Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu²⁺ binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization. The results of our work show that the presence of Mg²⁺ at the high-affinity cation binding site of Cu-modified actin polymerized with MgCl₂ strongly enhances the rate of filament subunit exchange and promotes the filament instability. In the presence of 0.1 M KCl, the filament subunit exchange was 2–3-fold lower than that in the MgCl₂-polymerized F-actin. This effect correlates with the reduced accessibility of the D-loop and Segment 227-235 on opposite filament strands, consistent with an ionic-strength-dependent conformational change that modulates involvement of Segment 227-235 in stabilization of the intermonomer interface. KCl may restrict the mobility of the α-helix encompassing part of Segment 227-235 and/or be bound to Asp236 at the boundary of Segment 227-235. These results provide experimental evidence for the involvement of Segment 227-235 in salt-induced stabilization of contacts within the actin filament and suggest that they can be weakened by mutations characteristic of actin-associated myopathies.     

The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization—12 h post contamination. Based on the obtained model we performed a metabolomics analysis by ¹H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.     

Bubble Formation at a Rotating Cylindrical Surface in Cross‐Flowing Liquid

A novel method of gas sparging from a rotating cylinder is proposed, which prevents against formation of large attached gas cavities in cross‐flowing liquids including those flowing downwards. Experimental and theoretical results regarding critical rotation speed necessary to remove the attached cavity, bubble formation process and size distribution of the produced bubbles in a low viscosity system (air‐water) are presented in this study.     

Emission of excited potassium species from an industrial iron catalyst for ammonia synthesis

The angular dependence of potassium emission-desorption is studied from a fused iron catalyst of the type used for ammonia synthesis. The excited species (K^*, K _n ^* , etc.) and positive ions K⁺ have strongly different angular distributions. The bilobular distribution measured for ion desorption is concluded to be either due to excited atoms, so-called Rydberg atoms, or excited clusters. Both types of species have to desorb from the edges of the sample and become field ionized and deexcited just outside the sample, as reported in previous studies on an iron oxide catalyst. The peak in the normal direction measured for excited species is due to excited cluster formation outside the catalyst surface. Similarities with previous results for other catalysts are observed. The possibility that the promoter function of potassium in the ammonia synthesis is due to excited species is pointed out.On leave from Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Cracow, Poland.     

Asphalts as inhibitors of radical polymerization

The results of investigations concerning the effect of asphalts on radical polymerization of a monomer for model systems composed of styrene and two petroleum asphalts originated from two different crude oils: low sulphur nonparaffinic crude oil and medium sulphur paraffinic crude oil. Based on determination of styrene conversion in the radical polymerization process in the presence of asphalts, it was found that both asphalts act as inhibitors of this reaction. The inhibiting effect of asphalts is weakened by using higher temperatures and prolongation of polymerization time or application of greater initiator rates. On the basis of the analysis of group and elementary chemical composition of both asphalts as well as electron paramagnetic resonance, it was found that the inhibiting effect to radical polymerization is caused mainly by the presence of structures which are stable free radicals in character. The probability of inhibiting effect by sulphur compounds present in asphalts is also discussed.     

Spectroscopic EPR and IR studies of monomeric and dimeric species formed upon adsorption of nitric oxide on Ce0.75Zr0.25O2 and their reactivity with dioxygen

The interaction of nitric oxide with Ce_0.75Zr_0.25O₂ solid solution were investigated by means of EPR and IR spectroscopies. The influence of adsorption parameters such as adsorption temperature and pressure, presence of the O₂ co-reactant on the nature and relative surface abundance of the resultant mono- and dimeric NO species was elucidated. The thermal stability of the surface nitrosyl complexes and their reactivity toward dioxygen were also examined.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidasefrom Streptomyces and that from Brevibacterium, both enzymesexhibit different characteristics, such as catalytic activity,optimum pH and temperature. In attempts to define the molecularbasis of differences in catalytic activity or stability, substitutionsat six amino acid residues were introduced into cholesteroloxidase using site-directed mutagenesis of its gene. The aminoacid substitutions chosen were based on structural comparisonsof cholesterol oxidases from Streptomyces and Brevibacterium.Seven mutant enzymes were constructed with the following aminoacid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357Nand S379T. All the mutant enzymes exhibited activity with theexception of that with the L117P mutation. The resulting V145Qmutant enzyme has low activities for all substrates examinedand the S379T mutant enzyme showed markedly altered substratespecificity compared with the wild-type enzyme. To evaluatethe role of V145 and S379 residues in the reaction, mutantswith two additional substitutions in V145 and four in S379 wereconstructed. The mutant enzymes created by the replacement ofV145 by Asp and Glu had much lower catalytic efficiency forcholesterol and pregnenolone as substrates than the wild-typeenzyme. From previous studies and this study, the V145 residueseems to be important for the stability and substrate bindingof the cholesterol oxidase. In contrast, the catalytic efficiencies(k_cat/K_m) of the S379T mutant enzyme for cholesterol and pregnenolonewere 1.8- and 6.0-fold higher, respectively, than those of thewild-type enzyme. The enhanced catalytic efficiency of the S379Tmutant enzyme for pregnenolone was due to a slightly high k_catvalue and a low K_m value. These findings will provide severalideas for the design of more powerful enzymes that can be appliedto clinical determination of serum cholesterol levels and assterol probes.     

Monte Carlo simulation of radiation-induced solid state polymerization

A Monte Carlo method was used for a computer simulation of radiation-induced solid state polymerization. The propagation of polymer chains was simulated by means of self-avoiding random walks on a tetrahedral lattice. The initiation and termination of the chains were modelled by pseudorandom processes. The influence of conditions of the in-source process on the post-polymerization kinetics and on the degree of polymerization of the polymers was studied.     

Quality assessment for a visual and automatic license plate recognition

Video transmission and analysis is often utilized in applications outside of the entertainment sector, and generally speaking this class of video is used to perform specific tasks. Examples of these applications include security and public safety. The Quality of Experience (QoE) concept for video content used for entertainment differs significantly from the QoE of surveillance video used for recognition tasks. This is because, in the latter case, the subjective satisfaction of the user depends on achieving a given functionality. Recognizing the growing importance of video in delivering a range of public safety services, we focused on developing critical quality thresholds in license plate recognition tasks based on videos streamed in constrained networking conditions. Since the number of surveillance cameras is still growing it is obvious that automatic systems will be used to do the tasks. Therefore, the presented research includes also analysis of automatic recognition algorithms.