activity of Tilapia mossambica scale extracts. Hydrolysates of tilapia scales were prepared by enzymatic extraction using five proteases (${\alpha}$-chymotrypsin, Alcalase, Kojizyme, Protamex and trypsin) after scales were treated with hot water for 3 hr. Scale enzymatic hydrolysates prepared using both hot water and enzyme treatments exhibited elevated hydrolysis (about 25%-55%) compared to only enzyme treatment (about 15%-45%). Enzymatic hydrolysates (1 mg/mL) prepared by both hot water and enzyme treatments also showed significantly increased ACE inhibitory activities from about 20%-75%. The pattern of ACE inhibitory activities was similar to the degree of hydrolysis. Alcalase and ${\alpha}$-chymotrypsin hydrolysates displayed the highest ACE inhibitory activities ($IC_{50}$ = 0.83 mg/mL and 0.68 mg/mL, respectively). In addition, the ACE inhibitory effects of $IC_{50}$-chymotrypsin hydrolysates increased with decreasing molecular weight (5 kDa>, 10 kDa> and 30 kDa>), with the 5 kDa> fraction displaying the highest ACE inhibitory activity (about 89.9% and $IC_{50}$ = 0.1 mg/mL). We suggest that the peptide compounds of enzymatic hydrolysates prepared from tilapia scale enhances ACE inhibitory activity and might be useful as an antihypertensive material.

The Characteristics of Particulate PAHs Concentrations at a Roadside in Seoul (서울시 도로변에서 입자상 다환방향족탄화수소의 농도 특성)

  • Lee, Ji-Yi;Kim, Yong-Pyo;Bae, Gwi-Nam;Park, Su-Mi;Jin, Hyun-Chur
    • Journal of Korean Society for Atmospheric Environment
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    • v.24 no.2
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    • pp.133-142
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    • 2008
  • Five intensive measurements of particulate PAHs were made at a roadside in Seoul from May 2005 to June 2006. The average concentration of particulate PAHs was $15.1{\pm}10.6ng\;m^{-3}$. The high concentrations of particulate fluoranthene and pyrene were observed in November 2005 due to the influence of the lower ambient temperature. Compared to the previous results at tunnel and ambient sites in Seoul, larger fraction of the high molecular PAH compounds which consist with five or six benzene rings, was observed at a roadside. This might indicate high influence of vehicle emission at a roadside. The distribution of diagnostic ratios for specific PAH compounds indicated that the influence of vehicular emission, especially diesel vehicular emission seems to be high at a roadside.