Charge-induced pretransition in phosphatidylethanolamine multilayers. The occurrence of ripple structures

The influence of pli and ionic strength on the phase transition behaviour of 1,2-dihexadecylphosphatidylethanolamine was studied calorimetrically. In the range of ionic strength from 0.75 to 1.5 M NaC1 at $\text{pH}\text{? 13}$, where the amino group of the phosphatidylethanolarnine is in the deprotonated state, resulting in one negative charge per lipid molecule, the calorimetric scan shows a pretransition before the main transition. Accompanying freeze-fracture electron microscopic studies on these preparations in the temperature range between the pre- and main transitions show a regular surface, the so-called ripple structure. These are comparable with the structures seen in phosphatidylcholine-water systems af temperatures between the pre- and main transition.     

Effect of n-alcohols and glycerol on the pretransition of dipalmitoylphosphatidylcholine

We have systematically investigated the effect of short-chain n-alcohols and glycerol on the pretransition of 1,2-dipalmitoylphosphatidylcholine (DPPC) by spectrophotometry. It is found that the n-alcohols and glycerol remove the pretransition above a critical concentration for each ligand. In addition, the short-chain n-alcohols below the critical concentration decrease the pretransition temperature. The longer the aliphatic chain length of the n-alcohol (up to butanol) (a) the greater the decrease in the pretransition temperature, and (b) the lower the concentration necessary to remove the pretransition. However, glycerol differs from the short-chain n-alcohols in that it has no significant effect on either the pretransition or the main transition, but it is also capable of removing the pretransition above a critical concentration. It has previously been shown that alcohols have a biphasic effect on the main transition temperature of phosphatidylcholines (Rowe, E.S. (1983) Biochemistry 22, 3299–3305). At high alcohol concentrations, the main transition is not thermodynamically reversible (Rowe, E.S. (1985) Biochim. Biophys. Acta 813, 321–330). Recently, Simon and McIntosh (Biochim. Biophys. Acta (1984) 773, 169–172) have identified that at high ethanol concentration DPPC exists in the interdigitated phase. The critical ligand concentration at which the pretransition disappears coincides with the induction of main transition hysteresis and the biphasic alcohol effect in the main transition. These three effects appear to correlate with the induction of the interdigitated gel state by alcohols and glycerol.     

Gap junctions between the follicle cells and the oocyte during oogenesis in an insect,Tribolium destructor/Coleoptera

Summary Oocyte-follicle cell gap junctions inTribolium occur in all oogenetic stages studied. During early previtellogenesis the junctions are found exclusively between lateral membranes of oocyte microvilli and the membrane of prefollicle cells. In late previtellogenesis and vitellogenesis the junctions are located between the tips of oocyte microvilli and the flat membranes of the follicle cells. During previtellogenesis gap junctions are infrequent, whereas in the phase of yolk accumulation their number increases considerably, exceeding 17 junctions/m² of the follicle cell membrane. It could be shown by microinjection of a fluorescent dye that gap junctions are in a functional state during vitellogenesis. Possible roles of heterologous gap junctions in oogenesis are discussed.     

Effect of potassium perfluorooctanesulfonate, perfluorooctanoate and octanesulfonate on the phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse effects by inhibiting pulmonary surfactant. To gain further insights in this potential mechanism of toxicity, we investigated the interaction of PFOS potassium salt with dipalmitoylphosphatidylcholine (DPPC) - the major component of pulmonary surfactant - using steady-state fluorescence anisotropy spectroscopy and DSC (differential scanning calorimetry). In addition, we investigated the interactions of two structurally related compounds, perfluorooctanoic acid (PFOA) and octanesulfonic acid (OS) potassium salt, with DPPC. In the fluorescence experiments a linear depression of the main phase transition temperature of DPPC (T_m) and an increased peak width was observed with increasing concentration of all three compounds, both using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as fluorescent probes. PFOS caused an effect on T_m and peak width at much lower concentrations because of its increased tendency to partition onto DPPC bilayers, i.e., the partition coefficients decrease in the K(PFOS) > K(PFOA) >> K(OS). Similar to the fluorescence anisotropy measurements, all three compounds caused a linear depression in the onset of the main phase transition temperature and a significant peak broadening in the DSC experiments, with PFOS having the most pronounced effect of the peak width. The effect of PFOS and other fluorinated surfactants on DPPC in both mono- and bilayers may be one mechanism by which these compounds cause adverse biological effects.     

Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine bilayers: X-ray diffraction study

The structural changes accompanying the recently described sub-transition of hydrated dipalmitoylphosphatidylcholine (Chen, S.C., Sturtevant, J.M. and Gaffney, B.J. (1980) Proc. Natl. Acad. Sci. USA 77, 5060–5063) have been defined using X-ray diffraction methods. Following prolonged storage at ?4°C the usual L_β′ gel form of hydrated dipalmitoylphosphatidylcholine (DPPC) is converted into a more ordered stable ‘crystal’ form. The bilayer periodicity is 59.1 Å and the most striking feature is the presence of a number of X-ray reflections in the wide angle region. The most prominent of these are a sharp reflection at $\text{1}\text{4.4}\text{A}\text{?}{}^{\mathrm{?1}}$ and a broader reflection at $\text{1}\text{3.9}\text{A}\text{?}{}^{\mathrm{?1}}$. This diffraction pattern is indicative of more ordered molecular and hydrocarbon chain packing modes in this low temperature ‘crystal’ bilayer form. At the sub-transition (T_rmsub = 15–20°C) an increase in the bilayer periodicity occurs ($\text{d=63.6}\text{A}\text{?}$) and a strong reflection at approx. $\text{1}\text{4.2}\text{A}\text{?}{}^{\mathrm{?1}}$ with a shoulder at approx. $\text{1}\text{4.1}\text{A}\text{?}{}^{\mathrm{?1}}$ is observed. This diffraction pattern is identical to that of the bilayer gel (L_β′) form of hydrated DPPC. Thus, the sub-transition corresponds to a bilayer ‘crystal’ → bilayer L_β′ gel structural rearrangement accompanied by a decrease in the lateral hydrocarbon chain interactions. Differential scanning calorimetry and X-ray diffraction show that on further heating the usual structural changes L_β′ → P_β′ and P_β′ → L_α occur at the pre- and main transitions, at approx. 35°C and 41°C, respectively.     

Halothane-induced membrane reorganization monitored by DSC,freeze fracture electron microscopy and 31 P-NMR techniques

The effect of the volatile anaesthetic halothane on the structure and dynamics of lipid multilayers (dimyristoyl- and dipalmitoylphosphatidylcholine, DM-and DP-PC, aqueous dispersions) was studied using Differential Scanning Calorimetry (DSC), Freeze Fracture Electron Microscopy and solid state phosphorus-31 Nuclear Magnetic Resonance (³¹P-NMR). The action of the drug depends upon the halothane-to-lipid molar ratio, Ri, and temperature. With DPPC lipids, three main regions can be distinguished: i) 0 < Ri < 0.7, ii) 0.7 < Ri < 2 and iii) Ri > 2. As Ri increases in the first region, a linear decrease in the main gel-to-fluid phase transition temperature (T _c, a broadening in the DSC transition peak and a lowering in the enthalpy variation (H), are observed. A minimum in H is reached at Ri=0.7. In this region, ³¹P-NMR spectra indicate that the multibilayer structure is maintained. In the second region, T _c still decreases with the same slope, but H increases up to a plateau value for Ri=2. In the lipid fluid phase, an isotropic NMR line appears superimposed on the powder pattern that corresponds to a lamellar phase. For Ri > 2, T _c and H remain almost constant. At values of temperature that are greater than T _c a growing isotropic line occurs in ³¹P-NMR spectra. This means a new supramolecular structure made of lipids and halothane is stabilized. This structure has been characterized as small vesicles of about 400 Å to 600 Å diameter by Freeze Fracture electron microscopy observations. With DMPC and low ratios (Ri < 2), DSC and NMR results are similar to those obtained for DPPC. However, the minimum H is reached at Ri=0.2 and the decrease in T _c is faster than for DPPC when Ri increases from 0. For Ri > 2, while T _c and H remain constant as in the case of DPPC, ³¹P-NMR spectra of DMPC systems show a superimposition of an isotropic line and two powder patterns, which correspond to small tumbling vesicles, a possible hexagonal phase and a lamellar phase respectively. Halothane, thus acts on model membranes in two different steps: at low Ri the bilayer is disturbed but keeps its structure. Whereas for higher drug concentrations, a new organization of lipids seems to be stabilized for T > T _c.Abbreviations DPPC Dipalmitoylphosphatidylcholine - DMPC Dimyristoylphosphatidylcholine - DSC Differential scanning calorimetry - NMR Nuclear magnetic resonance - EDTA Ethylenediaminetetraacetic acid - DMSO Dimethyl sulfoxide - Ri Halothaneto-lipid molar ratio - T _c Main gel (L )-to-fluid (L ) phase transition temperature - T _m Maximum temperature of the transition - H Enthalpy variation - C _{p max} excess heat capacity at the maximum temperature of the transition T _m - n number of phospholipid molecules per cooperative unit Offprint requests to: J.-P. Renou     

Molecular study of the diffusional process of DMSO in double lipid bilayers

As a way to quantify the diffusion process of molecular compounds through biological membranes, we investigated in this study the dynamics of DMSO through an 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) bilayer system. To properly account for the diffusion of DMSO due to a concentration gradient, a double DPPC bilayer was setup for our simulations. In such configuration, the aqueous phases can be explicitly associated with the extra and intracellular domains of the membrane, which is seldom the case in studies of single lipid bilayer due to the periodicity imposed by the simulations. DMSO molecules were initially contained in one of the aqueous phases (extracellular region) at a concentration of 5 wt.%. Molecular dynamics simulation was performed in this system for 95 ns at 350 K and 1 bar. The simulations showed that although many DMSO molecules penetrated the lipid bilayer, only about 10% of them crossed the bilayer to reach the other aqueous phase corresponding to the intracellular region of the membrane. The simulation time considered was insufficient to reach equilibrium of the DMSO concentration between the aqueous phases. However, the simulations provided sufficient information to estimate parameters to apply Fick's Law to model the diffusion process of the system. Using this model, we predicted that for the time considered in our simulation, the concentration of DMSO in the intracellular domain should have been about half of the actual value obtained. The model also predicted that equilibrium of the DMSO concentration in the system would be reached after about 2000 ns, approximately 20 times longer than the performed simulation.     

Determination of the Role of Cold Acclimation-Induced Diverse Changes in Plant Cells from the Viewpoint of Avoidance of Freezing Injury

Received 4 January 1999/ Accepted in revised form 7 April 1999     

Heterogeneity of tight junctions along the collecting duct in the renal medulla

Summary The tight junctions along the medullary collecting duct in the kidneys of the rat and the rabbit were studied with freeze-fracture electron microscopy and quantitated according to the number of strands and the apico-basal depth (nm) of the junctions.The most elaborate tight junctions were found in the inner stripe of the outer medulla; rat: 10.6±0.8 strands and 205±24nm; rabbit: 11.6±2.4 strands and 291±55 nm.The elaboration of the tight junctions decreased continuously towards the papillary tip. Inner zone I; rat: 9.3±2.6 strands and 186±38nm, rabbit: 9.5±2.3 strands and 247±59nm. Inner zone II; rat: 7.1±2.2 strands and 129±32nm, rabbit: 8.5±1.4 strands and 199±26nm. Inner zone III; rat: 6.0±1.6 strands and 111 + 19 nm, rabbit: 7.0±1.5 strands and 183±43 nm. In the inner zone III comprising the papillary tip tight junctions with only 1–3 strands were not infrequently seen. Preliminary findings in the kidney of the golden hamster indicate a similar decline of junctional tightness along the collecting duct.These morphological observations suggest that the permeability of the paracellular pathway of the medullary collecting duct increases towards the tip of the papilla, especially in the rat. The functional implications for the medullary recycling of urea and electrolytes, and for the urinary concentrating mechanism are discussed.In addition, the tight junctions of the papillary epithelium are described.     

The permeability barrier in the epidermis of the grass snake during the resting stage of the sloughing cycle

Summary Tracer and freeze-fracture replication techniques show that there are two morphologically and topographically distinct permeability barriers in the epidermis of the grass snake. Tight junctions interconnect the apico-lateral plasma membranes of the uppermost living cells, establishing an ionic or osmotic gradient between the stratum germinativum and alpha layer. The second barrier is formed by intercellular lipid sheets in the overlying mesos layer, which are very similar to the barrier found in the stratum corneum of mammals.     

Effects of reserpine administration on the fine structure of the rat pars intermedia

Summary Hormone release from the pars intermedia is under inhibitory control of the hypothalamus. Control may be mediated via dopaminergic fibers which directly contact secretory cells. Administration of reserpine in vivo to adult male rats for four consecutive days results in major alternations in pars intermedia cytology. Cells show expanded areas and whorls of rough endoplasmic reticulum, as well as extensive Golgi zones with numerous dense granules. Some nerve fibers exhibit alterations in vesicle content, while others retain a more normal appearance. Freeze-fracture of glands from reserpine-treated animals provides evidence for exocytosis of granules, although such phenomena are not observed in thin sections. The ultrastructural findings suggest that reserpine alters the content of local inhibitory neurotransmitters in the pars intermedia, leading to unrestrained hormone release, followed by renewed granule synthesis.My thanks to Ms. Judi DeLongo for skillful technical assistance     

The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.     

Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii

Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1′s gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts.     

On the Lamellar Structure of the Tracheid Cell Wall

Abstract: It is clear that cross sections of wood cells show a lamellar structure. This paper investigates the orientation of this lamellar structure of spruce (Picea abies) tracheids using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cross sections of spruce wood were produced through fracturing in longitudinal bending and tensile testing. When investigated with SEM, the fracture surfaces show a structure of mostly larger radial lamellae, in the order of 30 - 100 nm, i.e., agglomerations of a few cellulose aggregates. Thin transverse sections of the fracture zones investigated with atomic force microscopy show concentric lamellae with a width in the order of a single cellulose aggregate, i.e., 15 - 25 nm. No structural connection to the splinters in the radial direction can be seen. It is suggested that the radial lamellar structure is a consequence of the energy released during fracturing of the wood samples and that the undistorted wood has a concentric lamellar structure on a smaller structural level.     

Drying techniques for the visualisation of agarose‐based chromatography media by scanning electron microscopy

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub‐micron level. Achieving suitable drying conditions is especially important with agarose‐based chromatography resins, as over‐drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under‐drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect?/d _w ≈85 µm and Capto? Adhere/d _w ≈75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualization of both resins under SEM. Under this protocol both the polymer fibers (thickness ≈20 nm) and the pore sizes (diameter ≈100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favorable option for ultrastructural visualization of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose‐based chromatography media.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Investigations of the possible role of proteases in altering surface proteins of virally transformed hamster fibroblasts

Virally transformed fibroblasts have on their surfaces zero or reduced amounts of a large external transformation-sensitive (LETS) glycoprotein. This protein is extremely sensitive to proteolysis. When prelabeled normal fibroblasts are cocultivated with transformed cells, the LETS glycoprotein of the normal cells shows an increased rate of turnover. Experiments are described which investigate the possibility that this phenomenon and the absence of LETS glycoprotein are due to proteolysis by the transformed cells. In particular, the role of plasminogen activation is examined by the use of protease inhibitors and plasminogen-depleted serum. It is concluded that activation of plasminogen is not required for the disappearance of the LETS glycoprotein although the involvement of other proteases cannot be ruled out. The role of proteases in affecting cell growth and behavior is discussed.     

Extracellular coats on the surface of Strongylocentrotus purpuratus eggs: stereo electron microscopy of quick-frozen and deep-etched specimens

Summary Sea urchin (Strongylocentrotus purpuratus) eggs were fixed, quick-frozen, deep-etched, and rotary-replicated, and the three-dimensional structure of the external surface of the egg visualized using stereo electron microscopy. The cell surface is coated with three layers of filaments: the sheetlike vitelline layer adhering closely to the plasma membrane, a second layer of oblique fibrils extending from microvillar tips to the vitelline layer below, and a third, outermost layer of horizontal filaments coursing in bundles over the microvillar tips. After fertilization, the newly elevated vitelline envelope is transformed into a three-layered structure, the central layer being a tightly knit network of fine filaments decorated on each side with a loose network of thicker fibrils. Subsequently, the envelope becomes coated with paracrystalline protein released from the cortical granules, and microvillar casts are reshaped into angular, jagged peaks having two to five sides. The final structure of the fertilization envelope consists of a thick central layer of compact fibrillar material that is coated on each side with thin plates of paracrystalline protein.     

Pollen morphology of the Dimorphandra group (Leguminosae,Caesalpinioideae)

The Dimorphandra group, as traditionally circumscribed, is a rather diverse assemblage of genera in Leguminosae subfamily Caesalpinioideae that share certain morphological characteristics with the basally branching lineages of subfamily Mimosoideae. It currently comprises 51 species in seven genera: Burkea (1 species), Dimorphandra (26 species), Erythrophleum (10 species), Mora (6 species), Pachyelasma (1 species), Stachyothyrsus (2 species) and Sympetalandra (5 species). This study investigates the pollen morphology of 25 samples from 19 species of all seven genera. Pollen of the Dimorphandra group is small, isopolar, trizonocolporate and released in monads. Apertures are almost equal to polar length, with correspondingly small apocolpial areas. The shape of the aperture apices varies from acute to wide and rounded. Surface ornamentation is psilate, perforate, microreticulate, or perforate‐rugulate. The wall structure is usually columellate with a well developed foot layer. The pollen is small and unspecialised, agreeing with a previously noted pattern of more fixed and homogenous pollen structure in the more derived clades in subfamily Caesalpinioideae, compared with the great diversity of pollen types found in the basally branching lineages.