Transport across homoporous and heteroporous membranes in nonideal, nondilute solutions. II. Inequality of phenomenological and tracer solute permeabilities.

The phenomenological solute permeability (omega p) of a membrane measures the flux of solute across it when the concentrations of the solutions on the two sides of the membrane differ. The relationship between omega p and the the conventionally measured tracer permeability (omega T) is examined for homoporous and heteroporous (parallel path) membranes in nonideal, nondilute solutions and in the presence of boundary layers. In general, omega p and omega T are not equal; therefore, predictions of transmembrane solute flux based on omega T are always subject to error. For a homoporous membrane, the two permeabilities become equal as the solutions become ideal and dilute. For heteroporous membranes, omega p is always greater than omega T. An upper bound on omega p- omega T is derived to provide an estimate of the maximum error in predicted solute flux. This bound is also used to show that the difference between omega P and omega T demonstrated earlier for the sucrose-Cuprophan system can be explained if the membrane is heteroporous. The expressions for omega P developed here support the use of a modified osmotic driving force to describe membrane transport in nonideal, nondilute solutions.     

Pyrazolopyranopyrimidines as a class of anti-inflammatory agents

Pyrazolopyranopyrimidines 6a-c and 8a-c were prepared from the reaction of compounds 4a-c or 7a-c with methylamine or ammonium hydroxide solutions. Treatment of compounds 6a-c or 8a-c with 2-chloroethyl methyl ether afforded their corresponding acyclonucleosides 9a-c or 10a-c, respectively, as a new class of acyclonucleosides. All prepared compounds were tested as anti-inflammatory agents and some of them revealed moderate to potent anti-inflammatory activity.     

Downstream separation and purification of bio-based alpha-ketoglutaric acid from post-fermentation broth using a multi-stage membrane process

In this study, a multi-stage membrane process, assisted by vacuum evaporation and crystallization, for recovery of bio-based alpha-ketoglutaric acid from the actual post-fermentation broth was designed and investigated. In the first part of this study, pre-treatment of crude fermentation broth (centrifugation-ultrafiltration-nanofiltration) was carried out to remove biomass, proteins, sugars, part of inorganic ions and color compounds. The commercial ceramic UF membrane (15 kDa) and nanofiltration ceramic membrane (200 Da or 450 Da) were applied. Electrodialysis with a bipolar membrane was proposed for separation of ionic compounds and simultaneous electro-acidification to the acid form. During bipolar membrane electrodialysis carried out under acidic conditions, it was possible to remove close to 90 % of alpha-ketoglutaric acid. Moreover, the migration of other acids present in the fermentation broth (lactic and acetic) was significantly limited. The calculated specific energy consumption was low and equal to 0.6 kW h/kg. The final purification using crystallization assisted vacuum evaporation allowed obtaining alpha-ketoglutaric acid in solid form. Analysis of the final product showed that the proposed method of alpha-ketoglutaric acid recovery gives the acid of high purity equal to 94.8 %. Furthermore, the presented results have practical relevance and may in future be the basis for the development of separation technologies of alpha-ketoglutaric acid from the fermentation broth on industrial scale.     

Membrane currents in retinal bipolar cells of the axolotl

By whole-cell patch-clamping bipolar cells isolated from enzymatically dissociated retinae, we have studied the nonsynaptic ionic currents that may play a role in shaping the bipolar cell light response and in determining the level of voltage noise in these cells. Between -30 and -70 mV, the membrane current of isolated bipolar cells is time independent, and the input resistance is 1-2 G omega. Depolarization past -30 mV activates an outward current (in less than 100 ms), which then inactivates slowly (approximately 1 s). Inactivation of this current is removed by hyperpolarization over the range -20 to -80 mV. This current is carried largely by K ions. It is not activated by internal Ca2+. The membrane current of isolated bipolar cells is noisy, and the variance of this noise has a minimum between -40 and -60 mV. At its minimum, the standard deviation of the voltage noise produced by nonsynaptic membrane currents is at least 100 microV. The membrane currents of depolarizing bipolar cells in slices of retina were investigated by whole-cell patch-clamping. Their membrane properties were similar to those of isolated bipolar cells, but with a larger membrane capacitance and a smaller input resistance. Their membrane current noise also showed a minimum near -40 to -60 mV. The time-dependent potassium current in axolotl bipolar cells is not significantly activated in the physiological potential range and can therefore play little role in shaping the bipolar cells' voltage response to light. Differences in the waveform of the light response of bipolar cells and photoreceptors must be ascribed to shaping by the synapses between these cells. The noise minimum in the bipolar membrane current is near the dark potential of these cells, and this may be advantageous for the detection of weak signals by the bipolar cells.     

Molecular packing parameters of bipolar lipids.

The bipolar lipid fractions extracted from the thermophilic archaeobacterium Sulfolobus solfataricus have different chemical structures and geometrical shapes. The conditions which lead to the formation of vesicles were investigated in order to study the self-assembly of these molecules. Such conditions are fulfilled when an appropriate mixture of two different molecular species (both bipolar or bipolar and monopolar) is used. According to the theory introduced by Israelachvili and co-workers, lipid self-assembly results from the balance of interaction free energy, entropy and molecular geometry. We have shown that this theory can be extended to bipolar lipids, in spite of their more complex nature, and the experimental results obtained combining 1H-NMR, light scattering and entrapped volume techniques closely match theoretical expectations. To carry out calculations, it was necessary to introduce hypotheses about the disposition of bipolar molecules in the vesicle membrane. These hypotheses have been tested indirectly by measuring the transport properties mediated by carriers or channels, whose transport mechanism can be considered to be a probe of the membrane structure.     

Transport across homoporous and heteroporous membranes in nonideal, nondilute solutions. I. Inequality of reflection coefficients for volume flow and solute flow.

The Kirkwood formulation of the Stefan-Maxwell equations is used to develop the transport equations for a membrane bounded by nonideal, nondilute solutions. The reflection coefficients for volume flow and solute flow are not equal but are related by a simple expression that depends on the concentration of the bounding solutions. The ratio of the two coefficients is independent of heteroporous membrane structure and the thickness of adjacent boundary layers. Experimental measurements of these reflection coefficients for sucrose transport across Cuprophan verify this relationship; this indicates that the Onsager reciprocal relation, which is assumed by the theory, holds for nonideal, nondilute solutions. The two reflection coefficients may be made operationally identical by a simple redefination of the osmotic driving force.     

The role of retinal bipolar cell in early vision: an implication with analogue networks and regularization theory

A linear analogue network model is proposed to describe the neuronal circuit of the outer retina consisting of cones, horizontal cells, and bipolar cells. The model reflects previous physiological findings on the spatial response properties of these neurons to dim illumination and is expressed by physiological mechanisms, i.e., membrane conductances, gap-junctional conductances, and strengths of chemical synaptic interactions. Using the model, we characterized the spatial filtering properties of the bipolar cell receptive field with the standard regularization theory, in which the early vision problems are attributed to minimization of a cost function. The cost function accompanying the present characterization is derived from the linear analogue network model, and one can gain intuitive insights on how physiological mechanisms contribute to the spatial filtering properties of the bipolar cell receptive field. We also elucidated a quantitative relation between the Laplacian of Gaussian operator and the bipolar cell receptive field. From the computational point of view, the dopaminergic modulation of the gap-junctional conductance between horizontal cells is inferred to be a suitable neural adaptation mechanism for transition between photopic and mesopic vision. Received: 20 December 1996 / Accepted in revised form: 16 May 1997     

The critical role of detergents in the crystallization of membrane proteins

Significant progress in the elucidation of folding patterns of membrane proteins has been made over the past 10 years; yet, the scope of our knowledge remains extremely limited. Several difficulties beset the rational selection of crystallization conditions, of which the problem of the colloidal properties of detergent solutions is only one. In this report, specific critical parameters of colloidal solutions are considered: the shape of surfactant micelles and their clustering near phase transitions. If recognized, these properties may be exploited for crystallization. Yet, a systematic search of parameters remains necessary if the chances of obtaining a better view of the scope of membrane protein folding patterns are to be increased.     

2-ns Electrostimulation of Ca2+ Influx into Chromaffin Cells: Rapid Modulation by Field Reversal

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca²⁺ levels due to Ca²⁺ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca²⁺ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca²⁺ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.     

Induced capacitance in the squid giant axon. Lipophilic ion displacement currents

Voltage-clamped squid giant axons, perfused internally and externally with solutions containing 10(-5) M dipicrylamine (DpA-), show very large polarization currents (greater than or equal to 1 mA/cm2) in response to voltage steps. The induced polarization currents are shown in the frequency domain as a very large voltage-and frequency-dependent capacitance that can be fit by single Debye-type relaxations. In the time domain, the decay phase of the induced currents can be fit by single exponentials. The induced polarization currents can also be observed in the presence of large sodium and potassium currents. The presence of the DpA- molecules does not affect the resting potential of the axons, but the action potentials appear graded, with a much-reduced rate of rise. The data in the time domain as well as the frequency domain can be explained by a single-barrier model where the DpA- molecules translocate for an equivalent fraction of the electric field of 0.63, and the forward and backward rate constants are equal at -15 mV. When the induced polarization currents described here are added to the total ionic current expression given by Hodgkin and Huxley (1952), numerical solutions of the membrane action potential reproduce qualitatively our experimental data. Numerical solutions of the propagated action potential predict that large changes in the speed of conduction are possible when polarization currents are induced in the axonal membrane. We speculate that either naturally occurring substances or drugs could alter the cable properties of cells in a similar manner.     

Identification and characterization of the principal proteins of the fat-globule membrane from guinea-pig milk

The milk-fat-globule membrane (MFGM) was isolated from guinea-pig milk and the membrane-associated proteins and glycoproteins characterized by electrophoretic techniques. Major components of the membrane included PAS-I, a sialoglycoprotein of Mr greater than or equal to 200000, the redox enzyme xanthine oxidase and the glycoprotein, butyrophilin. Membrane preparations also contained two other glycoproteins, GP-80 and GP-55, of Mr 80000 and 55000, respectively. Comparison of guinea-pig xanthine oxidase and butyrophilin with proteins from bovine MFGM by peptide mapping procedures, showed that the two proteins in both species were similar, but not identical. GP-55 may also be related to glycoproteins of Mr 45000 and 48000 in the bovine membrane. The integral and peripheral components of guinea-pig MFGM were identified by treating membrane preparations with sodium carbonate solutions at high pH and by partitioning the membrane proteins in solutions of Triton X-114. By these criteria xanthine oxidase and GP-55 appeared to be peripheral components and GP-80 an integral protein of the membrane. PAS-I and butyrophilin displayed hydrophilic properties in Triton X-114 solutions, but could not be removed from membrane preparations with sodium carbonate. Possible reasons for these ambiguous data are discussed. The observed similarity between several of the proteins of guinea-pig and bovine MFGM implies that these proteins may have specific functions related to milk secretion in mammary tissue, e.g. in the budding of milk-fat globules or the exocytosis of milk protein and lactose at the apical surface.     

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.     

Membrane transport of electrolyte ions and time-dependent membrane potential

Equations are derived for the transport of a symmetrical electrolyte, consisting of cations and anions of equal valency, through a neutral membrane that separates two solutions of finite volume under quasi-steady-state conditions. The time-dependent membrane potential produced by the flow of ions is taken into account. Deviation of the time course of the solute concentrations from that of neutral solutes is found to be determined by the permeability ratio of cations and anions (when this ratio equals unity, the derived membrane transport equations reduce to those for neutral substances). Simple approximate expressions for the solute concentrations and of the membrane potential as functions of time are proposed, which are in excellent agreement with the exact numerical results.     

Distribution of protein kinase C immunoreactivity in rat retina

Summary A polyclonal antiserum to protein kinase C has been used to study the distribution of the enzyme antigenic sites in rat retina. The results indicate that the kinase is concentrated in photoreceptor outer segments as well as in the outer and inner plexiform layers. In identified components of retinal neuronal circuits, the kinase immunoreactivity is present in photoreceptor presynaptic terminals, in bipolar cell dendrites and axons, and probably in bipolar cell presynaptic terminals impinging on retinal ganglion cell dendrites. Thus, protein kinase C is positioned to play a role in specialized compartments of photoreceptor membrane and at both pre- and postsynaptic levels in the function of retinal neuronal circuits. Label in the nucleus is observed in retinal ganglion cells, but not bipolar or horizontal cells and probably not in amacrine cells. A role for protein kinase C in neuronal function at the level of the cell nucleus is therefore not likely to be universal, but to be determined by the particular properties of individual neuronal types.     

Improved method to evaluate the ability of compounds to destabilize the cellular plasma membrane

In the paper, we present an improved method for evaluation of a compound ability to destabilize erythrocyte plasma membrane. The proposed method is based on the continuous monitoring of the light scattered by erythrocytes exposed to osmotic pressure differences. The kinetics of hemolysis depends on the plasma membrane mechanics and the extent of the osmotic stress. Generally, the osmotic pressure difference of approximately 150 mOsm is taken for measurements, as a result of the equal volume mixing with the physiological salt solutions. In this approach the hemolytic process completion is not established which may result in poor quality and reproducibility of the experimental data. In consequence, inaccurate parameters of the kinetic are determined due to the low quality fitting to the, widely used, single exponential model. In the paper we propose a new experimental protocol allowing to determine the extended set of parameters for kinetics of hemolysis. Namely, the method of the minimal osmotic pressure difference determination is proposed which ensures the completeness of the hemolytic process. This step allows improving the quality and exactness of the calculated parameters. The developed methodology was tested on two qualitatively different, biologically relevant, experiments; evaluation of the peptide effect on the plasma membrane properties and differentiating between human and rabbit erythrocytes.     

Dual-acting agents that possess free radical scavenging and antithrombotic activities: design, synthesis, and evaluation of phenolic tetrahydro-beta-carboline RGD peptide conjugates

A new approach to construct a single dual-acting agent is described. Compounds 6a-c are potent free radical scavengers as demonstrated by the EC(50) values in PC12 cell survival assay in term of NO, H(2)O(2), and ()OH scavenging activity. The Ach-induced vaso-relaxation assay further confirms the potent NO scavenging activity of compounds 6a-c. In addition, 6a-c are efficacious in a rat arterial thrombosis, and are active in ADP- or PAF-induced in vitro platelet aggregation assay, suggesting that compounds 6a-c also possess anti-thrombotic activities. Since both free radical and thrombogenesis are important risk factors in myocardial ischemic/reperfusion injuries, these dual-acting agents having both free radical scavenging and antithrombolic activities may potentially be beneficial toward their treatment.     

Synthesis and biological evaluation of some acyclic alpha-(1H-pyrazolo-[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides

The chemical synthesis of some acyclic alpha-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides (10-12)a-c is described. The treatment of IH-pyrazolo[3,4-d]pyrimidin-4-thione 1 with compounds 2a-c gave, regioselectively, ethyl alpha-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylates 3a-c, respectively. These heterocycles were alkylated, separately, with alkylating agents 4, 5 and 6 to give, regioselectively, the N1-acyclic nucleosides (7-9)a-c which were deprotected to afford the desired products (10-12)a-c. All synthetic compounds were characterized on the basis of their physical and spectroscopic properties. The products (10-12)a-c were evaluated for their inhibitory effects against the replication of HIV-1 (III(B)), HIV-2 (ROD), various DNA viruses, a variety of tumor-cell lines and M. tuberculosis. No marked biological activity was found.     

Molecular dynamics study of bipolar tetraether lipid membranes

Membranes composed of bipolar tetraether lipids have been studied by a series of 25-ns molecular dynamics simulations to understand the microscopic structure and dynamics as well as membrane area elasticity. By comparing macrocyclic and acyclic tetraether and diether archaeal lipids, the effect of tail linkage of the two phytanyl-chained lipids on the membrane properties is elucidated. Tetraether lipids show smaller molecular area and lateral mobility. For the latter, calculated diffusion coefficients are indeed one order-of-magnitude smaller than that of the diether lipid. These two tetraether membranes are alike in many physical properties except for membrane area elasticity. The macrocyclic tetraether membrane shows a higher elastic area expansion modulus than its acyclic counterpart by a factor of three. Free energy profiles of a water molecule crossing the membranes show no major difference in barrier height; however, a significant difference is observed near the membrane center due to the lack of the slip-plane in tetraether membranes.     

APPLICATION OF THE DIFFUSION HYPOTHESIS TO MEMBRANE POTENTIALS

A system consisting of two aqueous solutions, containing equal concentrations of lactic acid, but different concentrations of Na lactate, separated by a layer of amyl alcohol has been described. This system exhibits electrical properties ranging (as the concentration of NaL is increased) from those characteristic of a simple Donnan equilibrium to those characteristic of simple diffusion. The fact that the Donnan P.D. can be treated as a special case of a diffusion potential has been emphasized. The experiments call attention to the effect of the thermodynamic properties of the membrane, and it is suggested that such properties as conductivities, ionic mobilities, and distribution coefficients in membranes of any sort should be investigated. The experiments afford an interesting example of "phase reversal."     

Carrier Model for Active Transport of Ions across a Mosaic Membrane

The central purpose of this paper is to elucidate in a well defined system the meaning of certain phenomena and concepts associated with the active transport of ions. To this end a specific model for a carrier system which actively transports a single ionic species is analyzed and discussed in detail. It is assumed in this model that the carrier-mediated ionic transport occurs in regions of the membrane physically separate from those regions in which free ionic movement takes place,—coupling between the active and passive regions of the membrane occurring through local current flows. The model is seen to display the following characteristics: (a) Starting from identical solutions on the two sides of the membrane, there is produced a redistribution of ions; (b) with identical solutions on the two sides of the membrane there exists a potential difference across the membrane, i.e., the “pump” is electrogenic; (c) the “short circuit” current for symmetrical solutions is equal to the flux of the neutral ion carrier complex; (d) the rate of active transport (and hence of metabolism) is dependent on the ionic concentrations in the surrounding solutions. Throughout the paper comparison is made between features of the model and properties displayed by biological active transport systems, but there is no claim of an identity between the two.