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

Objective To look for more serum biomarkers supporting the diagnosis of multiple system atrophy ( MSA) and providing more evidence for early treatment.Methods All patients and healthy controls were enrolled from January 2011 to March 2015 in the First Affiliated Hospital of Zhengzhou University.Demographic features and biochemical examination results were collected.The t test was used to compare the lipid levels between MSA patients and controls.LSD-t test was used to compare the lipid levels among subtypes of MSA patients.Multivariate Logistic regression analysis was conducted to analyze the influencing factors.The relevance between lipid levels and onset age, disease duration and Hoehn & Yahr stage was calculated by Spearman correlation coefficients.Results Participants included 195 MSA patients and 195 age-and gender-matched controls with no neurological diseases.The levels of total cholesterol ((4.33 ±0.90) mmol/L), triglyceride ((1.27 ±0.71) mmol/L), low-density lipoprotein (LDL;(2.70 ±0.76) mmol/L) were significantly lower in patients than in controls ((4.52 ±0.85), (1.47 ± 0.86), (2.85 ±0.71) mmol/L ,t=2.056,2.528 and 2.149 respectively, all P<0.05).The levels of total cholesterol ((4.28 ±0.96) mmol/L) and triglyceride ((1.20 ±0.64) mmol/L) were significantly lower in MSA-P patients than in control group ((4.52 ±0.85), (1.47 ±0.86) mmol/L;LSD-t=1.983, 2.566, both P<0.05).After adjusting for age, gender and histories, the odds ratio ( OR) was 0.31 (95%CI 0.15-0.65, P =0.002 ) for MSA patients in the highest quartile of triglyceride and 0.38 (95%CI 0.17 -0.83,P=0.016) for those in the highest quartile of high-density lipoprotein (HDL), compared with the lowest quartiles.And HDL level was in a significantly positive correlation with onset age (r=0.15, P=0.039).Conclusion Our data suggest that triglyceride and HDL may be associated with the prevalence of MSA, and the lower levels of HDL, the earlier onset of MSA.

Objective:To investigate the effect of tumor necrosis factor-α (TNF-α) on the differentiation of orbital fibroblasts (OF) in thyroid-associated ophthalmopathy (TAO) and its regulation mechanism.Methods:Six patients (six eyes) diagnosed with TAO were collected in Tianjin Medical University Eye Hospital from December 2019 to August 2020.Adipose connective tissue was collected during the orbital decompression surgery.OF was isolated and cultured using the tissue block method and vimentin was identified by immunofluorescence.Lipogenic differentiation of OF was induced and identified by oil red O staining.Complete culture medium containing 0, 0.1, 1.0 and 10.0 μg/L TNF-α was used to induce the dedifferentiation of orbital mature adipocytes.Primary culturing cells, 14-day differentiation cells and 20-day dedifferentiation cells were collected.The relative mRNA expression levels of peroxisomal proliferation-activated receptor (PPARγ), extracellular regulatory protein kinase1 (ERK1), ERK2 and fat-coated protein1 (perilipin1) were detected by real-time fluorescent quantitative PCR.The relative protein expression levels of PPARγ, P-ERK1/2 and perilipin1 were detected by Western blot.Results:Human TAO-derived OF were successfully cultured in vitro, spindle-shaped or polygonal, tightly arranged in a vortex pattern, and immunofluorescence staining for vimentin was positive.After OF adipogenic differentiation, lipid droplet structures could be seen in the cytoplasm of some cells, and the stained lipid droplet structures in the cytoplasm could be seen by oil red O staining, which confirmed that the cells obtained after differentiation were adipocytes.Dedifferentiation of adipocytes was induced by 0.1, 1.0, and 10.0 μg/L TNF-α.With the extension of induction time, the volume of lipid droplets in the cytoplasm and the number of cells containing lipid droplets decreased.Lipid droplets disappeared in the cytoplasm on the 20th day of dedifferentiation, and the cells became long spindle-shaped and tightly arranged, dedifferentiated into fibroblast-like cells.Real-time fluorescence quantitative PCR detection results showed that the relative expression levels of PPARγ, ERK1, ERK2 and perilipin1 mRNA in 14-day differentiation group were 4.26±0.09, 2.01±0.09, 3.23±0.10 and 8.69±0.33, respectively, which were significantly higher than 1.00±0.09, 1.05±0.19, 1.00±0.10 and 1.05±0.07 in primary group, and 1.06±0.03, 1.15±0.11 and 6.27±0.09 in 20-day dedifferentiation group (all at P<0.05). Western blot analysis showed that the expression levels of PPARγ, ERK1/2 and perilipin1 proteins in 14-day differentiation group were 1.07±0.03, 1.00±0.03 and 1.13±0.02, respectively, which were significantly higher than 0.37±0.02, 0.29±0.02 and 0.00±0.00 in primary group, and 0.20±0.02, 0.38±0.06 and 0.00±0.00 in 20-day dedifferentiation group (all at P<0.001). Conclusions:TNF-α has a dedifferentiation effect on TAO orbital adipocytes.The mechanism may be related to the downregulation of ERK1/2-PPARγ-perilipin1 signaling pathway.

[Abstract] Objective: To explore the mechanism of glucose transport protein-1(Glut-1) promoting the migration of osteosarcoma MG63 cells through Wnt/β-catenin pathway. Methods: RNA interference recombinant adenovirus targeting Glut-1 gene (Ad-Glut-siRNA) and control recombinant adenovirus (Ad-GFP) were constructed and transfected into MG63 cells to silence Glut-1 gene expression. The cell migration ability of Blank group, Ad-AFP group, Ad-Glut-siRNA group and AZD2858 (inhibitor of GSK-3) group were detected by Transwell chamber migration assay. Immunofluorescence assay was used to detect the expression of E-cadherin and vimentin in each group and the nuclear translocation of β-catenin. The expression of MMP-2 and MMP-9 in each group and FZD7, β-catenin, Dsh protein in Blank group, Ad-AFP group, Ad-Glut-siRNA group were detected by Western blotting assay. Results: The migration ability of MG63 cells was significantly decreased (P<0.05) after Glut-1 gene silencing, which was restored afterAZD2858 treatment (P <0.05). Compared with Blank group and Ad-GFP group, the E-cadherin level in MG63 cells in Ad-Glut-siRNA group was significantly increased (P<0.05), while the expressions of vimentin, MMP-2, MMP-9, FZD7, β-catenin and Dsh protein were significantly reduced (all P<0.05). Compared with Ad-Glut-siRNA group, E-cadherin expression of AZD2858 group was significantly reduced, while the expressions of vimentin, MMP-2, MMP-9 were significantly up-regulated (P<0.05). Conclusion: The high expression of Glut-1 gene is closely related to the invasion and metastasis of MG63 cells. The possible mechanism is that the high expression of Glut-1 leads to the activation of Wnt/β-catenin pathway, which leads to the decrease of EMT-related protein E-cadherin, and the increase of vimentin and MMP-2, MMP-9, and further promotes the migration of MG63 cells.