Establishing the impact of LAP and LIP on gene expression under ER stress will further clarify the mechanism by which LAP attenuates cell death

ORF corresponding to Hcl Protein Expression and Purification of CfpE. coli TOP Protein Expression and Purification of HclE. coli BL In Vitro Protein-Protein Interaction EsatNovember Anti-Mycobacterial Peptides washed with pre-warmed medium to remove extracellularly present bacteria. Additionally, cells were treated with gentamicin for Growth Curve Studies of M. tuberculosis Mycobacteria harbouring hcl Construction of Expression Vectors pVV Transmission Electron Microscopy M. tuberculosis H Total RNA Isolation from M. tuberculosis Total RNA was isolated as described previously with some modifications. Cells were harvested from exponentially growing mycobacteria harbouring either hcl Expression of HclMycobacteria harbouring either hcl Infection of Human Macrophages with M. tuberculosis Labeling of cDNA with Cy Dyes Equal amounts of RNA from both control and treated samples were used for preparing labeled cDNA for microarray experiment. Total RNA was labeled November Anti-Mycobacterial Peptides with Cy difference. Test of significance for changes in gene expression was measured by calculating the Z-ratios and p values in a standard T-test. Z-ratios were calculated by measuring the difference between the average Zscore of genes from treated samples and from control samples and dividing them by the standard deviation of the difference. Genes having a Z-ratio. Microarray Slides Hybridization M. tuberculosis H Real Time PCR Microarray Data Analysis and therefore, the ratio between CoA and its thioester derivatives is important for maintaining cellular homeostasis. This is further corroborated by the fact that all attempts to disrupt the genes coding for the enzymes in the CoA biosynthetic pathway have either failed or have resulted in lethal phenotypes in many organisms. CoA biosynthesis, a five-step pathway which utilizes the precursors, pantothenate, cysteine and ATP, is invariant in all organisms. It begins with the phosphorylation of pantothenate by the enzyme CoaA. CoaB condenses the October Mycobacterial CoaE to generate the final CoA precursor, dephosphocoenzyme A. CoaE then phosphorylates the charged Ni- NTA affinity resin and eluted with Enzyme Assays All enzyme assays were carried out at ITC Experiments Since we assayed the enzyme using coupled assays which could be prone to errors that could MedChemExpress Dansyl chloride account for the high affinity of the mycobacterial enzyme for its substrates and the higher catalytic efficiency, compared to its other prokaryotic counterparts, we chose to quantify the kinetic parameters also using a direct, thermodynamic approach. A thermodynamically favorable reaction is accompanied by a decrease in free energy, which is composed of enthalpic and entropic terms. Calorimetry measures the heat exchanged during any reaction. The high sensitivity of the technique allows experiments to be carried out with low amounts of enzyme and a single experiment allows rate calculations multiple times as several injections of the substrate into the cell containing the enzyme can be carried out. ITC experiments were performed using a VP-ITC titration microcalorimeter. The reference cell was filled with water and the calorimeter was calibrated using standard electrical pulses as recommended by the manufacturer. For all ITC experiments, the CoaE protein was dialyzed overnight in Materials and Methods Plasmid Construction, Expression and Purification of CoaE Determination of the Kinetic Parameters of ATP Hydrolysis by ITC To evaluate the kineti