PAMAM-calix-dendrimers: Synthesis and Thiacalixarene Conformation Effect on DNA Binding

A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.

Spectroscopic and electrochemical approaches for the Analysis of interaction between textile dye Reactive Red 231 and salmon sperm DNA

DNA is one of the most critical targets of many artificial agents listed as carcinogens. Most of them irreversibly bind to the DNA inducing genome mutation; therefore, it is vital to study the nature of binding of these molecules to anticipate their toxicity. The interaction between textile dye reactive red 231 and salmon sperm double-strand DNA (ss-dsDNA) was investigated applying cyclic voltammetry (CV), differential pulse voltammetry (DPV) and ultraviolet-visible spectroscopy (UV/vis spectroscopy). The changes in the anodic current signals of dye were observed in the presence and absence of ss dsDNA at a glassy carbon electrode (GCE) using CV. The diffusion coefficient (D) was found to be 2.2× 10-7 and 9.5× 10-8 cm2s-1 from the CV data for the free dye and the dye-DNA complex, respectively. The electrochemical and UV/vis spectroscopy indicated a 1:1 complex formation of dye with DNA. The binding constant (kb) between the dye and DNA was calculated to be 5.4×105M-1 and 4.9×105M-1 in pH 4.0, using CV and UV/vis spectroscopy, respectively. The overall results suggest that dye binds to DNA through the combined effect of intercalation and electrostatic interaction. The damage of the DNA was also detected through the changes in the voltammetric behaviour of the dye.

Ethidium Binding to Salmonella enterica ser. Typhimurium Cells and Salmon Sperm DNA

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-β-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. The results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measurements of Et+ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably decreases in the presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.

Ethidium binding to Salmonella enterica ser. Typhimurium Cells and Salmon sperm DNA

Bacterial resistance to antibiotics due to an increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for the efflux analysis are needed. Here we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenyl-alanyl-arginyl-β-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. Results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measure-ments of Et+ interaction with the purified DNA demonstrated that affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably de-creases in presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.