Molecular modeling of immersion optical clearing of biological tissues

The interaction of six low-molecular tissue-clearing agents (1,2 and 1,3-propanediol, ethylene glycol, glycerol, xylitol, sorbitol) with the collagen mimetic peptide (GPH)₃ was studied by applying the methods of classical molecular dynamics (GROMACS), molecular docking (AutoDock Vina) and quantum chemistry (PM6 and B3LYP). The spatial configurations of intermolecular complexes were determined and interaction energies calculated. The dependence of the volume occupied by the collagen peptide on the clearing agent concentration in an aqueous solution was calculated. This dependence is not linear, and has a maximum for almost all the agents in the study. The correlations between the optical clearing potential and intermolecular interactions parameters, such as the time of an agent being in a hydrogen-bonded state, and the relative probability of formation of double hydrogen bonds and interaction energies, were determined. Using the correlations determined, we predicted the numeric value of the optical clearing potential of dextrose molecules in rat skin, which correlates with experimental data. A molecular mechanism of tissue optical clearing within the post-diffusion stage is suggested.

Open image in new window

Graphical abstract The molecular modeling of the interaction between clearing agents and collagen

     

Synthesis, cloning and expression of genes for antibacterial peptides: Cecropin, magainin and bombinin

Summary Three DNA sequences encoding the antimicrobial peptides bombinin, cecropin and magainin were synthesised. DNA fragments were cloned into pET-21d plasmid under T7 promoter for expression in vivo and in vitro and into pRIT-2T plasmid for expression as a fusion product with protein A. The polypeptides synthesised in both systems possess antibacterial activity.     

Modelling of the prebiotic synthesis of oligopeptides: silicate catalysts help to overcome the critical stage

On the basis of experimental studies of the initial stages of glycine oligomerization in aqueous suspension of zeolite and kaolinite catalysts, a model is suggested for the prebiotic synthesis of oligopeptides from -amino acids. The formation of linear dipeptides by hydrolysis of one amide bond in the cyclic piperazinedione intermediate (formed from glycine spontaneously) is found to be the critical stage of the reaction. This stage is base catalyzed and its rate increases when pH of the medium goes up. The linear glycyl-glycine yield rises under effect of hydroxyl anions generated from different sources including insoluble silicates and soluble sodium bicarbonate. During prebiotic evolution silicates capable of cation-exchange can serve as local sources of the hydroxyl anions which dramatically accelerate formation of linear dipeptides from cyclic ones. Oligopeptides of higher molecular weight are then easily formed from the linear dipeptides at neutral pH, even in the absence of catalysts or sources of energy (e.g. such as light). The described catalytic synthesis could occur in the proximity of submarine hydrothermal vents.     

Calcium signaling complexes in microdomains of polarized secretory cells

The highly polarized nature of epithelial cells in exocrine glands necessitates targeting, assembly into complexes and confinement of the molecules comprising the Ca(2+) signaling apparatus, to cellular microdomains. Such high degree of polarized localization has been shown for all Ca(2+) signaling molecules tested, including G protein coupled receptors and their associated proteins, Ca(2+) pumps, Ca(2+) influx channels at the plasma membrane and Ca(2+) release channels in the endoplasmic reticulum. Although the physiological significance of polarized Ca(2+) signaling is clear, little is known about the mechanism of targeting, assembly and retention of Ca(2+) signaling complexes in cellular microdomains. The present review attempts to summarize the evidence in favor of polarized expression of Ca(2+) signaling proteins at the apical pole of secretory cells with emphasis on the role of scaffolding proteins in the assembly and function of the Ca(2+) signaling complexes. The consequence of polarized enrichment of Ca(2+) signaling complexes at the apical pole is generation of an apical to basal pole gradient of cell responsiveness that, at low physiological agonist concentrations, limits Ca(2+) spikes to the apical pole, and when a Ca(2+) wave occurs, it always propagates from the apical to the basal pole. Our understanding of Ca(2+) signaling in microdomains is likely to increase rapidly with the application of techniques to controllably and selectively disrupt components of the complexes and apply high resolution recording techniques, such as TIRF microscopy to this problem.     

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.