Highly efficient production of diverse rare ginsenosides using combinatorial biotechnology

The rare ginsenosides are recognized as the functionalized molecules after the oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by methyl jasmonate-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six β-glycosidases and their combination with yields ranging from 5.54 to 32.66 mg L⁻¹. The yield of Rh2 was furthermore increased by 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature conditions, with the highest yield reaching 51.17 mg L⁻¹ (17.06% of protopanaxadiol-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.     

DcR3在甲状腺乳头状癌中的表达及临床意义

应用RT-PCR、Westem blot、免疫组化分别检测甲状腺乳头状癌组织与癌旁正常甲状腺组织标本中DcR3mRNA及蛋白的表达情况,探讨DcR3在甲状腺乳头状癌组织中的表达及,临床意义。RT-PCR检测显示,甲状腺乳头状癌中DcR3 mRNA的表达明显高于正常甲状腺组织（P〈0．05）：Western blot提示,DcR3蛋白在甲状腺乳头状癌中表达比正常甲状腺组织高（P〈0．05）;免疫组化显示,DcR3蛋白在甲状腺乳头状癌中高表达（P〈0．05）。DcR3mRNA及蛋白质在甲状腺乳头状癌及正常甲状腺组织间的表达差异有统计学意义（P〈0．05）。DcR3基因及蛋白在甲状腺乳头状癌中高表达,提示DcR3可能促进了甲状腺乳头状癌的发生发展。     

Identification of DNA-binding proteins by incorporating evolutionary information into pseudo amino acid composition via the top-n-gram approach

DNA-binding proteins are crucial for various cellular processes and hence have become an important target for both basic research and drug development. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to establish an automated method for rapidly and accurately identifying DNA-binding proteins based on their sequence information alone. Owing to the fact that all biological species have developed beginning from a very limited number of ancestral species, it is important to take into account the evolutionary information in developing such a high-throughput tool. In view of this, a new predictor was proposed by incorporating the evolutionary information into the general form of pseudo amino acid composition via the top-n-gram approach. It was observed by comparing the new predictor with the existing methods via both jackknife test and independent data-set test that the new predictor outperformed its counterparts. It is anticipated that the new predictor may become a useful vehicle for identifying DNA-binding proteins. It has not escaped our notice that the novel approach to extract evolutionary information into the formulation of statistical samples can be used to identify many other protein attributes as well.     

Toxicity of Binary Mixtures of Enantiomers in Chiral Organophosphorus Insecticides: The Significance of Joint Effects Between Enantiomers

The existence of enantiomer‐enriched mixtures of chiral pesticides in the environment is overwhelmingly positive. However, interactions between enantiomers have not been considered so far in risk assessments. Here, we chose three organophosphorus pesticides as representative chiral pesticides to investigate the possible interaction mode between each pair of enantiomers both in in vivo and in vitro. Data show that the enantiomers of methamidophos and profenofos have a simple additive effect, <zaq;1> whereas fensulfothion acts as an antagonist in AChE‐inhibition model. In contrast, enantiomers of methamidophos and fensulfothion had an additive effect in an acute toxicity test against Daphnia magna. A synergistic effect was observed in the joint toxicity of the profenofos enantiomers. The ability for enantiospecific biodegradation in the in vivo model contributed to the different interaction observed between the in vitro and in vivo models. Moreover, binding affinities were suspected as another reason for the different mode of action of mixture enantiomers. Our study recommends using a joint research model to treat chiral compounds in the real environment. Chirality 25:787–792, 2013. © 2013 Wiley Periodicals, Inc.     

Sequence characterization and computational analysis of the non-ribosomal peptide synthetases controlling biosynthesis of lipopeptides, fengycins and bacillomycin D, from Bacillus amyloliquefaciens Q-426

Lipopeptides secreted by bacteria attract interest because of their uses in biomedicine, biotechnology and food technology; however, harnessing their megasynthases (non-ribosomal peptide synthetases, NRPSs) has met with some difficulties in heterologous expression and crystallization. Here, we used similarity and phylogenetic analysis of NRPS sequences, including the fengycin and iturin family synthetases from Bacillus spp., and have developed a novel approach for delineating the length and boundaries of NRPS domains from Bacillus amyloliquefaciens strain Q-426. The sequences were further characterized (including specific residues and conserved motifs) that gave insight into the basis of the substrate specificity. Data from the prediction of the NRPS domains, obtained by the self-optimized prediction method with Alignment program, showed they are all structurally unstable, making it difficult to determine their crystal structures.     

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.     

EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

Activation of EP2 receptors by prostaglandin E2 (PGE₂) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gα_s and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE₂ had no effect on expression of CXCL10, TGF-β1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1β but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE₂ or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.     

Phenazine-1-carboxylic acid production in a chromosomally non-scar triple-deleted mutant Pseudomonas aeruginosa using statistical experimental designs to optimize yield

We constructed a non-scar triple-deleted mutant Pseudomonas aeruginosa to improve phenazine-1-carboxylic acid (PCA) yield and then optimized the culture conditions for PCA production. Using a non-scar deletion strategy, the 5′-untranslated region of the phz1 gene cluster and two genes, phzM and phzS, were knocked out of the P. aeruginosa strain M18 genome. The potential ability for high-yield PCA production in this triple-deleted mutant M18MSU1 was successfully realized by using statistical experimental designs. A 2^5–1 fractional factorial design was used to show that the three culture components of soybean meal, corn steep liquor and ethanol had the most significant effect on PCA production. Using a central composite design, the concentration of the three components was optimized. The maximum PCA production was predicted to be 4,725.1 mg/L. With the optimal medium containing soybean meal 74.25 g/L, corn steep liquor 13.01 g/L and ethanol 21.84 ml/L, a PCA production of 4,771.2 mg/L was obtained in the validation experiments, which was nearly twofold of that before optimization and tenfold of that in the wild-type strain. This non-scar triple-deleted mutant M18MSU1 may be a suitable strain for industrial production of this biologically synthesized fungicide due to its high PCA production, presumed safety, thermal adaptability and cost-effectiveness.