Decrease in gyrase A protein expression in _E. coli_ cells inhibited by antisense ribozymes

RNase P complexed with external guide sequence (EGS) represents a novel nucleic-acid-based gene interference approach to modulate gene expression. Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of protein expression of _E. coli_ gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of _E. coli_, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. Western blot was done to investigate the downregulation of gyrase A protein. The results showed a significant decrease of gyrase A suggesting the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins

Identification of XMRV Infection-Associated microRNAs in Four Cell Types in Culture

INTRODUCTION: XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture. METHODS: Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray. RESULTS: MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types. DISCUSSION: The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types

Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR

Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins

A peptide derived from phage display library exhibits antibacterial activity against E. coli and Pseudomonas aeruginosa.

Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents. Antimicrobial peptides (AMPs) are receiving attention as alternatives to antibiotics. In this study, we used phage-display random peptide library to identify peptides binding to the cell surface of E. coli. The peptide with sequence RLLFRKIRRLKR (EC5) bound to the cell surface of E. coli and exhibited certain features common to AMPs and was rich in Arginine and Lysine residues. Antimicrobial activity of the peptide was tested in vitro by growth inhibition assays and the bacterial membrane permeabilization assay. The peptide was highly active against gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log(10) CFU/ml. In homologous plasma and platelets, incubation of EC5 with the bacteria resulted in significant reduction of E. coli and P. aeruginosa, compared to the peptide-free controls. The peptide was non-hemolytic and non-cytotoxic when tested on eukaryotic cells in culture. EC5 was able to permeabilize the outer membrane of E. coli and P. aeruginosa causing rapid depolarization of cytoplasmic membrane resulting in killing of the cells at 5 minutes of exposure. The secondary structure of the peptide showed a α-helical conformation in the presence of aqueous environment. The bacterial lipid interaction with the peptide was also investigated using Molecular Dynamic Simulations. Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides

Structure and properties of EC5.

<p>A. Edmundson helical wheel presentation of 12-mer EC5. Hydrophobic residues are represented by diamonds and positive charge as pentagons. The most hydrophobic residue is green, with the amount of green decreasing proportionally to the hydrophobicity with least hydrophobic being yellow. B. Ribbon and surface representation of EC5. Ribbon model shows α-helical peptide as the conformation. Green-most hydrophobic. Secondary structure of the peptide was determined and viewed using PYMOLv0.99.</p

Hemolytic activity of EC5.

<p>1% suspension of chicken RBCs were made with PBS. 100 µl and 50 µl of this suspension were incubated with different concentrations of the peptide in 96 well microtiter plates. Results were read visually.</p

MIC of EC5 against bacteria (µg/ml).

<p>MIC of EC5 against bacteria (µg/ml).</p

Mechanism of action of EC5 against <i>E. coli</i> and <i>P. aeruginosa</i>.

<p>A. Outer-membrane permeabilization mediated by EC5 as assessed by NPN uptake. Effect of EC5 and Polymyxin B on NPN fluorescence. Value on y axis is the maximum fluorescence upon NPN uptake by the bacteria. B. EC5-induced permeability of bacterial cells studied using Syto9 and PI staining. Peptide treated cells had increased membrane permeability as seen by increase in red fluorescence whereas live or untreated cells showed increase in green fluorescence. C. Cytoplasmic membrane depolarization using the fluorescent dye diSC₃-5. Corresponding values on y axis represents maximum intensity upon release of the dye mediated by EC5 plotted against time (min). D. EC5 mediated inhibition of ATP synthesis. ATP concentration was measured after the addition of EC5 and polymyxin B at various concentrations and the luminescence units measured.</p

Effect of EC5 on different bacteria.

<p>A. Plasma, B. Platelets. Blood matrices were spiked with bacteria and incubated with different concentrations of EC5. Growth was monitored after 2 h by plating them onto NA plates.</p