Quantitative study of molecular transport due to electroporation: uptake of bovine serum albumin by erythrocyte ghosts.

Electroporation is believed to involve the creation of aqueous pathways in lipid bilayer membranes by transient elevation of the transmembrane voltage to approximately 1 V. Here, results are presented for a quantitative study of the number of bovine serum albumin (BSA) molecules transported into erythrocyte ghosts caused by electroportion. 1) Uptake of BSA was found to plateau at high field strength. However, this was not necessarily an absolute maximum in transport. Instead, it represented the maximum effect of increasing field strength for a particular pulse protocol. 2) Maximum uptake under any conditions used in this study corresponded to approximately one-fourth of apparent equilibrium with the external solution. 3) Multiple and longer pulses each increased uptake of BSA, where the total time integral of field strength correlated with uptake, independent of inter-pulse spacing. 4) Pre-pulse adsorption of BSA to ghost membranes appears to have increased transport. 5) Most transport of BSA probably occurred by electrically driven transport during pulses; post-pulse uptake occurred, but to a much lesser extent. Finally, approaches to increasing transport are discussed.     

Membrane electroporation: a molecular dynamics simulation

We present results of molecular dynamics simulations of lipid bilayers under a high transverse electrical field aimed at investigating their electroporation. Several systems are studied, namely 1), a bare bilayer, 2), a bilayer containing a peptide nanotube channel, and 3), a system with a peripheral DNA double strand. In all systems, the applied transmembrane electric fields (0.5 V.nm(-1) and 1.0 V.nm(-1)) induce an electroporation of the lipid bilayer manifested by the formation of water wires and water channels across the membrane. The internal structures of the peptide nanotube assembly and that of the DNA strand are hardly modified under field. For system 2, no perturbation of the membrane is witnessed at the vicinity of the channel, which indicates that the interactions of the peptide with the nearby lipids stabilize the bilayer. For system 3, the DNA strand migrates to the interior of the membrane only after electroporation. Interestingly enough, switching of the external transmembrane potential in cases 1 and 2 for few nanoseconds is enough to allow for complete resealing and reconstitution of the bilayer. We provide evidence that the electric field induces a significant lateral stress on the bilayer, manifested by surface tensions of magnitudes in the order of 1 mN.m(-1). This study is believed to capture the essence of several dynamical phenomena observed experimentally and provides a framework for further developments and for new applications.     

Transendothelial transport of serum albumin: a quantitative immunocytochemical study

The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody. Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation. Despite this large difference in albumin concentration between capillary lumen and interstitium, plasmalemmal vesicles labeling was uniformly distributed across the endothelial profile. 68% of the junctions displayed labeling for albumin, which was however low and confined to the luminal and abluminal sides. The scarce labeling of the endothelial cell surface did not confirm the fiber matrix theory. The kinetics of albumin transcytosis was evaluated by injecting radioiodinated and DNP-tagged BSA. At 3, 10, 30, and 60 min, and 3, 5, and 24 h circulation time, blood radioactivity was measured and diaphragms were fixed and embedded. Anti-DNP antibodies were used to map the tracer in aforementioned compartments. A linear relationship between blood radioactivity and vascular labeling density was found, with a detection sensitivity approaching 1 gold particle per DNP-BSA molecule. Tracer presence over endothelial vesicles reached rapidly (10 min) a saturation value; initially localized near the luminal front, it evolved towards a uniform distribution across endothelium during the first hour. An hour was also needed to reach the saturation limit within the subendothelial space. Labeling of the junctions increased slowly, out of phase with the inferred transendothelial albumin fluxes. This suggests that they play little, if any, role in albumin transcytosis, which rather seems to proceed through the vesicular way.     

Millisecond measurement of transport during and after an electroporation pulse.

Electroporation involves the application of an electric field pulse that creates transient aqueous pathways in lipid bilayer membranes. Transport through these pathways can occur by different mechanisms during and after a pulse. To determine the time scale of transport and the mechanism(s) by which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost membranes was measured with a fluorescence microscope photometer with millisecond time resolution during and after electroporation pulses several milliseconds in duration. One of four outcomes was typically observed. Ghosts were: (1) partially emptied of calcein, involving efflux primarily after the pulse; (2) completely emptied of calcein, involving efflux primarily after the pulse; (3) completely emptied of calcein, involving efflux both during and after the pulse; or (4) completely emptied of calcein, involving efflux primarily during the pulse. Partial emptying, involving significant efflux during the pulse, was generally not observed. We conclude that under some conditions transport caused by electroporation occurs predominantly by electrophoresis and/or electroosmosis during a pulse, although under other conditions transport occurs in part or almost completely by diffusion within milliseconds to seconds after a pulse.     

Driving activity. A quantitative study.

A comparative study of driving activity between normal subjects and neurological patients was performed. Driving activity was considered as the energy of the visual evoked potentials filtered at the same frequency of stimulation (1, 2, 4, 7, 10, 12 and 15 cps) using a CAT 400 C computer as a digital filter. The hemispheric symmetry of the responses was measured by the Pearson product moment correlation coefficient and the signal energy ratio. Each symmetry measure for every patient was compared with the normal values and considered abnormal when differences were greater than 3 SD from the normal mean. Of 25 patients 14 of them with a normal EEG, 23 presented severe alterations in the symmetry of the filtered visual evoked responses. Each patient showed a peculiar pattern of abnormality. It is concluded that the procedure described is a very powerful method in the discrimination of brain lesions.     

Solute transport in orthorhombic lysozyme crystals: a molecular simulation study

Long-time equilibrium molecular dynamics simulations were performed to study the passage of a substrate, l-arabinose, through nanopores of orthorhombic hen egg white lysozyme crystals. Cross-linked protein crystals (CLPC), as novel biological nanoporous media, consist of an extensive regular matrix of chiral solvent-filled nanopores via which ions and solutes, e.g. sugars and amino acids, travel in and out. We studied the diffusive motion of arabinose inside protein channels. The computed diffusion coefficients within the crystal were orders of magnitudes lower relative to the diffusion coefficient of the solute in water. This study is valuable for understanding the nature of solute–protein interactions and transport phenomena in CLPCs and provides an understanding of biocatalytic and bioseparation processes using CLPC.     

Glutamate transport,glutamine synthetase and phosphate-activated glutaminase in rat CNS white matter. A quantitative study

Glutamatergic signal transduction occurs in CNS white matter, but quantitative data on glutamate uptake and metabolism are lacking. We report that the level of the astrocytic glutamate transporter GLT in rat fimbria and corpus callosum was approximately 35% of that in parietal cortex; uptake of [3H]glutamate was 24 and 43%, respectively, of the cortical value. In fimbria and corpus callosum levels of synaptic proteins, synapsin I and synaptophysin were 15-20% of those in cortex; the activities of glutamine synthetase and phosphate-activated glutaminase, enzymes involved in metabolism of transmitter glutamate, were 11-25% of cortical values, and activities of aspartate and alanine aminotransferases were 50-70% of cortical values. The glutamate level in fimbria and corpus callosum was 5-6 nmol/mg tissue, half the cortical value. These data suggest a certain capacity for glutamatergic neurotransmission. In optic and trigeminal nerves, [3H]glutamate uptake was < 10% of the cortical uptake. Formation of [14C]glutamate from [U-14C]glucose in fimbria and corpus callosum of awake rats was 30% of cortical values, in optic nerve it was 13%, illustrating extensive glutamate metabolism in white matter in vivo. Glutamate transporters in brain white matter may be important both physiologically and during energy failure when reversal of glutamate uptake may contribute to excitotoxicity.     

Proline residues in transmembrane helices of channel and transport proteins: a molecular modelling study.

Proline residues are commonly found in putative transbilayer helices of many integral membrane proteins which act as transporters, channels and receptors. Intramembranous prolines are often conserved between homologous proteins. It has been suggested that such intrahelical prolines provide liganding sites for cations via exposure of the backbone carbonyl oxygen atoms of residues i-3 and i-4 (relative to the proline). Molecular modelling studies have been carried out to evaluate this proposal. Bundles of parallel proline-kinked helices are considered as simplified models of ion channels. The energetics of K+ ion-helix bundle interactions are explored. It is shown that carbonyl oxygens exposed by the proline-induced kink and at the C-terminus of the helices may provide cation-liganding sites. 'Hybrid' bundles of antiparallel helices, only some of which contain proline residues, are considered as models of transport proteins. Again, proline-exposed carbonyl oxygens are shown to be capable of liganding cations. The roles of alpha-helix dipoles and of the geometry of helix packing are considered in relation to cation-bundle interactions. Implications with respect to modelling of ion channel and transport proteins are discussed.     

Plasmid transformation by electroporation of Leuconostoc paramesenteroides and its use in molecular cloning.

In this report, we demonstrate the utility of electroporation as an efficient method for genetic transformation of Leuconostoc paramesenteroides. We optimized several factors which determine the transformation frequency, resulting in transformation efficiencies of up to 4 x 10(3) transformants per micrograms of pNZ12 DNA, which contains the promiscuous Lactococcus lactis pSH71 replicon. Slightly lower efficiencies were obtained with a deletion derivative of the broad-host-range plasmid pAM beta 1. These plasmids could be stably maintained in L. paramesenteroides NZ6009 for more than 100 generations, even in the absence of selective pressure. In order to show the use of the developed host-vector system, we cloned the Lactococcus lactis gene encoding phospho-beta-galactosidase in L. paramesenteroides. Expression of this heterologous gene in L. paramesenteroides under control of Lactococcus lactis expression signals was evident from the presence, in transformants, of phospho-beta-galactosidase activity and a specific phospho-beta-galactosidase protein band on Western blots (immunoblots). In addition, we transformed a lactose-deficient derivative of L. paramesenteroides with a plasmid carrying a Lactococcus lactis-Escherichia coli lacZ gene fusion. The resulting transformants synthesized high levels of beta-galactosidase, indicating the efficiency of heterologous gene expression signals in L. paramesenteroides.     

Axonal transport: a quantitative study of retained and transported protein fraction in the cat

The fast axonal transport of proteins was studied in the cat sciatic nerve after injection of [3H]leucine into the spinal ganglion or the ventral horn of the seventh lumbar segment. The amount of transported proteins after ganglion injection was linearly related to the amount of label present at the ganglion. At variable intervals after ganglion or spinal cord injection, the sciatic nerves were sectioned in some experiments. The transport of proteins continued in the peripheral nerve stump in a wavelike manner, but the advancing wave leaves a labeled trail behind. A fraction of this trail corresponds to proteins moving at slower velocities than the velocity of proteins in the wave front. Another fraction of the trail corresponds to molecules retained by the axons. Each nerve segment of 5 mm in length retains 1.5% of the transported proteins, and the profile of retained proteins along the sciatic nerves follows a single exponential function. From the proportion of retained proteins, the concentration of transported proteins at the terminals of branching axons as a function of the branching ratio was estimated. In the case of motor axons innervating the soleus muscle of the cat, the concentration of recently transported proteins at the nerve terminals would be approximately 0.83% of the proteins leaving the spinal cord. This low concentration of transported proteins at the nerve terminals may explain the lability of neuromuscular synapses when axonal transport is decreased or interrupted.     

Protein transport into canine pancreatic microsomes: a quantitative approach.

Transport of presecretory proteins into the mammalian rough endoplasmic reticulum involves a protein translocase that comprises the integral membrane proteins Sec61alphap, Sec61betap, and Sec61gammap as core components. Electron microscopic analysis of protein translocase in rough microsomal membranes suggested that between three and four heterotrimeric Sec61p complexes form the central unit of protein translocase. Here we analyzed the stoichiometry of heterotrimeric Sec61p complexes present in cotranslationally active protein translocases of canine pancreatic microsomes and various other lumenal and membrane components believed to be subunits of protein translocase and to be involved in covalent modifications. Based on these numbers, the capacity for cotranslational transport was estimated for the endoplasmic reticulum of the human pancreas.     

A new high molecular mass protein showing unique localization in desmosomal plaque

A high molecular mass protein of 680 kD was identified and purified from the isolated desmosomes in bovine muzzle epidermal cells. This protein, called "desmoyokin" (from the English, yoke) here, showed no binding ability with keratin filaments in vitro, and its molecule had a characteristic dumbell shape approximately 170 nm in length. We have succeeded in obtaining one monoclonal antibody specific to desmoyokin. By the use of this monoclonal antibody and antidesmoplakin monoclonal antibody, desmoyokin was shown to be colocalized with desmoplakin at the immunofluorescence microscopic level; desmoyokin occurred only in the stratified epithelium, not in the simple epithelium nor in the other tissues. At the electron microscopic level, these two proteins were clearly seen to be sorted out in the plaque of desmosomes with desmoyokin at the periphery and desmoplakin at the center of the disk- shaped desmosomal plaque, suggesting that these two plaque proteins play distinct roles in forming and maintaining the desmosomes in stratified epithelium.     

A kinetic study of mitochondrial calcium transport.

This report describes a kinetic analysis of energy-linked Ca2+ transport in rat liver mitochondria, in which a ruthenium red/EGTA [ethanedioxy-bis(ethylamine)-tetraacetic acid] quenching technique has been used to measure rates of 45Ca2+ transport. Accurately known concentrations of free 45Ca2+ were generated with Ca2+/nitrilotriacetic acids buffers for the determination of substrate/velocity relationships. The results show that the initial velocity of transport is a sigmoidal function of Ca2+ concentration (Hill coefficient = 1.7), the Km being 4 muM Ca4 at 0 degrees C and pH 7.4. These values for the Hill coefficient and the Km remain constant in the presence of up to 2 mM phosphate, but with 10 mM acetate both parameters are increased slightly. Both permeant acids increase the maximum velocity to an extent dependent on their concentration. The Ca2+-binding site(s) of the carrier contains a group ionizing at pH approximately 7.5 at 0 degrees C, which is functional in the dissociated state. The stimulatory effect of permeant acids is ascribed to their facilitating the release of Ca2+ from the carrier to the internal phase, an interpretation which is strengthened by the lack of effect of the permeant anion SCN- on Ca2+ transport. Studies on the time-course of Ca2+ uptake and of EFTA-induced Ca2+ efflux from pre-loaded mitochondria demonstrate the reversibility of the carrier in respiring mitochondria and the extent to which this property is influenced by permeant acids. These data are accommodated in a carrier mechanism based on electrophoretic transport of Ca2+ bound to pairs of interacting acidic sites.     

Ion-water and ion-polypeptide correlations in a gramicidin-like channel. A molecular dynamics study.

This work describes a molecular dynamics study of ion-water and ion-polypeptide correlation in a model gramicidin-like channel (the polyglycine analogue) based upon interaction between polarizable, multipolar groups. The model suggests that the vicinity of the dimer junction and of the ethanolamine tail are regions of unusual flexibility. Cs+ binds weakly in the mouth of the channel: there it coordinates five water molecules and the #11CO group with which it interacts strongly and is ideally aligned. In the channel interior it is generally pentacoordinate; at the dimer junction, because of increased channel flexibility, it again becomes essentially hexacoordinate. The ion is also strongly coupled to the #13 CO but not to either #9 or #15, consistent with 13C NMR data. Water in the channel interior is strikingly different from bulk water; it has a much lower mean dipole moment. This correlates with our observation (which differs from that of previous studies) that water-water angular correlations do not persist within the channel, a result independent of ion occupancy or ionic polarity. In agreement with streaming potential measurements, there are seven single file water molecules associated with Cs+ permeation; one of these is always in direct contact with bulk water. At the mouth of an ion-free channel, there is a pattern of dipole moment alteration among the polar groups. Due to differential interaction with water, exo-carbonyls have unusually large dipole moments whereas those of the endo-carbonyls are low. The computed potential of mean force for CS+ translocation is qualitatively reasonable. However, it only exhibits a weakly articulated binding site and it does not quantitatively account for channel energetics. Correction for membrane polarization reduces, but does not eliminate, these problems.