Large divalent cations and electrostatic potentials adjacent to membranes. A theoretical calculation

We have extended the Gouy-Chapman theory of the electrostatic diffuse double layer by considering the finite size of divalent cations in the aqueous phase adjacent to a charged surface. The divalent cations are modeled as either two point charges connected by an infinitely thin, rigid "rod" or two noninteracting point charges connected by an infinitely thin, flexible "string." We use the extended theory to predict the effects of a cation of length 10 A (1 nm) on the zeta and surface potentials of phospholipid bilayer membranes. The predictions of the rod and string models are similar to one another but differ markedly from the predictions of the Gouy-Chapman theory. Specifically, the extended model predicts that a large divalent cation will have a smaller effect on the potential adjacent to a negatively charged bilayer membrane than a point divalent cation, that the magnitude of this discrepancy will decrease as the Debye length increases, and that a large divalent cation will produce a negative zeta potential on a membrane formed from zwitterionic lipids. These predictions agree qualitatively with the experimental results obtained with the large divalent cation hexamethonium. We discuss the biological relevance of our calculations in the context of the interaction of cationic drugs with receptor sites on cell membranes.     

Semiconductor theory of ion transport in thin lipid membranes: I. Potential and field distributions

In the theory as presented in this paper and the following one, we shall attempt to apply the semiconductor principles and methods to the study of ion transport in thin lipid membranes. Detailed formulations are given on the potential energy barriers at the interfaces, voltage drops in the polar and non-polar regions, and potential and field distributions in the diffuse double layer and within a charged membrane. These results will be used mainly as the boundary conditions for the solution of ion flow as to be given in the following paper. The analysis clearly indicates that the ion transport is interface-limited and is profoundly influenced by the presence of surface charges. An explanation of Na⁺ extrusion in nerve membrane is given based on the field distribution analysis. The theory also suggests that the “membrane potential” depends mainly on surface charges but not necessarily on ion permeation through the membrane.     

Rapid analysis of membrane lipids using a combination of thin-layer chromatography and scanning of photographic negatives

A simple method is proposed for the analysis of the distribution and changes in membrane lipids subjected to different treatments (lipolytic, aging, etc.). This technique involves only one-thin layer chromatographic step followed by a scanning of the photographic negative of the charred thin layer. This method is time saving, inexpensive and does not require the technical skill usually demanded in lipidology. The precision of this method is compared with that obtained with the classical TLC-GLC method: its variability is roughly twice that of the TLC-GC method.     

Stability and phase separation in mixed monopolar lipid/bolalipid layers

The phase stability of a fluid lipid layer that is a mixture of conventional monopolar lipids and C20 bipolar bolalipids was studied using a mean field theory that explicitly includes molecular details and configurational properties of the lipid molecules. The effect of changing the fraction of bolalipids, as well as the length of the hydrocarbon chain of the monopolar lipids, was probed. A phase separation between two liquid lipid phases was found when a mismatch exists in the optimal hydrophobic thicknesses of the pure bolalipid and monopolar lipid layers. The lipid mixture phase separates into a thin bolalipid-rich layer and a thicker monopolar-rich layer. The thin membrane phase is mainly composed of transmembrane bolalipid molecules whose polar heads are positioned at opposite membrane-water interfaces. In the monopolar lipid-rich phase, bolalipids are the minor component and most of them assume a looping configuration where both headgroups are present at the same membrane-water interface. For mixed layers that form a single lipid phase across all bolalipid concentrations, the hairpin-transmembrane ratio strongly depends on the hydrocarbon chain length of the monopolar lipid and the bolalipid concentration. The C-D bond order parameters of the different species have been calculated. Our findings suggest that the concentration-dependent phase transition should be experimentally observable by measuring of the order parameters through quadrupolar splitting experiments. The driving force for the phase separation in the monopolar lipid/bolalipid mixture is the packing mismatch between hydrophobic regions of the monopolar lipid hydrocarbon chains and the membrane-spanning bolalipid chains. The results from the molecular theory may be useful in the design of stable lipid layers for integral membrane protein sensing.     

Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.     

Ultrastructure of the pellicular complex of Plasmodium fallax

The exoerythrocytic merozoites of Plasmodium fallax grown in a tissue-culture system have been investigated by negative staining and thin-sectioning techniques, and the respective results have been compared. Negative staining provided additional information, corroborated findings obtained with thin sectioning, and contributed particularly to the study of the pellicular complex of the merozoites which has been demonstrated as being composed of three layers: a thin outer membrane, a thick interrupted inner membrane, and a partial layer of microtubules. Observations made of negatively stained parasites revealed that the thick, interrupted inner membrane in thin sections is actually a labyrinthine structure and covers the entire surface of the merozoite, except at the regions of the conoid and the cytostome. The microtubules which radiate from the conoid to the posterior end demonstrated a transverse periodicity and filamental subunits parallel to the axis of the microtubule. The detailed structure of the conoid and the cytostome is also described.     

AMPHIESMAL ULTRASTRUCTURE OF DINOFLAGELLATES: A REEVALUATION OF PELLICLE FORMATION1

The ultrastructure of the amphiesma during pellicle formation was investigated in two species of Dinophyceae, Amphidinium rhynchocephalum Anissimowa and Heterocapsa niei (Loeblich) Morrill & Loeblich using thin sections. In both species the amphiesma consists of an outermost membrane (i.e. the plasma membrane) underlain by amphiesmal vesicles. In A. rhynchocephalum the latter appear empty whereas each amphiesmal vesicle in H. niei contains a thecal plate and a thin, amorphous layer (dark-staining layer) located between, the thecal plate and the inner amphiesmal vesicle membrane. When cells of both taxa are carefully fixed, amphiesmal vesicles are always separate entities (i.e. the sutures are undisrupted). During ecdysis the following amphiesmal components are shed: the plasma membrane, the outer amphiesmal vesicle membrane, and in H. niei the thecal plates. The inner membranes of the amphiesmal vesicles then fuse with each other and form a continuous membrane (termed pellicle membrane) that remains tightly oppressed to an underlying amorphous layer (pellicular layer). In A. rhynchocephalum the pellicular layer is already present in vegetative non-ecdysed cells, whereas in H. niei it forms during ecdysis beneath the pellicle membrane. During ecdysis in H. niei, material from the dark-staining layer precipitates on the outer surface of the pellicle membrane, where it forms a characteristic honeycomb pattern. The new observations are incorporated into a revised model of pellicle formation in dinoflagellates and contrasted with earlier proposals.     

Water transport in the arterial wall--a theoretical study

Water transport in the arterial wall is studied using a mathematical model based on the theory for the consolidation of water saturated soils (Biot, 1941; Kenyon, 1976a). The intimal pressure is considered to be harmonic in time. Analytical results are obtained for both large and small consolidation times since both the situations are of physiological relevance. For large consolidation times, the filtration is confined to a thin boundary layer. Large pressure gradients exist within the boundary layer while the pressure gradient is negligible in the intermediate layer. Thus, the pulsatile flow is found to be confined to the boundary layer while a smaller mean flow exists throughout the wall.     

Laser interferometric investigation of solute transport through membrane-concentration boundary layer system

We investigate diffusive transport in a membrane system with a horizontally mounted membrane under concentration polarization conditions performed by a laser interferometry method. The data obtained from two different theoretical models are compared to the experimental results of the substance flux. In the first model, the membrane is considered as infinitely thin, while in the second one as a wall of finite thickness. The theoretical calculations show sufficient correspondence with the experimental results. On the basis of interferometric measurements, the relative permeability coefficient (ζ_s) for the system, consisting of the membrane and concentration boundary layers, was also obtained. This coefficient reflects the concentration polarization of the membrane system. The obtained results indicate that the coefficient ζ_s of the membrane-concentration boundary layer system decreases in time and seems to be independent of the initial concentration of the solute.     

The fine structure of the pellicle ofEuglena gracilis as revealed by freeze-etching

Summary The ultrastructure of the pellicle ofEuglena gracilis (Klebs) Z strain was studied using the freeze-etching technique and the results were correlated with data obtained from thin sections of fixed material.Examination of freeze-etched pellicles reveals an outer particulate layer and an inner striated layer. The particles of the outer layer measure approximately 150 Å in diameter. The striations of the inner layer are about 50 Å wide and are separated from each other by about 35 Å. A broad repeating pattern is also visible with a periodicity of about 450 Å. When deep etching is employed, a smooth outer layer is seen covering the particulate layer. This is probably the outer surface of the plasma membrane. Mucilage is present on the outer surface of the cell and is seen as a substructure of threads superposed on the smooth layer of plasma membrane.Thin sectioning also shows a striated layer interior to the plasma membrane. This appears to be identical to the striated layer seen after freeze-etching.     

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca. 10-nm-thick peptidoglycan-containing layer and a ca. 10-nm-thick S layer. Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan. This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria. Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells. In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain. In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca. 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer. The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant. It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane. Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside.     

Epidermal ultrastructure of Seison nebaliae and Seison annulatus, and a comparison of epidermal structures within the Gnathifera

 The epidermis of both species of Seison is syncytial and has a characteristic internal layer divided into two distinct sublayers. Sublayer I is very thin (0.03 μm) and bounded to the outer cell membrane of the epidermis. Sublayer II is 0.5 μm thick and separated from sublayer I by a thin layer of cytoplasm. Intrusions of the outer cell membrane of the epidermis perforate the internal layer, before terminating within the cytoplasm. The intrusions of the cell membrane of S. annulatus are coated by an electron-dense material, the annulus, when passing through the internal layer. Bundles of filaments are present in the epidermis of S. nebaliae. A comparison of epidermal structures within the Gnathifera Ahlrichs, 1995, confirms phylogenetic relationships earlier proposed by the author. Accepted: 19 December 1996     

Studies on microplasmodia ofPhysarum polycephalum: II. Fine structure and function of the mucous layer

Summary Microplasmodia of the acellular slime moldPhysarum polycephalum have developed an extensive extracellular slime layer which amounts to 75% of the volume of the total biomass. The slime layer covers the entire plasma membrane including the cell surface invagination system and represents a humid compartment protecting the organism against damaging influence from the surrounding environment.Histochemical treatment with cationic substances revealed that the slime is mostly composed of acid mucopolysaccharides: intensive staining was obtained by colloidal or dialyzed iron, Ruthenium Red, acriflavine and lanthanum hydroxide.At the macromolecular level the slime layer is composed of thin filaments showing a parallel course to the plasma membrane. The slime filaments are very sensitive to fixation and dehydration procedures and tend to form thicker aggregates.From feeding experiments using different fine-particulated markers (Thorotrast, Aerosil) it can be concluded that the slime layer exhibits an important function with regard to the controlled transport and uptake of substances. In particular, the indentations of the cell membrane invagination system running perpendicular from the microplasmodial surface to the cell interior represent preferred pathways for the selective diffusion of macromolecules.     

Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.     

Location of the Fracture Faces Within the Cell Envelope of Acinetobacter Species Strain MJT/F5/5

The cell wall of the gram-negative bacterium Acinetobacter species strain MJT/F5/5 shows in thin section an external “additional” layer, an outer membrane, an intermediate layer, and a dense layer. Negatively stained preparations showed that the additional layer is composed of hexagonally arranged subunits. In glycerol-treated preparations, freeze-etching revealed that the cell walls consist of four layers, with the main plane of fracture between layers cw 2 and cw 3. The surface of [Formula: see text] 2 consisted of densely packed particles, whereas [Formula: see text] 3 appeared to be fibrillar. In cell envelopes treated with lysozyme by various methods, the removal of the dense layer has detached the outer membrane and additional layer from the underlying layers, as shown in thin sections. When freeze-etched in the absence of glycerol, these detached outer membranes with additional layers fractured to reveal both the faces [Formula: see text] 2 and [Formula: see text] 3 with their characteristic surface structures, and, in addition, both the external and internal etched surfaces were revealed. This experiment provided conclusive evidence that the main fracture plane in the cell wall lies within the interior of the outer membrane. This and other evidence showed that the corresponding layers in thin sections and freeze-etched preparations are: the additional layer, cw 1; the outer membrane, cw (2 + 3); and the intermediate and dense layers together from cw 4. Because of similarities in structure between this Acinetobacter and other gram-negative bacteria, it seemed probable that the interior of the outer membrane is the plane most liable to fracture in the cell walls of most gram-negative bacteria.     

Improved antigen retrieval in freeze-fracture cytochemistry by evaporation of carbon as first replication layer

The recently developed freeze-fracture replica immunolabeling technique uses sodium dodecyl sulfate to clean replicas obtained from chemically unfixed, rapidly frozen cells by evaporation of platinum as first and carbon as second replication layer. The detergent dissolves remains of cellular material with the exception of components which are in direct contact to the replica film. Membrane lipids and membrane protein complexes of the protoplasmic and the exoplasmic membrane halves remain attached to the replica film and are accessible for cytochemical localization. We immunolabeled the membrane proteins caveolin-1 and connexin 43 in mouse cell lines as well as the membrane attached protein tetrachloroethene reductive dehalogenase (PceA) in bacterial cells at freeze-fracture replicas generated by different evaporation parameters. The labeling experiments for caveolin-1 and the PceA showed that freeze-fracture replication of cellular membranes accomplished with thin platinum layers as well as replication with carbon as first evaporation layer lead in these cases to an improved antigen retrieval, whereas the labeling efficiency of connexin 43 was not affected by different evaporation conditions.     

Microfluidic chips controlled with elastomeric microvalve arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.     

Fine Structure of Thermus aquaticus, an Extreme Thermophile

Electron microscopic studies using thin sections revealed that Thermus aquaticus has a structure similar to that of most other gram-negative bacteria. The cell envelope is tripartite: plasma membrane, thin middle layer, and a thicker and irregular outer layer. The outer layer appears to be joined to the plasma membrane by a series of connections and, when seen in tangential section, the outer layer appears as a series of parallel bands. The cell division mechanism resembles that of typical gram-negative bacteria. Large spherical bodies designated “rotund bodies” are formed as a result of the association of a number of separate cells. In this association the outer envelope layers of the cells fuse and pull away from the middle layer. The rotund body thus appears as a series of rods, usually lying in parallel around the periphery of the sphere, completely connected by means of the fused outer layer.     

Comparative observations on corneas,with special reference to Bowman's layer and Descemet's membrane in mammals and amphibians

Corneas of tadpole, mouse, rat, guinea pig, rabbit, cat, cattle, and human were examined by TEM and SEM in a comparative study. The differences between species were noted mainly by using TEM. Bowman's layer showed a tendency to be well developed in higher mammals. Tadpoles lack a Bowman's layer, lower mammals have a thin Bowman's layer, and higher mammals have a thick Bowman's layer. The boundary between the substantia propria and Descemet's membrane was distinct in higher mammals. On the other hand, there are no differences in thickness of the collagen fibrils that constitute Bowman's layer and those of the substantia propria. NaOH digestion was utilized for SEM preparation. SEM imaging revealed a textured appearance of the epithelial side of Bowman's layer. In Descemet's membrane, fibrous long spacing (FLS) fiber-like structures, which are arranged in parallel to the endothelium, were observed by both TEM and SEM. To our knowledge, this is the first report of SEM observations of FLS fiber-like structures on the endothelial surface of Descemet's membrane. SEM at a plane normal to the plane of the cornea showed that Descemet's membrane has a piled laminar structure. Descemet's membrane is closely associated with the collagen layer of the substantia propria. Collagen fibrils invading from the substantia propria into Descemet's membrane were observed with both TEM and SEM.     

Contractile elements of Lemna trisulca L. glycerinated cell models during chloroplast translocations.

Electron microscopy of Lemna glycerinated cell models depicts contractile elements during chloroplast translocations. One contractile element, the thin ectoplasmic layer, is < or = 0.4 microm thick, pressed against plasma membrane-cell wall. Thin ectoplasmic layer contains numerous oriented filaments and some appear to be actin and myosin. Another contractile element is the outer chloroplast membrane which envelops each chloroplast and joins or fuses with the thin ectoplasmic layer. Chloroplast interconnections are formed between two or more chloroplasts by outer chloroplast membranes; they enhance chloroplast communications, translocations, and molecular exchanges.