Larvicidal activity of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or B.t.i toxin Cyt2Ba against mosquito larvae and formulations for enhancing the effects / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the larvicidal effects of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or Bacillus thuringiensis subsp israelensis (B.t.i) toxin Cyt2Ba against the second instar larvae of Culex pipiensquinquefasciatus and Aedes albopictus and compare different formulations for their larvicidal effects.</p><p><b>METHODS</b>The AaIT- or Cyt2Ba-coding sequences were cloned into pET28a(+) and the recombinant plasmids were transformed into E. coli BL21(DE3). After induction with IPTG, the recombinant proteins expressed by the recombinant E. coli were detected and identified by SDS-PAGE and Western blotting, respectively. The larvicidal activity of the bacterial suspension was tested at different concentrations against mosquitoes. The effective engineered bacteria were prepared into dry powder with different formulations, and their larvicidal activity was tested.</p><p><b>RESULTS</b>AaIT and Cyt2Ba proteins were successfully expressed in E. coli. The recombinant AaIT protein showed no virulence to the mosquito larvae. The suspension of the recombinant E. coli expressing Cyt2Ba protein exhibited a stronger killing effect on Aedes albopictus larvae than on Culex pipiens quinquefasciatus larvae at 48 h (P<0.001) with LCof 3.00×10cells/mL and 1.25×10cells/mL, respectively. The dry powder of the engineered bacteria formulated with yeast extract, wheat flour or white pepper powder at the mass ratio of 1:1 showed the strongest killing effect on mosquito larvae (P=0.044), and the formulation with white pepper powder produced a stronger killing effect than formulations with yeast extract or wheat flour (P=0.002).</p><p><b>CONCLUSION</b>The B.t.i Cyt2Ba protein expressed in E. coli BL21(DE3) shows a good larvicidal activity against mosquitoes, and appropriate formulations of the engineered bacteria can enhance its efficiency in mosquito control.</p>

Hydrogen dioxide increases the expression of vascular endothelial growth factor in human bronchiolar epithelium cell via a PI3K-dependent pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of hydrogen dioxide (H(2)O(2)) on the expression of vascular endothelial growth factor (VEGF) in human bronchiolar epithelial (HBE) cells.</p><p><b>METHODS</b>MTT assay was used to assess HBE cell viability after exposure to different concentrations of H(2)O(2). VEGF/beta-actin gene fragments were amplified simultaneously by RT-PCR from the total HBE cell RNA, and VEGF protein expression in the cells was detected using ELISA.</p><p><b>RESULTS</b>The exposure to 200 micromol/L H(2)O(2) did not obviously affected the cell viability. Compared with those in the control cell, VEGF165/beta-actin and VEGF189/beta-actin ratios were significantly increased in the cells after treatment with 50, 200, and 600 micromol/L H(2)O(2) (P<0.05). The protein expression of VEGF significantly increased after 50 micromol/L H(2)O(2) treatment (P<0.05), but significantly decreased with pretreatment with the PI3K inhibitor Ly294002 (P>0.05).</p><p><b>CONCLUSION</b>Oxidative stress increases the expression of VEGF via a PI3K-dependent pathway in human bronchiolar epithelial cells, which may play an important role in the onset and maintenance of chronic inflammation in asthma.</p>

Antibacterial activity of recombinant thanatin expressed by E.coli ER2566 / 南方医科大学学报

<p><b>OBJECTIVE</b>To express antibacterial peptide thanatin in the prokaryotic expression system and test its antibacterial activity.</p><p><b>METHODS</b>The DNA sequence coding for the 21 peptides of thanatin was synthesized using the preferred genetic codes of E. coli, cloned into pTYB11 plasmid, and transformed into E.coli ER2566. The expression of thanatin fused with intein was induced by IPTG in E.coli, and intein-thanatin specifically bound to the column through intein tag was cleaved overnight at 4 degrees celsius; in DTT/cysteine buffer.</p><p><b>RESULTS</b>The cleaved thanatin was eluted with a protein concentration of 245 microg/ml in the first 4 ml. The purified thanatin had showed strong antibacterial activities against G- bacteria such as Shigella flexneri, Klebsiella pneumoniae, Shigella snnei, Escherichia coli O157, toxin producing Escherichia coli, Pseudomonas aeruginosa, and fungi such as Candida albicans, with especial potency in killing drug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and extended-spectrum beta-lactamases (ESBL)-producing E.coli. Eighty strains of drug-resistant (ESBL-producing) and 30 strains of sensitive E. coli were used for anti-bacterial assay, and no significant differences in the antibacterial activity of thanatin were found between the sensitive and drug-resistant E. coli (P>0.05).</p><p><b>CONCLUSION</b>The recombinant thanatin obtained shows strong antibacterial activity against drug-resistant and sensitive bacteria, and can be a potential substitute for routine antibiotics in the treatment of G- bacterial infections.</p>

Toluene diisocyanate increases vascular endothelial growth factor expression in human bronchial epithelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of toluene diisocyanate (TDI) on the expression of vascular endothelial growth factor (VEGF) in human bronchial epithelial (HBE) cells.</p><p><b>METHODS</b>TDI-human serum albumin (TDI- HSA) conjugate was prepared using a modified Son's method. MTT assay was used to examine the viability of HBE135-E6E7 cells cultured in serum-free medium after treatment with HSA or TDI-HSA at different concentrations. VEGF mRNA expression of the HBE cells treated with HSA or TDI-HSA at 10, 20, 30 and 40 microg/ ml, respectively, was detected using semi-quantitative RT-PCR.</p><p><b>RESULTS</b>Treatment with 40 microg/ml HSA and 40 microg/ml TDI-HSA did not result in significant changes in the viability of HBE135-E6E7 cells. RT-PCR revealed the constitutive expression of two VEGF isoforms, namely VEGF189 and VEGF165, in cultured HBE135-E6E7 cells. After exposure to TDI-HSA at the different concentrations (except for 10 microg/ml), a significant increase occurred in both VEGF189 and VEGF165 mRNA expressions in HBE135-E6E7 cells as compared with the expressions in the control group and the HSA-treated cells (P<0.05), and significant dose dependence was noted in the effect of TDI-HSA (P<0.05). No significant difference was found in the expressions between the control cells and the HAS-treated cells (P>0.05).</p><p><b>CONCLUSION</b>TDI induces significant increase in VEGF expression in HBE cells, and VEGF overexpression may play an important role in the pathogenesis of TDI-induced asthma.</p>

Value of the hypointensive internal septation on MR for the differentiation between benign and malignant breast lesions / 中华放射学杂志

Objective To investigate the diagnostic value of internal septation for differentiating benign from malignant breast lesions.Methods A total of 26 patients were included in the study,in which 12 patients had 20 lesions of breast carcinoma and 14 patients had 25 lesions of fibroadenoma diagnosed either pathologically or clinically.The differential diagnoistic value of the hypointensive internal septation was analyzed.Results The signal intensity of fibroadenomas and malignant lesions on T_2-weighted fat- suppressed images could be classified as iso- to hyper- intensity,hypointensity and mixed intensity. According to the signal intensity classification,there were 5,11 and 4 cases in patients with breast carcinoma respectively,while 11,10,4 cases in patients with fibroadenoma respectively.There was no statistical difference in the distribution between the two patient groups(?~2=1.764,P=0.414).The shape of fibroadenomas and malignant lesions could be classified as irregular、roundish or lobulated.According to the morphological classification,there were 12,7 and 1 case in patients with breast carcinoma respectively, while 1,7,17 cases in patients with fibroadenoma respectively.There was statistical difference in the distribution between the two patient groups(?~2=23.262,P=0.000).The typical features of fibroadenomas were as follows:lobulated shape,hypointensive internal septations on T_2-weighted or postcontrast images. The diagnostic sensitivity of the three imaging features for fibroadenoma was 68%(17/25),52%(13/25), and 72%(18/25)respectively;and the diagnostic specificity was 95%(19/20),90%(18/20),95% (19/20)respectively.Conclusion The internal septation is a rather specific sign for diagnosis of fibroadenomas.