Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.     

SUBMICROSCOPIC ORGANIZATION OF THE POSTSYNAPTIC MEMBRANE IN THE MYONEURAL JUNCTION : A Polarization Optical Study

Cross-striated muscles of frogs and rats were fixed in 3.3 per cent lead nitrate solution. Frozen sections 30 micra thick were mounted in different media and observed by polarization microscopy. The subneural apparatus of myoneural junctions exhibits a strong birefringence in these sections. Birefringence is exerted by a highly organized lipoprotein framework (postsynaptic material) which builds up the "organites" (junctional folds) of the postsynaptic membrane. Synaptic cholinesterase is closely associated with this material. Freezing and/or formalin fixation results in a destruction of the molecular organization of the postsynaptic material, but does not influence the synaptic enzyme activity. It is hypothesized from this study that the junctional folds (postsynaptic "organites") consist of regularly arranged, sheet-like lamellar micellae in the frog and of less regular, mainly radially arranged submicroscopic units in the rat. The micellar organization as revealed by polarization analysis is in good agreement with the electron microscopic findings reported in the literature. Intramicellar protein molecules of the resting postsynaptic membrane are arranged longitudinally, lipids transversely. Supramaximal stimulation or treatment with acetylcholine + eserine results in a disorganization of proteins and a rearrangement of lipids. Denervation results in a rearrangement of lipids without any significant alterations of proteins. All these functional stresses influence only the molecular and not the micellar structure of the membrane. The function of the organized lipoprotein framework as an acetylcholine receptor is suggested.     

Indirect evidence of submicroscopic pores in giant unilamellar [correction of unilamelar] vesicles

Formation of pore-like structures in cell membranes could participate in exchange of matter between cell compartments and modify the lipid distribution between the leaflets of a bilayer. We present experiments on two model systems in which major lipid redistribution is attributed to few submicroscopic transient pores. The first kind of experiments consists in destabilizing the membrane of a giant unilamellar vesicle by inserting conic-shaped fluorescent lipids from the outer medium. The inserted lipids (10% of the vesicle lipids) should lead to membrane rupture if segregated on the outer leaflet. We show that a 5-nm diameter pore is sufficient to ease the stress on the membrane by redistributing the lipids. The second kind of experiments consists in forcing giant vesicles containing functionalized lipids to adhere. This adhesion leads to hemifusion (merging of the outer leaflets). In certain cases, the formation of pores in one of the vesicles is attested by contrast loss on this vesicle and redistribution of fluorescent labels between the leaflets. The kinetics of these phenomena is compatible with transient submicroscopic pores and long-lived membrane defects.     

Studies of the thermotropic phase behavior of phosphatidylcholines containing 2-alkyl substituted fatty acyl chains: a new class of phosphatidylcholines forming inverted nonlamellar phases.

We have synthesized a number of 1,2-diacyl phosphatidylcholines with hydrophobic substituents adjacent to the carbonyl group of the fatty acyl chain and studied their thermotropic phase behavior by differential scanning calorimetry, 31P-nuclear magnetic resonance spectroscopy, and x-ray diffraction. Our results indicate that the hydrocarbon chain-melting phase transition temperatures of these lipids are lower than those of the n-saturated diacylphosphatidylcholines of similar chain length. In the gel phase, the 2-alkyl substituents on the fatty acyl chains seem to inhibit the formation of tightly packed, partially dehydrated, quasi-crystalline bilayers (Lc phases), although possibly promoting the formation of chain-interdigitated bilayers. In the liquid-crystalline state, however, these 2-alkyl substituents destabilize the lamellar phase with respect to one or more inverted nonlamellar structures. In general, increases in the length, bulk, or rigidity of the alkyl substituent result in an increased destabilization of the lamellar gel and liquid-crystalline phases and a greater tendency to form inverted nonlamellar phases, the nature of which depends upon the size of the 2-alkyl substituent. Unlike normal non-lamella-forming lipids such as the phosphatidylethanolamines, increases in the length of the main acyl chain stabilize the lamellar phases and reduce the tendency to form nonlamellar structures. Our results establish that with a judicious choice of a 2-alkyl substituent and hydrocarbon chain length, phosphatidylcholines (and probably most other so-called "bilayer-preferring" lipids) can be induced to form a range of inverted nonlamellar structures at relatively low temperatures. The ability to vary the lamellar/nonlamellar phase preference of such lipids should be useful in studies of bilayer/nonbilayer phase transitions and of the molecular organization of various nonlamellar phases. Moreover, because the nonlamellar phases can easily be induced at physiologically relevant temperatures and hydration levels while avoiding changes in polar headgroup composition, this new class of 2-alkyl-substituted phosphatidylcholines should prove valuable in studies of the physiological role of non-lamella-forming lipids in reconstituted lipid-protein model membranes.     

Oxidative depolymerization of polysaccharides by reactive oxygen/nitrogen species

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly produced and are tightly regulated to maintain a redox balance (or homeostasis) together with antioxidants (e.g. superoxide dismutase and glutathione) under normal physiological circumstances. These ROS/RNS have been shown to be critical for various biological events including signal transduction, aging, apoptosis, and development. Despite the known beneficial effects, an overproduction of ROS/RNS in the cases of receptor-mediated stimulation and disease-induced oxidative stress can inflict severe tissue damage. In particular, these ROS/RNS are capable of degrading macromolecules including proteins, lipids and nucleic acids as well as polysaccharides, and presumably lead to their dysfunction. The purpose of this review is to highlight (1) chemical mechanisms related to cell-free and cell-based depolymerization of polysaccharides initiated by individual oxidative species; (2) the effect of ROS/RNS-mediated depolymerization on the successive cleavage of the glycosidic linkage of polysaccharides by glycoside hydrolases; and (3) the potential biological outcome of ROS/RNS-mediated depolymerization of polysaccharides.     

Swelling of intercellular lipid lamellar structure with short repeat distance in hairless mouse stratum corneum as studied by X-ray diffraction

Lamellar structures of intercellular lipids in stratum corneum of hairless mouse were studied at various water contents by small-angle X-ray diffraction. At room temperature there are at least two lamellar structures, long and short lamellar structures, with repeat distances of 13.6 and around 6 nm, respectively. The long lamellar spacing is almost constant over the water content from 0% w/w to 80% w/w that is consistent with the previously reported results. For the short lamellar structure we found that with increasing the water content the lamellar spacing becomes larger, that is, from 12 to 50% w/w the short lamellar spacing increases from 5.8 to 6.6 nm. In addition to the previously reported result that at the water content of about 20% w/w the X-ray diffraction peak for the long lamellar structure becomes sharp, we found that this is also the case for the short lamellar structure. Below the water content of about 12% w/w the X-ray diffraction peak for the short lamellar structure dies out and conversely above the water content of about 50% w/w it becomes weak and finally merges into the second-order diffraction peak for the long lamellar structure. Considering the matching of the long lamellar spacing that is unchanged with the water content and twice the short lamellar spacing that changes as a function of the water content, it is likely that the swelling of the short lamellar structure plays an important role in the regulation of water stored in stratum corneum.     

Intracytoplasmic membrane structures in the L forms of the hemolytic streptococcus]

In studying the submicroscopic structure of the L-form of streptococcus, group A, isolated from the heart tissue of rabbit there were revealed intracytoplasmic membrane structures. Ring lamellar structures were most frequently revealed in the spheroid cells with dense and loose cytoplasm. They were also found in dense cytoplasm of elementary bodies. Myelin-like structures or those resembling a bundle of microtubules were less incident. Fibrillar structures collected into bands, 64--140 nm in with, and located on one or both sides of the cells beside the cytoplasmic membrane were revealed in the spheroid cells. Individual fibrillae, 8 to 10 nm in diameter, adhered one to another, interlaced, and were sometimes located in parallel. The fibrillar band was loose in the lysed cells.     

A CYTOCHEMICAL STUDY OF EMBRYO DEVELOPMENT IN STELLARIA MEDIA

The caryophyllad type of embryogenesis in Slellaria media was investigated using topological cytochemical methods for the demonstration of nucleic acids, proteins and polysaccharides. The primary suspensor cell initially contained high levels of cytoplasmic RNA, but these declined rapidly after the first few cell divisions. Cytoplasmic protein levels were high throughout the existence of the primary suspensor cell, becoming concentrated into proteinoplasts at the time of cotyledon initiation in the embryo. These plastids were RNA- and DNA-negative, and only slightly positive with methods for polysaccharides and lipids. Cytoplasmic and nucleolar RNA and protein levels were high in the embryo throughout its development. The cotyledons and endosperm also showed high RNA and protein levels. Cytoplasmic DNA was present in the embryonic apical meristematic cells in the form of Feulgen-positive, deoxyribonuclease-removable granules. Cytoplasmic histones were present in the embryo and endosperm. Polysaccharides were demonstrable in the cotyledons, cortex, endosperm, and nucellus.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Glycolipid storage material in Fabry's disease: a study by electron microscopy, freeze-fracture, and digital image analysis

The glycolipid storage material in Fabry's disease was studied by electron microscopy of thin-sectioned (TS) and freeze-fractured (FF) specimens. In the kidney all deposits were found to be located in lysosomes, arranged as lamellar stacks. Deposits in the heart consisted of intracytoplasmic concentric whirls or folded lamellar structures. High resolution TS micrographs disclosed various defects in the lamellar structure. For stabilization, such defects require additional amphiphilic, surface-active molecules. These molecules could interact with other cellular constituents. The lamellar periodicity of the deposits in FF specimens was determined by reconstruction of the three-dimensional fracture face by digital image analysis. Homogeneous multilamellar deposits exhibited a periodicity of 14-15 nm, contrasting with the conventional estimates of 4-5 nm on TS micrographs. This difference is explained by better preservation of the physiologic hydrated state in FF specimens, with 1 vol of lipids binding 2 vol of water. Inhomogeneous structures with an even higher state of hydration included water lenses between the sheets. The strong hydration obviously contributes to the enlargement of the intracellular glycolipid deposits.     

Physical properties of glycosyl diacylglycerols. 2. X-ray diffraction studies of a homologous series of 1,2-Di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

X-ray diffraction methods were used to characterize the thermotropic polymorphism exhibited by aqueous dispersions of a homologous series of 1,2-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols. Upon cooling from temperatures at which the acyl chains of these lipids are melted, all of these compounds form structures that exhibit both low-angle and wide-angle diffraction patterns consistent with the formation of lamellar L beta gel phases. After a suitable protocol of low-temperature annealing, complex diffraction patterns consistent with the formation of highly ordered, lamellar, crystal-like phases are obtained. These patterns are similar for all of the compounds studied, suggesting that the unit cell structure is invariant. The assumption that the unit cell structure is invariant permits the assignment of phases to the diffraction orders, thereby making possible the construction of electron density profiles. These electron density profiles indicate that the crystal-like phases of these lipids are poorly hydrated structures with the hydrocarbon chains inclined at 35 degrees to the bilayer normal. The diffraction patterns of the crystal-like phases of these lipids changed abruptly at the calorimetrically determined phase transition temperatures to those characteristic of either lamellar liquid crystalline phases (N less than or equal to 17) or inverted nonbilayer phases. With these X-ray diffraction data we demonstrate that, at elevated temperatures, the shorter chain homologues (N less than or equal to 16) form cubic phases of the Pn3m space group, whereas the longer chain compounds form inverted hexagonal phases.     

Models of haloadaptation in bacterial membranes

Abstract Cell membranes consist of a complex assortment of amphipathic lipids. These lipids exist in one of three phases in aqueous systems at the growth temperature of the organism: namely, lamellar gel, lamellar liquid-crystalline or hexagonal-II. The phase behaviour is modified by interaction of the lipids with other membrane components and electrolytes. A stable membrane structure is achieved when the polar and non-polar interactions are balanced such that a durable bilayer arrangement is formed into which the various membrane proteins are integrated. The effect of surface charge on phase domain behaviour of the membrane lipids and the modulation by electrolytes is crucial to understanding how halophiles adapt to high-salt environments.     

Lipoprotein charge and vascular lipid metabolism

The internal lipids were extracted from untreated hair without surface lipids. Liposomes were formed with the internal lipids at different hydration levels to determine the organization of these lipids and the influence of the water content on the lamellar structure of the hair fibres by X-ray Scattering (SAXS). Two structures of hair lipids were observed at 4.5 and approximately 9.0nm with a different behaviour as a function of water content: the largest bilayer being the one that showed a capacity to retain water inside its structure. SAXS was also applied directly to three samples: a packed swatch of hair fibres at 60% RH, fibres soaked in water and delipidized fibres. Only the lamella at 9.0nm was slightly affected by water content. Moreover, there was a small diminution in intensity probably due to a high permeability of wet fibres which could give rise to a disorder of the lipid structure. These two lamellar rearrangements are probably made up of lipids with a different and specific hydrophilic/hydrophobic balance.     

Characterisation of bacterial polysaccharides: steps towards single-molecular studies

Techniques used in studies of polysaccharides, including chemical composition, linkage pattern, and higher order structures are in constant development. They provide information necessary for understanding of the polysaccharide properties and functions. Here, recent advancements in studies of the polysaccharides at the single-molecule level are highlighted. Over the last few years, single-molecule techniques such as force spectroscopy have improved in sensitivity and can today be used to detect forces in the pN range. In addition, these techniques can be used to investigate properties of single molecules close to physiological conditions. The challenges in the interpretation of the observations are aided by control experiments using well-characterised polysaccharides and by data provided by complementary methods. This field is expected to have increasing impact on the further advancement of the molecular understanding of the role of polysaccharides in various biological processes such as recognition and cell adhesion.     

Functional domain-assembly in hairpin ribozymes

Functional structures of hairpin ribozymes have been investigated by constructing various chemically modified molecules. Domain-exchange and linker insertion experiments were performed to find active conformations of the RNA enzyme showing cleavage activity. Our experiments and other evidence suggest that the active structure has a bent conformation, and that domain-interactions are essential for the cleavage activity.     

Surface charge markedly attenuates the nonlamellar phase-forming propensities of lipid bilayer membranes: calorimetric and (31)P-nuclear magnetic resonance studies of mixtures of cationic, anionic, and zwitterionic lipids

The lamellar/nonlamellar phase preferences of lipid model membranes composed of mixtures of several cationic lipids with various zwitterionic and anionic phospholipids were examined by a combination of differential scanning calorimetry and (31)P NMR spectroscopy. All of the cationic lipids utilized in this study form only lamellar phases in isolation. Mixtures of these cationic lipids with zwitterionic strongly lamellar phase-preferring lipids such as phosphatidylcholine form only the lamellar liquid-crystalline phase even at high temperatures, as expected. Moreover, mixtures of these cationic lipids with strongly nonlamellar phase-preferring zwitterionic lipids such as phosphatidylethanolamine exhibit a markedly reduced propensity to form inverted nonlamellar phases, again as expected. However, when mixed with anionic lipids such as phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid, a marked enhancement of nonlamellar phase-forming propensity occurs, despite the fact both components of the mixture are nominally lamellar phase-preferring. An examination of the lamellar/nonlamellar phase transition temperatures and the nature of the nonlamellar phases formed, as a function of temperature and of the composition of the mixture, indicates that the propensity to form inverted nonlamellar phases is maximal in mixtures where the mean surface charge of the membrane surface approaches neutrality and decreases markedly with increases in the density of positive or negative charge at the membrane surface. Moreover, the onset temperatures of the reversed hexagonal phase rise more steeply than do those of the inverted cubic phase as the ratio of cationic and anionic lipids is varied, suggesting that the formation of inverted hexagonal phases is more sensitive to this surface charge effect. These results indicate that surface charge per se is a significant and effective modulator of the lamellar/nonlamellar phase preferences of membrane lipids and that charged group interactions at membrane surfaces may have a major role in regulating this particular membrane property.     

Immunomodulating polysaccharides from the lichen Thamnolia vermicularis var. subuliformis

Three heteroglycans Ths-4, Ths-5 and thamnolan and a beta-glucan, Ths-2, isolated from the lichen Thamnolia vermicularis var. subuliformis were tested for in vitro immunomodulating activities and shown to have various influences on the immune system. All the polysaccharides except Ths-4 caused a stimulation of rat spleen cell proliferation. In contrast, Ths-4 caused cell death early in the culture, probably due to over-stimulation. Moreover, the galactofuranomannans, Ths-4, Ths-5 and the beta-glucan Ths-2, induced rat spleen cells to secrete IL-10 significantly above background levels. In addition, Ths-4 and Ths-5 stimulated significant TNF-alpha secretion by rat peritoneal macrophages. The galactofuranomannans Ths-4 and Ths-5 have similar structures apart from the molecular weight. Thus, it may be concluded that the molecular size might influence the potency but not the pattern of activity for Ths-4 and Ths-5. The galactofuranorhamnan thamnolan had less mitogenic effect than Ths-5 and Ths-2 and neither induced IL-10 secretion by rat spleen cells nor TNF-alpha secretion by peritoneal macrophages to significant levels. This shows that thamnolan with its unusual galactofuranorhamnan structure differs from the other Thamnolia polysaccharides in its immunomodulatory activity.     

Relationship between internucleotide linkage geometry and the stability of RNA

The inherent chemical instability of RNA under physiological conditions is primarily due to the spontaneous cleavage of phosphodiester linkages via intramolecular transesterification reactions. Although the protonation state of the nucleophilic 2'-hydroxyl group is a critical determinant of the rate of RNA cleavage, the precise geometry of the chemical groups that comprise each internucleotide linkage also has a significant impact on cleavage activity. Specifically, transesterification is expected to be proportional to the relative in-line character of the linkage. We have examined the rates of spontaneous cleavage of various RNAs for which the secondary and tertiary structures have previously been modeled using either NMR or X-ray crystallographic data. Rate constants determined for the spontaneous cleavage of different RNA linkages vary by almost 10,000-fold, most likely reflecting the contribution that secondary and tertiary structures make towards the overall chemical stability of RNA. Moreover, a correlation is observed between RNA cleavage rate and the relative in-line fitness of each internucleotide linkage. One linkage located within an ATP-binding RNA aptamer is predicted to adopt most closely the ideal conformation for in-line attack. This linkage has a rate constant for transesterification that is approximately 12-fold greater than is observed for an unconstrained linkage and was found to be the most labile among a total of 136 different sites examined. The implications of this relationship for the chemical stability of RNA and for the mechanisms of nucleases and ribozymes are discussed.