Heterologous expression, characterization and evolution prediction of a diaphorase from Geobacillus sp. Y4.1MC1.

ß-hydroxybutyric acid is the most sensitive indicator in ketoacidosis detection, and accounts for nearly 78% of the ketone bodies. Diaphorase is commonly used to detect the ß-hydroxybutyric acid in clinical diagnosis. However, the extraction of diaphorase from animal myocardium is complex and low-yield, which is not convenient for large-scale production. In this study, a diaphorase from Geobacillus sp. Y4.1MC1 was efficiently heterologous expressed and purified in E. coli with a yield of 110 mg/L culture. The optimal temperature and pH of this recombinant diaphorase (rDIA) were 55 °C and 6.5, respectively. It was proved that rDIA was a dual acid- and thermo-stable enzyme, and which showed much more accurate detection of ß-hydroxybutyric acid than the commercial enzyme. Additionally, we also investigated the molecular interaction of rDIA with the substrate, and the conformation transition in different pH values by using homology modeling and molecular dynamics simulation. The results showed that 141-161 domain of rDIA played important role in the structure changes and conformations transmission at different pH values. Moreover, it was predicted that F105W, F105R, and M186R mutants were able to improve the binding affinity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were benefit for the stability of rDIA.

Bostrycin inhibits growth of tongue squamous cell carcinoma cells by inducing mitochondrial apoptosis.

BACKGROUND: Bostrycin is a natural pigment product with anthraquinone skeleton, which has excellent inhibitory effect on a variety cancer cells. However, its suppression on tongue squamous cell carcinoma has not been reported. METHODS: We studied the effects of bostrycin on the cell growth and apoptosis of tongue squamous cell carcinoma in vitro, and explored its mechanism. RESULTS: Bostrycin could inhibit the proliferation and migration of tongue squamous cell carcinoma by arresting cell cycle at G2/M phase, and inducing cell apoptosis with mitochondrial membrane potential changes. West-blotting analysis also showed that bostrycin could inhibit the growth of tongue squamous cell carcinoma cells by activating the apoptosis-related signal proteins especially in mitochondrial apoptotic pathway. CONCLUSIONS: Bostrycin can be used as a new anti-tumor candidate drug for further research. Our study provides reference for the potential application of bostrycin in the treatment of the tongue squamous cell carcinoma.

Antimicrobial peptide CAMA-syn expressed in pulmonary epithelium by recombination adenovirus inhibited the growth of intracellular bacteria.

BACKGROUND: Intracellular bacteria, especially Mycobacterium tuberculosis, are important pathogenic microorganisms that endanger human health. Purified and synthesized cecropin A-magainin 2 (CAMA-syn) can exhibit a higher antibacterial activity and lower cytotoxicity. To enhance such antimicrobial potential, it would be desirable to deliver CAMA-syn expressed in lung epithelial cells by an adenovirus vector using gene therapy. METHODS: A549 cells in vitro and lung epithelial cells in vivo were used to express CAMA-syn by transducing recombinant adenovirus Ad-SPC-CAMA/GFP, and the expression of CAMA-syn was determined by a reverse transcriptase-polymerase reaction (RT-PCR) and immunofluorescence. The antimicrobial activity in cells was investigated by colony-forming rate and growth curve. Forty Kunming mice of a Bacillus Calmette-Guerin (BCG) infection animal model were randomly divided into three groups: adenoviruses delivery of Ad-SPC-CAMA/GFP, Ad-CMV-CAMA/GFP and empty-virus Ad-CMV-GFP. The expression of CAMA-syn in mice was confirmed by RT-PCR and immunofluorescence. After tracheal injection of adenoviral vector for 3 days, lungs from the mouse model were extracted and homogenized for detection of colony-forming efficiency. RESULTS: CAMA-syn expressed in lung epithelial cells A549 conferred antimicrobial activity against a series of bacteria, including Salmonella abortusovis and BCG. The results obtained in vivo showed that the colony-forming rate of Ad-SPC-CAMA/GFP (74.54%) and Ad-CMV-CAMA/GFP (62.31%) transduced into mice was significantly lower than that of the control group. CONCLUSIONS: Lung epithelial-specific expression of antimicrobial peptide CAMA-syn mediated by adenovirus suppressed the growth of intracellular bacteria, providing a promising approach for the control of refractory intracellular infection.

Targeting expression of antimicrobial peptide CAMA-Syn by adenovirus vector in macrophages inhibits the growth of intracellular bacteria.

Although purified and synthesized Cecropin A-magainin 2 (CAMA-syn) shows potent antibacterial activity in vitro, its ability to inhibit bacteria within mammal cells mediated by virus vector has not yet been investigated. To enhance its antimicrobial potential and reduce systemic side effects, it would be desirable to deliver CAMA-syn in macrophages by adenovirus vector. In this study,recombinant adenovirus Ad-MSP-CAMA/GFP were used to infect macrophages RAW264.7 cells in vitro and macrophages cells of lungs in vivo and the expression of CAMA-syn was detected by RT-PCR and observation of co-expression of GFP. Antimicrobial activity in cells was evaluated by colony enumeration. The results showed that expression of CAMA-syn in macrophages conferred antimicrobial activity against a series of bacteria, including E. coli and BCG(Bacillus Calmette-Guérin). To establish BCG infection animal model, 40 Kunming mice were randomly divided into the following four groups: adenoviral delivery of Ad-MSP-CAMA/GFP, Ad-CMV-CAMA/GFP, empty-virus Ad-GFP, and control PBS, respectively. The expression of CAMA-syn in mouse was confirmed by real-time PCR and GFP co-expression. In brief, 3 days after injection of adenoviral vector, mice were scarified, different tissues were sectioned and homogenized and colony-forming efficiency by these treated tissues was determined. The colony-forming efficiency of Ad-MSP-CAMA/GFP (78.31%) and Ad-CMV-CAMA/GFP (61.68%) showed significant reduction compared to control groups. No inhibition of bacterial colony was observed from tissues treated by the PBS or empty-virus control. In conclusion, our results demonstrated that macrophages-specific expression of antimicrobial peptide CAMA-syn in macrophages inhibited the growth of intracellular bacteria, providing a promise approach for the control of refractory intracellular infection.