Osmotic Depletion Research Articles

PH Dependence of the Adhesion Property of Lipid Bilayers and a Method of Measuring the Adhesion Energy

We found that phospholipid bilayers are adhesive to each other at pH values lower than 5, while they are not adhesive at pH values higher than 6. This is significant to membrane fusion occurring at low pH, and to membrane experiments using lipid vesicles. We used the experimental method invented by Evans and collaborators in which one flaccid GUV was released to adhere to one tensed GUV. We developed a new analysis method to measure the adhesion energy per unit area. This new method is independent of how the adhesion state was reached. The order of magnitude of the adhesion energy is ∼0.01 to ∼0.02 erg/cm2 for SOPC. The addition of SOPE slightly decreases the adhesion energy of pure SOPC, while the addition of cholesterol has little effect. The same method of measurement was applied to a case where two lipid bilayers underwent the first step of membrane fusion, called hemifusion. Hemifusion was induced by injecting 5 wt % PEG8000 solution at pH 4. The PEG injection was used to produce a transient osmotic depletion attraction between the two GUVs. The energy of hemifusion is one order of magnitude larger than the adhesion energy, about ∼0.3 erg/cm2 for DOPC/DOPE/cholesterol (4:4:2). This is the first time the free energy of the membrane fusion intermediate state was experimentally measured.

Adhesion and Merging of Lipid Bilayers: A Method for Measuring the Free Energy of Adhesion and Hemifusion

Lipid bilayers can be induced to adhere to each other by molecular mediators, and, depending on the lipid composition, such adhesion can lead to merging of the contacting monolayers in a process known as hemifusion. Such bilayer-bilayer reactions have never been systematically studied. In the course of our studies of membrane-active molecules, we encountered such reactions. We believe that they need to be understood whenever bilayer-bilayer interactions take place, such as during membrane fusion. For illustration, we discuss three examples: spontaneous adhesion between phospholipid bilayers induced by low pH, polymer-induced osmotic depletion attraction between lipid bilayers, and anionic lipid bilayers cross-bridged by multicationic peptides. Our purpose here is to describe a general method for studying such interactions. We used giant unilamellar vesicles, each of which was aspirated in a micropipette so that we could monitor the tension of the membrane and the membrane area changes during the bilayer-bilayer interaction. We devised a general method for measuring the free energy of adhesion or hemifusion. The results show that the energies of adhesion or hemifusion of lipid bilayers could vary over 2 orders of magnitude from −1 to −50 × 10 −5 J/m 2 in these examples alone. Our method can be used to measure the energy of transition in each step of lipid transformation during membrane fusion. This is relevant for current research on membrane fusion, which focuses on how fusion proteins induce lipid transformations.

Steric and Electrostatic Complementarity in the Assembly of Two-Dimensional Virus Arrays

A highly ordered assembly of biological molecules provides a powerful means to study the organizational principles of objects at the nanoscale. Two-dimensional cowpea mosaic virus arrays were assembled in an ordered manner on mica using osmotic depletion effects and a drop-and-dry method. The packing of the virus array was controlled systematically from rhombic packing to hexagonal packing by modulating the concentrations of poly(ethylene glycol) surfactant in the virus solutions. The orientation and packing symmetry of the virus arrays were found to be tuned by the concentrations of surfactants in the sample solutions. A phenomenological model for the present system is proposed to explain the assembly array morphology under the influence of the surfactant. Steric and electrostatic complementarity of neighboring virus capsids is found to be the key factors in controlling the symmetry of packing.

Hemifusion of Giant Lipid Vesicles by a Small Transient Osmotic Depletion Pressure

We observed hemifusion of giant unilamellar vesicles (GUVs) by injecting a small volume of 5% polyethylene glycol solution in the vicinity for a short time. The induced transient osmotic depletion attraction between two bilayers was smaller than the corresponding van der Waals force, yet hemifusion readily occurred for some lipid compositions. This might imply that the energy barrier for hemifusion is very low if the lipid compositions were primed for the reaction. The barrier for hemifusion is then the task of bringing two membranes into contact. We found that hemifusion is a stochastic event very much like pore formation by tension or by pore-forming peptides. This method provides an operational definition for hemifusogenicity which so far has been defined only qualitatively. Surprisingly the configuration of hemifusion depends on the lipid composition, ranging from a stalk-like hemifusion to a large hemifusion diaphragm. The formation of a large hemifusion diaphragm requires trans-bilayer lipid mixing in the participating GUVs. We also found that hemifusogenicity correlates with the water activity for the stalk phase in the lipid water mixture. The effect of cholesterol on hemifusogenicity was clearly demonstrated.

Electrostatic depletion forces between planar surfaces

The interaction between two dielectric plates immersed in an electrolyte solution is examined by using a variational perturbation approximation for the grand partition function. This approach differs from previous treatments in that the screening length between the plates is treated as a variational parameter. A key finding is that adjacent to each plate is a layer of ion depletion with thickness given by about one-half of a Bjerrum length. Consequently, for plate-plate separations less than the Bjerrum length, nearly all the electrolyte is excluded from between the plates, and the interaction is given by the sum of a van der Waals interaction and an attractive osmotic depletion force. In contrast to the predictions of previous theories, the interaction between the plates at short range increases with increasing electrolyte concentration and may provide an important contribution to the salt-induced attraction, commonly referred to as salting out. Because the range of the osmotic depletion force is roughly equal to the Bjerrum length, it increases with the square of the valency of the electrolyte. At larger plate-plate separations, the van der Waals interaction is screened as electrolyte enters the space between the plates, leading to an exponential decay of the interactions, as has been previously observed. However, this interaction is slightly stronger than that previously predicted, due to ion depletion from the surface of the interface, also this effect increases with increasing electrolyte concentration.

Entropy and Fluid−Fluid Separation in Nonadditive Hard-Sphere Mixtures: The Asymmetric Case

We report Monte Carlo simulation results for the thermodynamic and structural properties of binary nonadditive mixtures of hard spheres with not too dissimilar sizes. The simulations were carried out over a range of parameters where fluid-fluid separation is known to compete with freezing. We investigated the onset of structural ordering in a one-phase scheme as that provided by the residual multiparticle entropy, an integrated measure of the weight of spatial correlations involving more than two particles in the configurational entropy of the system. This approach has been already tested in the symmetric case [Saija, F.; Pastore, G.; Giaquinta, P. V. J. Phys. Chem. B 1998, 102, 10368]. The present study confirms the indication that a rather small amount of nonadditivity may significantly influence the phase behavior of the mixture also in a thermodynamic regime where osmotic depletion effects do not play a leading role.

Postural Control and Glycerol Test in Me´nie`re's Disease

Ménière's disease is a clinical disorder, characterized by fluctuating hearing loss, recurrent spontaneous episodic vertigo, tinnitus and aural fullness, which may be defined as the idiopathic syndrome of endolymphatic hydrops. The most important test for diagnosis of Ménière's disease is the glycerol test. This is a simple and rapid method and several authors have confirmed its efficiency for identifying endolymphatic hydrops. This test provides information on the cochlear response to the osmotic changes produced by glycerol in the inner ear, whereas modifications in the vestibular labyrinth are usually not evaluated. The aim of this study was to evaluate the effects of glycerol on postural control during attacks of Ménière's disease, and to correlate this data with data on cochlear function. After the glycerol test, an improvement in postural control was recorded in 70% of patients, with all patients reporting a recovery of vertigo. The impairment of postural control during endolymphatic hydrops could be related to a pressure increase in the labyrinth, which interferes with the normal dynamics of the endolymph, and a rapid functional recovery could occur during an osmotic depletion. Dynamic posturography improves the sensitivity of the glycerol test and may therefore be useful in the diagnosis and staging of Me´nie ¤ re's disease.

Electrostatic Nanotitration of Weak Biochemical Bonds

We introduce a new method to measure the energetics and range of weak biochemical bonds using functionalized vesicles. Large bilayer regions are held in molecular proximity by osmotic depletion forces to enable rapid specific bonding. By fixing an electrical charge to the tethering site of the functional group on one surface, persistent adhesion of the vesicles after removal of the depletion stress is titrated against the clamped electrostatic potential of the opposite surface. We demonstrate the method with DNA bases and obtain new information on the range of their specific interactions.

Osmotic depletion, non-additivity and phase separation

A brief discussion is given of the subtle interplay between non-additivity of hard sphere diameters and the osmotic depletion effect, and their influence on phase separation in bidisperse colloidal suspensions.

Depletion of glutathione from brain cells in hyponatremia

In response to hyponatremia, brain cells extrude electrolytes and organic osmolytes, thereby minimizing brain edema. We demonstrate that rat brain is depleted of the antioxidant glutathione in response to hyponatremia and that osmotically-induced loss of glutathione makes neuronal cells more susceptible to oxidative injury. Total glutathione content of brain tissue decreased from 6.80 +/- 0.14 mumol/g dry wt in normonatremic controls to 5.00 +/- 0.31 mumol/g dry wt after 72 hours of hyponatremia. Following slow correction of hyponatremia, brain glutathione content returned to control values (6.77 +/- 0.34 mumol/g dry wt). Brain content of taurine, a beta-amino acid with antioxidant properties, similarly decreased in hyponatremia (29.6 +/- 0.9 to 17.1 +/- 1.2 mumol/g dry wt), then increased with slow correction (24.8 +/- 1.3 mumol/g dry wt). Although taurine served as an osmolyte in rat heart, liver and brain, osmotically-induced changes in glutathione content were found only in brain. We also studied osmotically-induced changes in glutathione and taurine content in C6 glioma and SK-N-SH neuroblastoma cells. In both cell lines, adaptive decreases in glutathione and taurine content were found in response to lowering medium sodium concentration from 140 mM to 100 mM. The cell content of these solutes increased after returning to media containing 140 mM sodium. Following exposure of both cell lines to hypoosmolar media, there was no increase in media content of glutathione. This suggest that osmotic depletion of glutathione is not due to cellular efflux of intact glutathione. We questioned if osmotic depletion of glutathione and taurine renders brain cells more susceptible to oxidative stress. Incubation of SK-N-SH cells with 1.0 mM H2O2 for four hours induced greater cytolytic injury in cells adapted to hypoosmolar media than in isoosmolar controls. Hypoosmolar C6 glioma cells were not significantly more sensitive to cytolytic injury from H2O2 than were cells grown in isosmolar media. We conclude that hypoosmolality induces glutathione depletion in rat brain in vivo and in cultured brain cells in vitro. Osmotic depletion of this antioxidant renders SK-N-SH neuronal cells more susceptible to oxidative injury.

Entropy driven phase separation in binary emulsions.

We have studied experimentally phase separation in binary mixtures comprised of nearly monodisperse emulsion droplets of two different sizes. For three values of \ensuremath{\xi}, where $\ensuremath{\xi}l1$ is the size ratio, we have determined phase diagrams and find that segregation occurs for $\ensuremath{\xi}\ensuremath{\le}0.30\ifmmode\pm\else\textpm\fi{}0.02$. The phase boundaries are strongly dependent on \ensuremath{\xi}, and phase-separated samples show nearly complete segregation of the two components, as a result of the osmotic depletion effect which drives the phase segregation. Our results agree qualitatively with recent model calculations.