Role of solute drag in intestinal transport

This study presents experiments related to the role of solvent drag and solute drag in the transmembrane movement of nonelectrolytes in a perfused rat intestine preparation. Conditions were chosen to simulate the effects of luminal hyperosmolarity on the permeability of tracer solutes. Data are presented on net water flux, transepithelial potentials, and lumen-to-blood and blood-to-lumen tracer solute movements during control electrolyte perfusion and after making the perfusate hyperosmotic. The results indicate that both solvent drag and solute drag can play significant roles in the transepithelial movement of solute and solute permeabilities in the rat ileum preparation. It is suggested that the potential roles of solvent drag and solute drag should be accounted for or considered during the characterization of the mechanisms of biological membrane function.     

Effect of tryptophan derivatives on the phase properties of bilayers

Binding of several tryptophan derivatives and tryptophan-containing peptides to bilayers is examined by monitoring fluorescence enhancement as a function of lipid concentration. The thermodynamic and spectral parameters of the solutes in the bilayers of vesicles and liposomes do not exhibit any anomalous dependence upon the gel or the liquid-crystalline phase state of the bilayer. Effects of these solutes on the phase-transition profiles of the bilayers of liposomes and vesicles are examined, and the lowering of the phase-transition temperature is correlated with the mole fraction of the solute in the bilayer. The partition coefficients do not change at the main phase-transition temperature. These observations contradict the thermodynamic explanation of the solute-induced lowering of the phase-transition temperature which is based on the Van't Hoff relationship for distribution of the solute in the two coexisting phases at the phase-transition temperature. It is postulated that solute molecules bound to defect sites in bilayers modulate the phase properties of bilayers. These defect sites are induced in the gel phase of bilayers of liposomes above the subtransition temperature.     

Molecular dynamics simulations of peptides and proteins with amplified collective motions

We present a novel method that uses the collective modes obtained with a coarse-grained model/anisotropic network model to guide the atomic-level simulations. Based on this model, local collective modes can be calculated according to a single configuration in the conformational space of the protein. In the molecular dynamics simulations, the motions along the slowest few modes are coupled to a higher temperature by the weak coupling method to amplify the collective motions. This amplified-collective-motion (ACM) method is applied to two test systems. One is an S-peptide analog. We realized the refolding of the denatured peptide in eight simulations out of 10 using the method. The other system is bacteriophage T4 lysozyme. Much more extensive domain motions between the N-terminal and C-terminal domain of T4 lysozyme are observed in the ACM simulation compared to a conventional simulation. The ACM method allows for extensive sampling in conformational space while still restricting the sampled configurations within low energy areas. The method can be applied in both explicit and implicit solvent simulations, and may be further applied to important biological problems, such as long timescale functional motions, protein folding/unfolding, and structure prediction.     

The Balance of Fluid and Osmotic Pressures across Active Biological Membranes with Application to the Corneal Endothelium

The movement of fluid and solutes across biological membranes facilitates the transport of nutrients for living organisms and maintains the fluid and osmotic pressures in biological systems. Understanding the pressure balances across membranes is crucial for studying fluid and electrolyte homeostasis in living systems, and is an area of active research. In this study, a set of enhanced Kedem-Katchalsky (KK) equations is proposed to describe fluxes of water and solutes across biological membranes, and is applied to analyze the relationship between fluid and osmotic pressures, accounting for active transport mechanisms that propel substances against their concentration gradients and for fixed charges that alter ionic distributions in separated environments. The equilibrium analysis demonstrates that the proposed theory recovers the Donnan osmotic pressure and can predict the correct fluid pressure difference across membranes, a result which cannot be achieved by existing KK theories due to the neglect of fixed charges. The steady-state analysis on active membranes suggests a new pressure mechanism which balances the fluid pressure together with the osmotic pressure. The source of this pressure arises from active ionic fluxes and from interactions between solvent and solutes in membrane transport. We apply the proposed theory to study the transendothelial fluid pressure in the in vivo cornea, which is a crucial factor maintaining the hydration and transparency of the tissue. The results show the importance of the proposed pressure mechanism in mediating stromal fluid pressure and provide a new interpretation of the pressure modulation mechanism in the in vivo cornea.     

Modeling of neutral solute transport in a dynamically loaded porous permeable gel: implications for articular cartilage biosynthesis and tissue engineering

A primary mechanism of solute transport in articular cartilage is believed to occur through passive diffusion across the articular surface, but cyclical loading has been shown experimentally to enhance the transport of large solutes. The objective of this study is to examine the effect of dynamic loading within a theoretical context, and to investigate the circumstances under which convective transport induced by dynamic loading might supplement diffusive transport. The theory of incompressible mixtures was used to model the tissue (gel) as a mixture of a gel solid matrix (extracellular matrix/scaffold), and two fluid phases (interstitial fluid solvent and neutral solute), to solve the problem of solute transport through the lateral surface of a cylindrical sample loaded dynamically in unconfined compression with frictionless impermeable platens in a bathing solution containing an excess of solute. The resulting equations are governed by nondimensional parameters, the most significant of which are the ratio of the diffusive velocity of the interstitial fluid in the gel to the solute diffusivity in the gel (Rg), the ratio of actual to ideal solute diffusive velocities inside the gel (Rd), the ratio of loading frequency to the characteristic frequency of the gel (f), and the compressive strain amplitude (epsilon 0). Results show that when Rg > 1, Rd < 1, and f > 1, dynamic loading can significantly enhance solute transport into the gel, and that this effect is enhanced as epsilon 0 increases. Based on representative material properties of cartilage and agarose gels, and diffusivities of various solutes in these gels, it is found that the ranges Rg > 1, Rd < 1, correspond to large solutes, whereas f > 1 is in the range of physiological loading frequencies. These theoretical predictions are thus in agreement with the limited experimental data available in the literature. The results of this study apply to any porous hydrated tissue or material, and it is therefore plausible to hypothesize that dynamic loading may serve to enhance solute transport in a variety of physiological processes.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

Using high-performance liquid chromatography to measure the effects of protein-stabilizing cosolvents on a model protein and fluorescent probes

The effect of cosolvents on the fluorescence of solutes was measured manually and in an automated high-performance liquid chromatography (HPLC) system that eliminates fluorescent contaminants on-line. The HPLC system was used to show that the effect of cosolvents on the fluorescence spectrum of heated chymotrypsin (a measure of unfolding) correlates with the effect of the solutes on the heat stabilization of catalytic activity; r2=0.73 with 12 example cosolvents. Changes in the fluorescence of model probes showed that known counteracting solutes slightly decrease the polarity of the solvent. Different cosolvents affect the proton transfer indicator, 2-naphthol (a model for tyrosinyl residues) differently, polyhydric alcohols enhance the protonated naphthol emission whereas zwitterionic solutes enhance naphthoxide fluorescence. The results with the automated system are consistent with the known stabilizing effects of the cosolvents and validate it as a tool to explore the development of novel cosolvents and their effects on multiple biological systems.     

Role of water in some biological processes.

The state of intracellular water has been a matter of controversy for a long time for two reasons. First, experiments have often given conflicting results. Second, hitherto, there have been no plausible grounds for assuming that intracellular water should be significantly different from bulk water. A collective behavior of water molecules is suggested here as a thermodynamically inevitable mechanism for generation of appreciable zones of abnormal water. At a highly charged surface, water molecules move together, generating a zone of water perhaps 6 nm thick, which is weakly hydrogen bonded, fluid, and reactive and selectively accumulates small cations, multivalent anions, and hydrophobic solutes. At a hydrophobic surface, molecules move apart and local water becomes strongly bonded, inert, and viscous and accumulates large cations, univalent anions, and compatible solutes. Proteins and many other biopolymers have patchy surfaces which therefore induce, by the two mechanisms described, patchy interfacial water structures, which extended appreciable distances from the surface. The reason for many conflicting experimental results now becomes apparent. Average values of properties of water measured in gels, cells, or solutions of proteins are often not very different from the same properties of normal water, giving no indication that they are averages of extreme values. To detect the operation of this phenomenon, it is necessary to probe selectively a single abnormal population. Examples of such experiments are given. It is shown that this collective behavior of water molecules amounts to a considerable biological force, which can be equivalent to a pressure of 1,000 atm (1.013 x 10(5) kPa). It is suggested that cells selectively accumulate K+ ions and compatible solutes to avoid extremes of water structure in their aqueous compartments, but that cation pumps and other enzymes exploit the different solvent properties and reactivities of water to perform work of transport or synthesis.     

Diffusion coefficient in biomembrane critical pores

Diffusion is fundamental to the random movement of solutes in solution throughout biological systems. Theoretical studies of diffusing solutes across cell membranes confined in a microscopic size of pores have been an interesting subject in life and medical sciences. When a solute is confined in a critical area of membrane pores, which shows a quite different behavior compared to the homogeneous-bulk fluid whose transport is isotropic in all directions. This property has novel features, which are of considerable physiological interest.     

Compatible solute addition to biological systems treating waste/wastewater to counteract osmotic and other environmental stresses: a review

This study reviews the addition of compatible solutes to biological systems as a strategy to counteract osmolarity and other environmental stresses. At high osmolarity many microorganisms accumulate organic solutes called “compatible solutes” in order to balance osmotic pressure between the cytoplasm and the environment. These organic compounds are called compatible solutes because they can function inside the cell without the need for special adaptation of the intracellular enzymes, and also serve as protein stabilizers in the presence of high ionic strength. Moreover, the compatible solutes strategy is regularly being employed by the cell, not only under osmotic stress at high salinity, but also under other extreme environmental conditions such as low temperature, freezing, heat, starvation, dryness, recalcitrant compounds and solvent stresses. The accumulation of these solutes from the environment has energetically a lower cost than de novo synthesis. Based on this cell mechanism several studies in the field of environmental biotechnology (most of them on biological wastewater treatment) employed this strategy by exogenously adding compatible solutes to the wastewater or medium in order to alleviate environmental stress. This current paper critically reviews and evaluates these studies, and examines the future potential of this approach. In addition to this, a strategy for the successful implementation of compatible solutes in biological systems is proposed.     

Countercurrent flows,water movements and nutrient absorption in the locust midgut

Using amaranth dye as a marker solute, the movements of fluids in the gut of Schistocerca gregaria was studied, either by feeding a meal containing the dye or by injecting the dye into the haemolymph, and by comparing the distribution of amaranth with those of naturally-occurring solutes in the alimentary tract.In animals deprived of food for more than 2–4 hr, some of the fluid from the Malpighian tubules moves forward through the solid food matrix in the midgut carrying solutes into the anterior midgut and gastric caeca, where water is absorbed. After a meal the crop empties at a rate which saturates the absorptive capacity of the anterior caeca, producing a net movement of fluid down the midgut and so such a countercurrent system is not observed in animals fed ad lib., where dye introduced into the gut always moves posteriorly.A countercurrent fluid movement confers several advantages on the alimentary system which act to maximise the efficiency of nutrient absorption: the principal disadvantage of the countercurrent system is that noxious solutes, as well as nutrients, will accumulate at high concentrations near the permeable site of nutrient uptake. Thus a countercurrent flow of solutes is observed only when the insect is deprived of food and the need to conserve nutrient resources exceeds that of excretion of noxious substances. Ways in which the site of nutrient absorption may be protected from noxious solutes are discussed.The anterior caeca gradually become bloated with dark fluid as digestion proceeds; this is expelled into the midgut when a fresh meal is ingested.     

Biomolecular-solvent stereodynamic coupling probed by deuteration

Thermodynamic interpretation of experiments with isotopically perturbed solvent supports the view that solvent stereodynamics is directly relevant to thermodynamic stability of biomolecules. According with the current understanding of the structure of the aqueous solvent, in any stereodynamic configuration of the latter, connectivity pathways are identifiable for their topologic and order properties. Perturbing the solvent by isotopic substitution or, e.g., by addition of co-solvents, can therefore be viewed as reinforcing or otherwise perturbing these topologic structures. This microscopic model readily visualizes thermodynamic interpretation. In conclusion, the topologic stereodynamic structures of connectivity pathways in the solvent, as modified by interaction with solutes, acquire a specific thermodynamic and biological significance, and the problem of thermodynamic and functional stability of biomolecules is seen in its full pertinent phase space.     

Extraction and Quantification of Solutes in Solidified Agar Culture Media

A method is described for determining the concentration of certain solutes in solidified culture media. The method is based upon the finding that under specified conditions the concentration of solute in an agar gel (C_g) is related to the concentration of solute in a centrifugally extracted gel supernatant (C_s) by the ratio, C_g/C_s, which is characteristic for each solute. The method avoids direct assays of the gels and instead involves assaying the supernatants from inoculated and uninoculated (control) gels with conventional liquid assay techniques and then calculating solute concentrations in the inoculated gels by use of the C_g/C_s ratios determined from the controls. Uninoculated agar gels containing known concentrations of various solutes and similar gels inoculated with Neurospora crassa or Escherichia coli were centrifuged at various times, and the supernatants were assayed for solute concentrations. The solute concentrations in the supernatants from the inoculated gels multiplied by the C_g/C_s ratios for those solutes determined at the same times for the uninoculated controls gave calculated solute concentrations in the inoculated gels. The differences between these calculated solute concentrations and those initially present in the inoculated gels indicated the amounts of solutes utilized from the gels by the microorganisms at various incubation times.     

Exclusion and retention of compensatory kosmotropes by HPLC columns

With water as the elution solvent, zwitterionic solutes and polyols were retained on HPLC columns, more than was water, by totally hydrophobic packing materials. Relative retentions were systematically affected by oxygen functional groups in the packing material, explicable as specific retention of water. Reproducible elution sequences of 20 solutes at a variety of hydrophobic surfaces (aromatic and both long- and short-alkyl aliphatic surfaces) showed there is a general process, consistent with interactions with hydration water at the surface having solvent properties distinct from bulk water. Early eluting solutes included glycine, sarcosine and taurine. Glycine betaine followed both these and N,N-dimethylglycine. The natural betaines propionobetaine and dimethylsulfoniopropionate also preceded glycine betaine. Dimethylsulfoxide was strongly retained, as (to a lesser extent) was proline betaine. Polyols eluted in the sequence sorbitol, trehalose, glycerol. Changes in the chemical nature of the surface or base material affected relative retentions of water and solutes. The presence of hydrogen-bonding functions increased retention of polyols, as well as water, relative to zwitterionic solutes. Specific effects with some solutes may be related to inconsistencies seen in biological systems. Pressures up to 8 MPa did not affect relative retention, constraining models based on the formation of low-density water.     

A general model for the dynamics of cell volume,global stability,and optimal control

Cell volume and concentration regulation in the presence of changing extracellular environments has been studied for centuries, and recently a general nondimensional model was introduced that encompassed solute and solvent transmembrane flux for a wide variety of solutes and flux mechanisms. Moreover, in many biological applications it is of considerable interest to understand optimal controls for both volume and solute concentrations. Here we examine a natural extension of this general model to an arbitrary number of solutes or solute pathways, show that this system is globally asymptotically stable and controllable, define necessary conditions for time-optimal controls in the arbitrary-solute case, and using a theorem of Boltyanski prove sufficient conditions for these controls in the commonly encountered two-solute case.     

Biomolecular-Solvent Stereodynamic Coupling Probed by Deuteration

Abstract

Thermodynamic interpretation of experiments with isotopically perturbed solvent supports the view that solvent stereodynamics is directly relevant to thermodynamic stability of biomolecules. According with the current understanding of the structure of the aqueous solvent, in any stereodynamic configuration of the latter, connectivity pathways are identifiable for their topologie and order properties. Perturbing the solvent by isotopie substitution or, e.g., by addition of co-solvents, can therefore be viewed as reinforcing or otherwise perturbing these topologie structures.

This microscopic model readily visualizes thermodynamic interpretation. In conclusion, the topologie stereodynamic structures of connectivity pathways in the solvent, as modified by interaction with solutes, acquire a specific thermodynamic and biological significance, and the problem of thermodynamic and functional stability of biomolecules is seen in its full pertinent phase space.     

A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding.

To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.     

Location and dynamics of tryptophan in transmembrane alpha-helix peptides: a fluorescence and circular dichroism study

Amphiphilic and hydrophobic peptides play a key role in many biological processes. We have developed a reference system for evaluating the insertion of such peptides bearing Trp fluorescent reporter groups into membrane mimetic systems. This system involves a set of six 25-amino acid synthetic peptides that are models of transmembrane alpha-helices. They are Lys-flanked polyLeu sequences, each containing a single Trp residue at a different position (P i, with i=3, 5, 7, 9, 11 and 13). These peptides were inserted into micelles of a non-ionic detergent, dodecylmaltoside (DM). We analyzed this system by use of circular dichroism and steady-state and time-resolved fluorescence in combination with Trp quenching with two brominated DM analogs. We found significant variations in the Trp emission maximum according to its position in each peptide (from 327 to 313 nm). This is consistent with the radial insertion of the peptides within DM micelles. We observed characteristic patterns of fluorescence quenching of these peptides in mixed micelles of DM, with either 7,8-dibromododecylmaltoside (BrDM) or 10,11-dibromoundecanoylmaltoside (BrUM), that reflect differences in the accessibility of the Trp residue to the bromine atoms located on the detergent acyl chain. In the isotropic reference solvent, methanol, the alpha-helix content was high and identical (approximately 76%) for all peptides. In DM micelles, the alpha-helix content for P9 to P13 was similar to that in methanol, but slightly lower for P3 to P7. The fluorescence intensity decays were heterogeneous and depended upon the position of the Trp. The Trp dynamics of each peptide are described by sub-nanosecond and nanosecond rotational motions that were significantly lower than those observed in methanol. These results, which precisely describe structural, dynamic and microenvironment parameters of peptide Trp in micelles according to its depth, should be useful for describing the interactions of peptides of biological interest with micelles.     

Water-coupled low-frequency modes of myoglobin and lysozyme observed by inelastic neutron scattering.

Conformational changes of proteins often involve the relative motion of rigid structural domains. Normal mode analysis and molecular dynamics simulations of small globular proteins predict delocalized vibrations with frequencies below 20 cm(-1), which may be overdamped in solution due to solvent friction. In search of these modes, we have studied deuterium-exchanged myoglobin and lysozyme using inelastic neutron scattering in the low-frequency range at full and low hydration to modify the degree of damping. At room temperature, the hydrated samples exhibit a more pronounced quasielastic spectrum due to diffusive motions than the dehydrated samples. The analysis of the corresponding lineshapes suggests that water modifies mainly the amplitude, but not the characteristic time of fast protein motions. At low temperatures, in contrast, the dehydrated samples exhibit larger motional amplitudes than the hydrated ones. The excess scattering, culminating at 16 cm(-1), is suggested to reflect water-coupled librations of polar side chains that are depressed in the hydrated system by strong intermolecular hydrogen bonding. Both myoglobin and lysozyme exhibit ultra-low-frequency modes below 10 cm(-1) in the dry state, possibly related to the breathing modes predicted by harmonic analysis.     

ON THE DISSOLVED SOLIDS OF THE PONGOLO FLOOD PLAIN PANS

SUMMARY

The water retained in the Pongolo flood plain pans differs from that of the Pongolo River not only in having a higher TDS, but also in the composition of the solutes, which approximate to seawater in their equivalent ionic proportions at high concentrations. These solutes are shown to originate from very highly mineralised seepage water characteristic of soils overlying Cretaceous sediments of marine origin found elsewhere in South Africa. This feature of the soils of the flood plain precludes the use of irrigation seepage water to maintain the water levels in the pans, and stresses the need for the release of simulated floods from the Pongolo-poort Dam to flush the system if the present biological characteristics of the pans are to be maintained.