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ABSTRACT: The budC gene encoding the meso-2,3-BDH from Klebsiella pneumoniae XJ-Li was expressed in E. coli BL21 (DE3) pLys. Hypothetical amino acid sequence alignments revealed that the enzyme belongs to the short chain dehydrogenase/reductase family. After purification and refolding, the recombinant enzyme had activities of 218 U/mg for reduction of acetoin and 66 U/mg for oxidation of meso-2,3-butanediol. Highest activities were at pH 8.0 and 9.0 respectively. These are higher than other meso-2,3-butanediol dehydrogenases from K. pneumoniae. The low K (m) value (0.65 mM) for acetoin indicated that the enzyme can easily reduce acetoin to meso-2,3-butanediol. There were no significant activities towards 2R,3R-2,3-butanediol, 1,4-butanediol and 2S,3S-2,3-butanediol, suggesting that the enzyme has a high stereospecificity for the meso-dihydric alcohol.
Biotechnology Letters 05/2012; 34(8):1519-23. · 1.68 Impact Factor
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ABSTRACT: Genes dhaF and dhaG encoding the α and β subunits of glycerol dehydratase reactivation factor (GDHtR) were amplified from the genomic DNA of
Klebsiella pneumoniae XJPD-Li. The identity of the deduced amino acid sequence of the β subunit was relatively low compared with that of K. pneumoniae (U30903), where the 96th amino acid residue was found to be the more active amino acid histidine instead of glutamine in
K. pneumoniae (U30903). A specific GDHtR activity of approximately 30U/mg was attained in Escherichia coli BL21 (pET-28a (+)-dhaFG). His6-tagged GDHtR was purified by Ni-nitrilotriacetate chromatography, and the enzyme was purified 2.6-fold in a yield
of 20.7%. The study showed that both glycerol and O2-inactivated glycerol dehydratase (GDHt) could be quickly reactivated by GDHtR in the presence of ATP, Mg2+ and coenzyme B12. However, the glycerol-inactivated GDHt was more easily reactivated than O2-inactivated GDHt. In the first 10min of the reactivation reaction, the average reactivation rate was 0.18 and 0.12μmol/min
for glycerol and O2-inactivated GDHt, respectively.
World Journal of Microbiology and Biotechnology 04/2012; 25(11):1947-1953. · 1.53 Impact Factor
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ABSTRACT: Although polymeric magnetic resonance imaging (MRI) agents have significantly improved relaxivity and prolonged circulation time in vivo compared with current imaging agents, the potential for long-term toxicity prevents their translation into the clinic. The aim of this study was to develop a new biodegradable, nonionic polymeric blood pool MRI contrast agent with efficient clearance from the body. We synthesized PHPG-DTPA, which possesses two potentially degradable sites in vivo: protein amide bonds of the polymer backbone susceptible to enzymatic degradation and hydrolytically labile ester bonds in the side chains. After chelation with Gd(3+), PHPG-DTPA-Gd displayed an R(1) relaxivity of 15.72 mm(-1)⋅sec(-1) (3.7 times higher than that of Magnevist(T)). In vitro, DTPA was completely released from PHPG polymer within 48 h when incubated in mouse plasma. In vivo, PHPG-DTPA-Gd was cleared via renal route as shown by micro-single photon emission computed tomography of mice after intravenous injection of (111)In-labeled PHPG-DTPA-Gd. MRI of nude rats bearing C6 glioblastoma showed significant enhancement of the tumor periphery after intravenous injection of PHPG-DTPA-Gd. Furthermore, mouse brain angiography was clearly delineated up to 2 h after injection of PHPG-DTPA-Gd. PHPG-DTPA-Gd's biodegradability, efficient clearance, and significantly increased relaxivity make it a promising polymeric blood pool MRI contrast agent.
Biomaterials 04/2012; 33(21):5376-83. · 7.40 Impact Factor
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ABSTRACT: Sparganosis, caused by the plerocercoid larvae of members of the genus Spirometra, can cause significant public health problem and considerable economic losses. In the present study, the complete mitochondrial DNA (mtDNA) sequence of Spirometra erinaceieuropaei from China was determined, characterized and compared with that of S. erinaceieuropaei from Japan. The gene arrangement in the mt genome sequences of S. erinaceieuropaei from China and Japan is identical. The identity of the mt genomes was 99.1% between S. erinaceieuropaei from China and Japan, and the complete mtDNA sequence of S. erinaceieuropaei from China is slightly shorter (2 bp) than that from Japan. Phylogenetic analysis of S. erinaceieuropaei with other representative cestodes using two different computational algorithms [Bayesian inference (BI) and maximum likelihood (ML)] based on concatenated amino acid sequences of 12 protein-coding genes, revealed that S. erinaceieuropaei is closely related to Diphyllobothrium spp., supporting classification based on morphological features. The present study determined the complete mtDNA sequences of S. erinaceieuropaei from China that provides novel genetic markers for studying the population genetics and molecular epidemiology of S. erinaceieuropaei in humans and animals.
International journal of biological sciences 01/2012; 8(5):640-9. · 2.70 Impact Factor
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ABSTRACT: To observe whether the impairment of endothelial nitric oxide synthase (eNOS) activity by elevated level of free fatty acid (FFA) is associated with activation of protein kinase C (PKC).
The cultured human umbilical vein endothelial cells were used for four groups: control group, oleic acid (OA) group (10, 50, 100, 150, 200 micromol/L), GFX group and OA+GFX group. The activity of eNOS in each group was examined, and the activity and expression of PKC were determined in both control and OA groups. The method of immunocytochemistry was performed to detect the expression of p-eNOS(1177) in control, OA (100 micromol/L), GFX and OA+GFX groups respectively.
Compared with control group, the eNOS activity of endothelial cells in OA group was significantly reduced in a dose-dependent manner, and the expression of p-eNOS(1177) were also impaired (0.0854 +/- 0.017 vs. 0.0134 +/- 0.023, P < 0.05). PKC transposition from endochylema to cytomembrane was observed by confocal microscopy. The PKC activity was degraded in endochylema, while enhanced in cell membrane inversely, and the gross activity of PKC increased after all. When GFX, an inhibition of PKC, was added to endothelial cells at the present of OA or not, the impaired eNOS activity were partly improved.
Elevated level of FFA (here is oleic acid) leading to endothelial dysfunction results from a loss of eNOS activity and its serine phosphorylation, and it may be related to activation of PKC.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 03/2010; 41(2):208-11, 225.
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ABSTRACT: The genes encoding glycerol dehydratase were cloned and characterized by genomic DNA from Klebsiella pneumoniae XJPD-Li, and the assigned accession number EF634063 was available from the GenBank database. The DNA sequence analysis showed that the clone included three ORFs (dhaB, dhaC and dhaE, encoding α, β and γ subunit of glycerol dehydratase, respectively). Among three subunits of glycerol dehydratase, amino acid residues H13, S193, N359, E407, and M515 of α subunit, N47, L150, V189 of β subunit are different with what had been reported. Subsequently, the expression vector was constructed and transformed into E. coli BL21, and the colony carried genes of glycerol dehydratase were selected. SDS-PAGE examination showed that the three subunits were well expressed. The specific activity of recombined glycerol dehydratase reached to 0.299 U mg−1, which was about 3 times comparing with that of the wild strain. The research also displayed that both glycerol and O2 could inactive the glycerol dehydratase expressed in E. coli quickly in 10 min. The inactivated glycerol dehydratase could be effectively reactivated under the system as follows: the concentration of ATP, Mg2+ and coenzyme B12 were 50 mM, 10 mM and 3 μM, respectively, when the ratio (W/W) of glycerol dehydratase to reactivation factor was 4:1. The O2-inactivated and glycerol-inactivated dehydratase could be reactivated to 97.3% and 98.9% of initial activity in 10 min in above-mentioned conditions, respectively. The reactivation factor together with ATP was considered as the “ON/OFF” reactivating condition.
Journal of Molecular Catalysis B Enzymatic 56:108-114. · 2.73 Impact Factor
