Insight into enzyme-catalyzed aziridine formation mechanism in ficellomycin biosynthesis.

Ficellomycin is an aziridine-containing antibiotic, produced by Streptomyces ficellus. Based on the newly identified ficellomycin gene cluster and the assigned functions of its genes, a possible pathway for aziridine ring formation in ficellomycin was proposed, which is a complex process involving at least 3 enzymatic steps. To obtain support for the proposed mechanism, the targeted genes encoding sulfate adenylyltransferase, adenylsulfate kinase, and a putative sulfotransferase were respectively disrupted and the subsequent analysis of their fermentation products revealed that all the three genes were involved in aziridine formation. To further confirm the mechanism, the key gene encoding a putative sulfotransferase was over expressed in Escherichia coli Rosseta (DE3). Enzyme assays indicated that the expressed sulfotransferase could specifically transfer a sulfo group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto the hydroxyl group of (R)-(-)-2-pyrrolidinemethanol. This introduces a good leaving group in the form of the sulfated hydroxyl moiety, which is then converted into an aziridine ring through an intramolecular nucleophilic attack by the adjacent secondary amine. The sulfation/intramolecular cyclization reaction sequence maybe a general strategy for aziridine biosynthesis in microorganisms. Discovery of this mechanism revealed an enzyme-catalyzed route for the synthesis of aziridine-containing reagents and provided an important insight into the functional diversity of sulfotransferases.

The immunomodulatory effect of docosahexaenoic acid (DHA) on the RAW264.7 cells by modification of the membrane structure and function.

BACKGROUND: DHA can regulate various physiological functions of cells. Our group has clarified the immunomodulatory activity and molecular mechanism of DHA on RAW264.7 cells. However, the effect of DHA on the membrane fatty acid environment and the activation of signaling pathways on the cell membrane is still not clear. METHODS: In this study, we evaluated the fluidity, the potential and the fatty acid, phospholipid and protein composition of the RAW264.7 cell membrane by DHA treatment. RESULTS: The fluidity of the RAW264.7 cell membrane was increased by DHA treatment. The results of membrane potential analysis suggested that DHA (2.4 µM) significantly reduced the surface potential of the cell membrane, which might influence the fluidity of cell membranes. In addition, the fatty acids and phospholipids were measured and the results indicated that DHA treatment (2.4 µM) altered the lipid environment and the composition of phospholipids on the RAW264.7 cell membrane. Then the LC-MS/MS-based label free quantitative proteomics approach was applied to identify a total of 86 differential proteins in the 2.4 µM DHA and control groups (>2.0-fold change or <0.5-fold change in protein expression); these proteins are most frequently related to the cell response to stimuli and the response to stress. These results suggested that DHA could alter the fluidity, the potential, the fatty acid and phospholipid composition of the RAW264.7 cell membrane, eventually affecting the proteins of the cell membrane, especially the changes in Siglec 1, iNOS, GPR120, Ras and MEK expressions (validated by western blot analysis), which are likely associated with the activation of the intracellular signaling pathway in RAW264.7 cells by DHA treatment.

Divalent oseltamivir analogues as potent influenza neuraminidase inhibitors.

A panel of divalent oseltamivir and guanidino oseltamivir analogues with esterification on the carboxyl acid group as potent inhibitors of influenza virus neuraminidase was prepared via click reaction. The primary structure activity relationship study demonstrated that appropriate distance between two oseltamivir monomers around 30 Šcan crosslink two adjacent neuraminidase tetramers on the virion surface and result in highly effective NA inhibitors against three strains of influenza virus and H7N9 virus like particle. This strategy also provides a basis for the multivalent modification on oseltamivir.

Enhancement of bioelectricity generation via heterologous expression of IrrE in Pseudomonas aeruginosa-inoculated MFCs.

Low electricity power output (EPT) is still the main bottleneck limited the industrial application of microbial fuel cells (MFCs). Herein, EPT enhancement by introducing an exogenous global regulator IrrE derived from Deinococcus radiodurans into electrochemically active bacteria (EAB) was explored using Pseudomonas aeruginosa PAO1 as a model strain, achieving a power density 71% higher than that of the control strain. Moreover, IrrE-expressing strain exhibited a remarkable increase in the total amount of electron shuttles (majorly phenazines compounds) and a little decrease in internal resistance, which should underlie the enhancement in extracellular electron transfer (EET) efficiency and EPT. Strikingly, IrrE significantly affected substrate utilization profiling, improved cell growth characterization and cell tolerance to various stresses. Further quantitative RT-PCR analysis revealed that IrrE led to many differentially expressed genes, which were responsible for phenazines core biosynthesis, biofilm formation, QS systems, transcriptional regulation, glucose metabolism and general stress response. The results substantiated that targeting cellular regulatory network by the introduction of exogenous global regulators could be a facile and promising approach for the enhancement of bioelectricity generation and cell multiple phenotypes, and thus would be of great potential application in the practical MFCs.

Identification and characterization of the ficellomycin biosynthesis gene cluster from Streptomyces ficellus.

Ficellomycin is a peptide-like antibiotic which exhibits potent in vitro activity against Penicillium oxalicum and Staphylococcus aureus, even against strains resistant to most clinically used antibiotics. The gene cluster responsible for ficellomycin biosynthesis was cloned from Streptomyces ficellus and sequenced. It was found to contain 26 ORFs and is located within 30 kb of contiguous DNA. Targeted disruption of the encoding genes revealed that most were involved in the functional section of ficellomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of ficellomycin 2-[4-guanidyl-1-azabicyclo[3.1.0]hexan-2-yl] glycine (2-GAHG). Within the 2-GAHG synthesis pathway, a sulfate adenylyltransferase appears to be involved in the synthesis of the pharmaceutically important 1-azabicyclo[3.1.0]hexane ring moiety, which has been reported to cause DNA cross-linking or impairment of semiconservative DNA replication.

Access to 8-Azachromones via Activation of C-H in N-Oxides.

A concise and practical synthetic method has been developed for 8-azachromones, including 8-azaflavones, which have emerged as a promising class of compounds. Using commercially available nicotinates as the starting material, 8-azachromones were obtained in only three steps. The key intramolecular O-arylation reaction was achieved by nucleophilic attack of enolates to C2 of N-oxides under PyBrop or Ac2O activation conditions. These studies provide the basis for the access to 8-azachromones, enabling future work including the discovery and development of novel chromonoid drugs or other functional materials.

A Shortcut to the Optimization of Cellulase Production Using the Mutant Trichoderma reesei YC-108.

Trichoderma reesei YC-108, a strain isolated by a kind of newly invented plate was found to over produce cellulase and it was then used as a cellulase producer. To get the maximum amount of cellulase, the combination of the medium ingredients, which has a profound influence on metabolic pathway was optimized using response surface methodology. The optimum composition was found to be 24.63 g/L wheat bran, 30.78 g/L avicel, and 19.16 g/L soya-bean cake powder. By using the optimized medium, the filter paper activity (FPA) increased nearly five times to 15.82 IU/mL in a 30 L stirred fermenter, carboxymethyl cellulase activity (CMCase) was increased from 83.02 to 628.05 IU/mL and the CMCase/FPA ratio was nearly doubled compared with the parent strain at initial medium.

[A multi-step distillation method for sample preparation in gas chromatographic analysis].

A sample pretreatment device based on multi-step distillation principle was constructed and evaluated. Trace components in the sample were extracted, separated and enriched at the cold receptor of the molecular diffusion distillation device. The cold receptor was designed as an exchangeable part to avoid the cross interferences from one sample to another one. A model sample containing four kinds of organophosphorus pesticides in ethyl actate was tested in the device, and the distillated and enriched solution was analyzed by a gas chromatograph. It was proven that the interferential components to gas chromatography such as protein, lipid and pigment could be removed effectively, and the enrichment factor for trace target components was more than 150 times. The recoveries of the method were from 90% to 117%, and the relative standard deviations (RSDs) from 1.3% to 10%. The limit of detection (LOD) of this method was 1 ng/mL without further concentration. The reused ratio of the organic reagents was more than 95%. Practical application was performed by using a spiked tea sample. The device can treat more than 100 g of samples with a boiling point range of 120 - 600 degrees C.