Monolayer Fusion Research Articles

Chitosan-gentamicin conjugate attenuates heat stress-induced intestinal barrier injury via the TLR4/STAT6/MYLK signaling pathway: In vitro and in vivo studies

Heat stress (HS) has a negative impact on animal health. A modified chitosan-gentamicin conjugate (CS-GT) was prepared to investigate its potential protective effects and mechanism of action on heat stress-induced intestinal mucosa injury in IPEC-J2 cells and mouse 3D intestinal organs in a mouse model. CS-GT significantly (P < 0.01) reversed the decline in transmembrane resistance and increased the FITC-dextran permeability of the IPEC-J2 monolayer fusion epithelium caused by heat stress. Heat stress decreased the expression of the tight binding proteins occludin, claudin1, and claudin2. However, pretreatment with CS-GT significantly increased (P < 0.01) the expression of these tight binding proteins. Mechanistically, CS-GT inhibited the activation of the TLR4/STAT6/MYLK signaling pathway induced by heat stress. Molecular docking showed that CS-GT can bind effectively with TLR4. In conclusion, CS-GT alleviates heat stress-induced intestinal mucosal damage both in vitro and in vivo. This effect is mediated, at least partly, by the inhibition of the TLR4/STAT6/MYLK signaling pathway and upregulation of tight junction proteins. These findings suggest that CS-GT may have therapeutic potential in the prevention and treatment of heat stress-related intestinal injury.

Membrane fusion. Two possible mechanisms underlying a decrease in the fusion energy barrier in the presence of fusion proteins

Here we explored a contribution of fusion proteins to stalk formation, the first stage of membrane fusion, and considered two likely mechanisms, by which these proteins could influence the membrane transformation. One mechanism represents the induction of spontaneous membrane curvature, while another is membrane disturbance by a force generated by attached proteins. The energy barrier arising due to the deformation of approaching membranes and hydration repulsion between them was calculated. In addition, a dependence of an energy barrier height on certain protein features, such as spontaneous curvature, was analyzed. It was found that if fusion proteins do not produce a force directly applied to fusing membranes, they negligibly affect the barrier height irrespective of a value of spontaneous protein curvature. Thus, the overall results provide evidence that if fusion proteins are unable to exert force, they cannot provide monolayer fusion of the membranes.

A Molecular Mechanism of Lipid Membrane Fusion

Membrane fusion is an essential molecular event involved in many cellular processes, such as exocytosis, endocytosis, intracellular vesicle trafficking, fertilization, and viral infection to target cells. In spite of extensive studies of membrane fusion, however, the basic molecular mechanisms in biological systems are not well understood. Probably, it is due to the complex nature of biological membranes and the variety of possible molecular pathways for membrane fusion. We have studied the membrane fusion process, particularly ion-induced membrane fusion. Biological membrane fusion seems to occur with either ion-induced or non-ion-induced membrane process, particularly the later case is for virus membrane fusion system. Dr. Chernomordik and his co-workers have studied on non-ion-induced lipid membrane fusion and developed the so-called “Stalk-intermediate model” before total membrane fusion. That fusion model has been well received by many membrane fusion investigators, particularly in the virus fusion field. Stalk formation between two lipid membranes may occur due to undulation of lipid molecules or local binding of the lipid bilayers, which results in the formation of a local region of outer monolayer fusion. The stalk hypothesis can be described by macroscopic models treating bilayers and monolayers as homogeneous elastic surfaces. We have also studied non-ion-induced bilayer membrane fusion. Our membrane fusion theory is based on the interaction energies between the two membranes due to alternation of the membrane surface properties, e.g., hydrophilicity and hydrohobicity of interacting membranes, and then lipid membrane close approach and due to membrane curvature. Although the membrane interaction processes are different between the two models, these membrane fusion properties are the same as those of our ion-induced lipid membrane fusion.

Effect of cations/polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane

We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5 ± 1.8 Ǻ, in contrast to only 2 ± 1.5 Ǻ usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca 2+ the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na + was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors.

Surface wound healing: a new, general function of eukaryotic cells.

The ability to repair surface wounds is a property, necessary for long-term survival, expressed to various extents by all eukaryotic cell types except erythrocytes. The process is based on the rapid Ca(2+)-induced exocytosis of various types of specific organelles, such as lysosomes and enlargeosomes, that decreases surface tension and makes possible the spontaneous fusion of lipid monolayers at the lesion edges. The recognized importance of the process in physiology and in several cases of pathology is discussed.

Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.

The influence of cosurfactants on the solubilization of phenylalanine in water-in-oil microemulsion

The influence was studied of cosurfactants (a series of n-alkanols) on the equilibria of phenylalanine (Phe) solubilization in a water-in-oil (w/o) microemulsion (Winsor II system) as well as on the kinetics of phenylalanine mass transfer from an aqueous phase to a micellar phase. The distribution coefficient of phenylalanine was independent of the alcohol type and concentration. The resulting values of the water ratio and the interfacial tension as well as alcohol partitioning between the microemulsion and solvent phases were measured, also. The kinetics results (obtained from stirred cell measurements) are discussed from the view point of the three-step bud mechanism previously proposed for interfacial solubilization in w/o microemulsions. The observed “catalytic” effect of the added alcohol is discussed with respect to its influencing the incorporation of Phe molecules into the Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) layer as well as influencing the dynamics of the fusion of monolayers in the neck of the buds. Generally, the short-chain alkanols (pentanol, hexanol) accelerate the mass transfer and enable the easy reorganization of Aerosol OT molecules during the Phe incorporation, while the long-chain alkanols ( n >/ 9) retard the mass transfer from the aqueous to the micellar phase. These effects seem to be connected with the mechanical state of the surfactant/cosurfactant interfacial layer: rigid or fluid.

The influence of gangliosides on the hydrophilic pore edge line tension and monolayer fusion of lipid membranes

It is shown that inclusion of gangliosides into planar phospholipid bilayers leads to an essential suppression of their monolayer fusion. In contrast to phospholipid lysoforms, the gangliosides do not cause an associative decrease of the specific energy of the hydrophilic pore edge determined in the experiments on the electrical breakdown of membranes. This phenomenon depends on the effective size of the polar headgroup of the gangliosides. It is supposed that the effect of gangliosides on the parameters mentioned is based on the fact that they have large hydrated polar headgroups which create steric hindrances, which prevent (i) the membranes to come into contact during monolayer fusion; (ii) the gangliosides to take part in hydrophilic pore formation.

Stalk mechanism of vesicle fusion

A mechanism for rupture of a separating bilayer, resulting from vesicle monolayer fusion is investigated theoretically. The stalk mechanism of monolayer fusion, assuming the formation and expansion of a stalk between two interacting membranes is considered. The stalk evolution leads to formation of a separating bilayer and mechanical tension appearance in the system. This tension results in rupture of the separating bilayer and hydrophilic pore formation. Competition between the mechanical tension and hydrophilic pore energy defines the criteria of contacting bilayer rupture. The tension increases with an increase of the absolute value of the negative spontaneous curvature of the outer membrane monolayer, Kos. The pore edge energy decreases with an increase of the positive spontaneous curvature of the inner membrane monolayer, Kis. The relations of spontaneous curvatures of outer and inner monolayers, leading to separating bilayer rupture, is calculated. It is demonstrated that his process is possible, provided spontaneous curvatures of membrane monolayers have opposite signs: Kos less than O, Kis greater than O. Experimental data concerning the fusion process are analysed.

Membrane fusion: Overcoming of the hydration barrier and local restructuring

The early stages of membrane fusion have been investigated theoretically. It has been shown that the hydration repulsion, operating between apposed membranes, is overcome locally under the action of out-of-plane thermal fluctuations of the bilayers. The fluctuations lead to the formation of close (less than 0.5 nm) contact between the membranes within a small area (approximately 10 nm2). Increasing hydration repulsion between apposed polar heads of lipid molecules in this area causes the rupture of interacting monolayers. The rupture results in monolayer fusion of the membranes, i.e. in the formation of a bridge connecting the monolayers, which is usually named the monolayer stalk. The influence of degree of hydration of the monolayers and their spontaneous curvature on conditions of monolayer fusion have been analysed. The proposed mechanism of early stages of fusion process can proceed without preliminary formation of tight dehydrated contact between the membranes and even without any dehydration.

The shape of lipid molecules and monolayer membrane fusion

The contact between two bilayer membranes results in their monolayer fusion comprising the formation of a trilaminar structure (a single bilayer connected to two bilayers over the whole perimeter) in the contact region. The time required for monolayer fusion was measured and irreversible electrical breakdown was studied for membranes of different compositions. A theoretical model of the monolayer fusion is suggested to explain the results. It assumes that the structural reorganization underlying the process involves the formation of a stalk between bilayers as a result of local bending of the interacting monolayers. This structural reorganization is similar to the hydrophilic pore formation in a bilayer under irreversible breakdown. However, the directions of the monolayer bending are different in the two processes and, therefore, the bending energies depend oppositely on the effective shape of lipid molecules. Theoretical predictions agree well with experimental data. The applicability of the suggested mechanism to biomembrane fusion is discussed.

A simple method for fusing human lymphocytes with rodent cells in monolayer by polyethylene glycol.

Human lymphocytes were efficiently hybridized with three rodent cell lines growing attached to a substrate, by a procedure similar to the monolayer fusion technique. Lymphocytes in suspension were directly added to the monolayer and cocultured for a few hours. Treatment with polyethylene glycol (PEG) induced close contact between the two cell types, and extensive cell fusion occurred. The rate of hybridization differed markedly for the different cell lines used.

Exchange and interactions between lipid layers at the surface of a liposome solution

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. p] Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.

A comparison of the susceptibility to fusion of cell monolayers of human origin by inactivated Sendai virus

Cell monolayers of human foetal liver and lung, SV40 transformed foetal liver, HeLa cells and tonsils, showed a wide range of susceptibility to fusion by inactivated Sendai virus. Monolayers from human leucocytes, thyroid, foetal skin and thymus were also susceptible to fusion. All cells tested were found to be more susceptible to fusion in monolayers than in suspension.

THE HOST RANGE AND PLAQUE MORPHOLOGY OF RABBITPOX VIRUS (RPU+) AND ITS U MUTANTS ON CHICK FIBROBLAST, PK-2A, AND L929 CELLS.

SummaryRabbitpox virus (RPu+), 18 white pock (u) mutants derived from it and several strains of vaccinia virus were examined in respect to their cytopathology in chick embryo fibroblast cultures, and several continuous lines of mammalian cells; PK‐2a (pig kidney), L929 (mouse fibroblast), FAF (Chinese hamster fibroblast), AT (Chinese hamster, epithelial), KB (human epithelial), FL (human amnion) and Chang liver (human epithelial). The plaquing characteristics of the viruses, and the capacity to multiply exhibited by those which did not produce plaques, were investigated in chick fibroblast, PK‐2a, and L929 cells.Cytopathic changes were characterized by early toxic effects, cellular fusion, and progressive destruction of monolayers, the pattern of changes being a function of specific virus‐cell combinations. With input inocula of 1‐5 PFU per cell, most of the virus strains and mutants produced toxic changes in most of the epithelial type cells (PK‐2a, FL, Chang liver) but not in the fibroblastic cells, and to a very slight degree in KB cells. Three mutants which failed to multiply in PK‐2a cells nevertheless produced moderate toxic changes in them.Pronounced fusion of cells was produced in the epithelial type cells, but not in fibroblastic cells, by most of the viruses which multiplied in them, but not by mutants which failed to multiply.All the mutants produced plaques on chick embryo fibroblasts, and these could be distinguished as turbid or clear, and differed in size and in cultural requirements for maximal plating efficiency. A standard plaque assay method is described which gives good plaques with the majority of mutants.Conditions for optimal plaque assays on PK‐2a and L929 cells are also described, and several distinctive plaque types were recognized. Some mutants failed to produce plaques on PK‐2a or L929 cells, and in all cases except one this was shown to be due to their failure to multiply in the cells concerned.