Antibacterial mechanism of high-mobility group nucleosomalbinding domain 2 on the Gram-negative bacteria Escherichia coli

Objective： To investigate the antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 （HMGN2） on Escherichia coil K12, focusing on the antibacterial and antibiofilm formation effects. Its chemotactic activity on human neutrophils was also investigated. Methods： Human tissue-derived HMGN2 （tHMGN2） was extracted from fresh uterus fiber cystadenoma and purified by HPl100 reversed-phase high-performance liquid chromatography （RP-HPLC）. Recombinant human HMGN2 （rHMGN2） was generated in E. coil DE3 carrying PET-32a- c（＋）-HMGN2. Antibacterial activity of HMGN2 was determined using an agarose diffusion assay and minimum inhibitory concentration （MIC） of HMGN2 was determined by the microdilution broth method. Bacterial membrane permeability assay and DNA binding assay were performed. The antibiofilm effect of HMGN2 was investigated using a crystal violet assay and electron microscopy scanning. The activating effect and chemotactic activity of HMGN2 on neutrophils were determined using a nitroblue tetrazolium （NBT） reduction assay and Transwell chamber cell migra- tion assay, respectively. Results： HMGN2 showed a relatively high potency against Gram-negative bacteria E. coli and the MIC of HMGN2 was 16.25 μg/ml. Elevated bacterial membrane permeability was observed in HMGN2-treated E. coil K12. HMGN2 could also bind the bacterial plasmid and genomic DNA in a dose-dependent manner. The antibiofilm effect of HMGN2 on E. coil K12 was confirmed by crystal violet staining and scanning electron microscopy. However, the activating effects and chemotactic effects of HMGN2 on human neutrophils were not observed. Con- clusions： As an antimicrobial peptide （AMP）, HMGN2 possessed a good capacity for antibacterial and antibiofilm activities on E. coil K12. This capacity might be associated with disruption of the bacterial membrane and combination of DNA, which might affect the growth and viability of E. coil.

Bioactive proteins in healthy pregnancies and preeclampsia： relevance to hypertension and proteinuria

Background Bioactive proteins, such as cytokines and chemokines, have not been systematically evaluated in healthy and preeclamptic pregnancies. We aimed to investigate the difference of these proteins between healthy and preeclamptic pregnancies in order to help clarify their potential roles in the pathogenesis of hypertension and proteinuria in preeclampsia. Methods Samples of amniotic fluid and maternal/umbilical cord blood were collected from normal pregnancies and women with preeclampsia for examination of bioactive proteins. Fifty-three pregnant women were enrolled in this study. Of them, 30 pregnant women were recruited as healthy controls, and 23 pregnant women were diagnosed with preeclampsia. An antibody array was used to screen for higher levels of cytokines and related proteins in amniotic fluid than in the blood samples, and these proteins were then selected for quantification by immunoassay. Results Interleukin-1 receptor 4, hepatocyte growth factor, and urokinase plasminogen activator receptor were significantly elevated in the blood of preec^ampsia patients. In particular, intedeukin-1 receptor 4 was 8-fold higher in preeclampsia patients than in the healthy pregnancies. Moreover, in cord blood samples hepatocyte growth factor and interleukin-8 were significantly higher in preeclampsia patients. Conclusions Because of the biologic activities, Interleukin-1 receptor 4, hepatocyte growth factor, urokinase plasminogen activator receptor and interleukin-8 in maternal and/or cord blood could play a role in the pathogenesis of hypertension and proteinuria in preeclampsia.