Attila Bóta

Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones

Background: Neutrophil extracellular traps (NETs) composed of DNA and proteins form a scaffold in thrombi, supplementing the fibrin matrix. Results: DNA and histones modify the structure of fibrin and render it resistant to mechanical and enzymatic destruction. Conclusion: NET components are essential factors in thrombus stability. Significance: Therapeutic strategies could be optimized to enhance fibrinolysis in clots containing DNA and histones. Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator.

Comparative adsorption study on carbons from polymer precursors

Abstract The effect of the precursor polymer on nitrogen adsorption properties, pore size distribution and the hydrophilic/hydrophobic character of both the pyrolyzed char and the activated carbon was studied in the case of three basically different polymers, i.e. polyacrylonitrile, polyethyleneterephthalate and cellulose. Pyrolyzed samples were produced from these polymers and activated by steam at 900°C. After a 50% burn-off, the pore volume and the specific surface area increase significantly and the pore size distribution is determined by that of the pyrolyzed char, i.e. the starting polymer. The activated sample derived from polyacrylonitrile is microporous, while the other two carbons contain both micro- and mesopores. Activation creates pores which are equally available for methanol and benzene and the heterogeneous surfaces become more benzophilic in all three polymers. The N-content has limited effect on the surface properties studied.

Fractal approach of activated carbons from solid waste materials

Abstract Carbonaceous composite materials were produced from various pyrolyzed organic waste materials by steam activation. Fractal dimensions derived from SAXS and adsorption measurements were used to explain the changes in the matrix and the surface microstructure, due to the activation. The comparison of the fractal dimensions from SAXS and nitrogen adsorption data can be successfully applied to understand the development of the porous structure. In these materials, of heterogeneous matrix, no correlation was found between the B.E.T. specific surface area and the fractal dimensions derived from various methods. The adsorption of different substrates is disturbed by the chemical heterogeneity of the surface. In these cases, the applicability of aqueous solutions in the description of the surface geometry might be questioned.

A closer look at the structure of sterically stabilized liposomes: a small-angle X-ray scattering study.

The evaluation of the radial electron density profile of a drug containing a sterically stabilized liposomal system is described. Using synchrotron small-angle X-ray scattering, we were able to characterize the hydrophilic shell of the polyethylene glycol chains. Using a Gaussian model for describing the electron density profile along the normal of the bilayer, we got an asymmetric distribution of PEGylated lipids in accordance with theoretical considerations. Moreover, we used anomalous X-ray scattering to study the localization of a hydrophobic drug (a kinase inhibitor), which revealed that these molecules are mainly located in the hydrocarbon chain region of the phospholipid bilayer.

Adsorbents from Waste materials

The possibility of using pyrolyzed wastes produced in already working incineration plants, as adsorbents for waste water treatment, was studied. Showing very poor adsorption properties, they were improved by steam activation technique used in the conventional activated carbon manufacturing. It is concluded that various organic waste materials can be converted to carbonaceous final products with a character similar to activated carbon. Their adsorption properties and pore size distribution are determined by the structure of the starting material. Although most of these samples have a low specific surface area, their pore volume is not negligible in the meso-and micropore range. Adsorption tests with model waste waters confirmed that adsorption properties are strongly influenced by the character of the suface. The adsorption capacity of these samples can be utilized for the treatment of strongly polluted industrial waste waters. Considering that the raw material ‘needed’ to manufacture these adsorbent is produced permanently and the adsorbents do not have to be regenerated, it might be worthwhile using these kinds of adsorbents in the primary treatment of industrial waste waters.

Pigment Interactions in Light-Harvesting Complex II in Different Molecular Environments

Background: Light-harvesting complex II (LHCII) displays different spectroscopic properties in thylakoid membranes and in detergents. Results: Circular dichroism and fluorescence lifetimes disclose structural effects of protein-protein, lipid-protein, and detergent interactions. Conclusion: The native state of complexes is perturbed by detergents and best retained in lipid:LHCII assemblies. Significance: The lipid environment is important for the proper function of LHCII, the most abundant of membrane proteins. Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions, at (−)-437 and (+)-484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids, at (+)-447 and (−)-494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at (−)-494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggregation-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spectral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, whereas a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.

Characterization of the PEG layer of sterically stabilized liposomes: a SAXS study

Synchrotron small-angle X-ray scattering analysis of the bilayer structure of a pharmacologically relevant sterically stabilized liposome system is presented. Describing the electron density profile of the bilayer with the superposition of Gaussian functions, the contribution of the poly(ethylene glycol) (PEG) layers to the total electron density was identified. The changes in the thickness of the PEG layer as well as the distribution of the PEG chains among the outer and inner leaflets of the bilayers were followed by changing the molar ratio of the PEG-lipid and the molar weight of the PEG molecule.

Temperature dependence of the lipid packing in thylakoid membranes studied by time- and spectrally resolved fluorescence of Merocyanine 540.

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above -30 degrees C and that a phase transition occurs only above 45 degrees C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases--upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 degrees C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 degrees C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.

Excitation energy transfer between Light-harvesting complex II and Photosystem i in reconstituted membranes

Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latters absorption cross-section - for example in the well-known state 1 - state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI-LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.

A mechanistic view of lipid membrane disrupting effect of PAMAM dendrimers

The effect of 5th generation polyamidoamine (PAMAM G5) dendrimers on multilamellar dipalmitoylphosphocholine (DPPC) vesicles was investigated. PAMAM was added in two different concentrations to the lipids (10(-3) and 10(-2) dendrimer/lipid molar ratios). The thermal behavior of the evolved systems was characterized by DSC; while the structure and the morphology were investigated with small- and wide-angel X-ray scattering (SWAXS), freeze-fracture electron microscopy (FFTEM) and phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, respectively. IR spectroscopy was used to study the molecular interactions between PAMAM and DPPC. The obtained results show that the dendrimers added in 10(-3) molar ratio to the lipids generate minor perturbations in the multilamellar structure and thermal character of liposomes, while added in 10(-2) molar ratio dendrimers cause major disturbance in the vesicular system. The terminal amino groups of the dendrimers are in strong interaction with the phosphate headgroups and through this binding dendrimers disrupt the regular multilamellar structure of DPPC. Besides highly swollen, fragmented bilayers, small vesicles are formed.