Impact of tetracycline-clay interactions on bacterial growth.

Antibiotics are extremely effective against bacterial infections due to their selective toxicity for bacteria rather than the host. Extensive use and misuse of antibiotics resulted in significant increases in antibiotic levels in aquatic and soil environments. Bacteria exposed to antibiotics with low concentrations may develop antibiotic resistance. In this study a swelling 2:1 clay mineral montmorillonite (MMT) and a non-swelling 1:1 clay mineral kaolinite were premixed with tetracycline (TC) of varying concentrations. The gram-negative bacteria Escherichia coli (E. coli) and Salmonella enterica (S. enterica) of both TC sensitive and TC resistant strains were tested for their growth in the presence TC-loaded clay minerals of different amounts and under different physico-chemical conditions. The antimicrobial activity of TC was significantly decreased in the presence of MMT. In the absence of MMT, no bacteria growth was found at a TC concentration 0.25mg/mL and above. On the contrast, in the presence of MMT, 50% growth was still found for a TC resistant E. coli at a TC concentration of 5mg/g. The influence of kaolinite was to a lesser degree. These results suggest that antimicrobial agents present in clayey soils could be responsible for possible mutation of bacteria of high antibiotic resistance.

Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology.

With the advent of the recent determination of high-resolution crystal structures of bovine rhodopsin and human beta2 adrenergic receptor (beta2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and beta2AR. Structure determination of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biology becomes routine: over-expression, solubilization, and purification of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purification efforts, we review published conditions for solubilization and purification using detergents and additives. A discussion of sample preparation of GPCRs in detergent phase, bicelles, nanodiscs, or low-density lipoproteins is presented in the context of potential structural biology applications. In addition, a review of the solubilization and purification of successfully crystallized bovine rhodopsin and beta2AR highlights tools that can be used for other GPCRs.