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Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus (가물치(Channa argus) 젖산탈수소효소 동위효소들의 정제 및 특성)

  • Park, Eun-Mi;Yum, Jung-Joo
    • Journal of Life Science
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    • v.20 no.2
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    • pp.260-268
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    • 2010
  • The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Channa argus were purified and characterized by biochemical, immunochemical and kinetic methods. The activity of LDH in skeletal muscle was the highest at 380.4 units and those in heart, eye and brain tissues were 13.4, 3,5 and 5.4 units, respectively. Citrate synthase (EC 4.1.3.7, CS) activity in heart tissue was the highest at 20.7 units. LDH/CS in skeletal muscle, heart, eye and brain tissues were 172.9, 0.6, 0.32 and 0.47. Protein concentration in skeletal muscle tissue was 14.7 mg/g and specific activities of LDH in skeletal muscle, heart, eye and brain tissues were 25.88, 0.79, 0.31 and 1.38 units/mg, respectively. Therefore, skeletal muscle tissue was anaerobic and heart tissue was aerobic. The LDH isozymes in tissues were identified by polyacrylamide gel electrophoresis, immunoprecipitation and Western blot with antiserum against $A_4$, $B_4$, and eye-specific $C_4$. LDH $A_4$, $A_3B$, $A_2B_2$. $AB_3$ and $B_4$ isozymes were detected in every tissue, $C_4$, $AC_3$, $A_2C_2$ and $A_3C$ were detected in eye tissue, and $A_3C$ was found in brain tissue. LDH $A_4$, $A_3B$, $A_2B_2$, $AB_3$, $B_4$, eye-specific $C_4$ isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH $A_4$ isozyme was purified in the fraction from elution with $NAD^+$ containing buffer of affinity chromatography. Eye-specific $C_4$ isozyme was eluted right after $A_4$, after which $B_4$ isozyme was eluted with plain buffer. As a result, one part of molecular structures in $A_4$, $B_4$ and eye-specific $C_4$ were similar, but were different from each other in $B_4$ and $C_4$. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The activity of LDH $A_4$, $A_2B_2$, $B_4$, and eye-specific $C_4$ isozymes remained at 39.98, 21.28, 19.67 and 16.87% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The $Km^{PYR}$ values were 0.17, 0.27 and 0.133 mM in $A_4$, $B_4$ and eye-specific $C_4$ isozymes, respectively. The optimum pH of LDH $A_4$, $B_4$, eye-specific $C_4$, $A_2B_2$, $A_3B$, and $AB_3$ were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The $A_4$ and heterotetramer isozymes stabilized a broad range of pH. Especially, LDH activities in skeletal muscle tissue were high, resulting in a high degree of muscle activity.LDH metabolism in eye tissue seems to be converted faster from pyruvate to lactate by eye-specific $C_4$ isozyme as eye-specific $C_4$ have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by $A_4$ isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of $C_4$ isozyme were different according to the ecological environmental and feeding capturing patterns.

The Isolation of Bacillus sphaericus 366M-9 Producing New Cephalosporin-C Deacetylase (CAH) and its Enzymatic Characterization (신규 Cephalosporin-C Deacetylase(CAH) 생산 균주인 Bacillus sphaericus 366M-9의 선발 및 그 효소학적 특성)

  • 이승훈;권태종;이동희
    • Microbiology and Biotechnology Letters
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    • v.32 no.3
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    • pp.224-229
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    • 2004
  • Several microorganisms (esterase-producing group) were isolated by the solid selective media containing-naphtylacetate. Among them, strain 366M-9 having a high activity of cephalosporin-C deacetylase (CAH; EC 3.1.1.41) was selected. The strain 366M-9 was identified as Bacillus sphaericus on the basis of morphological, physiological, and biochemical characteristics. The production of CAH reached at maximum value after 32 hrs, when cultivated in the optimal medium containing dextrin 2.5%, peptone 2.5%, sodium chloride 0.5%, dipotassium phosphate 0.25%, ferrous sulfate 0.02%, and 7-ACA 0.1% at $30^{\circ}C$ with initial pH 6.0. The CAH was purified by 3 steps with ammonium sulfate precipitation, adsorption chromatography on hydroxyapatite column, and Sephadex G-200 gel chromatography. The final enzyme preparation was homogeneous as judged by the analysis of SDS-PAGE and HPLC. Optimum temperature and pH for CAH activity were $50{\circ}C$ and around 7.0, respectively. And the enzyme was stable at pH 6.0~8.0, up to $50^{\circ}C$. The Michaelis-Menten constants ($K_{m}$ ), $V_{max}$ were 0.87 mM and 1.22 unit/ml, respectively.

A Study on Exposure Indices for Diesel Engine Exhaust in Forklift Operating Areas (지게차 사용 사업장에서 디젤엔진배출물질 노출지표에 관한 연구)

  • Kim, Sangil;Park, Ji Young;Lee, Kyeongmin;Kim, Seung Won
    • Journal of Korean Society of Occupational and Environmental Hygiene
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    • v.26 no.1
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    • pp.38-47
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    • 2016
  • Objectives: The objective of this study was to determine the exposure levels of forklift operators to diesel engine exhaust(DEE) using black carbon(BC), elemental carbon(EC), and nitrogen dioxide($NO_2$) as indicators. Methods: A total of eight forklift operators in six collection companies were assessed over a period of two months from July to September 2015. BC was measured using a real-time monitor and respirable EC samples were analyzed using the NIOSH method 5040. $NO_2$ samples were collected using a passive badge-type sampler. Results: The geometric mean of BC, EC and $NO_2$ were $3.1-19.1{\mu}g/m^3$, $2.1-23.8{\mu}g/m^3$, and 12.5-166.6 ppb at all companies. When forklifts were operating both outside and inside, BC concentrations increased 2.0-5.6 times. The highest increase was observed when forklifts were operating indoors. The increase in BC concentrations varied by company(company A: 2.0 times, B: 3.2 times, C: 5.6 times, D: 2.1 times, E: 5.1 times, F: 2.6 times). The geometric mean of BC, EC, and $NO_2$ for the forklift operators was $9.6{\mu}g/m^3$, $7.9{\mu}g/m^3$, and 48.9 ppb, respectively. The geometric mean of BC, EC, and $NO_2$ for manufacturing workers was $9.3{\mu}g/m^3$, $0.9{\mu}g/m^3$, and 85.2 ppb, respectively. The mean BC and EC exposure levels for the forklift operators were slightly higher than those for manufacturing workers, but $NO_2$ levels for manufacturing workers were higher than those for the forklift operators(p>0.05). Multiple regression analysis revealed that diesel exhaust emissions standard, forklift weight and forklift manufacturer were the most influential factors in determining worker exposure. Conclusions: In the DEE work environment, workers who perform tasks within the workplace as well as inside forklifts as operators are likely to be exposed to a lack of ventilation. Further study of forklift operators' exposure to DEE indicators should be conducted to include a wider range of occupational and environmental situations, such as collection procedures, seasonal situations, types of fuel used, and number of forklifts.

NF-κB Activation and PPAR Transactivational Effects of a New Aliphatic Acid Amide from Pericarps of Zanthoxylum piperitum

  • Yang, Seo Young;Tai, Bui Huu;Song, Seok Bean;Li, Wei;Yan, Xi Tao;Sun, Ya Nan;Nguyen, Phuong Thao;Kim, Young Ho
    • Bulletin of the Korean Chemical Society
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    • v.35 no.8
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    • pp.2361-2366
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    • 2014
  • A new aliphatic acid amide, ZP-amide F (1), and eight known compounds, including bungeanumamide A (2), tumuramide C (3), ZP-amide A (4), ZP-amide B (5), ZP-amide D (6), hyperin (7), quercitrin (8), and (-)-sesamin (9), were isolated from pericarps of Zanthoxylum piperitum. The effects of these compounds on $TNF{\alpha}$-induced NF-${\kappa}B$ activation and transactivational activity of PPARs, including $PPAR{\alpha}$, $PPAR{\beta}({\delta})$ and $PPAR{\gamma}$ subtypes, were evaluated. Compounds 7 and 9 exhibited potent inhibitory effects on $TNF{\alpha}$-induced NF-${\kappa}B$ activation with $IC_{50}$ values of 5.50 and $8.10{\mu}M$, respectively. Aliphatic acid amide compounds 3, 4 and 6 displayed enhanced effects on PPAR transactivational activity with $EC_{50}$ values of 47.12, 19.13 and $12.02{\mu}M$, respectively. Among them, compound 4 demonstrated an increase in $PPAR{\alpha}$ transactivational activity, compound 3 showed a moderate increase on all PPAR subtypes, whereas compound 6 displayed weak PPAR transactivational activity.

2'-Spirocyclopropyl-carbocyclic Nucleoside as a Novel Scaffold for Potent Anti-HCV Agents

  • Li, Hua;Yoo, Jin-Cheol;Hong, Joon-Hee
    • Bulletin of the Korean Chemical Society
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    • v.32 no.4
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    • pp.1146-1152
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    • 2011
  • The discovery of 2'-spirocyclopropyl-ribocytidine (J. Med. Chem. 2010, 53, 8150-8160) as a potent inhibitor of RNA synthesis by NS5B ($IC_{50}=7.3{\mu}M$), the RNA polymerase encoded by hepatitis C Virus (HCV), has led to the synthesis and biological evaluation of several carbocyclic versions of 2'-spiropropyl-nucleosides. The cyclopentenol intermediate 7 was successfully constructed via ring-closing metathesis (RCM) from divinyl 6. Spirocyclopropanation of enone 8 was effected by using (2-chloroethyl)-dimethylsulfonium iodide and potassium tert-butoxide to form the desired intermediate 9. The synthesized nucleoside analogues 21-24 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line. Among them, the cytosine nucleoside analogue 22 exhibited significant anti-HCV activity ($EC_{50}= 8.2{\mu}M$).

Thelephoric acid and Kynapcin-9 in Mushroom Polyozellus multiflex Inhibit Prolyl Endopeptidase In Vitro

  • Kwak, Ju-Yeon;Rhee, In-Koo;Lee, Kyung-Bok;Hwang, Ji-Sook;Yoo, Ick-Dong;Song, Kyung-Sik
    • Journal of Microbiology and Biotechnology
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    • v.9 no.6
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    • pp.798-803
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    • 1999
  • Prolyl endopeptidase [PEP; EC 3.4.21.26], a serine protease which is known to cleave peptide bonds on the carboxy side of a proline residue, plays an important role in the degradation of proline-containing neuropeptides that have been suggested to participate in learning and memory processes. An abnormal increase in the level of PEP, which can lead to generation of $A{\beta}$, is also suggested to be involved in Alzheimer's type senile dementia. In the course of screening PEP inhibitors from Basidiomycetes, the mushroom Polyozellus multiplex exhibited a high inhibitory activity against PEP. Two active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification, using silica gel, Sephadex LH-20, and Lobar RP-18 chromatography. The chemical structures of these compounds were identified as thelephoric acid and 12-acety1-2,3,7,8-tetrahydroxy-[12H]-12-hydroxymethylbenzobis[I.2b,3.4b'] benzofuran-11-one (kynapcin-9) by spectral data including UV, IR, MS, HR-MS, $^1H-,{\;}^{13}C-$, and 2D-NMR. The $IC_{50}$ values of the thelephoric acid and kynapcin-9 were 0.157 ppm (446nM) and 0.087 ppm (212nM) and their inhibitor constants ($K_i$) were 0.73ppm ($2.09{\;}\mu\textrm{m}$) and 0.060 ppm (146 nM), respectively. Furthermore, they were non-competitive with a substrate in Dixon plots.

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Parthenogenetic Activation and Development of Freshly Matured Bovine IVM Oocytes (체외성숙 직후 소 난포란의 단위발생과 체외발육능)

  • 정희태;임석기;오세훈;박춘근;양부근;김정익
    • Korean Journal of Animal Reproduction
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    • v.21 no.1
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    • pp.71-78
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    • 1997
  • This study was conducted to investigate the activation condition of freshly matured bovine IVM oocytes for use as a cytoplasmic recipient in nuclear transfer. Bovine oocytes matured in vitro for 22-24 h were treated with various activation conditions. In Experiment 1 in vitro matured oocytes were treated with electric stimuIus (ES; 2 pulses of 1.25 kV/cm for 70 ${\mu}{\textrm{s}}$ec, each pulse 1 sec apart), ethanol (ET; 7%, 5min) , Ca$^2+$-ionophore(A23187; 10$\mu\textrm{g}$/ml, 5min) and cycloheximide(CH; 10$\mu\textrm{g}$/ml, 6 h). Activation rates were similar in treatments with ES, ET and A23187(48.8~54.3%), however, significantly reduced with CH treatment(15.9%, P

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Developmental characteristics of Hemiptarsenus sp.(Hymenoptera : Eulophidae), a parasitoid of Liriomyza trifolii (Burgess) and effect of the insectcides (아메리카잎굴파리 기생봉, Hemiptarsenus sp.의 발육특성 및 살충제의 영향)

  • Moon, Hyung-Chol;Choi, Jeong-Sik;Hwang, Chang-Yeon
    • The Korean Journal of Pesticide Science
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    • v.4 no.4
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    • pp.72-76
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    • 2000
  • The study was conducted to investigate the developmental periods and effect of several insecticides on Hemiptarsenus sp., ectoparasitoid of Liriomyza trifolii. The mean length and width of egg were 0.5mm and 0.1mm. The mean length of larva, pupae, abult female, and abult male were 1.9mm, 2.0mm, 2.2mm, and 1.8mm, respectively. Developmental periods of Hemiptarsenus sp. from egg to larva at 15, 20, 25, $30^{\circ}C$ were 16.9, 8.8, 5.9, and 4.5 days, and those of pupa were 20.7, 9.7, 5.6, and 3.4 days, respectively. Based on these results, developmental threshold temperatures and effective temperatures were $9.5^{\circ}C$, 91.5 degree-days in egg-larval stage, $10.9^{\circ}C$, 142.3 degree-days in pupal stage. When several insecticides were evaluated to Hemiptarsenus sp. at the recommended concentrations, B.t WP, diflubenzuron WP, and cyromazin were negligiblly effective all life stages. Fipronil SC, cartap SP, spinosad GW were less toxic to larva and pupa, but highly toxic to adults. Abamectin EC was less toxic to all life stages, but inhibited oviposition of 50% more to Hemiptarsenus sp. female.

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Effects of Excess Lead Addition on Sol-Gel Derived ($Pb_{0.9}La_{0.1}$)$Ti_{0.975}O_3$(PLT (10)) Thin Film

  • Kim, Seong-Jin;Jeong, Yang-Hui;Yun, Yeong-Seop
    • Journal of the Institute of Electronics Engineers of Korea SD
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    • v.39 no.3
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    • pp.1-8
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    • 2002
  • In order to study electric properties of (Pb$\_$0.9/La$\_$0.1/)Ti$\_$0.975/O$_3$(PLT (10)) films with varying excess lead concentration (7.5, 10, 12.5, 15 ㏖% excess lead), the PLT films were deposited by sol-gel process. DTA analyses reveal that the crystallization temperature of the precursor powers decreased with increasing amount of excess lead. XRD patterns of PLT reveal pure perovskite structure and the preferred orientation increased with increasing Pb content in the films. With increasing amount of excess P$\_$b/, the relative permittivity ($\xi$$\_$r/) increased and leakage current density at 100 ㎸/cm transformed 4.01$\times$10$\^$-5/, 2.42$\times$10$\^$-6/, 1.27$\times$10$\^$-6/, 1.56$\times$10$\^$-6/A/㎠ respectively. In the results of hysteresis loops measured at 166 kV/cm, the remanent polarization (P$\_$r/) and the coercive field (E$\_$c) are 6.36$\mu$C/cm and 58.7 ㎸/cm, respectively (at 12.5 ㏖% excess P$\_$b/) With increasing amount of excess Pb, the remanent polarization for PLT thin film degraded to about 44%, 27%, 15%, 16% of the initial value after 10$\^$9/ cycles./TEX>) With increasing amount of excess Pb, the remanent polarization for PLT thin film degraded to about 44%, 27%, 15%, 16% of the initial value after $10^{9}$ cycles.

Polymorphisms in Heat Shock Proteins A1B and A1L (HOM) as Risk Factors for Oesophageal Carcinoma in Northeast India

  • Saikia, Snigdha;Barooah, Prajjalendra;Bhattacharyya, Mallika;Deka, Manab;Goswami, Bhabadev;Sarma, Manash P;Medhi, Subhash
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.18
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    • pp.8227-8233
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    • 2016
  • Background: To investigate polymorphisms in heat shock proteins A1B and A1L (HOM) and associated risk of oesophageal carcinoma in Northeast India. Materials and Methods: The study includes oesophageal cancer (ECA) patients attending general outpatient department (OPD) and endoscopic unit of Gauhati Medical College. Patients were diagnosed based on endoscopic and histopathological findings. Genomic DNA was typed for HSPA1B1267 and HSPA1L2437 SNPs using the polymerase chain reaction with restriction fragment length polymorphisms. Results: A total of 78 cases and 100 age-sex matched healthy controls were included in the study with a male: female ratio of 5:3 and a mean age of $61.4{\pm}8.5years$. Clinico-pathological evaluation showed 84% had squamous cell carcinoma and 16% were adenocarcinoma. Dysphagia grades 4 (43.5%) and 5 (37.1%) were observed by endoscopic and hispathological evaluation. The frequency of genomic variation of A1B from wild type A/A to heterozygous A/G and mutant G/G showed a positive association [chi sq=19.9, p=<0.05] and the allelic frequency also showed a significant correlation [chi sq=10.3, with cases vs. controls, OR=0.32, $p{\leq}0.05$]. The genomic variation of A1L from wild T/T to heterozygous T/C and mutant C/C were found positively associated [chi sq=7.02, p<0.05] with development of ECA. While analyzing the allelic frequency, there was no significant association [chi sq=3.19, OR=0.49, p=0.07]. Among all the risk factors, betel quid [OR=9.79, Chi square=35.0, p<0.05], tobacco [OR=2.95, chi square=10.6, p<0.05], smoking [OR=3.23, chi square=10.1, p<0.05] demonstrated significant differences between consumers vs. non consumers regarding EC development. Alcohol did not show any significant association [OR=1.34, chi square=0.69, p=0.4] independently. Conclusions: It can be concluded that the present study provides marked evidence that polymorphisms of HSP70 A1B and HSP70 A1L genes are associated with the development of ECA in a population in Northeast India, A1B having a stronger influence. Betel quid consumption was found to be a highly significant risk factor, followed by smoking and tobacco chewing. Although alcohol was not a potent risk factor independently, alcohol consumption along with tobacco, smoking and betel nut was found to contribute to development of ECA.