Isolation of a triazophos-degrading strain Klebsiella sp. E6 effectively utilizing triazophos as sole nitrogen source

A triazophos-degrading strain, Klebsiella sp. E6, was isolated by enrichment technology from soil that had been exposed long-term to triazophos. The strain grew well at pH 7.0-8.0 with a broad temperature profile ranging from 32 to 37 degrees C. It could keep good growth on methanol as carbon source and TAP as additional carbon source or nitrogen source. The experiment on the degradation activities of strain E6 showed that it utilized TAP more effectively when TAP was supplied as the sole nitrogen source, as opposed to additional carbon source. The intermediates of triazophos metabolism indicated that degradation occurred through a hydrolysis mechanism, one of the products of which, 1-phenyl-3-hydroxy-1,2,4-triazole, was also mineralized by strain E6.     

Directed evolution and characterization of Escherichia coli glucosamine synthase

Glucosamine synthase (GlmS) converts fructose-6-phosphate to glucosamine-6-phosphate. Overexpression of GlmS in Escherichia coli increased synthesis of glucosamine-6-P, which was dephosphorylated and secreted as glucosamine into the growth medium. The E. coli glmS gene was improved through error-prone polymerase chain reaction (PCR) in order to develop microbial strains for fermentation production of glucosamine. Mutants producing higher levels of glucosamine were identified by a plate cross-feeding assay and confirmed in shake flask cultures. Over 10 mutants were characterized and all showed significantly reduced sensitivity to inhibition by glucosamine-6-phosphate. Ki of mutants ranged from 1.4 to 4.0 mM as compared to 0.56 mM for the wild type enzyme. Product resistance resulted from single mutations (L468P, G471S) and/or combinations of mutations in the sugar isomerase domain. Most overexpressed GlmS protein was found in the form of inclusion bodies. Cell lysate from mutant 2123-72 contained twice as much soluble GlmS protein and enzyme activity as the strain overexpressing the wild type gene. Using the product-resistant mutant, glucosamine production was increased 60-fold.     

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.     

Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms

Human liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserved between the human isozymes (15% identity). The three-dimensional structures of the beef mitochondrial and sheep cytosolic forms have virtually identical three-dimensional structures. Here, we solved the structure of the human mitochondrial enzyme and found it to be identical to the beef enzyme. The first 21 residues are found on the surface of the enzyme and make no contact with other subunits in the tetramer. A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from one isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic-like properties was produced. The other was expressed but was insoluble. It was possible to restore solubility and activity to the chimera that had the first 21 cytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N-terminal end. When residue 19 was changed from tyrosine to a cysteine, the residue found in the mitochondrial form, an active enzyme could be made though the Km for NAD+ was 35 times higher than the native mitochondrial isozyme and the specific activity was reduced by 75%. This residue interacts with residue 203, a nonconserved, nonactive site residue. A mutation of residue 18, which also interacts with 203, restored solubility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a soluble or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme.     

海马神经元的瞬间外向钾电流

瞬间外向钾电流(IA)具有快速激活和失活等特征,是动作电位复极化早期外向钾离子电流的主要成分,广泛分布在海马神经元,树突处尤为突出.该电流通过减慢去极化速度和延缓动作电位的产生等作用,调节突触的输入和动作电位的反向传播,从而在信号整合及突触可塑性等过程中扮演重要角色.很多人类疾病,如癫痫性疾病等,和海马神经元的IA电流有关.     

Transient expression of heterologous model gene in plants using <Emphasis Type="Italic">Potato virus</Emphasis><Emphasis Type="Italic">X</Emphasis>-based vector

To optimize the efficiency of expression of foreign proteins using Potato virus X (PVX) -- based vector, the gene for the coat protein (CP) of other virus (Potato virus A, PVA) was cloned into the vector, propagated in E. coli and subsequently inoculated or agroinfected into the host plants. Host range studies showed that the best host plant is N. benthamiana. By means of RT PCR the presence and the stability of the construct were tested. Both ELISA and Western blot analysis were applicable for expressed protein detection. Expression level of PVA CP achieved approximately 5--10 per mille of total soluble proteins. The results demonstrated that agroinfection is the most suitable method for the propagation of our model gene using PVX--based vectors.