Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection.

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na+ + K+)-stimulated Mg2-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61 degrees C, about 20 degrees C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28 degrees C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.     

Eimeria necatrix: Induced proteolytic sensitivity of infected chick crypt cells

While devising a protocol for the isolation of chick crypt cells infected with Eimeria necatrix, it was observed that infected cells were readily lysed by 0.25% trypsin. Time-course studies at 17 C with 5.5 × 10⁵ cells at 96 hr postinfection revealed that 0.001% trypsin effectively lysed >90% of infected cells within 10 min. Uninfected crypt cells were not lysed under these conditions. To determine the site of action of trypsin, the plasma membrane proteins from trypsin-treated and untreated infected cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the major proteins were unaffected by the trypsin treatment, some minor changes were noted: (1) three components (～-60, ～-52, and ～-20 KDa) were trypsin sensitive and (2) a new band (～-42 KDa) appeared in the membrane of trypsin-treated infected cells. Previously, it was found that the plasma membrane of infected cells, in contrast to uninfected cells, accumulated gel-phase lipid (J. E. Thompson, M. A. Fernando, and J. Pasternak, Biochimica et Biophysica Acta555, 472–487, 1979). Here, it was examined whether trypsin would perturb the physical state of the plasma membrane of infected cells. Both X-ray diffraction patterns and transition temperature studies revealed no difference between membranes from untreated and trypsin-treated infected cells. Thus, “trypsin sensitivity” may be a secondary phenomenon that is due primarily to the cellular leakiness that accompanies the accumulation of gel-phase lipid in the plasma membrane of infected cells. The uptake of trypsin may stimulate the release of catabolic enzymes that, consequently, lyse an infected cell.     

Interactions of Vesicular Stomatitis Virus Structural Proteins with HeLa Plasma Membranes

THE processes whereby nucleoprotein core particles of certain animal viruses become enveloped by and bud off from host cell membranes can be studied by preparing membrane^1,2 or “sedimentable”³ fractions from infected cells and examining them for the presence of virus proteins. We find that similar experiments designed to monitor assembly of vesicular stoma-titus virus (VSV) at sites along the plasma membranes of HeLa cells are best interpreted after first investigating the possibility that virus proteins adsorb to plasma membranes during cell fractionation and membrane isolation. In this report, we show that at 0° C the membrane protein of VSV, among other virus proteins, adsorbs to plasma membranes isolated from uninfected HeLa cells. With appropriate pulse-chase experiments, however, we are able to demonstrate the progressive association, in vivo, of VSV core protein with plasma membranes of infected HeLa cells.     

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lymphocytes and membranes exfoliated from their cell surface

The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, $\text{η}$, of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, $\text{η}$ was also determined in isolated plasma cell supernatants.For isolated plasma membranes of thymocytes and GRSL cells $\text{η}$ values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively.Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane.Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types $\text{η}$ (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane.     

Regulation of the plasma membrane during exposure to low temperatures in suspension-cultured cells from a cryophyte (Chorispora bungeana)

Summary. As the outermost boundary of the cell, the plasma membrane plays an important role in determining the stress resistance of organisms. To test this concept in a cryophyte, we analyzed alterations of several components in plasma membranes isolated from suspension-cultured cells of Chorispora bungeana Fisch. & C.A. Mey in response to treatment at 0 and −4 °C for 192 h. When compared with the controls growing at 25 °C, both the membrane permeability and fluidity showed recovery after the initial impairment. Linolenic acid and membrane lipid unsaturation increased by about 0.8-fold following cold treatments, although the kinetics of the increase varied with the temperatures examined. During the treatments, the plasma membrane H⁺-ATPase (EC 3.6.1.3) activity increased by 78.06% at 0 °C and 100.47% at −4 °C. However, the plasma membrane NADH oxidase (EC 1.6.99.3) activity only decreased when exposed to a lower temperature (−4 °C), and remained at 63.93% after being treated for 192 h. After the treatments, the physical properties of the plasma membranes of suspension-cultured cells, especially the −4 °C treated cells, were similar to those in the wild plants. These findings indicate that the specific mechanism of cold resistance of C. bungeana is tightly linked with the rapid and flexible regulation of membrane lipids and membrane-associated enzymes, which ensure the structural and functional integrity of the plasma membrane that is essential for withstanding low temperature. Correspondence: Lizhe An, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China.     

Spin label studies on rat liver and heart plasma membranes: Effects of temperature,calcium, and lanthanum on membrane fluidity

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(1 2,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl₂ to I(1 2,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)-labeled heart plasma membranes with either CaCl₂ or LaCl₃ decreased the lipid fluidity at 37°C, although the magnitude of the La³⁺ effect was larger and occurred at lower concentrations than that induced by Ca²⁺; addition of 0.2 mM La³⁺ or 3.2 mM Ca²⁺ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca²⁺ and La³⁺ at these concentrations decrease the activities of such plasma membrane enzymes as Na⁺, K⁺-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

Development of Plasmodium chabaudi in Mouse Red Blood Cells: Structural Properties of the Host and Parasite Membranes1

The structure of both the host and parasite membranes during stages in the asexual development of Plasmodium chabaudi in mouse red blood cells is examined by transmission electron microscopy of thin sections and freeze-fracture preparations. The erythrocyte's plasma membrane, the membrane of the parasitophorous vacuole, and the plasma membrane of the parasite exhibit different structural properties in terms of membrane width and the frequency and diameter of the typical intramembrane-particles (IMP) populating the membrane's fracture faces. The difference between the parasitophorous vacuolar membrane and host cell's plasma membrane is remarkable because the vacuolar membrane is formed from an invagination of the erythrocyte's plasma membrane. The vacuolar membrane has significantly reduced frequencies and diameters of IMP's on both faces and reveals a marked temperature response manifesting itself as large IMP-devoid domains emerging on both faces on chilling to 4°C. In contrast, cooling induces only some very small IMP-devoid patches on both faces of the host plasma membrane. Neither of these membranes changes significantly as parasite development progresses. In contrast, the parasite's plasma membrane shows local alterations during its development, forming compaction domains with the nuclear envelope in ca. 30% of the ring-stages and trophozoites. These compaction domains disappear in late uninuclear trophozoites and schizonts. Furthermore, the plasma membrane of the host cell, the vacuolar membrane, and the parasite's plasma membrane do not respond to externally applied Ca²⁺, and their temperature-response remains unaltered during the infection cycle. Thus, modification of these three membranes as a consequence of invasion and development of the parasites, as recently found in the primate malaria caused by P. knowlesi, can be detected neither directly nor indirectly via temperature- and/or Ca²⁺-response in the rodent malaria caused by P. chabaudi.     

Influence of lipid rafts on CD1d presentation by dendritic cells

Our main objective was to analyze the role of lipid rafts in the activation of Vα-14^? and Vα-14⁺ T hybridomas by dendritic cells. We showed that activation of Vα-14⁺ hybridomas by dendritic cells or other CD1d-expressing cells was altered by disruption of lipid rafts with the cholesterol chelator MβCD. However, CD1d presentation to autoreactive Vα-14^? anti-CD1d hybridomas which do not require the endocytic pathway was not altered. Using partitioning of membrane fractions with Brij98 at 37°C, we confirmed that CD1d was enriched in subcellular fractions corresponding to lipid rafts and we describe that α-GalCer enhanced CD1d amount in the low density detergent insoluble fraction. We conclude that the membrane environment of CD1d can influence antigen presentation mainly when the endocytic pathway is required. Flow cytometry analysis can provide additional information on lipid rafts in plasma membranes and allows a dynamics follow-up of lipid rafts partitioning. Using this method, we showed that CD1d plasma membrane expression was sensitive to low concentrations of detergent. This may suggest either that CD1d is associated with lipid rafts mainly in intracellular membranes or that its association with the lipid rafts in the plasma membrane is weak.     

A photobleaching recovery study of glucocorticoid effects on lateral mobilities of a lipid analog in S3G HeLa cell membranes

Treatment of the S3G strain of HeLa cells with dexamethasone inhibits cholesterol synthesis and thus results in decreased plasma membrane cholesterol-to-protein ratios. Incubation of HeLa cells with dexamethasone for 24 h lowers the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in intact cell plasma membranes and isolated plasma membranes (Johnston, D. and Melnykovych, G. (1980) Biochim. Biophys. Acta 596, 320–324). We have examined the effect of dexamethasone treatment of S3G HeLa cells on the lateral diffusion of the fluorescent lipid analogue 3,3′-dioctadecylindocarbocyanine iodide (DiI) by the fluorescence photobleaching recovery technique. The lateral diffusion of DiI was measured in cells 0, 2, 6, 12, and 24 h following treatment with dexamethasone and in cells identically handled without dexamethasone at 37°C. The diffusion constants of DiI in the treated and untreated cell membranes at zero time were (4.52±0.30) · 10^?9 cm²/s and (4.56±0.24) · 10^?9 cm²/s, respectively. There was no significant change in the lateral diffusion of DiI in the untreated cells over the 24 h period. The lateral diffusion of the lipid probe in the dexamethasone-treated cells began to increase 6 h following treatment and reached (6.43±0.27) · 10^?9 cm²/s at 24 h. The lateral diffusion of DiI was also measured at 25, 17, 10 and 4°C following 24 h incubation with and without dexamethasone. The effect of dexamethasone treatment on the lipid probe lateral diffusion observed at 37°C is decreased at 25°C and reversed in direction at 10 and 4°C. These results agree with those obtained in artificial systems containing varying amounts of cholesterol and support the suggestion that cholesterol acts to suppress phospholipid phase changes in animal cells. The lateral diffusion of DiI localized as a monolayer at a mineral oil-water interface was measured by fluorescence photobleaching recovery. The resulting data and the viscosity of the mineral oil were used to calculate the microviscosities of the plasma membranes of untreated and dexamethasone-treated cells at 25°C. Membrane microviscosities were also calculated from the fluorescence polarization studies cited above. In both cases the dexamethasone treatment reduced the apparent microviscosity by approximately 25%. However, the absolute microviscosity values obtained by the two techniques differ by a factor of 3.     

Intraerythrocytic Development of Plasmodium knowlesi: Structure,Temperature- and Ca2+-Response of the Host and Parasite Membranes1

Thin-sectioning and freeze-etching electron microscopy were applied to explore the structure and the temperature- and Ca²⁺-response of the different host and parasite membranes during intraerythrocytic development of Plasmodium knowlesi in Macacca mulatta. The plasma membrane of uninfected erythrocytes is temperature- and Ca²⁺-responsive: chilling to 4°C and exposure to 5 mM Ca²⁺ induces a slight decrease in IMP-frequency and the emergence of small IMP-devoid patches on P-faces. On parasite infection, the erythrocyte membrane becomes modified as indicated by an enhanced temperature-response and the appearance of caveolae, ca. 70–90 nm in diameter. The frequency of these caveolae is increased in schizont-infected erythrocytes. Moreover, electron dense plaques, ca. 40 nm in width, appear just beneath the erythrocyte membrane in late trophozoites and schizonts, thus indicating a further modification of the host cell membrane during parasite development. The membrane of the parasitophorous vacuole, derived from the host plasma membrane, dramatically reduces the IMP-frequency especially on the P-face upon parasite infection. This leads to an apparent reversal of the IMP-distribution persisting throughout the whole infection cycle. The parasite plasma membrane forms local compaction domains with the nuclear envelope in ca. 30% of the ring-stages and trophozoites, which disappear in late trophozoites and schizonts. Moreover, the IMP-frequency on plasma membrane fracture faces almost doubles during parasite development. Chilling induces a decrease in the IMP-frequency on P-faces of the plasma membrane. Surprisingly, however, the parasite plasma membrane and the vacuolar membrane respond to externally applied Ca²⁺ with almost a doubling of the IMP-frequency. The different parasite endomembranes also undergo characteristic changes during parasite development.     

Transport of fluorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum-infected cells

The asexual development of the human malaria parasite Plasmodium falciparum is largely intraerythrocytic. When 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]caproyl] phosphatidylcholine (NBD-PC) was incorporated into infected and uninfected erythrocyte membranes at 0 degrees C, it remained at the cell surface. At 10 degrees C, the lipid was rapidly internalized in infected erythrocytes at all stages of parasite growth. Our results indicate that the internalization of NDB-PC was not because of endocytosis but rapid transbilayer lipid flip-flop at the infected erythrocyte membrane, followed by monomer diffusion to the parasite. Internalization of the lipid was inhibited by (a) depleting cellular ATP levels; (b) pretreating the cells with N-ethyl maleimide or diethylpyrocarbonate; and (c) 10 mM L-alpha-glycerophosphorylcholine. The evidence suggests protein-mediated and energy dependent transmembrane movement of the PC analogue. The conditions for the internalization of another phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) were distinct from that of NBD-PC and suggest the presence of additional mechanism(s) of parasite-mediated lipid transport in the infected host membrane. In spite of the lack of bulk, constitutive endocytosis at the red cell membrane, the uptake of Lucifer yellow by mature infected cells suggests that microdomains of pinocytotic activity are induced by the intracellular parasite. The results indicate the presence of parasite-induced mechanisms of lipid transport in infected erythrocyte membranes that modify host membrane properties and may have important implications on phospholipid asymmetry in these membranes.     

A Plasmodium chabaudi chabaudi high molecular mass glycoprotein translocated to the host cell membrane by a non-classical secretory pathway

We have purified and characterized a novel high molecular mass glycoprotein of P. chabaudi chabaudi (Pc550gp) that is transported to the erythrocyte membrane during the intraerythrocytic cycle. Immuno fluorescence assays with polyclonal monospecific antibodies against Pc550gp show that the protein to be localized in the periphery of young trophozoite stages i.e., on the plasma membrane or parasitophorous vacuole membrane. However, in late trophozoites and schizonts the antigen is distributed in both parasite and host cell membranes. These results were confirmed by immunoblotting of isolated parasites and infected host cell membranes at different stages of parasite development. Moreover, alkali extraction of purified infected erythrocyte membranes at mature stages of parasite development does not solubilize Pc550gp, suggesting that it is an integral membrane protein. In addition proteinase K digestion of intact infected host cells induced the disappearance of Pc550gp. Further indicating its transmembrane nature and that it presents extracellular domains susceptible to proteolysis. Brefeldin A or low temperature (15 degrees C) treatment did not affect the translocation of Pc550gp from the parasite compartments to the erythrocyte membrane, indicating that the secretion of Pc550gp does not follow the classical transport pathway described in most eukaryotic cells.     

Regulation of H+Pumping by Plant Plasmalemma under Osmotic Stress: The Role of 14-3-3 Proteins

All higher plants have high-specific sites for binding fusicoccin (FCBS), a metabolite of the fungus Fusicoccum amygdaliDel. These sites are localized on the plasmalemma and produced by the association of the dimers of 14-3-3 proteins with the C-terminal autoinhibitory domain of H⁺-ATPase. Considering the fusicoccin binding to the plasmalemma as an index characterizing the formation of this complex, we studied the influence of osmotic stress on the interaction between 14-3-3 proteins and H⁺-ATPase in the suspension-cultured sugar beet cells and protoplasts obtained from them. An increase in the osmolarity of the extracellular medium up to 0.3 Osm was shown to enhance proton efflux from the cells by several times. The number of FCBS in isolated plasma membranes increased in parallel, whereas 14-3-3 proteins accumulated in this membrane to a lesser degree. The amount of H⁺-ATPase molecules did not change, and the ATP-hydrolase activity changed insignificantly. The data obtained indicate that osmotic stress affects H⁺-ATPase pumping in the plasmalemma through its influence on the coupling between H⁺-transport and ATP hydrolysis; 14-3-3 proteins are involved in this coupling. The interaction between the plasmalemma and the cell wall is suggested to be very important in this process.     

Activity of Plasma Membrane H<Superscript>+</Superscript>-ATPase in Coleoptile Cells during Development of Maize Seedlings

Hydrolytic activities of the H⁺-ATPase were compared for plasma membrane fractions isolated from coleoptile cells of 3-, 4-, and 5-day-old etiolated maize seedlings. The membrane preparations obtained by differential centrifugation were additionally purified in the gradient of sucrose density and in the polyethylene glycol-dextran system. The highest level of ATP-hydrolyzing activity was observed in the plasmalemma fraction obtained from 4-day-old seedlings. The pattern of age-dependent changes in H⁺-ATPase activity of the plasma membranes was clearly different from the monotonic deceleration of coleoptile cell elongation in the period examined. It is supposed that changes in ATPase activity reflect different regulatory roles of this principal ion-transporting enzyme of the plasma membrane at the stage of cell elongation and at a later developmental stage when the coleoptile has completed its physiological function.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 566–572.Original Russian Text Copyright © 2005 by Rudashevskaya, Kirpichnikova, Shishova.     

Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study

The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, v_j, within the lipid matrix. At T = 35°C a value of v_j = 1.6 · 10³ s^?1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and charge of the lipids we obtain jump frequencies between 0.8 · 10⁸ s^?1 and 1.5 · 10⁸ s^?1 at T = 35°C. The results are compared with jump frequencies yielded in model membranes.The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{min at}\text{T = 37°}\text{C}$) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 100}\text{min at}\text{T = 37°}\text{C}$.The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 23°C and T = 33°C we observed an additional fluidity change at T = 38°C in erythrocyte ghosts. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI⁺D⁺, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI⁺D^?, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI⁺D^? compared to the MGI⁺D⁺ clones. The MGI⁺D^? clones produced an unusual polyunsaturated C_20:5 fatty acid at 28°C, whereas the MGI⁺D⁺ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI⁺D^? clones than of the MGI⁺D⁺ clones. The cells of MGI⁺D⁺ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.     

Subcellular localization of binding sites for cytochalasin D: Evidence from activation energies

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at > 11.5 °C, 18 kcal/mol at < 11.5 °C) was taken as evidence that [³H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [³H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).     

Isolation of plasma membranes from the bovine retinal pigment epithelium

Retinal pigment epithelium plasma membranes have been isolated by differential and density gradient centrifugation of glass-bead-bound, collagenase-treated cells. Electron microscopic evidence indicates that the glass-bead-bound cells were devoid of red blood cells, rod outer segments and other ocular cell contaminants. The plasma membranes were recovered in 4–6 μg/eye yields and purified 10-fold by 5′-nucleotidase and alkaline phosphodiesterase 1, and 6.5-fold by (Na⁺ + K⁺)-ATPase. Plasma membrane purity as measured by covalent labeling of the epithelial cell plasma membrane proteins with p-(diazonium) benzene[³²S]sulfonic acid was 8–19-fold. In purified plasma membranes contamination by mitochondria was undetectable and lysosomal contamination reduced 100-fold, while endoplasmic reticulum was 2-fold enriched. SDS-polyacrylamide gel electrophoresis of the plasma membrane proteins revealed 23–26 major bands by Coomassie blue staining and 12–16 major bands by radioactive labeling. The plasma membranes exhibited a 3-fold lower concentration of docosahexaenoic acid, a 3-fold higher cholesterol/phosphate ratio, and were 10-fold enriched in cholesterol per μg protein when compared to the whole cell fraction. Retinal epithelial plasma membranes contain an average of 1 mol cholesterol per mol of lipid phosphorus, a high palmitic acid concentration (39 mol%) and a low concentration of docosahexaenoic acid (2 mol%). The lipid profile of the retinal pigment epithelial plasma membranes indicates that they are typical of plasma membranes from many other cell types and that they appear to be less fluid than total rod outer segment membranes.     

The fine structure and cytology of the association between the parasitic red algaHolmsella australis and its red algal hostGracilaria furcellata

Summary Holmsella australis Noble andKraft ms. is a colourless red algal parasite, forming whitish pustules on its photosynthetic red algal host,Gracilaria furcellata Harvey. In the infected region, host cortical tissue continues to grow and enclose the expanding pustule. Filaments of both host and parasite grow apically, the cells being connected by primary pit connections (PCs). Secondary PCs form between cells of the same species, and in addition,H. australis initiates the formation of secondary PCs with cells ofG. furcellata. All three types of secondary PC are morphologically distinct. In hostparasite PCs the surface adjoining the host cell is similar in structure to a host-host PC, while that adjoining the parasite cell has the structure of a parasite-parasite PC. The plasma membrane is continuous between the cells of the unrelated host and parasite. In addition, a cap membrane is typically produced only on the host surface, though occasionally the parasite side is enclosed by a cap membrane as well. Cap membranes are absent from parasite-parasite PCs (making them intracellular), while host-host PCs are typically extracellular, both cells producing cap membranes. The presence or absence of a cap membrane in certain positions appears to vary, and suggests that cells may be able to regulate its presence. Since transport of nutrients would be expected to occur from host to parasite cells, and between parasite cells, the morphological evidence presented here suggests the PCs may be the pathway.