Fluid Phase System Research Articles

No violations of critical-point wetting in ternary three fluid-phase systems with short range interactions

No violations of critical-point wetting in ternary three fluid-phase systems with short range interactions

The covalent binding story of the complement proteins C3 and C4 (I) 1972-1981

Alex Law and Paul Levine recall their work to establish the covalent bond between C3 and target surfaces. It started with a naive experiment by analyzing the membrane polypeptides of sheep erythrocytes bound with 125I-labelled C3. They found complexes with molecular weight higher than the individual C3 polypeptides. These complexes survived all conditions designed to disrupt non-covalent interactions. They then showed that the bond was an ester, with an active acyl group on C3 which reacted with a hydroxyl group on the acceptor molecule. With the discovery of an internal thioester by Jim Prahl, Jamila Janatova, Brian Tack and their colleagues, it became clear that the reaction was by an acyl transfer from the thioester of C3 to the target hydroxyl group. Later on they showed that C4 also bound covalently to target molecules. By establishing a fluid phase system to study the kinetics of the binding reactions of C3 and C4, Alex was able to continue the work in the MRC Immunochemistry Unit in Oxford from 1981, to eventually determine the chemical mechanism of the binding reaction. In order to give some sense of reality, this article is written as a narrative from Alex, who did the experiments. Both Alex and Paul are retired. Pauls lives on Martha's Vineyard where he writes occasional articles on science for one of the Island's newspapers. Alex lives in Hong Kong and tries to make some sense of the local politics.

Combined force spectroscopy, AFM and calorimetric studies to reveal the nanostructural organization of biomimetic membranes

In this work we studied a binary lipid matrix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), a composition that mimics the inner membrane of Escherichia coli. More specifically, liposomes with varying fractions of POPG were analysed by differential scanning calorimetry (DSC) and a binary phase diagram of the system was created. Additionally, we performed atomic force microscopy (AFM) imaging of supported lipid bilayers (SLBs) of similar compositions at different temperatures, in order to create a pseudo-binary phase diagram specific to this membrane model. AFM study of SLBs is of particular interest, as it is conceived as the most adequate technique not only for studying lipid bilayer systems but also for imaging and even nanomanipulating inserted membrane proteins. The construction of the above-mentioned phase diagram enabled us to grasp better the thermodynamics of the thermal lipid transition from a gel-like POPE:POPG phase system to a more fluid phase system. Finally, AFM force spectroscopy (FS) was used to determine the nanomechanics of these two lipid phases at 27°C and at different POPG fractions. The resulting data correlated with the specific composition of each phase was calculated from the AFM phase diagram obtained. All the experiments were done in the presence of 10mM of Ca2+, as this ion is commonly used when performing AFM with negatively charged phospholipids.

Determination of anti-PF4/heparin complex antibodies in the fluid phase (P4549)

Abstract Antibodies against the human platelet factor 4/heparin complex (PF4/H) are found in the immune-mediated adverse drug reaction heparin-induced thrombocytopenia. These antibodies are directed against PF4 within the complex due to conformational changes of PF4. To study the humoral immune response to PF4/H in greater detail, C57BL/6 mice were immunized with murine (mu) PF4/H by retro-orbital injection. Serum was obtained, and analyzed by ELISA either with muPF4/H directly plate-bound (solid phase), or in a fluid phase assay with muPF4/H that contained 10% biotinylated muPF4. The two approaches differed notably in their specificity and sensitivity to detect anti-muPF4/H antibodies. The solid phase ELISA could not distinguish between antibodies against plate-bound muPF4 alone and plate-bound muPF4/H. Only incubation with excess heparin allowed to distinguish these antibodies. In contrast, the fluid phase ELISA permitted the direct distinction between anti-muPF4 and anti-muPF4/H antibodies. The fluid phase system was also successfully applied to detect muPF4/H-specific B cells by ELISpot. Thus, the fluid phase ELISA for the detection of anti-muPF4/H antibodies was superior to the solid phase assay. Furthermore, our data suggest that surface binding of PF4 might induce conformational changes similar to those induced by heparin binding. Finally, fluid phase ELISAs might be preferable for the detection of antibodies that bind to conformation-dependent epitopes.

Chemical equilibrium in a heterogeneous fluid phase system: thermodynamic regularities and topology of phase diagrams

Singularities of the systems with simultaneous phase and chemical equilibria were examined. The thermodynamic regularities were revealed and topology of phase diagrams was presented for multicomponent heterogeneous systems including two or several equilibrium liquid phases (solution layering). The thermodynamic conditions for displacement of phase and chemical equilibrium are considered.

Transesterification of rapeseed oil over acid resins promoted by supercritical carbon dioxide

The methanolysis of rapeseed oil catalyzed by commercial styrene-divinylbenzene macroporous acid resins was performed in a batch reactor at 100–140°C and 10–46MPa to study the effect of supercritical carbon dioxide (scCO2) on the performances of the process. Reaction temperatures of 120–140°C were necessary to obtain high enough yields of fatty acid methyl esters. Upon addition of scCO2 faster transesterification kinetics was obtained also at the lowest investigated operating pressure (10–11MPa), working in two fluid phase systems. Experiments performed changing the reaction time indicated that most of the esters were formed during the first 3h. When the pressure was increased at 38–46MPa, the fluid phases merged in a single one without significant modification of the performances of the process.The enhancement effect of scCO2 on the transesterification kinetics is tentatively discussed in terms of modification of the phase behaviour of the reaction system and swelling of the polymeric acid resin.

Influence of Clay Lens on Migration of Light Nonaqueous Phase Liquid in Unsaturated Zone

This study concerns the control of movement of light nonaqueous phase liquid (LNAPL) in unsaturated zone in the presence of relatively low permeability clay lens. A two-dimensional, finite-difference numerical model for the simultaneous movement of LNAPL and water through the unsaturated zone of the soil has been developed. The system is a three fluid phase system (water, LNAPL, and air) but in the derivation of the model, air was treated as an immobile phase at constant atmospheric pressure. The flow equations for LNAPL and water were cast in terms of the wetting and non-wetting fluid pressure heads, respectively. The finite-difference equations were solved implicitly with explicitly scheme using Newton-Raphson iteration with Taylor series expansion to treat nonlinearity. A physical model to represent the infiltration of kerosene above the clay lens was constructed. The numerical results were compared with those observed experimentally. The results of all tests showed that the presence of clay lens controls the vertical movement of LNAPL in heterogeneous porous medium.

INFLUENCE OF LENS ON MIGRATION OF DENSE NON-AQUEOUS PHASE LIQUID IN SATURATED ZONE

This study concerns the control of movement of Dense Non-Aqueous Phase Liquid (DNAPL) in saturated zone in the presence of relatively low permeability lens. A two-dimensional, finite-difference numerical model for the simultaneous movement of the DNAPL and water through the saturated zone of the soil is developed. The system is, actually, a three fluid phase system (water, DNAPL and air) but in the derivation of the model, air was treated as an immobile phase at constant atmospheric pressure. The flow equations for Dense Non-Aqueous Phase Liquid and water are cast in terms of the wetting and non-wetting fluid pressure heads respectively. The finite- difference equations are solved fully implicitly using Newton-Raphson iteration scheme with a Taylor series expansion to treat the nonlinearity. The present numerical results are compared with results of Kueper and Frind (1991b). The results of all tests showed that the presence of lens controls the vertical movement of Dense Non-Aqueous Phase Liquid (DNAPL) in heterogeneous porous medium.

A modified light transmission visualization method for DNAPL saturation measurements in 2-D models

In this research, a light transmission visualization (LTV) method was used to quantify dense non-aqueous phase liquids (DNAPL) saturation in two-dimensional (2-D), two fluid phase systems. The method is an expansion of earlier LTV methods and takes into account both absorption and refraction light theories. Based on this method, DNAPL and water saturations can rapidly be obtained point wise across sand-packed 2-D flow chambers without the need to develop a calibration curve. A single point calibration step is, however, needed when dyed DNAPL is used to account for the change in the transmission factor at the dyed DNAPL–water interface. The method was applied to measure, for the first time, undyed DNAPL saturation in small 2-D chambers. Known amounts of DNAPL, modeled by tetrachloroethylene (PCE), were added to the chamber and these amounts were compared to results obtained by this LTV method. Strong correlation existed between results obtained based on this method and the known PCE amounts with an R 2 value of 0.993. Similar experiments conducted using dyed PCE showed a stronger correlation between results obtained by this LTV method and the known amounts of dyed PCE added to the chamber with an R 2 value of 0.999. The method was also used to measure dyed PCE saturation in a large 2-D model following sparging experiments. Results obtained from image analyses following each sparging event were compared to results obtained by two independent techniques, namely gas chromatography–mass spectrometry (GC/MS) analyses and carbon column extraction. There was a good agreement between the results obtained by this LTV method and those obtained by the two independent techniques when experiments were carried out under stable light source conditions and errors in mass balance were minor. The method presented here can be expanded to measure fluid contents in three fluid phase systems and provide a non-destructive, non-intrusive tool to investigate changes in DNAPL architecture and flow characteristics in laboratory experiments.

Fibrinolysis at the Fluid-Solid Interface of Thrombi

Thrombolysis is conventionally regarded as dissolution of the fibrin matrix of thrombi by plasmin, a protease generated by plasminogen activators from its inactive precursor, plasminogen. Typically plasminogen activation occurs on the surface of the clot, where fibrin behaves as a cofactor in this process, and plasmin also initiates its proteolytic action at the fluid-solid interface. Although the basic reactions of the plasminogen/plasmin system in fluid phase are well characterized in terms of classical enzymology, they cannot explain completely the interfacial fibrinolytic events. Recently new methods have been introduced for quantitative evaluation of plasminogen activation on gel-phase fibrin and heterogenous-phase proteolysis, an overview of the new methodology is presented. Following formation of an interfacial lytic zone, fibrin dissolution proceeds through propagation of this zone to the core of the clot, which depends on diffusion and permeation phenomena affected by the composition of thrombi. Phospholipids (originating from platelets) form a diffusion barrier to the thrombolytic agents and also bind some of them; structural cellular proteins (namely myosin) interact with the fibrin fibers masking their cofactor and plasmin-cleavage sites. The contribution of these recent findings to our understanding of the limitations of current thrombolytic therapy is discussed. Finally, attention is focused on the termination of thrombus-associated proteolytic action in an environment abundant in proteinase inhibitors. Thus, combining together the interfacial events in the initiation, progress and termination of thrombolysis, a concept for modeling the thrombus as a temporary fibrinolytic compartment is presented.

Toward rational design of powder processes

The past 25 years has seen particle technology grow from an under-funded and widely scattered research enterprise to a thriving globally recognized engineering discipline. Despite this change in the research environment, design and analysis of industrial particulate processes remain rooted in empiricism. Scale-up is largely heuristic, and quantitative design methods are non-existent. This is in stark contrast to fluid-phase systems, for which accurate scale-up and design methods have existed for decades. Why is this? One explanation lies in the traditional approaches to studying particle technology operations. Typically, these are studied at two length scales: the macroscale (unit operation level) and the microscale (particle level). Relatively little attention has been directed at an intermediate length scale, the mesoscale, which is characteristic of a “homogeneous” powder. This situation is analogous to ignoring classical thermodynamics and transport properties of fluids in the analysis of fluid-phase operations, instead trying to model unit operation performance directly using statistical mechanics or molecular dynamics. Advances in the design and analysis of particle technology operations requires filling of this “scale gap.” This can be accomplished by studying particulate systems in an analogous way to that used to study fluid-phase unit operations. This will require detailed investigation of mass, momentum, and energy transport in existing unit operations; development of experimental systems with well-defined and characterized flow fields; measurement of powder properties (transport properties and transformation kinetics) in these “simple” experimental systems; validation of theory and simulation against these data; and integration of these theories and simulations into system-level models.

Rat C-reactive protein activates the autologous complement system.

Activation of complement is a biological function of human C-reactive protein (hCRP), whereas rat CRP (rCRP) has been claimed to be unable to activate complement. As important biological functions of proteins are probably conserved among species, we re-evaluated, using various ligands, the capability of rCRP to activate complement. The activation of complement by hCRP and rCRP was investigated in solid- and fluid-phase systems. In the solid-phase system, purified CRP was fixed to enzyme-linked immunosorbent assay (ELISA) plates and incubated with human or rat recalcified plasma. Dose-dependent binding of human and rat C3 and C4 was observed to human and rat CRP, respectively. In the fluid-phase system, recalcified rat plasma, which contains about 500 mg/l of CRP, or human plasma supplemented with hCRP, were incubated with lyso-phosphatidylcholine. A dose-dependent activation of complement was observed upon incubation with this ligand, as reflected by the generation of activated C4 as well as of CRP-complement complexes. This activation was, in both cases, inhibited by preincubation of plasma with p-aminophosphorylcholine, a specific inhibitor of the interaction of CRP with its ligands, or by chelation of calcium ions. We conclude that rat CRP, similarly to human CRP, can activate autologous complement. These results support the notion that opsonization of ligands with complement is an important biological function of CRP.

A set of constitutive relationships accounting for residual NAPL in the unsaturated zone

Although laboratory experiments show that non-aqueous phase liquid (NAPL) is retained in the unsaturated zone, no existing multiphase flow model has been developed to account for residual NAPL after NAPL drainage in the unsaturated zone. We developed a static constitutive set of saturation–capillary pressure relationships for water, NAPL and air that accounts for both this residual NAPL and entrapped NAPL. The set of constitutive relationships is formulated similarly to the set of scaled relationships that is frequently applied in continuum models. The new set consists of three fluid-phase systems: a three-phase system and a two-phase system, that both comply with the original constitutive model, and a newly introduced residual NAPL system. The new system can be added relatively easily to the original two- and three-phase systems. Entrapment is included in the model. The constitutive relationships of the non-drainable residual NAPL system are based on qualitative fluid behavior derived from a pore scale model. The pore scale model reveals that the amount of residual NAPL depends on the spreading coefficient and the water saturation. Furthermore, residual NAPL is history-dependent. At the continuum scale, a critical NAPL pressure head defines the transition from free, mobile NAPL to residual NAPL. Although the P c– S relationships for water and total liquid are not independent in case of residual NAPL, two two-phase P c– S relations can represent a three-phase residual system of P c– S relations. A newly introduced parameter, referred to as the residual oil pressure head, reflects the mutual dependency of water and oil. Example calculations show consistent behavior of the constitutive model. Entrapment and retention in the unsaturated zone cooperate to retain NAPL. Moreover, the results of our constitutive model are in agreement with experimental observations.

Mechanism of anti-HIV activity of succinylated human serum albumin

In the present study, we described the interaction of succinylated human serum albumin (Suc-HSA), a negatively charged anti-HIV-1 active protein, with HIV-1 gp120 and in detail with the third variable domain of gp120 (V3 loop). To this end, different assay formats were tested in which gp120- and V3-related peptides were presented in various configurations in order to investigate the effect of the conformational structure of the V3 loop on the interaction with negatively charged albumins. When gp120 presented via a lectin was used, it was observed that Suc-HSA bound to native gp120. The binding site appeared to be located at or near the thrombin digestion site (GPGRAF sequence) in the V3 loop of gp120, since the cleavage of the loop resulted in decreased binding of Suc-HSA. In addition, Suc-HSA was able to protect the V3 region of gp120 from cleaving with thrombin. In contrast, significant binding of Suc-HSA to V3 loop or gp120 peptides was not observed when both were presented in a fluid phase system, suggesting the involvement of a monovalent-low affinity binding of Suc-HSA. Using overlapping peptides delineating the whole V3 loop immobilized to CNBr–Sepharose, we noticed that the interaction of the V3 loop with Suc-HSA was predominantly induced by electrostatic interactions between positively charged linearized peptide fragments and Suc-HSA and was positively influenced by the presence of hydrophobic amino in the V3 loop fragments as well. Moreover, the highest affinity site was located at sites near the GPGRAF sequence. These observations add to the evidence, collected earlier, that Suc-HSA interferes at the level of virus entry, independent of interaction with the CD4 receptor. Since the recently discovered chemokine receptors are negatively charged, we can hypothesize that Suc-HSA is able to prevent the positively charged V3 loop from interacting with these types of receptors, thereby inhibiting virus entry.

Interaction between serum amyloid P component and C4b-binding protein associated with inhibition of factor I-mediated C4b degradation.

Serum amyloid P component (SAP) is a member of the pentraxin protein family. Although present in all types of amyloid deposits, it is also a normal constituent of blood and extravascular tissues. In blood, SAP forms a calcium-dependent, noncovalent complex with C4b-binding protein (C4BP). C4BP regulates the classical complement pathway as it binds C4b and functions as cofactor in its degradation by factor I. Although SAP and C4b bind to distinct sites on C4BP, it is not known whether the SAP-C4BP interaction affects the function of C4BP. We report that in a fluid phase system, SAP inhibited degradation of C4(H2O) (which is C4b-like) in a dose-dependent manner. Phosphorylethanolamine was found to alleviate the inhibitory effect of SAP on C4(H2O) degradation. Because this compound is known to inhibit the SAP-C4BP interaction, this indicated direct binding of SAP to C4BP to be required for inhibition of C4(H2O) degradation. Even though C4BP, C4(H2O), and SAP form a multimolecular complex in fluid phase, SAP was found to inhibit binding of C4BP to immobilized C4(H2O). The inhibitory effect was calcium dependent and alleviated by phosphorylethanolamine. Heparin, which is known to inhibit the interaction between SAP and C4BP, was also found to counteract the inhibitory effect of SAP on C4BP binding to C4(H2O). However, the effect of heparin was biphasic because high concentrations of heparin directly inhibited binding of C4(H2O) to C4BP. The inhibition of C4BP function by SAP suggests that SAP may be involved in regulation of the classical complement pathway C3 convertase.

Biochemical studies on the interaction of fibronectin with Ig.

We have previously biochemically characterized three separate sites on the fibronectin (Fn) molecule that interact with IgG. These studies have been extended to examine the interaction of Fn with other classes and subclasses of Ig. By ELISA, a preferential quantitative binding order of Fn to the major Ig classes and subclasses was obtained as follows: IgG greater than IgM greater than IgA, IgG1 greater than IgG3 = IgG4 greater than IgG2, and IgA1 = IgA2. Using fragments of Fn obtained by subtilisin digestion followed by IgM and IgA affinity chromatography, immunoblot analysis using monospecific antisera to separate regions of the Fn molecule, and amino acid sequence analysis, these studies indicate that polyclonal IgA and IgM interact with Fn in the same three regions and under the same ionic conditions as previously described for IgG. Site 1 is a 22-kDa fragment that commences at residue 1 of the Fn molecule. Sites 2 (16 kDa) and 3 (26-29 kDa) begin at residues 588 and 1597, respectively. Under physiological conditions a monoclonal antibody that recognizes site 1 completely inhibited the interaction of intact Fn with IgG, IgM, and IgA. Therefore, this is the only physiologically active site in the intact molecule. Aggregated but not monomeric IgG competitively inhibited the binding of Fn to IgG-coated microtiter ELISA plates; thus, this interaction can take place in a fluid-phase system. These results indicate that Fn can potentially interact with immune complexes and aggregates of all Ig in the circulation and thus may play a significant role in both their clearance and deposition in Fn-containing tissues, such as occurs in immune-complex-related disorders.

Binding of iron to the 5th component of human complement directs oxygen radical-mediated conversion to specific sites and causes nonenzymic activation.

It was earlier found that hydroxyl radicals (OH.) generated in a fluid phase system, convert human C5 to an activated, C5b-like form without cleavage. For the conversion catalytic amounts of divalent iron were necessary; upon their oxidation OH. are generated. In view of the extraordinary reactivity of OH. their specific effect on C5 was surprising. It has now turned out that C5 binds iron, about 13-15 Fe ions per molecule C5. C5 was fully bound to iron-loaded Chelating Sepharose and in this way removed form the medium. Only at the sites where iron is bound, is OH. generation possible. The half-life of OH. is extremely short and hence the radicals can act on C5 only in the immediate environment of the binding sites for iron. In this way the oxidative change of C5, induced by the radicals, is restricted to some sites on the molecule determined by the iron binding. The hydroxyl radical scavenger, dimethylsulfoxide (DMSO), is unable to interfere with the conversion of C5 by OH. generated on iron-Sepharose, it does not reach the site of reaction in that system. In fluid phase systems DMSO does inhibit the conversion of C5 to C5b-like C5.

Determining three-phase permeability—saturation—pressure relations from two-phase system measurements

A mathematical model is outlined for fluid flow in porous media containing up to three fluid phases: air, water and nonaqueous phase liquid (NAPL). Nonhysteretic saturation-pressure relations are described using a scaled parametric function, assumed to be unique to the porous medium and fluid-pair dependent scaling coefficients which scale S w( h ow) and S t(( h ao) in three fluid-phase systems or S w( h aw) in two fluid-phase systems, where S w and S t are water and total liquid saturation and h ow, h ao and h aw are NPL-water, air-NAPL and air-water capillary pressures, respectively Expressions for fluid relative permeabilities are derived from the saturation-pressure functions using a generalized Kozeny-Carman-type analysis. Three methods are considered for calibration of the constitutive model from two-phase system measurements: (1) direct measurement of saturation-pressure relations in air-water, air-NAPL and NAPL-water systems, (2) direct measurement of saturation-pressure relations in a single two-phase system with scaling coefficients estimated from interfacial tension data and (3) numerical inversion of transient two-phase displacement experiments using a nonlinear optimization procedure. The three methods are evaluated for two porous media systems with p-cymene and trichloroethylene as the nonaqueous fluids.

The role of polarity in correlations of solid-supercritical fluid phase systems

Three complementary correlations capable of modelling binary SCF equilibrium data have been examined. The results of this study have shown that neither the purely empirical semi-log correlation of enhancement factor with solvent density nor the theoretical log-log relationship between the solute concentration and solvent density predicted experimental solubility data satisfactorily over a wide range of SCF conditions. The above mentioned correlations were suitable for nonpolar solutes where distinct “fine splitting” of the solubility isotherms was observed. These simple relationships break down as the solute polarity increases, indicating that the solute-solute and solute-solvent interactions such as hydrogen bonding, acid-base, and dipole-dipole interactions need to be considered. The semi-empirical semi-log correlation of Ziger and Eckert appeared to provide a distinct advance in handling of data for both the nonpolar and polar compounds studied.

The fluid-phase binding of human C4 and its genetic variants, C4A3 and C4B1, to immunoglobulins

Covalent binding of the fourth complement protein, C4, to immune complexes is an important first step in the complement mediated processing of the complexes. Many of the initial encounters between the proteins of the complement system and antigen and antibody occur in solution, and prior to this report, studies of the interactions between them have focused on complement binding to preformed immune precipitates that most likely are not found in vivo. We have characterized the covalent binding of C4b to immunoglobulin molecules in a fluid-phase system consisting only of antibody in solution and purified C4 and Cls. We demonstrate that human C4b binds to IgG in the fluid phase, that its covalent binding is predominantly to the heavy chain of IgG, and that the covalent linkage is by either amide or acyl ester bonds. In addition, we compare the covalent binding efficiencies of two genetic variants of C4, C4A3 and C4B1, to IgG. C4A3 binds 3–4 times more IgG than C4B1 over a range of C4 concentrations, and C4A3 has a higher binding efficiency than C4B1 for IgM, IgA, IgG2a and F(ab') 2 as well as for a protein antigen, BSA. Furthermore, we found that whereas C4A3 is bound to immunoglobulins in the fluid-phase predominantly by amide linkage, C4B1 is bound by either amide or acyl ester bonds. The results presented here suggest that the covalent binding efficiency of C4A3 and C4B1 to IgG is similar to that reported for their covalent binding to small molecules.