Tadeusz Janas

THE SELECTION OF APTAMERS SPECIFIC FOR MEMBRANE MOLECULAR TARGETS

A growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.

Voltammetric analysis of polyisoprenoid-containing bilayer lipid membranes

Abstract Voltammetric investigations of bilayer lipid membranes modified by polyisoprenols (PI) and phosphopolyisoprenols (phospho-PI) are described. The theoretical background of voltammetric analysis with application to a system is presented. It is shown that contrary to the behaviour of phospho-PI, PI increases membrane permeability and membrane deformability, decreases the activation energy of ionic transmembrane migration, decreases membrane stability and does not change membrane selectivity. The results also indicate that a transmembrane potential can facilitate the formation of local non-bilayer structures in dolichyl phosphate-phosphatidylcholine bilayer membrane and accelerate the transbilayer movement of the polar part of a dolichyl phosphate molecule. Our hypothesis of PI and phospho-PI ordering, dynamics and function in membranes is presented.

Interaction of undecaprenyl phosphate with phospholipid bilayers

The effect of undecaprenyl phosphate (C55-P) on dioleoylphosphatidylcholine (DOPC) bilayer lipid membranes has been studied. The current-voltage characteristics, steady-state diffusion potentials, membrane conductance-temperature relationships, membrane electric capacitance and membrane breakdown voltage have been measured for different mixtures of undecaprenyl phosphate and DOPC. The ratio of permeability coefficients for sodium and chloride ions, the activation energy for ion migration across the membrane and membrane thickness have been determined. The electrical measurements showed that undecaprenyl phosphate decreases membrane-normalized conductance, membrane ionic permeability, membrane hydrophobic thickness and membrane selectivity for chloride ions, and increases the activation energy for ion transport, membrane nonlinearity potential, membrane specific capacitance, membrane electromechanical stability and membrane selectivity for sodium ions. From the results, we suggest that the interaction of the gradient of electric transmembrane potential with the negative charge of the phosphate group of C55-P determines the dynamics, conformation and aggregation behaviour of undecaprenyl phosphate in phospholipid membranes. Some implications of these findings for a possible regulation of the C55-P-dependent expression of polysialic acid capsule in Escherichia coli K1 bacterial cells are indicated.

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.

Membrane potential-dependent binding of polysialic acid to lipid monolayers and bilayers

Polysialic acids are linear polysaccharides composed of sialic acid monomers. These polyanionic chains are usually membrane-bound, and are expressed on the surfaces of neural, tumor and neuroinvasive bacterial cells. We used toluidine blue spectroscopy, the Langmuir monolayer technique and fluorescence spectroscopy to study the effects of membrane surface potential and transmembrane potential on the binding of polysialic acids to lipid bilayers and monolayers. Polysialic acid free in solution was added to the bathing solution to assess the metachromatic shift in the absorption spectra of toluidine blue, the temperature dependence of the fluorescence anisotropy of DPH in liposomes, the limiting molecular area in lipid monolayers, and the fluorescence spectroscopy of oxonol V in liposomes. Our results show that both a positive surface potential and a positive transmembrane potential inside the vesicles can facilitate the binding of polysialic acid chains to model lipid membranes. These observations suggest that these membrane potentials can also affect the polysialic acid-mediated interaction between cells.

Electroporation of polyprenol-phosphatidylcholine bilayer lipid membranes

Abstract The electrochemical measurements showed that tetracosaprenol (C 120 ) isolated from leaves of Spermatophyta influenced some properties of phosphatidylcholine macrovesicular bilayer lipid membranes. The current–voltage characteristics, the membrane conductance–temperature relationships and the membrane breakdown voltage have been measured for different mixtures of tetracosaprenol and DOPC. The membrane conductance, the permeability coefficient for Cl − ions and the activation energy of ion migration across the membrane were determined. Tetracosaprenol decreases the membrane breakdown voltage and the activation energy, increases the membrane conductance and the membrane ionic permeability for Cl − ions. These results indicate that long-chain polyprenols modify bilayer lipid membranes by the formation of fluid microdomains and point to the importance of transmembrane electrical potential in the dynamics and aggregation behaviour of polyisoprenols in membranes. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lecithin–polyprenol bilayers at the microdomains border.

Voltage-dependent behaviour of dolichyl phosphate-phosphatidylcholine bilayer lipid membranes

The current-voltage steady-state characteristics, cyclic voltammograms and capacitance-voltage steady-state relationships of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl-12 phosphate have been studied. Sustained fluctuations of the capacitance of dolichyl phosphate modified bilayers under applied voltage were observed. The results suggest that the dynamics of dolichyl phosphate molecules in membranes can be regulated by transmembrane electrical potential.

The effect of hexadecaprenyl diphosphate on phospholipid membranes.

In the present study we investigated phospholipid bilayer membranes and phospholipid vesicles made from dioleoylphosphatidylcholine (DOPC) or its mixture with the phosphate ester derivative of long-chain polyprenol (hexadecaprenyl diphosphate, C(80)-PP) by electrophysiological and transmission electron microscopy (TEM) techniques. The membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C(80)-PP/DOPC. The current-voltage characteristics, the membrane conductance, the activation energy of ion migration across the membrane and the membrane breakdown voltage were determined. Hexadecaprenyl diphosphate decreases the membrane conductance, increases the activation energy and the membrane breakdown voltage for the various values of C(80)-PP/DOPC mole ratio. The analysis of TEM micrographs shows several characteristic structures, which have been described. The data indicate that hexadecaprenyl diphosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. The properties of modified membranes can result from the presence of the negative charges in the hydrophilic part of C(80)-PP molecules and can be modulated by the concentration of this compound in membranes. We suggest that the dynamics and conformation of hexadecaprenyl diphosphate in membranes depend on the transmembrane electrical potential.

Modulation of properties of phospholipid membranes by the long-chain polyprenol (C160).

The electrical measurements of phospholipid bilayers and the studies of phospholipid vesicles by using the transmission electron microscopy (TEM) showed that dotriacontaprenol (C(160)) isolated from leaves of Spermatophyta influences some properties of membranes. The current-voltage characteristics, the membrane conductance-temperature relationships, the membrane breakdown voltage and the membrane capacitance have been measured for different mixtures of C(160)/DOPC. The membrane conductance, the activation energy of ion migration across the membrane and the membrane thickness were determined. Dotriacontaprenol decreases the membrane breakdown voltage, the activation energy and the membrane capacitance, and increases the membrane conductance and the membrane hydrophobic thickness. The analysis of TEM micrographs shows several characteristic structures, which have been described. The results indicate that dotriacontaprenol increases the membrane elasticity and modulates the surface curvature of the membranes by the formation of fluid microdomains. We suggest that the long polyprenols facilitate the formation of transmembrane, ions-conductive pores.

Electromigration of polyion homopolymers across biomembranes: a biophysical model

The analysis of polyion transmembrane translocation was performed using membrane electrical equivalent circuit. The dependence of polyion flux across membranes on time, membrane electrical conductance, membrane electrical capacitance, degree of polymerization, water solution conductance and applied transmembrane potential is discussed. The changes in polyion flux were up to 88% after 1 ms. Both the increase of polyion chain length and the decrease of membrane conductance resulted in the diminution of this effect. Inversion of flux direction was observed as a result of external potential changes. Reversal curves, representing the values of considered parameters for zero-flux were also shown. The replacement of a polyanion by a polycation of the same chain length resulted in the same shape of the surface plot but with opposite orientation. The analysis describes the effect of transmembrane potential on the translocation rate of polyanionic polysialic acid and polynucleotides, and polycationic peptides across membranes.