Kinetic pathways of phase ordering in lipid raft model systems

We studied kinetic pathways of order-order transitions in bilayer lipid mixtures using a time-dependent Ginzburg-Landau (TDGL) approach. During the stripe-to-hexagonal phase transition in an incompressible two-component system, the stripe phase first develops a pearl-like instability along the phase boundaries, which grows and drives the stripes to break up into droplets that arrange into a hexagonal pattern. These dynamic features are consistent with recent experimental observations. During the disorder-to-hexagonal phase transition in an incompressible three-component system, the disordered state first passes through a transient stripelike structure, which eventually breaks up into a hexagonal droplet phase. Our results suggest experiments with synthetic vesicles where the stripelike patterns could be observed.     

Extended-ensemble docking to probe dynamic variation of ligand binding sites during large-scale structural changes of proteins

Proteins can sample a broad landscape as they undergo conformational transition between different functional states. At the same time, as key players in almost all cellular processes, proteins are important drug targets. Considering the different conformational states of a protein is therefore central for a successful drug-design strategy. Here we introduce a novel docking protocol, termed extended-ensemble docking, pertaining to proteins that undergo large-scale (global) conformational changes during their function. In its application to multidrug ABC-transporter P-glycoprotein (Pgp), extensive non-equilibrium molecular dynamics simulations employing system-specific collective variables are first used to describe the transition cycle of the transporter. An extended set of conformations (extended ensemble) representing the full transition cycle between the inward- and the outward-facing states is then used to seed high-throughput docking calculations of known substrates, non-substrates, and modulators of the transporter. Large differences are predicted in the binding affinities to different conformations, with compounds showing stronger binding affinities to intermediate conformations compared to the starting crystal structure. Hierarchical clustering of the binding modes shows all ligands preferably bind to the large central cavity of the protein, formed at the apex of the transmembrane domain (TMD), whereas only small binding populations are observed in the previously described R and H sites present within the individual TMD leaflets. Based on the results, the central cavity is further divided into two major subsites, first preferably binding smaller substrates and high-affinity inhibitors, whereas the second one shows preference for larger substrates and low-affinity modulators. These central subsites along with the low-affinity interaction sites present within the individual TMD leaflets may respectively correspond to the proposed high- and low-affinity binding sites in Pgp. We propose further an optimization strategy for developing more potent inhibitors of Pgp, based on increasing its specificity to the extended ensemble of the protein, instead of using a single protein structure, as well as its selectivity for the high-affinity binding site. In contrast to earlier in silico studies using single static structures of Pgp, our results show better agreement with experimental studies, pointing to the importance of incorporating the global conformational flexibility of proteins in future drug-discovery endeavors.

Functional states of P-glycoprotein formed during its full transition cycle (red to blue), captured by molecular dynamics simulations, form a structural framework for extended-ensemble docking of small-molecule ligands of diverse activities.     

Monitoring of neutron flux changes in short-lived neutron activation analysis

It is well known variations in neutron fluxes can adversely affect the final result in neutron activation analysis. The monitoring of neutron flux changes are usually described for medium and long-lived NAA using foils of cobalt, gold, zirconium, etc. However, for short-lived neutron activation analysis there appears to be no systematic study of the variations of the neutron flux. With our new automatic pneumatic system, where irradiation timing, decay and counting and position are very reproducible, we have performed a series of experiments using thermal and epithermal neutrons using aluminum wire as a monitor to monitor the neutron fluxes. Our experiments confirm that neutron flux fluctuations in the worst case can be up to ±12 % with a SD of 2–3 %. This effect can be seen regardless of the irradiation time and must be taken into consideration to achieve the best result.     

Kinetics of membrane raft formation: fatty acid domains in stratum corneum lipid models

The major barrier to permeability in skin resides in the outermost layer of the epidermis, the stratum corneum (SC). The major SC lipid components are ceramides, free fatty acids, and cholesterol. Ternary mixtures containing these constituents are widely used for physicochemical characterization of the barrier. Prior X-ray diffraction and IR spectroscopy studies have revealed the existence of ordered lipid chains packed in orthorhombic subcells. To monitor the kinetics of formation of regions rich in fatty acids, the current study utilizes a modification of the method (J. Phys. Chem. 1992, 96, 10008) developed to monitor component demixing in n-alkane mixtures. The approach is based on changes in the scissoring or rocking mode contours in the IR spectra of (orthorhombically packed) ordered chains. In the current study, equimolar mixtures of ceramides (either non-hydroxy fatty acid sphingosine ceramide or alpha-hydroxy fatty acid sphingosine ceramide) with chain perdeuterated fatty acids (either palmitic or stearic acid) and cholesterol reveal a time evolution of the scissoring contour of the deuterated fatty acid chains following quenching from relatively high temperatures where random mixing occurs. Segregation of domains enriched in the fatty acid component is observed. The kinetics of segregation are sensitive to the quenching temperature and to the chemical composition of the mixture. The kinetic regimes are conveniently catalogued with a power law of the form P=Ktalpha where P is a (measured) property related to domain composition. The time scales for demixing in these experiments are similar to times observed in several studies that have tracked the restoration of the in vivo permeability barrier following nonthermal challenges to SC integrity. Further evidence for the physiological importance of the current measurements is the detection of these phases in native SC. The current work constitutes the first direct, structure-based determination of the kinetics of barrier formation in relevant skin lipid barrier models.     

Ammonium nitrate changes during thermal analysis

A study was made of the difference occurring in ammonium nitrate during investigation by DTA and by DSC. The results show that the DTA of ammonium nitrate (p.a.) is accompanied by the crystal transformation IV III II I and melting. During DSC investigation of the same ammonium nitrate samples, the salt undergoes the transformations IV III and IV II in parallel; then follow the crystal transformations III II I and melting. The investigation conditions and the history of the samples do not essentially influence the crystal transformations during thermal analysis.

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Zusammenfassung Bei DTA und DSC von Ammoniumnitrat zu beobachtende Unterschiede wurden untersucht. Bei der DTA von p.a. Ammoniumnitrat verlaufen die Kristallumwandlungen IV III II I sowie der Schmelzvorgang. Während der DSC der gleichen Ammoniumnitratproben verlaufen die Übergänge IV III und IV II parallel, worauf die Kristallumwandlungen III II I und der Schmelzvorgang folgen. Die experimentellen Bedingungen und die Vorgeschichte der Proben hat keinen wesentlichen Einfluß auf die während der thermischen Analyse verlaufenden Kristallumwandlungen.

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. ( ..) IV III II I . , IV III IV II - , III II ar I . .

     

Impurity diffusion during activation a significant source of systematic errors in neutron activation analysis

Using a layer etching technique impurity profiles of Cu and Fe in silicon samples are measured by n.a.a. Radiation enhanced diffusion of Cu and Fe impurities from the surface into the bulk material has been observed. The samples were etched before irradiation to remove the impurity profiles caused by the sampling procedures.     

Modeling the effect of structural changes during dynamic separation processes on MOFs

A model able to describe the effect of structural changes in the adsorbent or adsorbed phase during the dynamic (breakthrough) separation of mixtures on metal-organic frameworks (MOFs) is presented. The methodology is exemplified for a few pertinent case studies: the separation of xylene isomers and ethylbenzene on the flexible MOF MIL-53 and the rigid MOF MIL-47. At low pressures, no preferential adsorption of any component occurs on both MOFs. Contrarily, at higher pressures separation of ethylbenzene (EB) from o-xylene (oX) occurs on MIL-53 as a result of the breathing phenomenon within the MIL-53 structure. The increase in selectivity, starting from the gate-opening pressure, could be modeled by using a pressure-dependent saturation capacity for the most strongly adsorbed component oX. In the separation of m-xylene (mX) from p-xylene (pX) on the rigid MOF MIL-47, separation at higher pressures is a result of preferential stacking of pX. Here, the selectivity increases once the adsorption of pX switches from a single to a double file adsorption. By implementing a loading dependent adsorption constant for pX, the different unconventional breakthrough profiles and the observed selectivity profile on MIL-47 can be simulated. A similar methodology was used for the separation of EB from pX on MIL-47, where the separation is a result from steric constraints imposed onto the adsorption of EB.     

Hydrophobic interaction chromatography of proteins: Thermodynamic analysis of conformational changes

For BSA and β-lactoglobulin adsorption to hydrophobic interaction chromatography (HIC) stationary phases leads to conformational changes. In order to study the enthalpy (ΔH_ads), entropy (ΔS_ads), free energy (ΔG_ads) and heat capacity (Δc_p,ads) changes associated with adsorption we evaluated chromatographic data by the non-linear van’t Hoff model. Additionally, we performed isothermal titration calorimetry (ITC) experiments. van’t Hoff analysis revealed that a temperature raise from 278 to 308 K increasingly favoured adsorption seen by a decrease of ΔG_ads from −12.9 to −20.5 kJ/mol for BSA and from −6.6 to −13.2 kJ/mol for β-lactoglobulin. Δc_p,ads values were positive at 1.2 m (NH₄)₂SO₄ and negative at 0.7 m (NH₄)₂SO₄. Positive Δc_p,ads values imply hydration of apolar groups and protein unfolding. These results further corroborate conformational changes upon adsorption and their dependence on mobile phase (NH₄)₂SO₄ concentration. ITC measurements showed that ΔH_ads is dependent on surface coverage already at very low loadings. Discrepancies between ΔH_ads determined by van’t Hoff analysis and ITC were observed. We explain this with protein conformational changes upon adsorption which are not accounted for by van’t Hoff analysis.     

Structural and dynamic analysis of residual dipolar coupling data for proteins.

The measurement of residual dipolar couplings in weakly aligned proteins can potentially provide unique information on their structure and dynamics in the solution state. The challenge is to extract the information of interest from the measurements, which normally reflect a convolution of the structural and dynamic properties. We discuss here a formalism which allows a first order separation of their effects, and thus, a simultaneous extraction of structural and motional parameters from residual dipolar coupling data. We introduce some terminology, namely a generalized degree of order, which is necessary for a meaningful discussion of the effects of motion on residual dipolar coupling measurements. We also illustrate this new methodology using an extensive set of residual dipolar coupling measurements made on (15)N,(13)C-labeled human ubiquitin solvated in a dilute bicelle solution. Our results support a solution structure of ubiquitin which on average agrees well with the X-ray structure (Vijay-Kumar, et al., J. Mol. Biol. 1987, 194, 531--544) for the protein core. However, the data are also consistent with a dynamic model of ubiquitin, exhibiting variable amplitudes, and anisotropy, of internal motions. This work suggests the possibility of primary use of residual dipolar couplings in characterizing both structure and anisotropic internal motions of proteins in the solution state.     

Metabolism,transformation and dynamic changes of alkaloids in silkworm during feeding mulberry leaves

Metabolism, transformation and dynamic changes of DNJ, 2-O-α-D-Gal-DNJ, fagomine, isofagomine and 4-O-β-d-Glc-fagomine from mulberry leaves in silkworms at different instars were observed. UPLC-Q/TOF-MS and UPLC-TQ/MS methods were adopted for qualitative and quantitative analysis respectively. Three species mulberry leaves were used to feed the silkworm as controls. By analyzing and comparing the content changes of DNJ, fagomine and their derivatives in silkworms and silkworm excrement at different instar, we revealed the dynamic changes, confirmed the enrichment effect of the polyhydroxy alkaloids by silkworm, and inferred the conversion process behind this effect. The experimental results indicated that DNJ and its derivatives turned into some intermediate substances in the metabolic process, and finally they converted back and the content increased. Fagomine and its derivatives interconverted into each other in the process, 4-O-β-d-Glc-fagomine transformed into fagomine, while fagomine transformed into isofagomine.     

Monitoring the status of T-cell activation in a microfluidic system

Leukocyte adhesion to the endothelium through surface molecules such as E-selectin and intercellular adhesion molecule-1 (ICAM-1) is a critical cellular event reflecting the physiological status of both cell types. Here we present a microfluidic system that can not only easily monitor the interaction between leukocytes and endothelial cells under physiological conditions, but also screen drug candidates for potential modulation of this interaction. Shear stress, which is an important factor for the binding of activated T cells to tumor necrosis factor-alpha (TNF-α)-treated human umbilical vein endothelial cells (HUVECs), was easily controlled by adjusting the flow rate in the microfluidic system. Whole blood of patients with systemic lupus erythematosus (SLE) who have auto-reactive T cells were infused into the activated HUVECs which subsequently showed a higher level of binding compared to a control blood sample from a person without SLE. When these autoreactive T cells were treated with immunosuppressors tacrolimus and cyclosporin A, the binding of the T cells to HUVECs was dramatically decreased. Therefore, this microfluidic system is capable of differentiating the physiological status of T cells or endothelial cells representing different disease conditions, as well as being useful for the identification of novel reagents that modulate the functions of leukocytes or endothelial cells.     

Structural changes in glassy polymers studied by thermal analysis and dynamic mechanical tests

Structural changes which take place in many amorphous polymers, when they are annealed at temperatures near the glass transition temperature, have important theoretical, physical, and mechanical consequences. In this paper the possible existence of some local ordering in highlycrosslinked epoxy resins is studied. Three kinds of tests—TMA, DSC, and dynamic experiments—are used for a type of epoxy resin, cured with six different amounts of curing agent. In order to study the effect of the thermal history on the behavior of the polymer at its transition region, as well as the morphology of the materials tested, three different thermal treatments have been followed. Interesting results were derived concerning the influence of these parameters to these parameters to the mechanical characterization of the polymer.     

Interaction of a Rhodococcus sp. trehalose lipid biosurfactant with model proteins: thermodynamic and structural changes

One major application of surfactants is to prevent aggregation during various processes of protein manipulation. In this work, a bacterial trehalose lipid (TL) with biosurfactant activity, secreted by Rhodococcus sp., has been identified and purified. The interactions of this glycolipid with selected model proteins have been studied by using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Bovine serum albumin (BSA) and cytochrome c (Cyt-c) have been chosen because of their quite different secondary structures: BSA contains essentially no β-sheets and an average 66% α-helix, whereas Cyt-c possesses up to 25% β-sheets and up to 45% α-helical structure. Differential scanning calorimetry shows that addition of TL to BSA at concentrations below the critical micelle concentration (cmc) shifts the thermal unfolding temperature to higher values. FTIR indicates that TL does not alter the secondary structure of native BSA, but the presence of TL protects the protein toward thermal denaturation, mainly by avoiding formation of β-aggregates. Studies on the intrinsic Trp fluorescence of BSA show that addition of TL to the native protein results in conformational changes. BSA unfolding upon thermal denaturation in the absence of TL makes the Trp residues less accessible to the quencher, as shown by a decrease in the value of Stern-Volmer dynamic quenching constant, whereas denaturation in the presence of the biosurfactant prevents unfolding, in agreement with FTIR results. In the case of Cyt-c, interaction with TL gives rise to a new thermal denaturation transition, as observed by DSC, at temperatures below that of the native protein, therefore facilitating thermal unfolding. Binding of TL to native BSA and Cyt-c, as determined by ITC, suggests a rather nonspecific interaction of the biosurfactant with both proteins. FTIR indicates that TL slightly modifies the secondary structure of native Cyt-c, but protein denaturation in the presence of TL results in a higher proportion of β-aggregates than in its absence (20% vs 3.9%). The study of Trp fluorescence upon TL addition to Cyt-c results in a completely opposite scenario to that described above for BSA. In this case, addition of TL considerably increases the value of the dynamic quenching constant, both in native and denatured protein; that is, the interaction with the glycolipid induces conformational changes which facilitate the exposure of Trp residues to the quencher. Considering the structures of both proteins, it could be derived that the characteristics of TL interactions, either promoting or avoiding thermal unfolding, are highly dependent on the protein secondary structure. Our results also suggest the rather unspecific nature of these interactions. These might well involve protein hydrophobic domains which, being buried into the protein native structures, become exposed upon thermal unfolding.     

Structural changes in Fe-doped SrSnO3 perovskites during thermal analysis

Perovskite-type strontium stannate, SrSnO₃, is of technological interest because of its potential applications. In the present work, Fe(III)-doped SrSnO₃ perovskites were synthesized using Sn metal and the polymeric precursor method, derived from the Pechini method. Thermogravimetric evaluations in N₂ and O₂ atmospheres were performed to evaluate the possible formation of oxygen vacancies resulting from Fe(III) doping. The X-ray diffraction patterns showed the formation of an orthorhombic structure with high crystallinity after calcination at 1,073 K. The first thermal analysis, in a N₂ atmosphere, led to the partial elimination of strontium carbonate and hydroxyl groups, while the thermal analysis in an O₂ atmosphere led to an increase in the mass, indicating O₂ adsorption on the lattice, which could be related to the presence of Fe(III) and Fe(IV) in the perovskite structure. Moreover, the thermal analysis changed the symmetry of the iron-doped perovskites.     

RP-HPLC analysis of Jirakadyarishta and chemical changes during fermentation

Jirakadyarishta, an Ayurvedic formulation prepared by the fermentation of a decoction of Cuminum cyminum (seeds) is traditionally used for intestinal disorders. RP-HPLC analysis of the decoction and the final processed formulation revealed that apigenin-7-O-[galacturonide (1 --> 4)-O-glucoside] and luteolin-4'-O-glucoside-7-O-galacturonide) were the two major constituents of the decoction of C. cyminum. Selective hydrolysis of 7-O-glucosides of luteolin and apigenin during fermentation resulted in an increase in the amount of luteolin and apigenin. The 4'-O-glucoside-7-O-galacturonide of luteolin and galacturonide derivative of apigenin were not hydrolyzed during fermentation. Monomeric phenolics, together with 5-hydroxymethyl furfural (5-HMF), were also introduced into the formulation through the jaggery and other plant materials during fermentation. This communication highlights the importance of the ancient processing methods used in Ayurveda.