Formation Of Hydrophilic Pores Research Articles

Effect of glycyrrhizin and its derivatives on integrity of human red blood cells

Introduction: The first and most prevailing cells that glycyrrhizin (GL) and glycyrrhetinic acid (GA) encounter are red blood cells (RBCs). However, what follows this event is poorly understood. This study aims to evaluate the effect of GL and its derivatives on the integrity of human RBCs. Methods: The integrity of human RBC was assessed under normal isotonic conditions and following osmotic and nystatin-induced colloid-osmotic stress by measuring the amount of hemoglobin released. The pore size was determined by the osmotic protection method. Results: GL was found to be virtually non-hemolytic. However, removal of the carbohydrate moiety of GL imparted significant RBC lytic activity to the cis-(beta-) but not to the trans-(alpha-) isoform of GA. The hemisuccinate radical at position C3 (carbenoxolone) greatly diminished the hemolytic property of GA. The RBC lysis occurred by colloid-osmotic mechanism due to the formation of hydrophilic pores with the radius of ~2.3 nm. At the sublytic doses, the two stereo-isoforms displayed opposite effects on the osmo-resistivity of human RBC: osmoprotection for alpha-GA and osmotic sensibilization for beta-GA. Similar osmotic sensibilization was also observed for GL and carbenoxolone. The two stereo-isoforms exhibited different but not opposite weakening effects on the resistivity of the RBC to the colloid-osmotic stress induced by nystatin, a pore-former. The weakening effect was found intermediate for GL and absent for carbenoxolone. Conclusion: Upon intestinal digestion and absorption, depending on the structure and dosage, the GL hydrolysis products interact with RBC with both beneficial and detrimental consequences.

Electroporation: An Effective Method For In Vivo Gene Delivery

Background: Gene therapy is a promising approach for the treatment of various diseases, including cancer, hereditary disorders, and some viral infections. The development of efficient and safe gene delivery systems is essential for facilitating gene trans-fer to various organs and tissues in vivo. Objective: In this review, we briefly describe the principal mechanisms of gene delivery systems, particularly electroporation, and discuss the latest advancements in the application of electro-poration for in vivo gene transfer. Methods: A narrative review of all the relevant publication known to the authors was conducted. Results: In recent years, electroporation-based strategies have emerged as an auspicious and versa-tile platform for efficient and controlled delivery of various biomolecules, including nucleic acids. Applying electric pulses of enough magnitude leads to the formation of hydrophilic pores in the cell membrane and allows the entry of otherwise membrane-impermeant molecules, such as DNA. Alt-hough electroporation has been initially developed for in vitro transfection of cells, it has recently advanced to preclinical in vivo applications and finally to clinical trials. Conclusion: Electroporation has already entered the clinical practice for antitumor therapy and may be an essential part of future personalized treatments. Given the ability of electroporation to deliver multiple genes in a single event, it will also certainly be further developed both as a stand-alone de-livery approach and when coupled with other technologies.

The good and the bad of cell membrane electroporation.

Electroporation is used to increase the permeability of the cell membrane through high-voltage electric pulses. Nowadays, it is widely used in different areas, such as medicine, biotechnology, and the food industry. Electroporation induces the formation of hydrophilic pores in the lipid bilayer of cell membranes, to allow the entry or exit of molecules that cannot otherwise cross this hydrophobic barrier. In this article, we critically review the basic principles of electroporation, along with the advantages and drawbacks of this method. We discuss the effects of electroporation on the key components of biological membranes, as well as the main applications of this procedure in medicine, such as electrochemotherapy, gene electrotransfer, and tissue ablation. Finally, we define the most relevant challenges of this promising area of research.

Towards standardization of electroporation devices and protocols

Cell exposure to high-voltage, short-duration electric pulses can lead to temporary formation of hydrophilic pores in the plasma membrane and an increase in the membrane's permeability which consequently increases the transmembrane transport of molecules that are otherwise unable to cross the membrane. This phenomenon, termed membrane electroporation, is currently an applicable technique in different areas such as biomedicine, biotechnology, food technology and environmental applications. Electroporation pulses are generated by pulse power generators known as electroporators and delivered to the cells (in tissue) via electrodes. The objective of this paper is to review and compare characteristics of electroporation applications and equipment described in the literature and/or present on the market. Since there are no specific standards or regulations that specifically refer to the safety of medical devices with intended medical uses for electroporation, we propose guidelines for the design of clinical electroporators and define minimal requirements for their safe and efficient use which can be incorporated within the particular standards in the future. In order to facilitate the comparison of data obtained by different research groups and to enable reproduction of results under the same conditions, we want to stress the necessity of defining the electroporator's output parameters and tolerances of electroporation parameters for electroporation-based therapies.

Effect of input voltage frequency on the distribution of electrical stresses on the cell surface based on single-cell dielectrophoresis analysis

Electroporation is defined as cell membrane permeabilization under the application of electric fields. The mechanism of hydrophilic pore formation is not yet well understood. When cells are exposed to electric fields, electrical stresses act on their surfaces. These electrical stresses play a crucial role in cell membrane structural changes, which lead to cell permeabilization. These electrical stresses depend on the dielectric properties of the cell, buffer solution, and the applied electric field characteristics. In the current study, the effect of electric field frequency on the electrical stresses distribution on the cell surface and cell deformation is numerically and experimentally investigated. As previous studies were mostly focused on the effect of electric fields on a group of cells, the present study focused on the behavior of a single cell exposed to an electric field. To accomplish this, the effect of cells on electrostatic potential distribution and electric field must be considered. To do this, Fast immersed interface method (IIM) was used to discretize the governing quasi-electrostatic equations. Numerical results confirmed the accuracy of fast IIM in satisfying the internal electrical boundary conditions on the cell surface. Finally, experimental results showed the effect of applied electric field on cell deformation at different frequencies.

Increase in the current variance in bilayer lipid membranes near phase transition as a result of the occurrence of hydrophobic defects

Most researchers associate the increase in the permeability of lipid bilayers of artificial and biological membranes observed in various experiments with the formation of hypothetical hydrophobic and hydrophilic pores. Although the existence of hydrophobic defects, as the first stage of the formation of a hydrophilic pore, was hypothesized decades ago from electroporation experiments, the difficulty of describing this stage is determined by the lack of experimental data confirming the existence or at least associated with hydrophobic pores. We explored the increase in the current variance through the lipid membrane, observed when approaching the phase transition from the side of high temperatures, and have associated it with capacitive currents arising in response to the formation of hydrophobic pores. Assuming that the number of hydrophobic pores in a membrane follows a Poisson distribution, and thus, the mean number of hydrophobic pores is equal to the variance of that number, we used the measurements of the membrane current variance to evaluate the number of hydrophobic pores. Analysis of experimental data within this model allows us to estimate the number of hydrophobic pores at or above the phase transition and shows that the number of hydrophobic pores in a membrane close to the phase transition increased 20 times compared to the number of hydrophobic pores existing in the membrane far from the melting transition.

The effects of SDS at subsolubilizing concentrations on the planar lipid bilayer permeability: Two kinds of current fluctuations.

Detergent effects on lipid bilayers of artificial and biological membranes at subsolubilizing concentrations are known to include the membrane permeabilization which manifests itself through both a flip-flop of detergent molecules from the outer monolayer to the inner monolayer and the membrane leakage of entrapped solutes. We have explored the current fluctuations occurring in planar BLM of asolectin in the presence of ionic detergent SDS at subsolubilizing concentration. Two groups of current fluctuations which the average duration differs by two orders of magnitude can be distinguished. We assume that these differences in the duration of current fluctuations are associated with a different number of SDS molecules in the walls of the putative toroidal hydrophilic pores. We associated short pulses with the formation of short-lived lipid hydrophilic pores. Impulses of greater duration (steps) were associated with the formation of hydrophilic pores, the walls of which contain detergent. Taking into account the characteristics of these pores, we estimated the pore energy, as well as the edge energy of these two kinds of pores. We believe that the flip-flop of SDS molecules in liposomes is provided by long-lived pores, and the contents of the liposome leakage occurs through all pores.

Acid post-treatment of sol-gel-derived ethylene-bridged organosilica membranes and their filtration performances

This study investigated the effect of acid post-treatment on the filtration performance of organosilica membranes. The membranes were prepared from 1,2-bis(triethoxysilyl)ethane (BTESE) via sol-gel processing with different H2O/BTESE molar ratios. Fourier-transform infrared spectra significant residual ethoxy groups within membranes derived from sols with low H2O/BTESE ratios, after firing at 300 °C in N2. The ethoxy groups within the membranes were hydrolyzed to hydroxide groups by immersing the membranes in hydrochloric acid (HCl). This rendered the membranes more hydrophilic. The hydroxyl groups may have promoted the formation of hydrophilic pores, which would in turn promote water permeation. The HCl-treated membranes showed higher water permeability than the original BTESE-derived membrane. The effect of HCl treatment was more pronounced for the membrane prepared with the lowest H2O/BTESE ratio of 1, resulting in approximately three times higher water permeability than the original membrane. Membranes prepared from sols with low H2O/BTESE ratios showed improved solute rejection and increased water permeability, upon HCl treatment. This was attributed to cross-linking in the organosilica matrix via the condensation of newly-formed hydroxyl groups, that caused the narrowing or blocking of large pores.

Erlang flow of hydrophilic pore formation and closure events in a lipid bilayer during phase transition resulting from diffusion in the radius space.

The Smoluchowski equation with an energy profile of a special type and an assumed hydrophobic ("half") pore source term is used to describe the process of hydrophilic pore formation in a lipid bilayer at the gel-liquid phase transition. The source term reflects the occurrence of molecule packing defects in a lipid bilayer at phase transition. The time sequences of the pore formation and closure events are treated as non-stationary, second-order Erlang flows whose characteristics depend on the equation solution. The computed distributions of the time intervals between hydrophilic pores, and pore lifetimes agree with the previously published experimental interpulse interval and pulse duration histograms for the current fluctuations through planar bilayer membranes of DPPC immersed in a LiCl aqueous solution containing polyethylene glycol. Thus, the statistical analysis of pore formation and closure times leads us to conclude that firstly, the increased permeability of a lipid bilayer during the gel-liquid phase transition is accounted for by the emergence of additional hydrophobic defects in the heterogeneous structure of the bilayer and secondly, that the non-exponential distributions of the lipid channel closed and open times observed in experiments are evidence that the process of hydrophilic pore formation is not a one-step process but involves at least two dependent events.

Bilayer permeability during phase transition as an Erlang flow of hydrophilic pores resulting from diffusion in the radius space

The formation of hydrophilic pores in a lipid bilayer during phase transition is described using the Smoluchowski equation with an additional term of the hydrophobic pore source. This term is added to account for defects in lipid packing during phase transition. We assume that the temporal sequence of the pores is a stochastic process, a non-stationary second-order Erlang flow. Flow characteristics depend on the equation solution and determine the formation times of the hydrophilic pores. The calculated distribution of the durations of intervals between hydrophilic pores is in a good agreement with experimental data published before. In the context of this model we describe the influence of poly(ethylene glycol) on the pore formation frequency.

Electroporation threshold of POPC lipid bilayers with incorporated polyoxyethylene glycol (C12E8).

Electroporation relates to a phenomenon in which cell membranes are permeabilized after being exposed to high electric fields. On the molecular level, the mechanism is not yet fully elucidated, although a considerable body of experiments and molecular dynamic (MD) simulations were performed on model membranes. Here we present the results of a combined theoretical and experimental investigation of electroporation of palmitoy-oleoyl-phosphatidylcholine (POPC) bilayers with incorporated polyoxyethylene glycol (C12E8) surfactants. The experimental results show a slight increase of the capacitance and a 22% decrease of the voltage breakdown upon addition of C12E8 to pure POPC bilayers. These results were qualitatively confirmed by the MD simulations. They later revealed that the polyoxyethylene glycol molecules play a major role in the formation of hydrophilic pores in the bilayers above the electroporation threshold. The headgroup moieties of the latter are indeed embedded in the interior of the bilayer, which favors formation of water wires that protrude into its hydrophobic core. When the water wires extend across the whole bilayer, they form channels stabilized by the C12E8 head groups. These hydrophilic channels can transport ions across the membrane without the need of major lipid head-group rearrangements.

Экологическая ниша как фактор, определяющий криорезистентность сперматозоидов рыб

In this research we have compared the spermatozoa cryoresistance of fish species from different ecological niches and explained the nature of differences based on our own results and published data. Differences in spermatozoa cryoresistance of fish species, spawning in fresh water at 0...10°C (6 species of salmonids), 18...25°C (5 species of carp) and in marine water at 16...25°C (mullet-golden mullet) were established using cryopreservation and visual assessment methods for sperm motility. Quantitative and qualitative differences in motility of frozen-thawed fish sperm from different ecological niches were established as stipulated by different strength of molecule-to-molecule bonds of membrane lipids and the ratio between components, determining the formation of hydrophilic pores at lipid phase transitions.Probl Cryobiol Cryomed 2014; 24(4):302-311.

Interactions of a Hydrophobically Modified Polycation with Zwitterionic Lipid Membranes

The interactions between synthetic polycations and phospholipid bilayers play an important role in some biophysical applications such as gene delivery or antibacterial usage. Despite extensive investigation into the nature of these interactions, their physical and molecular bases remain poorly understood. In this Article, we present the results of our studies on the impact of a hydrophobically modified strong polycation on the properties of a zwitterionic bilayer used as a model of the mammalian cellular membrane. The study was carried out using a set of complementary experimental methods and molecular dynamic (MD) simulations. A new polycation, poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3), was synthesized, and its interactions with liposomes composed of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were examined using dynamic light scattering (DLS), zeta potential measurements, and cryo-transmission electron microscopy (cryo-TEM). Our results have shown that polymer 3 can efficiently associate with and insert into the POPC membrane. However, it does not change its lamellar structure, as was demonstrated by cryo-TEM. The influence of polymer 3 on the membrane functionality was studied by leakage experiments applying a fluorescence dye (calcein) encapsulated in the phospholipid vesicles. The MD simulations of model systems reveal that polymer 3 promotes formation of hydrophilic pores in the membrane, thus increasing considerably its permeability.

Microbial inactivation by new technologies of food preservation

The increasing consumer demand for 'fresh-like' foods has led to much research effort in the last 20 years to develop new mild methods for food preservation. Nonthermal methods allow micro-organisms to be inactivated at sublethal temperatures thus better preserving the sensory, nutritional and functional properties of foods. The aim of this review is to provide an overview of the microbiological aspects of the most relevant nonthermal technologies for microbial inactivation currently under study, including irradiation, high hydrostatic pressure, pulsed electric field and ultrasound under pressure. Topics covered are the mechanisms of inactivation, sensitivity of different microbial groups and factors affecting it and kinetics of inactivation.

A Liposome-Permeating Activity From the Surface of the Carapace of the American Horseshoe Crab, Limulus polyphemus

Colonization by sessile fouling organisms (epibionts) is the usual fate for solid surfaces in the marine environment. The American horseshoe crab, Limulus polyphemus, ceases molting upon reaching maturity and lives several years as an adult in an epibiont-rich milieu, yet it typically maintains a cuticle that is largely free of macroscopic fl ora and fauna. Indeed, it is in the interest of the animal to maintain its carapace free from such organisms, as colonization of the cuticle by green and blue-green algae can be fatal (1). The mechanisms by which Limulus maintains a clean carapace are not well understood. We have investigated the antibiological properties of a potential anti-fouling system of Limulus, a viscous secretion of a system of dermal glands that discharge their product onto the surface of the carapace (2). We propose that this substance, dermal exudate (DE), contributes to maintaining the cleanliness, and thus the integrity, of the cuticle. In the past we have identifi ed and partially characterized a hemolytic activity present in DE (3, 4). It was shown that the presence of macromolecular osmolites in the hemolysis assay medium, dextran-8 and to a lesser extent dextran-4, prevented lysis of the red blood cells. This effect is attributed to the ability of these macromolecular osmolites to establish an osmotic balance between the interior and exterior of the cell so that no net water fl ow into the cell occurs, protecting the cell from cytolysis. The inhibitory effect of dextrans suggests that DE-induced lysis results from hydrophilic pore formation in the lipid bilayer of the red blood cell rather than from a detergent-like disruption of lipid packing or from phospholipase activity. To investigate the potential interactions of DE directly with lipid bilayers, we have utilized a model system in which a self-quenching fl uorescent dye has been trapped inside liposomes; an increase in the fl uorescence of the dye is a marker for membrane permeation. Here we report the ability of an acid-precipitable constituent of DE to permeabilize liposomes. The agent or agents responsible for these hemolytic and liposomepermeating activities may function as deterrents against potential colonizers of the Limulus cuticle. The secretion of DE can be stimulated by housing Limulus in stressful conditions such as a cage stocked with decaying fi sh material. Dermal exudate was collected by scraping the dorsal carapace. The exudate was stored at 20 °C or with 0.02% NaN3 to prevent bacterial growth. Liposomes were prepared with soybean type II phosphatidylcholine from Sigma. Lyophilized lipid was dissolved in chloroform and dried under a nitrogen stream to a thin fi lm. The fi lm was placed under vacuum overnight to remove any traces of solvent. The lipids were hydrated in 20 mM Tris pH 7.4, 100 mM carboxyfl uorescein (CF), a self-quenching fl uorescent dye. The resulting multilamellar liposomes were made unilamellar through fi ve successive freeze-thaw cycles. Untrapped CF was removed from the liposome suspension by passage over a Sephadex G-50 column. The release of CF from the interior of liposomes was monitored as an indicator of lipid bilayer permeation. Assays were performed with a 1:1000 dilution of liposomes in 20 mM Tris, pH 7.2. Fluorescence intensity was observed with a Photon technologies fl uorometer. Excitation and emission wavelengths were 460 nm and 550 nm respectively. The slit widths were adjusted such that the Raman scatter peak of distilled, deionized water at 397 nm gave an intensity of approximately 350,000 counts/s when excited at 350 nm. Release of entrapped CF from liposomes was seen as an increase in fl uorescence intensity. A unit of activity is arbitrarily de fi ned as an initial increase in fl uorescent intensity of 5% per minute. Intensity values corresponding to 100% lysis were obtained with the addition of Triton X-100.

Molecular features of the cytolytic pore-forming bacterial protein toxins.

The repertoire of the cytolytic pore-forming protein toxins (PFT) comprises 81 identified members. The essential feature of these cytolysins is their capacity to provoke the formation of hydrophilic pores in the cytoplasmic membranes of target eukaryotic cells. This process results from the binding of the proteins on the cell surface, followed by their oligomerization which leads to the insertion of the oligomers into the membrane and formation of protein-lined channels. It impairs the osmotic balance of the cell and causes cytolysis. In this review the molecular aspects of a number of important PFT and their respective encoding structural genes will be briefly described.

Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues.

Stonustoxin (SNTX) is a two-subunit protein toxin purified from the venom of the stonefish (Synanceja horrida), which induces potent haemolytic activity. We examined the pore-forming property of this non-enzymic protein by an osmotic protection assay. SNTX-induced haemolysis was completely prevented by osmotic protectants of adequate size [poly(ethylene) glycol 3000; molecular diameter approx. 3.2 nm]. Uncharged molecules of smaller size, such as raffinose and poly(ethylene) glycol 1000-2000, failed to protect against cell lysis. These findings indicate that SNTX induces the formation of hydrophilic pores in the cell membrane, which results in the lysis of erythrocytes. Since cationic residues contribute significantly to the cytolytic activity of several other pore-forming toxins, we examined the role of positively charged lysine and arginine residues in the haemolytic activity of SNTX. SNTX lost its haemolytic activity when the positively charged side chains of lysine residues were neutralized or converted into negatively charged side chains upon carbamylation or succinylation respectively. The haemolytic activity of SNTX was also inhibited by the modification of positively charged arginine residues using 2,3-butanedione. The loss of haemolysis showed strong correlation with the number of Lys or Arg residues modified. CD analyses, however, showed that the conformation of SNTX was not significantly affected by these chemical modifications. Further, the haemolytic activity of SNTX was competitively inhibited by various negatively charged lipids, such as phosphatidylserine, cardiolipin and monosialogangliosides. These results indicate that SNTX induces potent haemolytic activity through the formation of pores in the cell membrane, and that cationic residues play a crucial role in its cytolytic mechanism.

47]Assays for characterizing URF 13, the pathotoxin and methomyl receptor of cms-T maize

This chapter explains assays for characterizing URF 13, the Pathotoxin and Methomyl receptor of cms- T maize. Maize carrying the Texas cytoplasm is male sterile. Cytoplasmic male sterility (CMS) in cms-T maize is characterized by pollen abortion and failure to exert anthers, but female fertility is unaffected. Maize containing the cms-T cytoplasm is used extensively in hybrid seed production as it eliminates the need for manual emasculation. A 13-kDa protein—URF13—is the product of a mitochondrial gene called T-urf13, which is specific to cms-T maize and arises from a novel series of rearrangements within the mitochondrial genome. URF13 is an integral membrane protein that is located in the inner mitochondrial membrane. Escherichia coli cells expressing URF13 are also sensitive to T-toxin. T-toxin interacts with isolated cms-T mitochondria: cell respiration is inhibited, spheroplasts swell, and ion leakage is induced. Methomyl is often used in assays and in selective screens for the presence of functional URF13 as it is readily available (as Lannate), whereas T-toxin is more difficult to obtain. The effects of T-toxins or methomyl on cms-T mitochondria have led to the hypothesis that the interaction between these compounds and URF13 results in the formation of hydrophilic pores that permeabilize the inner mitochondrial membrane.

A model of amphiphilic-polypeptide-induced hydrophilic pores in membranes of dimyristoylphosphatidylcholine small unilamellar vesicles

Based on experimental results we propose a model of the complex between an amphiphilic polypeptides (LKKL) 4, and dimyristoyl phosphatidylcholine small unilamellar vesicles which can aid our understanding of the formation of both structural defects and hydrophilic pores in biological membranes. In this model the amphiphilic polypeptides lie parallel to the membrane surface and can penetrate down carbon C 5 into the outer leaflet of the bilayer. A domain of phospholipid heads is oriented around each polypeptide under the influence of the electric field generated by the α helix macrodipole. When two (or more) of these oriented domains, which can move freely on the membrane surface, approach each other closely the charge repulsion leads to the formation of structural defects. If an external or internal transmembrane potential is applied, the a helix macrodipoles reorient parallel to the electric field lines, resulting in the formation of hydrophilic pores.

Pore-formation by Escherichia coli hemolysin (HlyA) and other members of the RTX toxins family.

Escherichia coli hemolysin (HlyA) is a major cause of E. coli virulence. It lyses erythrocytes by a colloid osmotic shock due to the formation of hydrophilic pores in the cell wall. The size of these channels can be estimated using osmotic protectant of increasing dimensions. To show that the formation of pores does not depend critically on the osmotic swelling we prepared resealed human erythrocyte ghosts loaded with a fluorescent marker. When attacked by HlyA the internal marker was released, indicating the formation of toxin channels so large as to let it through. The channels can be directly demonstrated also in purely lipidic model systems such as planar membranes and unilamellar vesicles, which lack any putative protein receptor. HlyA has been recognised as a member of a large family of exotoxins elaborated by Gram-negative organisms including Proteus, Bordetella, Morganella, Pasteurella and Actinobacillus. These toxins have quite different target cell specificity and in many cases are leukocidal. When tried on planar membranes however, even specific leukotoxins open channels not dissimilar from those formed by HlyA, suggesting this might be a common step in their action. Comparison of the hydrophobic properties of six members of the toxin family indicates the presence of a conserved cluster of ten contiguous amphipathic helixes, located in the N-terminal half of the molecule, which might be involved in channel formation.