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

Morphological and growth altering effects of Cisplatin in C. albicans using fluorescence microscopy

Changes in morphology and growth curve of Candida albicans in response to treatment by Cisplatin has been studied using fluorescence staining with ethidium bromide. Treatment with Cisplatin was found to markedly inhibit hyphae and ovoid growth as revealed by ethidium bromide staining of drug treated cells. These changes were concomitant with inhibitory effects on the growth curve with respect to untreated cells Presence of Cisplatin not only caused suppression in the limiting values in the growth curve, but also caused a slight left shift in the EC50 values. Some of the ovoid cells undergoing poisoning with cisplatin were found to be unusually enlarged before undergoing their natural fate thus suggesting formation of similar cytotoxic end products with DNA

Spectral analysis of naturally occurring methylxanthines (theophylline, theobromine and caffeine) binding with DNA.

Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg(2+) and during helix-coil transitions of DNA by temperature (T(m)) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×10(3) M(-1), DNA-theobromine = 1.1×10(3) M(-1), and DNA-Caffeine = 3.8×10(3) M(-1). On the other hand T(m)/pH melting profiles showed 24-35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg(2+), methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg(2+). The spectral analyses indicated the order of binding affinity as "caffeine≥theophylline>theobromine" to the native double helical DNA, and "theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions

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

FTIR spectra of DNA, DNA-methylxanthines complexes in the presence of Mg²⁺ (30 mM).

<p>FTIR spectra of DNA, DNA-methylxanthines complexes in the presence of Mg²⁺ (30 mM).</p

Helix-coil transition analysis, <i>T_m-</i>profile.

<p>(<b>A</b>). UV spectra of <i>T_m</i>-DNA in the presence of methylxanthines at P/D 6. (<b>B</b>). A representative picture indicating the percentage of increased binding activity of methylxanthines with <i>T_m-</i>DNA at P/D 6. X1-theophylline, X2-theobromine and X3-caffeine. Values are mean ± SE with p<0.002 vs control.</p

The chemical structure of naturally occurring methylxanthines.

<p>The chemical structure of naturally occurring methylxanthines.</p

Methylxanthines binding with DNA.

<p>(<b>A</b>). The percentage of hyperchromicity with respect P/D 6, 3, 1 and 0.8 of theophylline (X1), theobromine (X2) and caffeine (X3) while interacting with DNA. (<b>B–D</b>): The double reciprocal plot of 1/(A−A₀) vs 1/[theophylline or theobromine or caffeine] (<b>B</b>) DNA-theophylline complexes; (<b>C</b>) DNA-theobromine complexes (<b>D</b>) DNA-Caffeine complexes, where A₀ = absorbance (260 nm) of free DNA, [theophylline or theobromine or caffeine] = concentration of the respective drug.</p