92 Novel interpretation of the isotherms of multimodal ligands binding with DNA

The binding of ligands with DNA is a key moment in a whole range of cellular processes that provide not only the normal cell vital activity but also the development of some pathological processes. Depending on ligand type, structure of DNA adsorption centers, and physical–chemical conditions of the surrounding, the ligand may bind to DNA by several modes [1]. Particularly, adsorption isotherm of multimodal ligands binding to DNA in Scatchard’s coordinates has a concave shape with two brightly expressed linear areas in the region of small fillings. The analysis of such type of adsorption isotherm for determining of important binding parameters such as binding constant and number of adsorption centers (the part of DNA polymer with which one ligand molecule binds) presents difficulties. Practically in all cases, the analysis of such adsorption isotherm is carried out by linear parts of curves. Such analysis mode of experimental points is approximate method, since all registered of experimental points are roughly divided into two groups and they are treated by linear binding isotherm and therefore the binding parameters are determined. In the present work, the non-linear adsorption isotherm in Scatchard‘s coordinates is obtained which allowed, provided, the more precise treatment of all experimental points by unique curve which includes linear regions as well. Such mode of treatment of experimental points makes more precise the determination of not only binding constant and number of adsorption centers that correspond to the one ligand molecule binding, but also additional binding parameter – a proportion of adsorption centers of each binding to DNA type of multimodal ligand.     

93 Spectroscopic investigation of methylene blue binding to DNA

The interaction of methylene blue (MB) with DNA has been investigated by UV absorption spectra, Fluorescence spectra and UV-melting method. Analysis of the results of the melting experiments shows that melting temperature (T _m) of the complexes increases with the [total ligand]: DNA ratio (r) at two concentrations of Na⁺ (2?mM Na⁺ and 20?mM Na⁺) providing support for conclusion that MB is a stabilizer of DNA helix structure. By contrast, the shapes of dependences of width of transition (ΔT) on r at low and high [Na⁺] are different which points to the existence of different types of binding modes of MB with DNA. UV-spectroscopy experiments and fluorescence spectra indicated that the binding modes of MB with DNA depended on r. At high r (r?>?0.25), remarkable hypochromic effect with no shift of λ _max in the absorption spectra of MB was observed. The fluorescence of MB was quenched which indicated that MB was bound to phosphate groups of DNA by electrostatic interaction. At low r ratios (r?<?0.2), the absorption spectra of MB upon increasing the concentration of DNA showed gradually decrease in the peak intensities with a red shift. This phenomenon is usually associated with molecular intercalation into the base stack of the ds-DNA. Using the Scatchard’s model, the complex formation constants for MB with DNA were determined: the binding constant K?≈?6.5?×?10⁵ and binding site size n?≈?4. Obtained data are not typical for intercalation model of ligands to DNA. Moreover, comparison between these data and our early experimental results of interaction of ethidium bromide with DNA made it possible to suggest that this binding type of MB is, more probably, semi-intercalation mode (Vardevanyan et al., 2003). This conclusion is in accordance with the analysis of the model structures of MB–DNA complexes which clearly shows the importance of solvent contributions in suggested structural form (Tong et al., 2010).     

Distribution and excretion of a phosphorothioate oligonucleotide in rats with experimentally induced renal injury

The effects of renal injury on the urinary excretion and tissue distribution of a 20-mer phosphorothioate oligonucleotide were investigated in male Sprague-Dawley rats. Renal injury was produced by treating the rats with either 5.0 mg/kg cisplatin or 2.5 mg/kg of a monoclonal antibody (mAb) directed toward Thy1.1. Controls received saline. Three days after cisplatin treatment or 2 days after anti- Thy1.1 treatment, the rats received 10 mg/kg ISIS 3521. Blood was collected at various times to assess the plasma concentrations of ISIS 3521, and rats were killed at various times from 6 to 48 hours after intravenous (i.v.) infusion of oligonucleotide to assess tissue concentrations by capillary gel electrophoresis (CGE). Cisplatin and anti-Thy1.1 antibody produced histologic and biochemical changes consistent with proximal tubular damage and glomerular damage, respectively. Urinary excretion of oligonucleotides was increased 2- to 4-fold of control; however, this amount accounted for only 1% to 2% of dose compared to 0.5% in controls. Proximal tubular damage reduced renal accumulations of ISIS 3521 and other oligonucleotide metabolites, but there were no obvious compensatory increases in concentrations in other organs except for a slight increase in spleen levels of total oligonucleotide. Glomerular damage was not associated with any change in oligonucleotide disposition. Immunohistochemical studies showed no evidence of alterations in the pattern of distribution within the injured kidney. The data suggest that acute renal dysfunction, either renal tubular or glomerular, does not markedly alter the urinary elimination and tissue deposition of a phosphorothioate oligonucleotide.     

The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot’s matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.     

Antibacterial activities of transient metals nanoparticles and membranous mechanisms of action

World Journal of Microbiology and Biotechnology - Karst caves, considering to be the “arks” of biodiversity, often contain high levels of endemism. In the present study, the...     

Interactive effects of chromate and arsenate on their uptake and speciation in Pteris ensiformis

Background and aims

Arsenate (AsV) and chromate (CrVI) inhibit each other’s uptake and translocation in As-hyperaccumulator Pteris vittata. In the present study, we extended the research to As-sensitive plant Pteris ensiformis to better understand the mechanism of their interactions.

Methods

Plants were exposed to 0, 0.75 or 7.5 mg L^?1 AsV and 0, 0.52, or 5.2 mg L^?1 CrVI for 7 d in hydroponics. Arsenic and Cr speciation were determined in nutrient solutions and plant biomass.

Results

P. ensiformis accumulated high levels of As and Cr in the rhizomes and roots with low levels in the fronds. However, P. ensiformis was much more effective in taking up Cr than As, as much more Cr was accumulated in the roots (306–6015 vs. 87–642 mg kg^?1). AsV and CrVI increased each other’s uptake in the rhizomes and roots when co-present. The AsV and CrVI taken up by P. ensiformis were reduced to arsenite (AsIII) and chromite (CrIII), possibly serving as detoxification mechanism.

Conclusions

Uptake of As and Cr induced oxidative stress as indicated by increased lipid peroxidation and electrical conductivity. Arsenic and Cr increased each other’s uptake by P. ensiformis.

     

P190RhoGAP prevents mitotic spindle fragmentation and is required to activate Aurora A kinase at acentriolar poles

Assembly of the mitotic spindle is essential for proper chromosome segregation during mitosis. Maintenance of spindle poles requires precise regulation of kinesin- and dynein-generated forces, and improper regulation of these forces disrupts pole integrity leading to pole fragmentation. The formation and function of the mitotic spindle are regulated by many proteins, including Aurora A kinase and the motor proteins Kif2a and Eg5. Here, we characterize a surprising role for the RhoA GTPase-activating protein, p190RhoGAP, in regulating the mitotic spindle. We show that cells depleted of p190RhoGAP arrest for long periods in mitosis during which cells go through multiple transitions between having bipolar and multipolar spindles. Most of the p190RhoGAP-depleted cells finally achieve a stable bipolar attachment and proceed through anaphase. The multipolar spindle phenotype can be rescued by low doses of an Eg5 inhibitor. Moreover, we show that p190RhoGAP-depleted multipolar cells localize Aurora A to all the poles, but the kinase is only activated at the two centriolar poles. Overall, our data identify an unappreciated connection between p190RhoGAP and the proteins that control spindle poles including Aurora A kinase and Eg5 that is required to prevent or correct spindle pole fragmentation.