Role of sterol structure in the thermotropic behavior of plasma membranes of Saccharomyces cerevisiae

Plasma membranes from Saccharomyces cerevisiae were prepared by a new procedure involving lyticase treatment of the yeast cells. The plasma membranes were right-side-out, closed vesicles of uniform appearance with a sterol to phospholipid molar ratio of 0.365. The thermotropic behavior of these plasma membranes from wild-type yeast and from sterol mutants was examined by differential scanning calorimetry, fluorescence anisotropy and Arrhenius kinetics of plasma membrane enzymes. While differential scanning calorimetry failed to demonstrate any lipid transition, fluorescence anisotropy data indicated that lipid transitions were occurring in the plasma membranes of the yeast sterol mutants but not the sterol wild-type. The temperature dependence of the plasma membrane enzymes, chitin synthase and Mg²⁺-ATPase, was also investigated. The Arrhenius kinetics of chitin synthase did not reveal any transitions in either the sterol mutant or wild-type plasma membranes, yet the Arrhenius kinetics of the Mg²⁺-ATPase suggested that lipid transitions were occurring in both cases.     

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.     

Lipid Reorganization Induced by Shiga Toxin Clustering on Planar Membranes

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb₃ in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb₃ clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb₃ lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb₃ (65∶30∶5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb₃ (40∶35∶20∶5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb₃, STxB-Gb₃ complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.     

No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism.We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb³⁺-containing liposomes. Upon chelation of Tb³⁺ by an aromatic carboxylic acid, the fluorescence of Tb³⁺ increases many fold. The CPP TAT(44-57)-SAM and TAT(37-53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol.In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44-57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44-57).Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44-57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.     

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.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

Effect of potassium phenosan on structure of plasma membranes of mice liver cells in vitro

The effect of synthetic anti-oxidant potassium phenosan (PP, potassium salt of β-(4-hydroxy-3,5-ditretbutil-phenyl)-propionic acid) on the structural state of the surface (8 Å) and deep (20–22 Å) lipid regions of plasma membranes of mice liver cells was studied by spin probes method in vitro in a wide range of concentrations (10^?5–10^?21 M). Two stable free radicals, 5- and 16-doxyl-stearic acids (C₅ and C₁₆), were used as spin probes. The nonlinear polymodal dose-effect dependences were obtained for parameters that characterize the microviscosity of the lipid bilayer (τ_c) in the site of localization of the probe C₁₆, and the order parameter (S), which characterizes the stiffness of the surface layers of lipids in the site of localization of the probe C₅. Statistically a reliable increase was observed for parameter τ_c after addition of PP at concentrations 10^?5–10^?7 M and 10^?18–10^?19 M, and for parameter S after addition of PP at concentrations 10^?6–10^?7 M and 10^?13–10^?15 M. Peaks on both dose-effect curves were separated by the intervals of concentrations where PP had no effect on the studied physico-chemical characteristics of biomembranes. For PP concentrations which caused maximal changes in τ_c and S, we investigated thermal dependence of these parameters and determined the thermally induced structural transitions. Comparing with control, ultra-low doses of PP (10^?13–10^?15 M) and (10^?18–10^?19 M) caused an appearance of additional thermally induced structural transition in the surface and deep regions of plasma membrane lipids. The possible role of the interaction of PP molecules with specific binding sites on plasma membranes and formation of nanoparticles of PP in very dilute aqueous solutions are discussed.     

Luminescence study of Dy or Ce activated LiCaBO3 phosphor for γ‐ray and C5+ ion beam irradiation

The photoluminescence and thermoluminescence characteristics of rare earths (Dy or Ce) activated LiCaBO₃ phosphors have been studied. Phosphors were synthesized by modified solid state synthesis. The phosphors were characterized by X‐ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL) and thermoluminescence (TL) for structural, morphological and luminescence studies. Dy³⁺ activated LiCaBO₃ shows emission at 486 and 577 nm due to ⁴ F_9/2 → ⁶H_15/2 and ⁴ F_9/2 → ⁶H_13/2 transition, respectively, whereas the PL emission spectra of Ce³⁺ activated LiCaBO₃ phosphor shows a broad band peaking at 432 nm, which is due to the transition from 5d level to the ground state of the Ce³⁺ ion. The thermoluminescence study was also carried out for both these phosphors for γ‐ray irradiation and carbon beam irradiation. Linearity was studied for a 0.4–3.1 Rad dose γ‐rays. Linear behaviour over this dose range was observed. Gamma ray‐irradiated phosphors were shown to be negligible fading upon storage. All the samples were also studied for 75 MeV C⁵⁺ ion beam exposure in the range of 3.75 × 10¹² – 7.5 × 10¹³ ion cm^–2 fluence. In addition to this, trapping parameters of all the samples were also calculated using Chen's peak shape method. Copyright © 2015 John Wiley & Sons, Ltd.     

STRUCTURE-FUNCTION RELATIONSHIPS IN THE ADIPOSE CELL : I. Ultrastructure of the Isolated Adipose Cell

A method is described for preparing isolated rat adipose cells for electron microscopy. The ultrastructure of such cells and their production of ¹⁴CO₂ from U-glucose-¹⁴C were studied simultaneously in the presence of insulin or epinephrine. Each adipose cell consists of a large lipid droplet surrounded by a thin rim of cytoplasm. In addition to typical subcellular organelles, a variety of small lipid droplets and an extensive system of membranes characterize the cell's cytoplasm. A fenestrated envelope surrounds the large, central lipid droplet. Similar envelopes surround cytoplasmic lipid droplets occurring individually or as aggregates of very small, amorphous droplets. Groups of individual droplets of smaller size also occur without envelopes. The system of membranes consists of invaginations of the cell membrane, vesicles possibly of pinocytic origin, simple and vesiculated vacuoles, vesicles deeper in the cytoplasm, flattened and vesicular smooth surfaced endoplasmic reticulum, and Golgi complexes. Neither insulin nor epinephrine produced detectable ultrastructural alterations even when cells were incubated under optimal conditions for the stimulation of ¹⁴CO₂ evolution. Structural responses of the isolated adipose cell to hormones, if such occur, must, therefore, be dynamic rather than qualitative in nature; the extensive system of smooth surfaced membranes is suggestive of compartmentalized transport and metabolism.     

High cytotoxicity and membrane permeability of Et3Pb+ in mammalian and plant cells

Cells of mammalian origin as well as those of higher plants appear to be very sensitive to triethyllead ion (Et₃Pb⁺). Neuroblastoma cells kept in the presence of 1 μM Et₃Pb⁺ lost their viability within 6 h. Growth of suspension culture cells of soybean (G. max(L.)Merr.) was inhibited by 1 μM Et₃Pb⁺, and finally the cells died. Morphologically, Et₃Pb⁺ caused the complete breakdown of microtubular structures in neuroblastoma cells; thus microtubules appeared to be the main target for the toxin. While in a previous study the effect of Et₃Pb⁺ on microtubules has been well documented at concentrations of 50–200 μM ¹, the present study demonstrates that the formation of microtubules from pig brain tubulin is disturbed at concentrations of Et₃Pb⁺ as low as 0.5 to 1 μM . We conclude from these data that Et₃Pb⁺ freely permeates the plasma membranes of mammalian as well as plant cells.     

Membrane Topology of Human Presenilin-1 in SK-N-SH Cells Determined by Fluorescence Correlation Spectroscopy and Fluorescent Energy Transfer

Presenilin-1 (PS1) protein acts as passive ER Ca²⁺ leak channels that facilitate passive Ca²⁺ leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer’s disease (FAD). FADPS1 mutations abrogate the function of ER Ca²⁺ leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487–500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca²⁺) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH₂-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP–PS1, and PS1–CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca²⁺ leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1–CFP was either expressed alone or together with YFP–PS1 into SK-N-SH cell line and the interaction between YFP–PS1 and PS1–CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP–PS1 and PS1–CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1–CFP and YFP–PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH₂-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH₂-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.     

Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: Relationship to expression of ANF-C receptors

Previous studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C-type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum-stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF_99–126, rANF_103–126, and rANF_103–125, were only poorly antimitogenic in serum-stimulated primary cultures, whereas des[Cys¹⁰⁵, Cys¹²¹] rANF_104–126 which binds selectively to the ANF-C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF_99–126, rANF_103–126, rANF_103–126, Cys¹¹⁶rANF_102–116, and des[Cys¹⁰⁵, Cys¹²¹]rANF_104–126 inhibited serum-induced [³H]thymidine incorporation (IC₅₀ in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP-elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric-oxide-vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [¹²⁵I]rANF_99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF-C-type type receptor. Covalent cross-linking studies with (¹²⁵I)rANF_99–126 confirmed that membranes prepared from fresh aortae predominantly expressed the ANF-A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross-linked protein was the ANF-C-type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased expression of the ANF-C-type receptors. © 1993 Wiley-Liss, Inc.     

RF discharge-based plasma emitter

An injector of hydrogen atoms for plasma diagnostics in modern tokamaks has been developed at the Budker Institute of Nuclear Physics (Novosibirsk). The ion source of the injector produces a proton (helium ion) beam with a current of up to 2 A (1 A), an ion energy of up to 55 keV, a beam divergence of ～0.6\deg, and a pulse duration of up to 10 s. An RF discharge-based plasma emitter, which is one of the main parts of the ion source, is described. The emitter diameter is 72 mm, the ion current density is 120 mA/cm², and the inhomogeneity of the current density is ±6%. The beam is formed by a four-electrode ionoptical system with 163 round apertures. At a current of 2 A, the ion beam consists of 67% protons, 18% H ₂ ⁺ ions, and 15% H ₃ ⁺ ions, the total content of heavier ions in the beam being no higher than 2–3%.     

pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen

Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K⁺ analogue, ⁸⁶Rb⁺, from prelabelled cells, and the influx of ²²Na⁺. The permeabilizing activity of PA was limited to a proteolytically activated form (PA_N) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PA_N facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PA_N permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.     

Changes in lipid peroxidation, the redox system and ATPase activities in plasma membranes of rice seedling roots caused by lanthanum chloride

Highly purified plasma membranes were isolated by aqueous two-phase partitioning from rice (Oryza sativa) seedling roots. The effects of lanthanum chloride (LaCl₃) on the activities of lipid peroxidation, the redox system and H⁺-ATPase, Ca²⁺-ATPase of plasma membranes were studied. The lipid peroxidation of plasma membranes could be depressed by certain low concentrations of LaCl₃ and enhanced by high concentrations of LaCl₃, while the lipid peroxidation was also dependent on the plasma membrane protein and incubation time. The relative activity of O₂ uptake of plasma membranes was inhibited by all tested LaCl₃ concentrations. In contrast, the reduction rate of Fe(CN)₆ ^3– by plasma membranes was stimulated below 40 M of LaCl₃, but was reduced above 60 M of LaCl₃. The relative activities of both H⁺-ATPase and Ca²⁺-ATPase increased constantly from control to LaCl₃ of concentration 60 M where the activities of both enzymes were the maximum, but decreased remarkably at 80 M LaCl₃ concentrations various LaCl₃ were added to culture solutions. In the other measurement case in which various LaCl₃ concentrations were added directly to reaction medium and the plasma membrane vesicles only came from the control cultured rice seedling roots, the response of H⁺-ATPase activity to La³⁺ was similar to the response in culture solution. However, the La³⁺ concentration was only 20 M when the activity of H⁺-ATPase was the maximum. In contrast to the case of LaCl₃ addition to culture solution, Ca²⁺-ATPase activity was inhibited by all concentrations of La³⁺ which were added directly to the reaction medium. The above results revealed that REEs inhibited electron transfer from NADH to oxygen in plant plasma membranes, depressed the production of active oxygen radicals, and reduced the formation of lipid peroxides through plasma membrane lipid peroxidation. REEs ions also enhanced the H⁺ extrusion by both standard redox system and H⁺-ATPase in plasma membranes at certain concentrations. A possible role for the plant cell wall in REEs effects on plasma membranes was also suggested.     

Surface Properties and Behavior of Lipid Extracts from Plasma Membranes of Cells Cultured as Monolayer and in Tissue-Like Conditions

The differences in the surface active properties of native lipids extracted from plasma membranes of cells cultured as a monolayer and in three-dimensional (3D) matrix were investigated. This experimental model was chosen because most of the current knowledge on cellular physiological processes is based on studies performed with conventional monolayer two-dimensional (2D) cell cultures, where cells are forced to adjust to unnaturally rigid surfaces that differ significantly from the natural matrix surrounding cells in living organisms. Differences between monolayer and 3D cells were observed in the lipid composition of plasma membranes and especially in the level of the two major microdomain-forming lipids—sphingomyelin (SM) and cholesterol, which were significantly elevated in 3D cells. The obtained results showed that culturing of cells in in vivo-like environment affected the surface active properties of plasma membrane lipids at interfaces which might influence certain membrane-associated interface processes. The detected differences in the lipid levels in 2D and 3D cell extracts affected significantly the behavior of the model lipid monolayers at the air–water interface (Langmuir monolayers) which resulted in different values of the monolayer equilibrium (γ_eq) and dynamic (γ_max, γ_min) surface tension and surface potential. Compensation of the SM content in extracts of 2D cell cultures up to a level close to the one measured in 3D cells approximated the monolayer properties to the values observed for 3D cells. These results implied that the interactions between the cells and the surrounding medium affected the level of plasma membrane SM and other lipids, which had a strong impact on the surface properties of lipid monolayers, such as γ_eq, γ_max, and γ_min, the compression/decompression curve shape, the hysteresis area during cycling of the monolayers, etc. We suggest that the elevated content of SM observed in plasma membranes of 3D fibroblasts could be responsible for an increased rigidity and possibly reduced permeability of cells cultured in 3D environment. The current results provide useful information that should be taken into account in the interpretation of the membrane physico-chemical properties of cells cultured under different conditions.     

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl₂ and 5 mM MgCl₂, and centrifuged at $\text{52 000 ×}\text{g}$ for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATpase}$, Mg²⁺-ATPase and 5′-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40°C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

Experimental study of the dynamics of neutron emission from the GOL-3 multimirror trap

In experiments on the plasma heating and confinement in the GOL-3 multimirror trap, a deuterium plasma with a density of ～10¹⁵ cm^?3 and an ion temperature of 1–2 keV is confined for more than 1 ms. The plasma is heated by a relativistic electron beam. The ion temperature, which was measured by independent methods, reached 1.5–2 keV after the beginning of the beam injection. Since such a fast ion heating cannot be explained by the classical energy transfer from electrons to ions through binary collisions, a theoretical model of collective energy transfer was proposed. In order to verify this model, a new diagnostics was designed to study the dynamics of neutron emission from an individual mirror cell of the multimirror trap during electron beam injection. Intense neutron bursts predicted by this model were detected experimentally. Periodic neutron flux modulation caused by the macroscopic plasma flow along the solenoid was observed. The revealed mechanism of fast ion heating can be used to achieve fusion temperatures in the multimirror trap.     

Subcellular distribution of prostaglandin and gonadotrop in receptors in bovine corpora lutea.

The subcellular distribution of prostaglandin (PG) E₁, F₂α and gonadotropin receptors in bovine corpora lutea was critically examined by preparing various subcellular fractions, assaying for various marker enzymes to assess the purity and examining ³H-PGE₁, ³H-PGF₂α and ¹²⁵I-human lutropin (hLH) specific binding. The marker enzyme data suggested that subcellular fractions were relatively pure with little or no cross contamination. The binding of ³H-PGs and ¹²⁵I-hLH was markedly enriched in plasma membranes with respect to homogenate. The other subcellular fractions also exhibited binding despite very little or no detectable 5′-nucleotidase activity. If 5′-nucleotidase was assumed to lack sensitivity and reliability to detect minor contamination with plasma membranes and ³H-PGs or ¹²⁵I-hLH binding were used as sensitive plasma membrane markers, it was still difficult to explain binding in other fractions based on plasma membrane contamination. Therefore, these results lead to the inevitable conclusion that plasma membranes were primary (or one of the primary) but not exclusive sites for PGE₁, PGF₂α and gonadotropin receptors.