AIM: To observe the protein expression of vascular endothelial growth factor 1 (VEGF-1) in pulmonary arterial endothelial cells and VEGF-1 gene expression in lung tissue in rats with hypoxia-induced pulmonary hypertension and treated with heparin. METHODS: Twenty four male adult SD rats were randomly divided into three groups (8 rats each): a control group (group A), a group with hypoxia for 4 weeks (group B) and a group with hypoxia for 4 weeks and injected with heparin to abdominal cavity simultaneously (group C). Mean pulmonary arterial pressure (mPAP), right ventricle hypertrophy index (RVHI) and vessel morphometry were measured. The morphology of pulmonary artery was observed by HE staining. The expression of VEGF-1 protein in pulmonary arterial endothelial cells was determined by immunohistochemistry. The level of VEGF-1 mRNA in lung tissue was measured by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: mPAP, RVHI, pulmonary artery remodeling parameters, VEGF-1 protein expression in pulmonary arterial endothelial cells and VEGF-1 gene expression in lung tissue of the three groups from high to low were group B, group C and group A. It was statistically significant when compared between either two groups of the three (P<0.01). Linear correlation analysis showed that VEGF-1 protein was positively correlated with pulmonary artery remodeling parameters (r=0.974, P<0.01), and VEGF-1 mRNA was positively correlated with VEGF-1 protein (VEGF 120 mRNA, r=0.919, P<0.01; VEGF164 mRNA, r=0.896, P<0.01). CONCLUSION: Heparin may down-regulate the expression of VEGF-1 at the levels of transcription and translation, resulting in the inhibitory effect on rats with hypoxia-induced pulmonary hypertension.

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.
Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Fungal Proteins ; genetics ; metabolism ; Glycosylation ; Hexosyltransferases ; genetics ; metabolism ; Molecular Sequence Data ; Molecular Weight ; Pichia ; Sucrose ; metabolism ; Trisaccharides ; metabolism

In this study, we investigated the mechanism of transformation by Bacillus subtilis strain 168 by proteomic analysis. B. subtilis strain 168 was able to stereoselectively transform cis-propenylphosphonic acid (cPPA) to fosfomycin. The maximal fosfomycin production was 816.6 microg/mL after two days cultivation, with a conversion rate of 36.05%. We separated the whole cellular proteins by two-dimensional gel electrophoresis (2-DE) method, and 562 protein spots were detected in the presence of cPPA in the medium, while 527 protein spots were detected in the absence of cPPA. Of them, 98 differentially expressed protein spots were found. Among them, 52 proteins were up-regulated whereas 20 were down-regulated in the presence of cPPA in the medium, and 26 induced at the presence of cPPA. The differentially expressed proteins were analyzed by combined MS and MS/MS methods. Eighty protein spots, including 45 up-regulated proteins, 17 down-regulated proteins, and 18 induced by cPPA were identified. Based on the results of proteomic analysis, we postulated two steps of transformation: in the first step, cPPA was hydrated to 2-hydroxypropylphosphonic acid; in the second step, 2-hydroxypropylphosphonic acid was transformed to fosfomycin via a dehydrogenation reaction.
Bacillus subtilis ; genetics ; growth & development ; metabolism ; Bacterial Proteins ; metabolism ; Biotransformation ; Fosfomycin ; metabolism ; Organophosphorus Compounds ; metabolism ; Proteome ; metabolism ; Proteomics

We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.
Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Mutation ; Paenibacillus ; enzymology ; Recombinant Proteins ; genetics ; gamma-Cyclodextrins ; metabolism