X-ray diffraction study of bilayer to non-bilayer phase transitions in aqueous dispersions of di-polyenoic phosphatidylethanolamines.

The low temperature phase properties of aqueous dispersions of di-18:2 and di-18:3 phosphatidylethanolamine are strongly influenced by the presence of ice. In the presence of cryoprotectants to inhibit ice formation, these lipids persist in the H(II) phase down to at least -50 degrees C. Ice formation, however, leads to a drastic reduction in the amount of available free water and a rapid reduction in the diameter of the inverted cylindrical micelles of the H(II) phase. The resulting increase in surface curvature of the micelles induces an imbalance in the forces acting in the lipid surface and the hydrophobic core which is relieved by formation of the L(alpha) phase. On reheating the lipid samples undergo an abrupt L(alpha) --> H(II) phase transition at about -20 degrees C. The radius of the water core of the inverted micelles at their point of formation is estimated to be 0.9 nm. This increases with temperature as more unfrozen water becomes available until the normal equilibrium radius of about 2.3 nm is reached at 0 degrees C when the bulk water in the sample finally melts. A small proportion of the H(II) phase lipid enters an as yet unidentified cubic phase on freezing. The spacings of the (10) planes of the H(II) phase, the (111) planes of the cubic phase and the d-spacing of the L(alpha) phase were found to be almost identical at the phase transition temperature. The cubic phase appears to disappear at low temperature but to reform on heating. Freeze-fracture studies revealed no unequivocal evidence for cubic phase lipid but the presence of residual non-bilayer lipid structures was observed even at temperatures as low as -80 degrees C. The presence of intersecting stacks of lamellar sheets in the replicas strongly suggest the existence of an epitaxial relationship between the L(alpha) and H(II) phases in these systems.

Phase separations in membranes of Anacystis nidulans grown at different temperatures.

Freeze fracture electron microscopy studies were performed on samples of Anacystis nidulans quenched from different temperatures. Membrane lipid phase separations were observed to take place over the ranges 15--30 degrees C, 5--25 degrees C and -5--15 degrees C for cultures grown at 38, 28 and 18 degrees C, respectively. Differential scanning calorimetry heating curves showed endotherms which coincided with these temperature ranges. Variations of phase separation temperatures with growth temperature, and hysteresis effects in the calorimetric measurements, were related to changes in the fatty acid composition of membrane lipids.

Structural characteristics of thylakoid membranes of Arabidopsis mutants deficient in lipid fatty acid desaturation.

The ultrastructure of thylakoid membranes from Arabidopsis thaliana wild-type, JB67 and LK3 fatty acid desaturation deficient mutants was studied by thin-section and freeze-fracture electron microscopy. There was a decrease in the amount of the appressed and non-appressed membranes in JB67 and LK3 Arbidopsis mutants when compared to the wild type, resulting in a reduction in the length of photosynthetic membrane per plastid. The results from freeze-fracture showed a decrease in size and a marked increase in packing density of membrane-associated particles on the exo- and endoplasmic fracture faces of the mutants. In addition, areas of the appressed membranes of the mutants contained particles in regular arrays under conditions where no such arrays were observed in wild-type thylakoid membranes. These observations suggest, that the decreased level of lipid fatty acid unsaturation affects the ability of the lipid matrix to mediate the assembly of chloroplast membrane components. The role of polyunsaturated membrane lipids is considered in terms of their ability to promote functional oligomeric assemblies of components of the photosynthetic apparatus.

Formation of inverted lipid micelles in aqueous dispersions of mixed sn-3-galactosyldiacylglycerols induced by heat and ethylene glycol.

The formation of 'lipidic' particles corresponding to inverted lipid micelles in freeze-fracture replicas of aqueous dispersions of mono- and digalactosyldiacylglycerols can be greatly enhanced either by increasing the temperature from which the samples are thermally quenched or by the addition of cryoprotectants such as ethylene glycol. In the case of the heated samples, the lipids tend to form quasi-crystalline structures consisting of sheets of 8-9 nm diameter particles organised on an orthorhombic lattice. The orientation of alternate sheets varies giving rise to a characteristic herring-bone pattern. Ethylene glycol-treated samples, in contrast, form more regular structures consisting of 13-16 nm diameter particles. Lowering the temperature from which the samples are quenched and/or decreasing the concentration of ethylene glycol reduces the frequency of formation of such structures. A number of intermediate states associated with the reincorporation of the lipid molecules of the inverted micelles into the lamella phase are also identified. The factors influencing particle formation are briefly discussed. It is concluded that the destabilisation of lipid-water interactions play a major role in this process.

The effect of chemical modification of Saccharomyces cerevisiae on electrophoretic mobility, cell-wall structure and amphotericin B uptake.

Saccharomyces cerevisiae NCYC 239 suspended in solutions of NaCl showed two distinct plateaus in plots of electrophoretic mobility vs. pH, corresponding to pKa values of approx. 2 and 5. This is in contrast to cells suspended in buffer where only a single pKa (4) can be determined. Modification of cells with KI/I2 or nitrous acid led to altered electrophoretic mobility, indicating the presence of sulphydryl and amino groups, respectively, in the yeast cell surface, whereas uranyl nitrate modification had little effect, suggesting phosphate groups to be absent. Electron micrographs showed visible effects of KI/I2 and nitrous acid modification on cell membrane structure, and in these modified cells amphotericin B uptake was rapid. It is suggested that diffusion through the cell wall is the rate-limiting step for amphotericin B uptake. An activation energy of 20 kJ X mol-1 was determined for uptake of amphotericin B by unmodified cells.

Scanning electron microscopic study of blood cell surfaces of the middle asian tortoise, Testudo horsfieldi.

The micromorphology and surface topography of spleen cells of the Middle Asian tortoise, Testudo horsfieldi, have been examined by scanning electron microscopy. Spleen cells comprise a population with surface characteristics similar to counterparts in mammalian spleen except for an, as yet undescribed, lymphocyte which is designated as lamellar type on the basis of its surface contours.

Structure of vesicles formed from non-ionic dialkylglycerol poly(oxyethylene) ether surfactants: effect of electrolyte and cholesterol.

Five non-ionic dialkylglycerol poly(oxyethylene) ether surfactants, designated 2C(m)E(n) (where m, the number of carbons in each alkyl chain=16 or 18, and n, the number of oxyethylene units=12, 16 or 17) have been examined for their ability to form vesicles when dispersed in water or in an aqueous solution of 154 mM NaCl, alone or in the presence of 50 mol% cholesterol. Freeze fracture electron microscopy and light scattering showed that regardless of the hydrating fluid, all the non-ionic surfactants, with the exception of 2C(16)E(17) and 2C(18)E(17), formed vesicles in the absence of cholesterol - 2C(16)E(17) and 2C(18)E(17) instead formed micellar aggregates. All surfactants, however, formed vesicles in the presence of 50 mol% cholesterol. Small angle neutron scattering studies of the surfactant vesicles enabled the bilayer thickness and repeat distance (d-spacing) to be determined. The bilayers formed by all the non-ionic surfactants in the absence of cholesterol were surprisingly thin (∼50 Å for the E(12) containing surfactants and ∼64 Å for 2C(18)E(16)) most likely due to the intrusion of oxyethylene groups into the hydrophobic core of the bilayers. In contrast, however, the non-ionic surfactants exhibited a relatively large d-spacing of around ∼130-150 Å. The addition of 50 mol% cholesterol had a dramatic effect on the thickness of the vesicle bilayer, increasing its size by 10-20 Å, most probably because of an extrusion of oxyethylene from the hydrophobic region of the bilayer and/or a reduction in the tilt on the surfactant alkyl chains. Additionally the presence of cholesterol in a vesicle tended to reduce slightly both the d-spacing and the thickness of the water layer separating the bilayers. The presence of NaCl, even at the low concentrations used in the study, did affect the properties of the bilayer such that it reduced the d-spacing and, in the case of cholesterol-containing systems, also reduced bilayer thickness.

Surface morphology of human airway mucosa: normal, carcinoma or cystic fibrosis.

The study presents preliminary qualitative findings of an investigation of grossly normal main and lobar bronchi at sites distant to well circumscribed tumour (n = 15), adjacent to tumour (n = 5) or of airways obtained during heart/lung transplantation in patients with cystic fibrosis (CF, n = 3). In the normal airways the surface epithelium was on average 50 micron thick, pseudo-stratified and rested on a roughly contoured basement membrane. A variety of cell types were identified although many were obscured by a dense covering of cilia, occasionally interrupted by foci of squamous metaplasia. Submucosal gland structure was observed in chance vertical fractures of the airway wall. Tissue adjacent to tumour showed sloughing, squamous metaplasia, pleomorphism and cell surface projections of stubby microvilli or tortuous microridges. The surface morphology of the three CF patients showed no feature unique to the condition, albeit secretions were found adherent to surface lining associated with isolated bacteria and groups of free cells (probably lymphocytes). In each of the three cases the epithelial surface was densely ciliated, interspersed with mucous (i.e., goblet) cells. Submucosal gland collecting ducts had dilated lumena.

Ultrastructure of the asexual apparatus in Mycotypha (Mucorales).

The development of the asexual apparatus of Mycotypha africana and Mycotypha poitrasii observed by means of scanning electron microscopy is reported. Using ultrathin sections and freeze-fracture replicas, sporangiole structure is described. Sporangiospore structure is compared with that of other members of the Thamnidiaceae.

Response of laryngeal and tracheo-bronchial surface lining to inhaled cigarette smoke in normal and vitamin A-deficient rats: a scanning electron microscopic study.

The effects on surface morphology of airway epithelium of cigarette smoke (CS) inhalation alone (experiments one and two) or of CS in combination with hypovitaminosis A (experiment two) was investigated using specific pathogen free rats. Eight morphologically distinct cell types were distinguished overall. Apart from atypical squamous lesions, each of the other cell types could be found in varying proportions in all experimental groups. CS alone caused an increase in the frequency with which intra-lumenal mucus was seen and an increase in the occurrence of secretory cells of types IV (i.e., 'merocrine') and V (i.e., 'apocrine'). In experiment one, the area of trachea covered by cilia as determined by point counting increased significantly (P less than 0.01). Hypovitaminosis A was induced by lowering the dietary intake of vitamin A to a minimum, defined level. Rats showed an approximately 75% decrease in plasma retinol levels and a 95-100% decrease in hepatic stores of vitamin A. At this level, hypovitaminosis A alone had no significant effect on airway epithelial morphology. Foci of squamous metaplasia (squamous cells of type VIIIa) were found in all groups but extensive squamous metaplasia of the larynx and squamous lesions of atypical appearance (type VIIIb) were found only in the vitamin deficient group exposed to CS. The results suggest the synergistic effects of reduced vitamin A and CS may be important in the induction of atypical squamous changes which may predispose the airway to the development of squamous carcinoma.

Influence of dehydration and watering on camel red cell size: a scanning electron microscopic study.

Three young female camels were subjected to an eleven day period of dehydration followed by a single large drink. Blood haematocrit and plasma protein concentration were monitored regularly during dehydration and for 48 h following watering. Blood samples were taken at regular intervals, before and after watering, and prepared for scanning electron microscopy (SEM) for analysis of red cell size and shape. Plasma protein concentration did not change rapidly on watering implying that the camel has homeostatic mechanisms which regulate plasma osmolarity by controlling the rate at which large volumes of water are absorbed on drinking. By SEM, camel red cells were elliptical in shape with mean surface dimensions of 6.5 microns x 4.0 microns with an estimated minimum thickness at mid point of 0.95 microns: neither their shape constant nor size changed on dehydration or watering corroborating the absence of major and rapid changes in osmolarity.

Association state of human red blood cell band 3 and its interaction with ankyrin.

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.

Delayed tail loss during the invasion of human skin by schistosome cercariae.

Schistosomiasis is initiated when cercarial larvae invade human skin. Contrary to long-held assumptions, most cercariae of Schistosoma mansoni do not shed their propulsive tails as they penetrate. Scanning electron microscopy studies and infection experiments with entire human skin and differentiated, stratum corneum-like, human keratinocyte cultures, have shown that most cercarial tails enter the skin along with their bodies. We propose that this behaviour is an adaptive trait linked with concomitant immunity.

A nystatin-resistant mutant of Saccharomyces cerevisiae: isolation and characterization by electron microscopy and chemical analysis of whole cells, cell-walls and protoplasts.

A mutant of Saccharomyces cerevisiae NCYC 239 with a high minimum inhibitory concentration (35 micrograms ml-1) for nystatin, compared to that of the parent strain (2 micrograms ml-1), was derived by a series of subcultures in media containing increasing antibiotic concentrations. In the absence of nystatin, the growth rate of the mutant was significantly lower than the parent strain, although mean cell-size and size-distribution were similar. No differences between strains were detectable by electron microscopy. Analysis of whole cells showed the total sterol present and the ratio of ergosterol:24(28)dehydroergosterol was similar. However, there were marked differences in amino acid content and chain-length of fatty acids in the cell wall, and protoplasts from resistant cells had decreased amounts of unsaturated fatty acids. It is suggested that alterations in cell wall components in the mutant may be directly linked to the mechanism of nystatin resistance.

Interaction of capsulate Haemophilus influenzae with human airway mucosa in vitro.

Two pairs of isogenic capsulate and noncapsulate and one pair of capsulate fimbriate and nonfimbriate strains of Haemophilus influenzae type b were studied in an organ culture of human respiratory mucosa. Over 24 h, the numbers of recovered bacteria increased from the original inoculum size of 10(5) to 10(8) CFU/ml. Transmission electron microscopy and scanning electron microscopy showed that noncapsulate organisms caused significant epithelial damage, whereas capsulate strains did not. Association of noncapsulate bacteria with damaged epithelial cells was observed by 14 h of incubation. In contrast, capsulate organisms were associated with a dense, thick, gel-like matrix which was observed above the epithelial surface. These capsulate organisms were not seen to associate with the epithelial surface (by transmission electron microscopy), though they were occasionally seen adhering to cells by scanning electron microscopy. Fimbriate capsulate H. influenzae showed increased adherence to buccal cells compared with nonfimbriate capsulate organisms. There was also association of fimbriate capsulate bacteria with damaged organ culture epithelium in one of four experiments. It is concluded that both capsule and fimbriae affect the interaction of H. influenzae with human airway mucosa in vitro by influencing adherence to and damage of the epithelium.

Interaction of nontypable Haemophilus influenzae with human respiratory mucosa in vitro.

One laboratory strain (SH9) (n = 12) and five clinical isolates of unencapsulated Haemophilus influenzae replicated from 10(4) to 10(8) cfu/ml over 24 h in an organ culture of human respiratory mucosa in which only the intact mucosal surface is exposed. By transmission electron microscopy (TEM), bacteria were not seen in association with normal respiratory epithelium, even after incubation for 24 h. Histology and TEM morphometry demonstrated patchy and occasionally confluent damage to epithelia at this time, with bacteria associated only with cells that were structurally damaged. Scanning electron microscopy revealed an increased quantity of mucus in infected preparations; H. influenzae were associated with mucus by 14 h of incubation and with damaged epithelial cells by 24 h. Fimbriation of H. influenzae increased buccal cell adherence but did not facilitate association with normal respiratory epithelium and failed to increase epithelial damage or association with damaged cells. Epithelial damage may be prerequisite for association of H. influenzae with respiratory epithelium in vitro.