Metastability of forest ecosystems infested by bark beetles

A simple two-species differential equation model is used to investigate the intrinsic metastability of forest ecosystems subjected to bark beetle infestations. We demonstrate that only one globally stable node or limit cycle is likely under biologically plausible conditions, but that, in the former case, this equilibrium is very sensitive to external perturbation.     

Metastability of microtubules induced by competing internal forces

Recent modeling efforts to estimate energies of tubulin-tubulin bonds shed light on a delicate balance between competing mechanical forces maintaining microtubule walls. Here we formulate two important refinements to the explanation of bond energetics. First, energy surface calculations in the elastic filament approximation reveal a finite stabilizing barrier assumed a simple Lennard-Jones-like potential for protein bonds. The presence of a guanosine triphosphate (GTP) cap represented by straight segments is necessary, as it is predicted for a long time. In the lack of such a cap, the protofilaments are either in an absolutely stable or absolutely unstable state. Second, our calculations show that this barrier appears only if the mechanical energy associated with the conformational change after GTP hydrolysis (curling energy) is larger than the strength of lateral bonds. The overall energy balance we propose supports continuous assembly of GTP dimers, a metastable state in the presence of a finite GTP cap and energetically driven disassembly of guanosine diphosphate protofilaments.     

Cryopreservation of keratinocytes in a monolayer.

The cryopreservation of cells in tissues is one of the major challenges in current cryobiology, especially with regard to the progressively increasing field of tissue engineering. It is very questionable whether protocols which were developed for the cryopreservation of isolated cells are also applicable for cells in more complex structures, such as tissues. As a starting point toward cryopreservation of these three-dimensional structures, the aim of this study was to find an optimum cryopreservation protocol for keratinocytes in a monolayer (two-dimensional structure). These epidermal cells can be transplanted as a monolayer grown on an appropriate matrix for the treatment of deep-dermal burns and leg ulcers. The successful cryopreservation of such transplants would offer the advantage of long-term storage and immediate availability of the transplant. In our study, the variables investigated were the cryoprotective solution and the cooling rate. In order to find a nontoxic cryoprotective agent (CPA) which could be transplanted without an additional washing step, we included hydroxyethyl starch (HES) as a possible CPA in our experimental protocol with the commonly used CPAs Me(2)SO, glycerol, and ethylene glycol. For the evaluation, the cell survival rate was determined by dye exclusion (trypan blue) and the cell metabolism was investigated by cell activity assay (alamarBlue). In conclusion, the cryopreservation protocol with 10 wt.-% HES resulted not only in the highest survival rate (72%) but also in the highest metabolic activity of the cells after thawing; comparable values for the other CPAs were: Me(2)SO, 48%; glycerol, 8%; and ethylene glycol, 10%.     

Quantitative Characterization of Metastability and Heterogeneity of Amyloid Aggregates

Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Aβ (TMR-Aβ) to characterize its stability and heterogeneity. Disaggregation of TMR-Aβ amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Aβ amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.     

Phase diagrams for a model of a lipid monolayer

A microscopic model of a lipid monolayer is proposed. It includes, within a single scheme, the following factors which are considered to be essential in phase transitions in lipid systems: formation of gauche rotamers, interactions between polar heads, interactions between hydrocarbon chains (depending on their conformation) and changes in the energy of the system due to a directional ordering of the chains. Phase diagrams are constructed and discussed and it is shown how the phase diagrams are modified by alterations of these parameters and the length of the hydrocarbon chains.     

Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells

Rates of fluid pinocytosis by bovine aortic endothelial cells were measured during various manipulations of growth status in vitro. Sparsely seeded cultures grew exponentially until a confluent monolayer was formed, at which time growth slowed. This change in growth rate coincided with a decline in the rate of pinocytosis to about one-third that in the growing cultures. During the subsequent attainment of maximal cell density in the confluent monolayer, the pinocytic rate remained constant. There was close correlation between ³H-thymidine labelling indices, as measured by autoradiography, and the rates of pinocytosis. Mechanical “wounding” of the confluent monolayer resulted in cell migration and proliferation. Twenty-four hours after “wounding,” rates of pinocytosis per mg. cell protein were significantly enhanced. When regeneration of the monolayer was blocked by cytochalasin B, pinocytosis remained at the same rate as in the uninjured, confluent monolayer. These experiments support, and extend to endothelium, earlier observations that in growing cells pinocytosis proceeds at a higher rate than in non-growing, quiescent cells. Furthermore, they raise the possibility that the transendothelial transport of macromolecules such as lipoproteins by receptor-in-dependent fluid pinocytosis in vivo may be altered by the growth status of the endothelium.     

Insertion of pea lectin into a phospholipid monolayer

Pea lectin (PSL) is a secretory sugar-binding protein, readily soluble in aqueous solutions of low osmolarity. However, PSL also appears to be associated with the plasma membrane at the tip of young pea root hairs. By using the Wilhelmy plate method, we found that PSL can insert into a lipid monolayer. This property appeared to be independent of the sugar-binding ability of the protein. This result suggests that PSL may be directly involved in membrane-mediated interactions with saccharide ligands, for example during root hair infection by symbiotic rhizobia.     

Lateral electrical conduction along a phosphatidylcholine monolayer

Lateral electrical conduction due to lipid-monolayers spread on the surface of pure water was observed under both d.c. and a.c. electrical fields. An apparent specific electrical conductivity is evaluated as high as approximately equal to 4.10(-2) mho/cm for the monolayer-water system of L-DPPC at 25 degrees C. Arrhenius plots of the apparent conductance show a deflection at a temperature corresponding to a crystalline-to-fluid phase transition of the surface monolayer. From the magnitude and temperature dependence of conductance and a comparison of results with those obtained by use of deuterated water, it is concluded that enhanced protonic conduction mediated by a network consisted of polar head groups of phosphatidylcholines and water molecules may be brought about near the lipid/water interface.     

Metastability in a temperature-dependent model system for predator-prey mite outbreak interactions on fruit trees

The nonlinear behavior of a particular Kolmogorov-type exploitation differential equation system assembled by May (1973,Stability and Complexity in Model Ecosystems, Princeton University Press) from predator and prey components developed by Leslie (1948,Biometrica 35, 213–245) and Holling (1973,Mem. Entomol. Soc. Can. 45, 1–60), respectively, is re-examined by means of the numerical bifurcation code AUTO 86 with model parameters chosen appropriately for a temperature dependent mite interaction on fruit trees. The most significant result of this analysis is that, in addition to the temperature ranges over which the single community equilibrium point of the system iseither globally stableor gives rise to a globally stable limit cycle, there can also exist a range wherein multiple stable states occur. These stable states consist of a focus (spiral point) and a limit cycle, separated from each other in the phase plane by an unstable limit cycle. The ecological implications of such metastability, hysteresis and threshold behavior for the occurrence of outbreaks, the persistence of oscillations, the resiliency of the system and the biological control of mite populations are discussed. It is further suggested that a model of this sort which possesses a single community equilibrium point may be more useful for representing outbreak phenomena, especially in the presence of oscillations, than the non-Kolmogorov predator-prey systems possessing three community equilibrium points, two of which are stable and the other a saddle point, traditionally employed for this purpose.     

Metastability in the inhibitory mechanism of human alpha1-antitrypsin

Metastability of the native form of proteins has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of influenza virus and the strained native structure of serpins (serine protease inhibitors) are typical examples. To understand the structural basis and functional role of the native metastability of inhibitory serpins, we characterized stabilizing mutations of alpha1-antitrypsin in a region presumably involved in complex formation with a target protease. We found various unfavorable interactions such as overpacking of side chains, polar-nonpolar interactions, and cavities as the structural basis of the native metastability. For several stabilizing mutations, there was a concomitant decrease in the inhibitory activity. Remarkably, some substitutions at Lys-335 increased the stability over 6 kcal mol-1 with simultaneous loss of activity over 30% toward porcine pancreatic elastase. Considering the location and energetic cost of Lys-335, we propose that this lysine plays a pivotal role in conformational switch during complex formation. Our current results are quite contradictory to those of previously reported hydrophobic core mutations, which increased the stability up to 9 kcal mol-1 without any significant loss of activity. It appears that the local strain of inhibitory serpins is critical for the inhibitory activity.     

Albert Galeev: The Problem of Metastability and Explosive Reconnection

Plasma Physics Reports - −Albert Abubakirovich Galeev is a Soviet and Russian expert in plasma physics who actively contributed to fusion research. In the early 1970s, he became a head of...     

Thermodynamic equilibrium of domains in a two-component Langmuir monolayer

This [corrected] article outlines the results from a combined experimental and theoretical study on the properties of circular domains in a mixed Langmuir monolayer at thermodynamic equilibrium. The mixed monolayer consisted of a binary mixture of dimyristoyl-phosphatidyl-choline and dihydrocholesterol. A long-term fluorescence microscopy study of these domains was carried out over the course of approximately 60 h. Image analysis of the domains over time revealed that the domains ripened slowly with an [corrected] increase in mean domain radius and a [corrected] decrease in domain number density. At the end of the measurement, the domains remained polydisperse, and true thermodynamic equilibrium was not reached. Theoretically, collective thermodynamic equilibrium properties such as mean domain size and size distribution were calculated by combining micelle self-assembly theory and the "equivalent dipole" model for the self-energy of two-dimensional domains. The calculations predicted existence of finite-sized circular domains at equilibrium. This suggests that equilibrium circular monolayer domains of single- or multicomponent lipids with a finite size distribution should form only at very limited experimental conditions. Both the predicted mean domain size and size distribution are strongly affected by line tension and dipole moment density difference. A comparison between the theoretical and experimental results is made.     

Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer

Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.     

Metastability of Papain and the Molecular Mechanism for its Sequential Acid-Denaturation

Acid unfolding of non-inhibited papain at pH 2 was studied by means of spectroscopic and electrophoresis techniques as well as activity assays. We found a molten globule like species (A state) similar to that previously reported for bromelain and S-carboxy-methyl-papain. We demonstrated that this A state is not thermodynamically stable but a metastable conformer which decays into an unfolded conformation in a few hours. The mechanism of acid unfolding to the A state proved to be completely irreversible, with a biphasic time evolution of spectroscopic signals characteristic of the existence of a kinetic intermediate. This latter species showed properties in-between native and A state such as secondary structure, exposition of hydrophobic area and tryptophan environment, but a native like hydrodynamic radius. Native papain seems to unfold at acid pH through at least two kinetic barriers, being its proregion mandatory to conduct and stabilize its active structure. Computer simulations of acid unfolding, followed by ANS docking, identified three regions of cavity formation induced by acid media which might be used as regions to be fortified by protein engineering in the quest for extreme-resistant proteases or as hot-spots for protease inactivation.     

Freeze-fracture of monolayer cultures

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.     

Kinetic analysis of antibody-antigen interactions at a supported lipid monolayer

Modified phospholipids possessing carboxyl head groups synthesized from phosphatidylethanolamine were incorporated into supported lipid monolayers on top of a thin gold film. A monoclonal antibody was chemically coupled to the modified lipids in these monolayers and the kinetics of antigen binding were determined by surface plasmon resonance. The binding could be analyzed using a conventional 1:1 binding algorithm and the derived kinetic and affinity constants were almost identical to those reported for the same interaction on a dextran hydrogel-based sensor chip. When an antigen was chemically coupled to a modified lipid monolayer, the binding of a monoclonal antibody to this surface was biphasic. A two-step algorithm describing the formation of a 1:2 antibody:antigen complex was developed which accurately described the data and enabled differentiation of the two binding steps. The binding was assayed varying both the concentration of antibody in solution and the density of antigen on the surface. The affinities determined by Scatchard analysis of equilibrium binding levels were similar to those values obtained from an ELISA.     

Three-dimensional organization of retroviral capsid proteins on a lipid monolayer

We have used a method for the two-dimensional crystallization of retroviral structural proteins to obtain a three-dimensional structure of negatively stained, membrane-bound, histidine-tagged Moloney murine leukemia virus (M-MuLV) capsid protein (his-MoCA) arrays. Tilted and untilted micrographs from crystals formed by purified his-MoCA proteins incubated beneath lipid monolayers containing nickel-chelating lipids were used in 3D reconstructions. The 2D crystals had unit cell dimensions of a=72.6 A, b=72.5 A and gamma=119.5 degrees, but appeared to have no intrinsic symmetry (p1) in 3D, in contrast to the trigonal or hexagonal appearance of their 2D projections. Membrane-bound his-MoCA proteins showed a strand-like organization, apparently with dimer building blocks. Membrane-proximal regions, or putative N-terminal domains (NTDs), dimerized with different partners than the membrane-distal putative C-terminal domains (CTDs). Evidence also suggests that CTDs can adopt alternate orientations relative to their NTDs, forming interstrand connections. Our results are consistent with helical-spiral models for retrovirus particle assembly, but are not easily reconcilable with icosahedral models.     

Mouse TSPO in a lipid environment interacting with a functionalized monolayer

Translocator protein TSPO is a membrane protein highly conserved in evolution which does not belong to any structural known family. TSPO is involved in physiological functions among which transport of molecules such as cholesterol to form steroids and bile salts in mammalian cells. Membrane protein structure determination remains a difficult task and needs concomitant approaches (for instance X-ray- or Electron-crystallography and NMR). Electron microscopy and two-dimensional crystallization under functionalized monolayers have been successfully developed for recombinant tagged proteins. The difficulty comes from the detergent carried by membrane proteins that disrupt the lipid monolayer. We identified the best conditions for injecting the histidine tagged recombinant TSPO in detergent in the subphase and to keep the protein stable. Reconstituted recombinant protein into a lipid bilayer favors its adsorption to functionalized monolayers and limits the disruption of the monolayer by reducing the amount of detergent. Finally, we obtained the first transmission electron microscopy images of recombinant mouse TSPO negatively stained bound to the lipid monolayer after injection into the subphase of pre-reconstituted TSPO in lipids. Image analysis reveals that circular objects could correspond to an association of at least four monomers of mouse TSPO. The different amino acid compositions and the location of the polyhistidine tag between bacterial and mouse TSPO could account for the formation of dimer versus tetramer, respectively. The difference in the loop between the first and second putative transmembrane domain may contribute to distinct monomer interaction, this is supported by differences in ligand binding parameters and biological functions of both proteins.     

Occluding junction structure-function relationships in a cultured epithelial monolayer

Electrical circuit analysis was used to study the structural development of occluding junctions (OJs) in cultured monolayers composed to T84 cells. The magnitude of the increments in transepithelial resistance predicted by such analysis was compared with the magnitude of the measured increments in resistance. Confluent sheets of epithelial cells were formed after cells were plated at high density on collagen-coated filters. Using Claude's OJ strand count-resistance hypothesis (1978, J. Membr. Biol. 39:219-232), electrical circuit analysis of histograms describing OJ strand count distribution at different time points after plating predicted that junctional resistance should rise in a proportion of 1:21:50 from 18 h to 2 d to 5 d. This reasonably paralleled the degree of rise in transepithelial resistance over this period, which was 1:29:59. The ability to predict the observed resistance rise was eliminated if only mean strand counts were analyzed or if electrical circuit analysis of OJ strand counts were performed using an OJ strand count-resistance relationship substantially different from that proposed by Claude. Measurements of unidirectional fluxes of inulin, mannitol, and sodium indicated that restriction of transjunctional permeability accounted for the observed resistance rise, and that T84 junctional strands have finite permeability to molecules with radii less than or equal to 3.6 A but are essentially impermeable to molecules with radii greater than or equal to 15 A. The results suggest that general correlates between OJ structure and OJ ability to resist passive ion flow do exist in T84 monolayers. The study also suggests that such correlates can be obtained only if OJ structural data are analyzed as an electrical circuit composed of parallel resistors.