Domain redistribution within ergosterol-containing model membranes in the presence of the antimicrobial compound fengycin

The cyclic lipopeptide fengycin, produced by Bacillus subtilis, exhibits its antimicrobial capabilities by altering the integrity of the cell membrane of plant pathogens. Previous work has correlated fengycin activity with membrane characteristics, such as sterol content. This work focused on the influence of fengycin on supported lipid bilayers containing varying levels of ergosterol. Total internal reflection fluorescence (TIRF) microscopy was used to visualize and distinguish ordered (L_β/L_o) and disordered (L_α/L_d) domains in the model membranes following exposure to low (50 μg) and high (500 μg) fengycin doses. Application of an initial low dose of fengycin to 0% and 3% ergosterol-containing bilayers resulted in redistribution of L_α/L_β and L_o/L_d domains, respectively, which the bilayers compensated and corrected for over time. These membranes were unable to tolerate a second 50 μg dose or a single high fengycin dose. The 6% ergosterol bilayers were able to tolerate sequential low doses of fengycin. Exposure of these bilayers to the high fengycin dose caused a decrease in the number of L_o domains, albeit less than that seen in the 0% and 3% ergosterol bilayers. Bilayers containing 12% ergosterol, exhibited the least amount of change after fengycin exposure. These were the only bilayer to exhibit an increase in area taken up by ordered domains. These results suggest fengycin may preferentially act on the L_β or L_o phase, the area in which ergosterol resides. Bilayers containing low levels of ergosterol appear to be more sensitive to the lipopeptide, suggesting ergosterol plays a role in buffering perturbations caused by fengycin.     

Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis

Hepatitis C virus (HCV) induced liver disease is the leading indication for liver transplantation (LTx). Reinfection and accelerated development of fibrosis is a universal phenomenon following LTx. The molecular events that lead to fibrosis following HCV infection still remains poorly defined. In this study, we determined microRNA (miRNA) and mRNA expression profiles in livers from chronic HCV patients and normals using microarrays. Using Genego software and pathway finder we performed an interactive analysis to identify target genes that are modulated by miRNAs. 22 miRNAs were up regulated (>2 fold) and 35 miRNAs were down regulated (>2fold) compared to controls. Liver from HCV patients demonstrated increased expression of 306 genes (>3 fold) and reduced expression of 133 genes (>3 fold). Combinatorial analysis of the networks modulated by the miRNAs identified regulation of the phospholipase C pathway (miR200c, miR20b, and miR31through cellular proto-oncogene tyrosine-protein kinase Src (cSrc)), response to growth factors and hormones (miR141, miR107 and miR200c through peroxisome proliferator-activated receptor alpha and extracellular-signal-regulated kinases, and regulation of cellular proliferation (miR20b, miR10b, and miR141 through cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 p21). Real time PCR (RT-PCR) validation of the miRNA in HCV infected livers demonstrated a 3.3 ±0.9 fold increase in miR200c. In vitro transfection of fibroblasts with miR200c resulted in a 2.2 fold reduction in expression of tyrosine-protein phosphatase non-receptor type 13 or FAS associated phosphatase 1 (FAP-1) and 2.3 fold increase in expression of cSrc. miR200c transfection resulted in significant increases in expression of collagen and fibroblast growth factor (2.8 and 3.4 fold, p<0.05). Therefore, we propose that HCV induced increased expression of miR200c can down modulate the expression of FAP1, a critical regulator of Src and MAP kinase pathway that play an important role in the production of fibrogenic growth factors and development of fibrosis.