Desiccation-induced alterations in surface topography of thylakoids from resurrection plant Haberlea rhodopensis studied by atomic force microscopy, electrokinetic and optical measurements.

With their ability to survive complete desiccation, resurrection plants are a suitable model system for studying the mechanisms of drought tolerance. In the present study, we investigated desiccation-induced alterations in surface topography of thylakoids isolated from well-hydrated, moderately dehydrated, severely desiccated and rehydrated Haberlea rhodopensis plants by means of atomic force microscopy (AFM), electrokinetic and optical measurements. According to our knowledge, so far, there were no reports on the characterization of surface topography and polydispersity of thylakoid membranes from resurrection plants using AFM and dynamic light scattering. To study the physicochemical properties of thylakoids from well-hydrated H. rhodopensis plants, we used spinach thylakoids for comparison as a classical model from higher plants. The thylakoids from well-hydrated H. rhodopensis had a grainy surface, significantly different from the well-structured spinach thylakoids with distinct grana and lamella, they had twice smaller cross-sectional area and were 1.5 times less voluminous than that of spinach. Significant differences in their physicochemical properties were observed. The dehydration and subsequent rehydration of plants affected the size, shape, morphology, roughness and therefore the structure of the studied thylakoids. Drought resulted in significant enhancement of negative charges on the outer surface of thylakoid membranes which correlated with the increased roughness of thylakoid surface. This enhancement in surface charge density could be due to the partial unstacking of thylakoids exposing more negatively charged groups from protein complexes on the membrane surface that prevent from possible aggregation upon drought stress.

Design and Concept of Polyzwitterionic Copolymer Microgel Drug Delivery Systems In Situ Loaded with Non-steroidal Anti-inflammatory Ibuprofen.

Nowadays, the modern pharmaceutical investigations are directed toward obtaining of new polymer micro- and nano-sized drug delivery carriers. In this respect, the use of hydrogel carriers based on polyzwitterions (PZIs) is an opportunity in the preparation of polymer drug delivery systems with desired characteristics. This paper describes the synthesis and characterization of micro-structured p(VA-co-DMAPS) systems with different compositions in situ loaded with Ibuprofen by emulsifier-free emulsion copolymerization (EEC) in water. The mean size of the prepared microparticles was measured by SEM and particles have been visualized by AFM. The inclusion of Ibuprofen in the polyzwitterionic copolymer microgel systems was established by using DSC. In vitro drug release experiments were carried out in order to estimate the ability of the obtained microgels to modify the release of water-insoluble Ibuprofen.

Phospholipase A2-Induced Remodeling Processes on Liquid-Ordered/Liquid-Disordered Membranes Containing Docosahexaenoic or Oleic Acid: A Comparison Study.

Vesicle cycling, which is an important biological event, involves the interplay between membrane lipids and proteins, among which the enzyme phospholipase A2 (PLA2) plays a critical role. The capacity of PLA2 to trigger the budding and fission of liquid-ordered (L(o)) domains has been examined in palmitoyl-docosahexaenoylphosphatidylcholine (PDPC) and palmitoyl-oleoylphosphatidylcholine (POPC)/sphingomyelin/cholesterol membranes. They both exhibited a L(o)/liquid-disordered (L(d)) phase separation. We demonstrated that PLA2 was able to trigger budding in PDPC-containing vesicles but not POPC ones. The enzymatic activity, line tension, and elasticity of the membrane surrounding the L(o) domains are critical for budding. The higher line tension of Lo domains in PDPC mixtures was assigned to the greater difference in order parameters of the coexisting phases. The higher amount of lysophosphatidylcholine generated by PLA2 in the PDPC-containing mixtures led to a less-rigid membrane, compared to POPC. The more elastic L(d) membranes in PDPC mixtures exert a lower counteracting force against the L(o) domain bending.

Low pH modulates the macroorganization and thermal stability of PSII supercomplexes in grana membranes.

Protonation of the lumen-exposed residues of some photosynthetic complexes in the grana membranes occurs under conditions of high light intensity and triggers a major photoprotection mechanism known as energy dependent nonphotochemical quenching. We have studied the role of protonation in the structural reorganization and thermal stability of isolated grana membranes. The macroorganization of granal membrane fragments in protonated and partly deprotonated state has been mapped by means of atomic force microscopy. The protonation of the photosynthetic complexes has been found to induce large-scale structural remodeling of grana membranes-formation of extensive domains of the major light-harvesting complex of photosystem II and clustering of trimmed photosystem II supercomplexes, thinning of the membrane, and reduction of its size. These events are accompanied by pronounced thermal destabilization of the photosynthetic complexes, as evidenced by circular dichroism spectroscopy and differential scanning calorimetry. Our data reveal a detailed nanoscopic picture of the initial steps of nonphotochemical quenching.

Evaluation of poly(2-ethyl-2-oxazoline) containing copolymer networks of varied composition as sustained metoprolol tartrate delivery systems.

Segmented copolymer networks (SCN) based on poly(2-ethyl-2-oxazoline) and containing 2-hydroxyethyl methacrylate, 2-hydroxypropyl acrylate, and/or methyl methacrylate segments have been evaluated as potential sustained release systems of the water soluble cardioselective ß-blocker metoprolol tartrate. The structure and properties of the drug carriers were investigated by differential scanning calorimetry, attenuated total reflectance Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. Swelling kinetics of SCNs in various media was followed, and the conditions for effective MT loading were specified. MT-loaded SCNs with drug content up to 80 wt.% were produced. The release kinetics of metoprolol tartrate from the systems was studied and it was shown that the conetworks of different structure and composition are able to sustain the metoprolol tartrate release without additional excipients.

Hho1p, the linker histone of Saccharomyces cerevisiae, is important for the proper chromatin organization in vivo.

Despite the existence of certain differences between yeast and higher eukaryotic cells a considerable part of our knowledge on chromatin structure and function has been obtained by experimenting on Saccharomyces cerevisiae. One of the peculiarities of S. cerevisiae cells is the unusual and less abundant linker histone, Hho1p. Sparse is the information about Hho1p involvement in yeast higher-order chromatin organization. In an attempt to search for possible effects of Hho1p on the global organization of chromatin, we have applied Chromatin Comet Assay (ChCA) on HHO1 knock-out yeast cells. The results showed that the mutant cells exhibited highly distorted higher-order chromatin organization. Characteristically, linker histone depleted chromatin generally exhibited longer chromatin loops than the wild-type. According to the Atomic force microscopy data the wild-type chromatin appeared well organized in structures resembling quite a lot the "30-nm" fiber in contrast to HHO1 knock-out yeast.

Surface pressure and elasticity of hydrophobin HFBII layers on the air-water interface: rheology versus structure detected by AFM imaging.

Here, we combine experiments with Langmuir trough and atomic force microscopy (AFM) to investigate the reasons for the special properties of layers from the protein HFBII hydrophobin spread on the air-water interface. The hydrophobin interfacial layers possess the highest surface dilatational and shear elastic moduli among all investigated proteins. The AFM images show that the spread HFBII layers are rather inhomogeneous, (i.e., they contain voids, monolayer and multilayer domains). A continuous compression of the layer leads to filling the voids and transformation of a part of the monolayer into a trilayer. The trilayer appears in the form of large surface domains, which can be formed by folding and subduction of parts from the initial monolayer. The trilayer appears also in the form of numerous submicrometer spots, which can be obtained by forcing protein molecules out of the monolayer and their self-assembly into adjacent pimples. Such structures are formed because not only the hydrophobic parts, but also the hydrophilic parts of the HFBII molecules can adhere to each other in the water medium. If a hydrophobin layer is subjected to oscillations, its elasticity considerably increases, up to 500 mN/m, which can be explained with compaction. The relaxation of the layer's tension after expansion or compression follows the same relatively simple law, which refers to two-dimensional diffusion of protein aggregates within the layer. The characteristic diffusion time after compression is longer than after expansion, which can be explained with the impedence of diffusion in the more compact interfacial layer. The results shed light on the relation between the mesoscopic structure of hydrophobin interfacial layers and their unique mechanical properties that find applications for the production of foams and emulsions of extraordinary stability; for the immobilization of functional molecules at surfaces, and as coating agents for surface modification.

On the growth of pneumatic foams.

This work reports on the behaviour of tenacious and transient pneumatic foams produced at large range of values of gas delivery rates (or superficial velocities) and surface tensions. Experimental data from the literature and produced in the course of this study were processed and analyzed. The tenacious foams were stabilized via Polyoxiethylene-2 sulfate (SDP(2)S) in presence of 0.024M NaCl and 0.003M AlCl(3) ( CMC = 1.83×10(-2) M) in the concentration range of 3.33×10(-3) M to 3.8×10(-2) M (0.18CMC -2.08CMC corresponding to values of the dynamic surface tension in the range of 42.7mN/m to 37.5mN/m. The range of gas delivery rates was from 20.5ml/min to 482.8ml/min. It was found out that the rate of foam generation coincides with the gas delivery rate until a certain critical value of the latter, beyond which the rate of foam growth exceeds the rate of gas delivery. The level of this exceeding depends on the dynamic surface tension. The lower the value of the dynamic surface tension the larger the level of this exceeding. This rule was found valid until a certain upper limit of the gas delivery rate, at which the dependence on the dynamic surface tension ceases to exist. The second set of experiments was conducted on transient foams. The latter were stabilized by three members of homologue surfactants series: sodium octylsulfate (SOS), sodium decylsulfate (SDeS), and sodium dodecylsulfate (SDS) in the concentration range of 0.01CMC -0.1CMC corresponding to 72.75mN/m to 68.18mN/m. The aqueous solutions of the three surfactant homologues had identical values of static surface tension at the same ratio C/CMC . Bikerman's "unit of foaminess" was measured for each particular case. It was shown that at identical equilibrium surface tensions both the foaminess and the rate of foam decay increase upon lengthening of the surfactant's hydrocarbon chain. It was indicated as well that the foaminess increases linearly upon raising the gas delivery rate until a certain critical value, above which substantial increase is observed. It was finally concluded that both the tenacious and the transient foams have completely different behaviour. For this reason they should be modeled separately but more experimental data are still needed.

Kinetics of degradation of dipalmitoylphosphatidylcholine (DPPC) bilayers as a result of vipoxin phospholipase A2 activity: an atomic force microscopy (AFM) approach.

In this paper we used AFM as an analytical tool to visualize the degradation of a phospholipid bilayer undergoing hydrolysis of the vipoxin's PLA(2). We obtained time series images during the degradation process of supported 1, 2-dipalmitoylphosphatidylcholine (DPPC) bilayers and evaluated the occurrence and the growth rate of the bilayer defects. The special resolution of the AFM images allowed us to measure the area and the perimeter length of these defects and to draw conclusions about the kinetics of the enzyme reaction. Moreover, we also report for some unique characteristics discovered during the vipoxin's PLA(2) action. Experimentally for the first time, we observed the appearance and the growth of three-dimensional (3D), crystal-like structures within the formed defects of the degraded bilayer. In an effort to explain their nature, we applied bearing image analysis to estimate the volume of these crystals and we found that their growth rate follows a similar kinetic pattern as the degradation rate of the supported bilayer.

Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A2 and Humicola lanuginosa lipase.

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A(2) (PLA(2)) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.

Effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC surface films.

Bestrophinopathies are ocular diseases caused by mutations in the human bestrophin-1 (hBest1) - transmembrane Ca2+-activated chloride channel protein, mainly expressed in the retinal pigment epithelium (RPE) cells. hBest1 is also an important transporter for neurotransmitters such as glutamate (Glu) and γ-aminobutyric acid (GABA) in the nervous system. Recently, a new biological role of hBest1, related to its possible involvement in the pathology of brain diseases (Alzheimer's, Parkinson's disease) has been proposed. Here, we report the effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) Langmuir and Langmuir-Blodgett monolayers based on surface dynamics (π/A isotherms, hysteresis and compressibility), morphology (Brewster angle microscopy, BAM) and visualization of protein molecular organization (Atomic force microscopy, AFM). Ca2+ ions and neurotransmitters Glu and GABA affect hBest1 topology at the air/water interface altering its surface activity, size, orientation and organization. In contrast, no significant changes were detected on π/A isotherms and hysteresis of the composite hBest1/POPC films but their effects on structure, aggregation state and orientation hBest1 established by BAM and AFM differentiate. We found that the binary films of hBest1 and POPC are phase separated at the air/water interface, suggesting stronger lipid-lipid and protein-protein interactions than lipid-protein interactions that can significantly alter the molecular organization and activity of hBest1 in cell membranes. Our data shed light on structure, surface behavior and organization of hBest1 that define relationship structure-functional activity of hBest1 as transport channel.

PlA2 Polymorphism in Glycoprotein IIb/IIIa Modulates the Morphology and Nanomechanics of Platelets.

Glycoprotein IIb/IIIa (GPIIb/IIIa) is the most abundant platelet surface receptor for fibrinogen and von Willebrand factor. Polymorphism PlA1/A2 in the gene of GPIIb/IIIa is among the risk factors for the development of arterial and venous thrombosis. The aim of this study is to evaluate the effect of the carriage of PlA1/A2 on the size, topographic features, and membrane stiffness of platelets from healthy controls and patients with deep venous thrombosis (DVT). Atomic force microscopy (AFM) imaging and nanoindentation (force-distance curves) were applied to investigate the morphological and nanomechanical properties (Young's modulus) of platelets immobilized on glass surface. The surface roughness ( Ra) and height ( h) of platelets from patients with DVT, carriers of mutant allele PlA2 ( Ra = 30.2 ± 6 nm; h = 766 ± 182 nm) and noncarriers ( Ra = 28.6 ± 6 nm; h = 865 ± 290 nm), were lower than those of healthy carriers of allele PlA2 ( Ra = 48.1 ± 12 nm; h = 1072 ± 338 nm) and healthy noncarriers ( Ra = 49.7 ± 14 nm; h = 1021 ± 433 nm), respectively. Platelets isolated from patients with DVT, both carriers and noncarriers, exhibit much higher degree of stiffness at the stage of spreading ( E = 327 ± 85 kPa and 341 ± 102 kPa, respectively) compared to healthy noncarriers ( E = 198 ± 50 kPa). In addition, more pronounced level of platelet activation was found in polymorphism carriers. In conclusion, the carriage of PlA2 allele modulates the activation state, morphology, and membrane elasticity of platelets.

Effects of Ca2+ ions on bestrophin-1 surface films.

Human bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein - member of the bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), ß-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160Å and 75Å height. Binding of calcium ions (Ca2+) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.

Microstructural investigations of carbon foams derived from modified coal-tar pitch.

This work reports the microstructural evaluation of carbon foams derived from coal-tar pitch precursors treated with H2SO4 and HNO3 and finally annealed at 1000°C and 2000°C. Our experimental investigations combine scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) imaging, X-ray photoelectron spectroscopy (XPS) and micro-spot near-edge X-ray absorption fine structure (µ-NEXAFS) spectroscopy. This set of complementary techniques provides detailed structural and chemical information of the surface and the bulk of the carbon foams. The high-resolution microscopy data indicate the formation of carbonaceous amorphous microspheres (average diameters of 0.28±0.01µm) embedded in the partially graphitized carbon foam matrix at 1000°C. The microspheres are enriched with sp-bonded species and their microstructural characteristics depend on the reagent (nitric vs. sulfuric acid) used for pitch treatment. A complete chemical transformation of the microspheres at temperatures >1000°C occurs and at 2000°C they are spectroscopically identical with the bulk material (sp(2)- and sp(3)-hybridised forms of carbon). The microstructure-property relationship is exemplified by the compressive strength measurements. These results allow a better description of coal-tar pitch-derived carbon foams at the atomic level, and may account for a better understanding of the processes during graphitization step.

Humicola lanuginosa lipase hydrolysis of mono-oleoyl-rac-glycerol at the lipid-water interface observed by atomic force microscopy.

A new type of planar lipid substrate for Humicola lanuginosa lipase (HLL) has been prepared by depositing a monolayer of 1-mono-oleoyl-rac-glycerol (MOG) on top of a monolayer of dipalmitoyl-phosphatidylcholine (DPPC) on mica by the Langmuir-Blodgett (LB) technique. The bilayer was subsequently exposed to HLL in a liquid cell of an atomic force microscope (AFM) allowing the time course of the lipolytic degradation to be observed. By analysing a series of AFM images, we find that enzymes are preferentially activated at the edge of nano-scale defects present in the bilayer prior to enzyme injection, while defect-free areas of the substrate are surprisingly stable towards enzyme degradation. The initial rate of hydrolysis is found to be proportional to the perimeter length, P, of the initial nano-scale defects as well as the bulk enzyme concentration, c(HLL); d(lipid)/dt=k P c(HLL). We estimate the specific rate of MOG hydrolysis by HLL to be 2.5x10(4) MOG molecules/(minute x molecule of HLL).

The linker histone in Saccharomyces cerevisiae interacts with actin-related protein 4 and both regulate chromatin structure and cellular morphology.

Chromatin structure promotes important epigenetic mechanisms that regulate cellular fate by organizing, preserving and controlling the way by which the genetic information works. Our understanding of chromatin and its functions is sparse and not yet well defined. The uncertainty comes from the complexity of chromatin and is induced by the existence of a large number of nuclear proteins that influence it. The intricate interaction among all these structural and functional nuclear proteins has been under extensive study in the recent years. Here, we show that Saccharomyces cerevisiae linker histone physically interacts with Arp4p (actin-related protein 4) which is a key subunit of three chromatin modifying complexes - INO80, SWR1 and NuA4. A single - point mutation in the actin - fold domain of Arp4p together with the knock-out of the gene for the linker histone in S. cerevisiae severely abrogates cellular and nuclear morphology and leads to complete disorganizing of the higher levels of chromatin organization.

Foam production--ratio between foaminess and rate of foam decay.

Foams are usually characterized by the foaminess of their surfactant solutions and the rate of foam decay. These two properties have been described many times separately in the literature. There is a certain correlation between them, which can vary depending on the type and the concentration of the surfactants, the method of foam generation, etc. We suggest with this work a new parameter unifying foaminess and rate of foam decay. The foam production is a parameter, which is a ratio between foaminess and rate of foam decay. It was shown an example how foaminess, rate of foam decay and foam production depends on C/CMC (C - surfactant concentration, CMC - critical micelle concentration) of aqueous solutions of sodium octylsulfate (SOS). In addition, it has been stressed that a number of scientific problems on transient foams can be solved by means of the approach pointed out by this study. An example, for which the foam production depends on the way of foam generation, is given. A new criterion for assessing the ability of the surfactants to stabilize foams has been suggested. Thus, the stronger surfactants do not always produce more stable transient foams than the weaker ones, as usually is assumed.

Delta-comb potential in modeling three-phase contact line (TPCL) on periodically patterned surfaces.

This work is a study of wetting of small water droplets on smooth glass surfaces with periodic patterns in the form of imprinted net with hydrophilic cells and hydrophobic bars. Microcover slides consisted of soda lime glass were used. The imprinted images of the net were with cell sizes in the range 40-200 µm, which corresponds to a quite narrow scope of hydrophilic surface fractions f(1)(30-36%) due to the relative increase in the size of the hydrophobic bars. The receding contact angles θ(R) of small water droplets, positioned on the patterned surfaces, were measured. The experiment showed significantly lower receding contact angles as compared to the theoretical expectations by the Cassie formula, which accounts for the contribution to the contact angle of the surface fraction of the imprinted hydrophobic/hydrophilic net. For this reason, we developed new theory accounting for the periodicity of the surface and the contribution of the three-phase contact line on the contact angle. This new theory considered delta-comb potential energy Δ(x,y) of the surface, effective line tension κ, and the lattice parameter a. The restriction of theory was discussed as well. It was pointed out that the theory is not valid for very small and very large lattice parameters.

Savinase action on bovine serum albumin (BSA) monolayers demonstrated with measurements at the air-water interface and liquid Atomic Force Microscopy (AFM) imaging.

We studied the enzymatic action of Savinase on bovine serum albumin (BSA) organized in a monolayer spread at the air/water interface or adsorbed at the mica surface. We carried out two types of experiments. In the first one we followed the degradation of the protein monolayer by measuring the surface pressure and surface area decrease versus time. In the second approach we applied AFM imaging of the supported BSA monolayers adsorbed on mica solid supports and extracted information for the enzyme action by analyzing the obtained images of the surface topography in the course of enzyme action. In both cases we obtained an estimate for the turnover number (TON) of the enzyme reaction.

Water in contact with extended hydrophobic surfaces: direct evidence of weak dewetting.

X-ray reflectivity measurements reveal a significant dewetting of a large hydrophobic paraffin surface floating on water. The dewetting phenomenon extends less than 15 A into the bulk water phase and results in an integrated density deficit of about one water molecule per 25-30 A(2) of water in contact with the paraffin surface. The results are supported by molecular dynamics simulations and related to the hydrophobic effect.