• Title/Summary/Keyword: Delta activity

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Aromaticity of Tyr-202 in the α4-α5 Loop Is Essential for Toxicity of the Bacillus thuringiensis Cry4A Toxin

  • Pornwiroon, Walairat;Katzenmeier, Gerd;Panyim, Sakol;Angsuthanasombat, Chanan
    • BMB Reports
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    • v.37 no.3
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    • pp.292-297
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    • 2004
  • The current model for the mechanism of action of the Bacillus thuringiensis Cry $\delta$-endotoxins involves the penetration of the ${\alpha}4-{\alpha}5$ hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the ${\alpha}4-{\alpha}5$ loop of the 130-kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the ${\alpha}4-{\alpha}5$ loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the ${\alpha}4-{\alpha}5$ loop of the Cry4A toxin.

Functional Studies of Tyrosine 108 Residue in the Active Site of Human Glutathione S-Transferase P1-1

  • Park, Hee-Joong;Koh, Jong-Uk;Ahn, So-Youn;Kong, Kwang-Hoon
    • Bulletin of the Korean Chemical Society
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    • v.26 no.3
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    • pp.433-439
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    • 2005
  • To gain further insight on the relationship between structure and functions of glutathione S-transferase (GST), the three tyrosine 108 mutants, Y108A, Y108F, and Y108W, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitution of Tyr 108 with alanine resulted in significant decrease of the GSH-conjugation activity and the GSH peroxidase activity, but approximately 63% increase of steroid isomerase activity toward ${\Delta}^5$–[androstene 3,17-dione. On the other hand, the substitution of Tyr 108 with phenylalanine resulted in decreases of $k_{cat}\;and\;k_{cat}/K_m{^{EPNP}}$ by 2 orders of magnitude, suggesting that Tyr 108 residue of hGSTP1-1 are considered to be important for the catalysis and the binding of the epoxide substrates. The substitution of Tyr 108 with tryptophan resulted in significant decreases of the specific activities toward EPNP, cumene hydroperoxide and ${\Delta}^5$–ndrostene 3,17-dione, but approximately 2-fold increase on the enzyme-catalyzed addition of GSH to DCNB. We conclude from these results that Tyr 108 in hGST P1-1 plays very different roles depending upon the nature of the electrophilic substrates.

Molecular Orbital Interpretation on the Inhibitory Effect of the Ni(Ⅱ) Complexes with Polyamines and Imidazole Derivatives (Polyamine류와 Imidazole 유도체가 배위된 Ni(Ⅱ) 착물의 저해 효과에 관한 분자궤도함수론적 해석)

  • Kim, Jung-Sung;Song, Young-Dae
    • Journal of the Korean Chemical Society
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    • v.48 no.2
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    • pp.123-128
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    • 2004
  • Quantum chemical quantities, enthalpy of formation(${\Delta}H_f$), HOMO and LUMO energy, and dipole moment(${\mu}_D$) were acquired by AM1, PM3, and ZINDO/1 methods for polyamines and imidazole derivatives. The investigation of the inhibitory activity on some Ni(II) complexes with polyamines and imidazole derivatives is performed by ZINDO/1 calculations. It was found that experimental inhibitory activity(IA) appeared when the value of net charge and enthalpy of formation were over 0.03 and -300 eV, respectively for Ni(II) complexes. These results showed that the Ni(II) complexes have exception on the following very unstable compounds: square pyramidal [Ni(dpt)(tn)])]$^{2+}$(dpt=3,3'-diaminodipropylamine)(tn=1,3-diaminopropane) and distorted tetrahedral [Ni(N-PropIm))$_2$(NCS))$_2$](N-PropIm=N-Propylimidazole).

Optimization in the Preparation of Aloe Vinegar by Acetobacter sp. and Inhibitory Effect against Lipase Activity (Acetobacter sp.를 이용한 알로에 식초의 발효조건 및 Lipase활성 저해효과)

  • Ryu, Il-Hwan;Lee, Kap-Sang;Shin, Yong-Seo;Chun, Seung-Ho;Lee, Do-Sang
    • Applied Biological Chemistry
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    • v.42 no.2
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    • pp.105-110
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    • 1999
  • Acetobacter sp. were isolated from persimmon vinegar. We studied about conditions of Aloe vinegar fermentation by an isolated strain and inhibitory effect against lipase activity. Strains DS-118 was strictly aerobic, motile, gram negative, non-spore-forming and short rod shaped. It reacted positively in catalase test, was oxidase test negative, was ureas negative, was produced acetic acid from alcohol. On the basis of these results, it was identified as a strain of Acetobacter sp. In the preparation of Aloe vinegar, optimun initial alcohol concentration, acidity, and fermentation temperature were 10%, $3{\sim}4%$ and $25^{\circ}C$, respectively. The major organic acid in Aloe vinegar was acetic acid (12%), but malic acid and ${\delta}-galactronic$ acid were also present in trace. The Aloe vinegar(acidity : 12%) inhibited lipase activity and it's $IC_{50}$ was 43%.

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Mesothermal Gold Mineralization in the Boseong-Jangheung area, Chollanamdo-province (전라남도 보성-장흥지역의 중열수 금광화작용)

  • 허철호;윤성택;소칠섭
    • Economic and Environmental Geology
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    • v.35 no.5
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    • pp.379-393
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    • 2002
  • Within the Boseong-Jangheung area of Korea, five hydrothermal gold (-silver) quartz vein deposits occur. They have the characteristic features as follows: the relatively gold-rich nature of e1ectrurns; the absence of Ag-Sb( -As) sulfosalt mineral; the massive and simple mineralogy of veins. They suggest that gold mineralization in this area is correlated with late Jurassic to Early Cretaceous, mesothermal-type gold deposits in Korea. Fluid inclusion data show that fluid inclusions in stage I quartz of the mine area homogenize over a wide temperature range of 200$^{\circ}$ to 460$^{\circ}$C with salinities of 0.0 to 13.8 equiv. wt. % NaCI. The homogenization temperature of fluid inclusions in stage II calcite of the mine area ranges from 150$^{\circ}$ to 254$^{\circ}$C with salinities of 1.2 to 7.9 equiv. wt. % NaCI. This indicates a cooling of the hydrothermal fluid with time towards the waning of hydrothermal activity. Evidence of fluid boiling including CO2 effervescence indicates that pressures during entrapment of auriferous fluids in this area range up to 770 bars. Calculated sulfur isotope composition of auriferous fluids in this mine area (${\delta}^34S$_{{\Sigma}S}$$\textperthousand$) indicates an igneous source of sulfur in auriferous hydrothermal fluids. Within the Sobaegsan Massif, two representative mesothermal-type gold mine areas (Youngdong and Boseong-Jangheung areas) occur. The ${\delta}^34S values of sulfide minerals from Youngdong area range from -6.6 to 2.3$\textperthousand$ (average=-1.4$\textperthousand$, N=66), and those from BoseongJangheung area range from -0.7 to 3.6$\textperthousand$ (average=1.6$\textperthousand$, N=39). These i)34S values of both areas are comparatively lower than those of most Korean metallic ore deposits (3 to 7TEX>$\textperthousand$). And, within the Sobaegsan Massif, the ${\delta}^34S values of Youngdong area are lower than those of Boseong-Jangheung area. It is inferred that the difference of ${\delta}^34S values within the Sobaegsan Massif can be caused by either of the following mechanisms: (1) the presence of at least two distinct reservoirs (both igneous, with ${\delta}^34S values of < -6 $\textperthousand$ and 2$\pm$2 %0) for Jurassic mesothermal-type gold deposits in both areas; (2) different degrees of the mixing (assimilation) of 32S-enriched sulfur (possibly sulfur in Precambrian pelitic basement rocks) during the generation and/or subsequent ascent of magma; and/or (3) different degrees of the oxidation of an H2S-rich, magmatically derived sulfur source ${\delta}^34S = 2$\pm$2$\textperthousand$) during the ascent to mineralization sites. According to the observed differences in ore mineralogy (especially, iron-bearing ore minerals) and fluid inclusions of quartz from the mesothermal-type deposits in both areas, we conclude that pyrrhotite-rich, mesothermal-type deposits in the Youngdong area formed from higher temperatures and more reducing fluids than did pyrite(-arsenopyrite)-rich mesothermal-type deposits in the Boseong-Jangheung area. Therefore, we prefer the third mechanism than others because the ${\delta}^34S values of the Precambrian gneisses and Paleozoic sedimentary rocks occurring in both areas were not known to the present. In future, in order to elucidate the provenance of ore sulfur more systematically, we need to determine ${\delta}^34S values of the Precambrian metamorphic rocks and Paleozoic sedimentary rocks consisting the basement of the Korean Peninsula including the Sobaegsan Massif.

Ammonia Decomposition Over Tantalum Carbides of Hydrogen Fuel Cell (수소연료전지용 탄탈륨 탄화물에 대한 암모니아 분해반응)

  • Choi, Jeong-Gil
    • New & Renewable Energy
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    • v.9 no.1
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    • pp.51-59
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    • 2013
  • Tantalum carbide crystallites which is to be used for $H_2$ fuel cell has been synthesized via a temperature-programmed reduction of $Ta_2O_5$ with pure $CH_4$. The resultant Ta carbide crystallites prepared using two different heating rates and space velocity exhibit the different surface areas. The $O_2$ uptake has a linear relation with surface area, corresponding to an oxygen capacity of $1.36{\times}10^{13}\;O\;cm^{-2}$. Tantalum carbide crystallites are very active for hydrogen production form ammonia decomposition reaction. Tantalum carbides are as much as two orders of magnitude more active than Pt/C catalyst (Engelhard). The highest activity has been observed at a ratio of $C_1/Ta^{{\delta}+}=0.85$, suggesting the presence of electron transfer between metals and carbon in metal carbides.

Saccharomyces cerevisiae Hsp30 is Necessary for Homeostasis of a Set of Thermal Stress Response Functions

  • Thakur, Suresh;Chakrabarti, Amitabha
    • Journal of Microbiology and Biotechnology
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    • v.20 no.2
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    • pp.403-409
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    • 2010
  • Saccharomyces cerevisiae Hsp30 is a plasma membrane heat shock protein that is induced by various environmental stress conditions. However, the functional role of Hsp30 during diverse environmental stressors is not presently known. To gain insight into its function during thermal stress, we have constructed and characterized a ${\Delta}hsp30$ strain during heat stress. $BY4741{\Delta}hsp30$ cells were found to be more sensitive compared with BY4741 cells, when exposed to a lethal heat stress at $50^{\circ}C$. When budding yeast is exposed to either heat shock or weak organic acid, it inhibits Pma1p activity. In this study, we measured the levels of Pma1p in mutant and Wt cells both during optimal temperature and heat shock temperature. We observed that $BY4741{\Delta}hsp30$ cells showed constitutive reduction of Pma1p. To gain further insights into the role of Hsp30 during heat stress, we compared the total protein profile by 2D gel electrophoresis followed by identification of differentially expressed spots by LC-MS. We observed that contrary to that expected from thermal-stress-induced changes in gene expression, the ${\Delta}hsp30$ mutant maintained elevated levels of Pdc1p, Trx1p, and Nbp35p and reduced levels of Atp2p and Sod1p during heat shock. In conclusion, Hsp30 is necessary during lethal heat stress, for the maintenance of Pma1p and a set of thermal stress response functions.

The expression analysis of mouse interleukin-6 splice variants argued against their biological relevance

  • Annibalini, Giosue;Guescini, Michele;Agostini, Deborah;De Matteis, Rita;Sestili, Piero;Tibollo, Pasquale;Mantuano, Michela;Martinelli, Chiara;Stocchi, Vilberto
    • BMB Reports
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    • v.45 no.1
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    • pp.32-37
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    • 2012
  • Alternative splicing generates several interleukin-6 (IL-6) isoforms; for them an antagonistic activity to the wild-type IL-6 has been proposed. In this study we quantified the relative abundance of IL-6 mRNA isoforms in a panel of mouse tissues and in C2C12 cells during myoblast differentiation or after treatment with the $Ca^{2+}$ ionophore A23187, the AMP-mimetic AICAR and TNF-${\alpha}$. The two mouse IL-6 isoforms identified, IL-6${\delta}$5 (deletion of the first 58 bp of exon 5) and IL-6${\delta}$3 (lacking exon 3), were not conserved in rat and human, did not exhibit tissue specific regulation, were expressed at low levels and their abundance closely correlated to that of full-length IL-6. Species-specific features of the IL-6 sequence, such as the presence of competitive 3' acceptor site in exon 5 and insertion of retrotransposable elements in intron 3, could explain the production of IL-6${\delta}$5 and IL-6${\delta}$3. Our results argued against biological significance for mouse IL-6 isoforms.

NICKEL INCORPORATION INTO Klebsiella aerogenes UREASE (Klebsiella aerogenes Urease로의 닉켈의 도입)

  • Lee, Mann-Hyung-
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1994.11a
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    • pp.69-80
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    • 1994
  • Although ureases play important roles in microbial nitrogen metabolism and in the pathogenesis of several human diseases, little is known of the mechanism of metallocenter biosynthesis in this Ni-Containing enzyme. Klebsiella aerogenes urease apo-protein was purified from cells grown in the absence of Ni. The purified apo-enzyme showed the same native molecular weight, charge, and subunit stoichiometry as the holo-enzyme. Chemical modification studies were consistent with histidinyl ligation of Ni. Apo-enzyme could not be activated by simple addition of Ni ions suggesting a requirement for a cellular factor. Deletion analysis showed that four accessory genes (ureD, ureE, ureF, and ureG) are necessary for the functional incorporation of the urease metallocenter. Whereas the $\Delta$ureD, $\Delta$ureF, and $\Delta$ureG mutants are inactive and their ureases lack Ni, the $\Delta$ureE mutants retain partial activity and their ureases possess corresponding lower levels of Ni. UreE and UreG peptides were identified by SDS-polyacrylamide gel comparisons of mutant and wild type cells and by N-terminal sequencing. UreD and UreF peptides, which are synthesized at ve교 low levels, were identified by using in vitro transcription/translation methods. Cotransformation of E. coli cells with the complementing plasmids confirmed that ureD and ureF gene products act in trans. UreE was purified and characterized. immunogold electron microscopic studies were used to localize UreE to the cytoplasm. Equilibrium dialysis studies of purified UreE with $^{63}$ NiC1$_2$ showed that it binds ~6 Ni in a specific manner with a $K_{d}$ of 9.6 $\pm$1.3 $\mu$M. Results from spectroscopic studies demonstrated that Ni ions are ligated by 5 histidinyl residues and a sixth N or O atom, consistent with participation of the polyhistidine tail at the carboxyl termini of the dimeric UreE in Ni binding. With these results and other known features of the urease-related gene products, a model for urease metallocenter biosynthesis is proposed in which UreE binds Ni and acts as a Ni donor to the urease apo-protein while UreG binds ATP and couples its Hydrolysis to the Ni incorporation process.ouples its Hydrolysis to the Ni incorporation process.s.

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Bioequivalence Evaluation of Two Brands of Cefixime 100 mg Capsule (Suprax and Alpha-Cefixime) in Korean Healthy Volunteers

  • Choi, Dong-Hyun;Burm, Jin-Pil
    • Biomolecules & Therapeutics
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    • v.15 no.3
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    • pp.182-186
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    • 2007
  • Cefixime is an orally absorbed cephalosporin with a broad spectrum of activity against Gram-negative bacteria and is highly resistant to beta-lactamase degradation. The purpose of the present study was to evaluate the bioequivalence of two cefixime capsules, Suprax capsule (Dong-A Pharmaceutical Co., reference drug) and Alpha-Cefixime capsule (Alpha Pharmaceutical Co., test drug), according to the guidelines of Korea Food and Drug Administration (KFDA). Twenty-four normal subjects, $23.5{\pm}3.72$ years in age and $68.3{\pm}8.89$ kg in body weight, were divided into two groups and a randomized $2{\times}2$ cross-over study was employed. There was one week washout period between the doses. After one capsule containing 100 mg of cefixime was orally administered, plasma was taken at predetermined time intervals and the concentrations of cefixime in plasma were determined using HPLC with UV detector. The pharmacokinetic parameters such as $AUC_{t}$, $C_{max}$ and $T_{max}$ were calculated and ANOVA test was utilized for the statistical analysis of the parameters. The results showed that the differences in $AUC_{t}$, $C_{max}$ and $T_{max}$ between two products were -3.91%, -2.23% and -3.18%, respectively, when calculated against the reference drug. The 90% confidence intervals using logarithmically transformed data were within the acceptance range of $log0.8{\leq}{\delta}{\leq}log1.25$ (e.g., $log0.8786{\leq}{\delta}{\leq}log1.0523$ and $log0.8889{\leq}{\delta}{\leq}log1.0512$ for $AUC_{t}$ and $C_{max}$, respectively). The 90% confidence intervals using untransformed data was within ${\pm}20%$(e.g., $-10.37%{\leq}{\delta}{\leq}6.73%$ for $T_{max}$). All parameters met the criteria of KFDA for bioequivalence, indicating that Alpha-Cefixime capsule (Alpha Pharmaceutical Co.) is bioequivalent to Suprax capsule (Dong-A Pharmaceutical Co.).