Chronotropic action of immobilized subtilisins during the perfusion of an isolated rat heart

The pharmacological experiments on isolated organs (ex vivo) are the preferred method for assessing the primary pharmacodynamics of the studied drugs, since this method is completely excluded the systemic influence of neurohumoral regulation. In the last decade, a new group of thrombolytic drugs based on immobilized subtilisins has been formed. At the stage of registrational preclinical and clinical studies, their pleiotropic pharmacological effects have not been studied. Meanwhile, there is a reason to consider that their pharmacological activity in the bloodstream is not limited to thrombolytic action, but may be extended to a systemic effect on the cardiovascular system. The aim of the study was to investigate the chronotropic effects of an isolated heart during its perfusion with solutions of immobilized subtilisins at different concentrations. Material and methods. The isolated rat heart model according to Langendorff was used in the study. The experiment included 50 Wistar rats, which were divided into 5 groups: isolated hearts perfused only with Krebs – Henseleit solution (control) or with immobilized subtilisins in 4 concentrations (170, 340, 510 и 1020 U/l). Results and discussion. The immobilized subtilisins have a negative chronotropic effect. The onset of the effect depends on the drug concentration in the solution: the higher concentration, the earlier effect. From 5 to 10 minutes of perfusion, a negative chronotropic effect is observed using of immobilized subtilisins at any dose. The duration of its increase is manifested up to 10–20 minutes, depending on the drug concentration in solution. After 20 minutes of perfusion, the achieved negative chronotropic effect remains at a plateau level up to 40 minutes. Conclusion. The immobilized subtilisins have an independent pharmacological effect on heart rate

The Superconductivity, Intragrain Penetration Depth and Meissner Effect of RuSr2(Gd,Ce)2Cu2O10+delta

The hole concentration (p)(delta), the transition temperature Tc, the intragrain penetration depth lambda, and the Meissner effect were measured for annealed RuSr2(Gd,Ce)2Cu2O10+delta samples. The intragrain superconducting transition temperature Tc} varied from 17 to 40 K while the p changed by only 0.03 holes/CuO2. The intragrain superfluid-density 1/lambda^2 and the diamagnetic drop of the field-cooled magnetization across Tc (the Meissner effect), however, increased more than 10 times. All of these findings are in disagreement with both the Tc vs. p and the Tc vs. 1/lambda^2 correlations proposed for homogeneous cuprates, but are in line with a possible phase-separation and the granularity associated with it.Comment: 7 pages, 6 figures, accepted for publication in Phys. Rev. B (May 2, 2002

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

The Escherichia coli NarL receiver domain regulates transcription through promoter specific functions

BACKGROUND: The Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function. RESULTS: NarL OD constructs containing different linker segments were examined for their ability to repress frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric. CONCLUSIONS: The NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0502-9) contains supplementary material, which is available to authorized users

Efficient Polysulfides Conversion Kinetics Enabled by Ni@CNF Interlayer for Lithium Sulfur Batteries

Recent advances in the development of lithium-sulfur batteries (Li-S) demonstrated their high effectiveness owing to their tremendous theoretical specific capacity and high theoretical gravimetrical energy. Nevertheless, the potential commercialization of Li-S is significantly held by the insulating nature of sulfur and complicated RedOx reactions during the electrochemical charge-discharge processes. This paper presents nickel nanoparticles embedded carbon nanofibers interlayer (Ni@CNF) between a cathode and a separator as an additional physical barrier against lithium polysulfides shuttle for their efficient conversion during the charge-discharge cycling. Furthermore, the interlayer provides an auxiliary electron pathway with subsequent lowering of the charge transfer resistance. The electrochemical analysis of a Li-S cell with the Ni@CNF interlayer demonstrated high initial discharge capacities of 1441.2 mAh g-1 and 1194.2 mAh g-1 at 0.1 and 1.0 C rates, respectively, with remarkable capacity retention of ~83% after 100 cycles. This study revealed the advantageous impact of Ni@CNF towards solving the major issues of lithium-sulfur batteries, i.e., sluggish kinetics and the shuttle effect