Objective:To explore the role of matrix metalloproteinase 12 (MMP12) in airway macrophages and pulmonary neu-rogenic substance P ( SP ) in the pathogenesis of asthma by analyzing their relationship in different categories of asthmatic patients.Methods:Twenty patients of asthma remission phase ( remission asthma group ) , twenty ones of mild acute exacerbation asthma (mild asthma group) and twenty healthy adults (normal control group) were included,respectively.After lung function was measured,the numbers of macrophage in induced sputum were counted.The expression levels of MMP12 mRNA and protein in sputum macrophages were detected by quantitative reverse transcription polymerase chain reaction and Western blot.The concentration of sputum SP was assayed by enzyme immunometric assay.Results: ( 1 ) Compared with the subjects in normal control group, forced expiratory volume in 1 second%predicted ( FEV1 ) and forced expiratory flow rates at 50% of the forced vital capacity % predicted ( FEF50 ) were much lower and the numbers of sputum macrophages were much higher in the patients in different asthmatic groups.Compared with the patients in remission asthma group,FEV1 and FEF50 were much lower in the ones in mild asthma group.(2) MMP12 expressions in the macrophages and the concentrations of SP in sputum were significantly increased in the patients in different asthmatic groups compared with those in normal control group;Furthermore,MMP12 and SP in mild asthma group were much higher than in remission asthma.(3) In all patients from different asthmatic groups,mRNA expressions of MMP12 in the macrophages were positively correlated with the levels of sputum SP or the numbers of sputum macrophages,whereas negative correlations between mRNA expressions of MMP 12 and FEV 1 or FEF50 were observed.Conclusion: The regulatory imbalance of macrophages′MMP12 and pulmonary neurogic SP may participate in the pathogenesis of asthma and become the potential targets for asthma therapy.

Auxotrophic strains of N1-37 (Phe-) and N2-27 (His-), screened from mutations of Paenibacillus polymyxa JSa-9 previously, were used as the parent strains to screen high-producing LI-F antibacterial lipopeptide fusion strain through protoplast fusion with polyethylene glycol as a promote agent. Fusion strain F5-15 was obtained. Then the product of LI-F antibacterial lipopeptide was quantified by HPLC, and the difference of expression of the key genes of lipopeptide synthase between wild strain JSa-9 and the fusion strain was analyzed by real-time PCR. LI-F antibacterial lipopeptide yield of the fusion strain F5-15 was 3.1-fold of the original strain JSa9's, and the expression levels of the target genes were 10.48, 2.48, 2.1 and 11.8 fold of the initial strain JSa-9, respectively.
Anti-Bacterial Agents ; biosynthesis ; Chromatography, High Pressure Liquid ; Lipopeptides ; biosynthesis ; Paenibacillus ; metabolism ; Protoplasts ; metabolism ; Real-Time Polymerase Chain Reaction

We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 degrees C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4 x 10(4) U/mg. The optimum reaction temperature and pH for ana-LOX were 45 degrees C and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+ Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.
Anabaena ; enzymology ; genetics ; Catalytic Domain ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; Lipoxygenase ; chemistry ; genetics ; Metals, Heavy ; chemistry ; Mutagenesis, Site-Directed ; Recombinant Proteins ; chemistry ; genetics

Lipases are important biocatalysts that are widely used in food processing and bio-diesel production. However, organic solvents could inactivate some lipases during applications. Therefore, the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application. In this work, we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 (GenBank Accession No. FJ979867), by PCR, which encoded a 31.6 kD polypeptide of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus (GenBank Accession No. AP008934). We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 (DE3), and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25 degrees C for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Lipase ; biosynthesis ; genetics ; Molecular Sequence Data ; Organic Chemicals ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; Solvents ; chemistry ; Staphylococcus saprophyticus ; enzymology

With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
Antifibrinolytic Agents ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fibrinolytic Agents ; metabolism ; Genetic Vectors ; genetics ; Paenibacillus ; chemistry ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

Clinical microbiology examination technology experiment is an important part of clinical microbiology examination technology teaching. In the experimental teaching of clinical microbiology examination technology, the virtual simulation technology was combined with traditional teaching to give full play to the advantages of the virtual experimental platform. As to experimental projects that couldn't be carried out in traditional teaching and some important experimental projects, students could learn on the virtual experimental platform, and after learning, they would participate in the corresponding assessment. The perfect combination of the two can solve the problem of high experimental cost and limited experimental content in the current experimental class, make up for the shortcomings of traditional teaching, realize the sharing of teaching resources. Besides, it can strengthen the students' experimental operation skills and enhance the interest of learning for cultivation of application-oriented medical talents.

For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803. The pFQ16, an RSF1010-derived broad host range shuttle vector, was constructed for conjugative transfer of genes to various cyanobacteria strains. All the four platforms constructed here applied the rbc (encodes Ribulose-1, 5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription. Besides, a "Shine-Dalgarno -AUG" fusion translation strategy was used to keep the high protein translation efficiency. Using lacZ as a reporter gene, the expression efficiency of pFQ20 was evaluated and showed a strong beta-galactosidase expression (109 Miller). Furthermore, the platform pFQ20 was used to express the E. coli tesA' gene and showed significant protein bands through the Western Blot test. The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future.
Genetic Vectors ; genetics ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Palmitoyl-CoA Hydrolase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Synechocystis ; genetics ; metabolism ; beta-Galactosidase ; biosynthesis ; genetics