A singular state of membrane lipids at cell growth temperatures.

Cells adjust their membrane lipid composition when they adapt to grow at different temperatures. The consequences of this adjustment for membrane properties and functions are not well understood. Our report shows that the temperature dependence of the diffusion of a probe molecule in multilayers formed from total lipid extracts of E. coli has an anomalous maximum at a temperature corresponding to the growth temperature of each bacterial preparation (25, 29, and 32 degrees C). This increase in the lateral diffusion coefficient, D, is characteristic of membrane lipids in a critical state, for which large fluctuations of molecular area in the plane of the bilayer are expected. Therefore, changes in lipid composition may be due to a requirement that cells maintain their membranes in a state where molecular interactions and reaction rates are readily modulated by small, local perturbations of membrane organization.     

Effect of temperature on nuclear membranes and nucleo-cytoplasmic RNA-transport in Tetrahymena grown at different temperatures.

The effect of temperature on the nuclear envelope structure and the transport of total RNA and ribosomal subunits from nucleus to cytoplasm was examined in Tetrahymena cells propagated at two different temperatures. Freeze-etch electron microscopy of cells grown at 23 and 18 degrees C detects the emergence of smooth areas on the fracture faces of the nuclear membranes upon lowering the temperature below approximately 15 and approximately 12 degrees C, respectively. Coincident with these freeze-etch changes, a discontinuous decrease is observed in the nucleocytoplasmic RNA-transport; this is probably not due to a cease in RNA-synthesis. Below the thermotropic discontinuity observed in the transport of total RNA in 18 degrees-cells the nucleocytoplasmic transport of the small and large ribosomal subunits is equally retarded. Recent temperature studies on the endoplasmic reticulum membranes of Tetrahymena suggest that the freeze-etch changes in the nuclear membranes are induced by a thermotropic clustering of the membrane lipids. We conclude that this lipid clustering induces the permanent protein constituents in the nuclear envelope pore complexes to change from a relatively "open" into a relatively "closed" state thus causing the observed decrease in RNA-transport.     

The physical state of quick-frozen membranes and lipids

Lipid bilayers and biomembranes produce nearly identical calorimeter scans regardless of whether they are slowly cooled under near-equilibrium conditions or rapidly frozen at rates used in freeze-fracture electron microscopy. Except for the melting of ice at 273 K, for both cooling regimens no significant thermal events occur from 100 K to the usual gel to liquid crystal transition. The gel to liquid crystal transition itself is somewhat altered by rapid cooling when bilayers contain mixed lipid species. Combined with X-ray diffraction studies, the results indicate that quickly frozen bilayers are crystalline, but that the crystalline domains are quite small or otherwise disordered. In contrast to the behavior of lipids in bilayers, hexagonal-phase calcium cardiolipid easily forms a glass upon cooling.     

Permeability and stability properties of membranes formed by lipids extracted from Lactobacillus acidophilus grown at different temperatures

Lactobacillus acidophilus CRL 640 grown at 25 and 37 degrees C showed a high content of cardiolipin, phosphatidylglycerol, and glycolipids. Cultures grown at 25 degrees C showed a twofold increase in glycolipids in relation to phospholipids, a twofold increase in the C16:0 and a fourfold increase in the C18:2 fatty acids. In contrast, the C19-cyc and the 10-hydroxy acid (C18:0-10 OH) species showed a noticeable decrease. Extracts of total lipids of bacteria grown at 25 and 37 degrees C dispersed in water yielded particles having a high negative surface potential as measured by electrophoretic mobility. Vesicles prepared by extrusion of these dispersions through polycarbonate membranes of 100-nm pore diameter showed high trapping of carboxyfluorescein (CF), which remained unchanged for at least 20 h. The fluorescence anisotropy measured with diphenylhexatriene (DPH) and the generalized polarization of Laurdan were significantly lower in vesicles prepared with lipids containing the highest glycolipid ratio, in comparison to those of bacteria grown at 37 degrees C. No phase transition was detected between 5 and 50 degrees C as measured with both probes. In accordance with these results, no significant release of the trapped CF in this range of temperature was detected. Bile salts and NaCl promoted an increase in the fluorescence, which is interpreted as a change in the permeability properties of the membrane. This effect was lower with KCl, while CaCl2 did not cause any change. The greater permeability change was observed in vesicles with a low glycolipid/phospholipid ratio. NaCl did not affect the packing of the interface as measured with Laurdan, in contrast to CaCl2. The action of Ca+2 may be ascribed to the binding to the negatively charged lipids, such as phosphatidyl glycerol and cardiolipin. It is concluded that the higher glycolipid/phospholipid ratio and the fatty acids C18:2 and C16:0 enhance the lipid membrane stability and decrease the organization in the interfacial and hydrocarbon zones. These results are congruent with the behavior of entire bacteria subject to osmotic and freeze/thaw stresses.     

Influence of dietary lipids on microsomal membranes from chick breast muscle

1. The effect of different dietary fat intake on the lipid composition and fluidity of microsomal membranes as well as in the enzymatic activity of the Ca2+-ATPase from chick breast muscle was investigated. 2. When a standard diet was supplemented with 10% sunflower seed oil, an increase in the relative amounts of unsaturated fatty acids and membrane fluidity and a decrease in the cholesterol content was observed. 3. The presence of 6% cholesterol in the diet does not modify the fatty acid composition and the fluidity of the membrane but increased, in a low extension, the cholesterol content. 4. The provision of the sunflower seed oil-rich diet supplemented with cholesterol just 48 hr before death promoted an increase in the relative amounts of unsaturated fatty acids and cholesterol content whereas the membrane fluidity decreased in a significant extent. 5. Despite that dietary lipids gave rise in some cases to changes in lipid composition and in the physical state of the microsomal membrane, neither the Ca2+ uptake capacity nor the ATPase activity were significantly affected.     

Molecular parameters and physical state of neutral glycosphingolipids and gangliosides in monolayers at different temperatures

The effect of temperature on the behaviour of four different gangliosides (GM3, GM1, GD1a and GT1b), sulphatide, ceramide (Cer) and three neutral glycosphingolipids (GalCer, Gg3Cer, Gg4Cer) was investigated in monolayers at the air-NaCl (145 mM) interface. GM1, GD1a and GT1b are liquid-expanded in the range of temperatures studied (5-65 degrees C). GM3, sulphatide, Cer and neutral glycosphingolipids show isothermal liquid-expanded----liquid-condensed transitions. The collapse pressure of ganglioside monolayers decreases with temperature, whereas neutral glycosphingolipids may show some maximum values at particular temperatures. The reduction of the molecular area of liquid-expanded glycosphingolipids under compression occurs with a favorable positive entropy change and an unfavorable negative enthalpy. By contrast, the compression of interfaces with a two-dimensional phase transition occurs with an unfavorable entropy but a favorable enthalpy change. From the temperature dependence of the surface pressure at which the two-dimensional phase transition takes place, a minimal temperature above which the isotherm becomes totally liquid-expanded can be obtained. For the different glycosphingolipids this temperature decreases in the order Cer greater than GalCer greater than sulphatide greater than Gg3Cer greater than Gg4Cer greater than GM3 greater than GM1 greater than GD1a greater than GT1b. This sequence is similar to that found for the calorimetrically determined transition temperatures (cf. Maggio, B., Ariga, T., Sturtevant, J.M. and Yu, R.K. (1985) Biochemistry 24, 1084-1092).     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

Studies on temperature adaptation in Tetrahymena. Positional distribution of fatty acids and species analysis of phosphatidylethanolamine from Tetrahymena pyriformis grown at different temperatures.

Phosphatidylethanolamine of 15 degrees C-grown Tetrahymena pyriformis (NT-I) cells contains more polyunsaturated fatty acids than 39.5 degrees C-grown cells. This increase in unsaturation is due to an increase in linoleic (C18 : 2) and linolenic (C18 : 3) acids, and a decrease in myristic (C14 : 0), palmitic (C16 : 0), palmitoleic (C16 : 1) and heptadecanoic (C17 : 0) acids. Compared with 39.5 degrees C-grown cells, the proportion of palmitic acid (C16 : 0) decreased in the 1-position as does at the 2-position in 15 degrees C-grown cells. On the contrary, there is a significant increase in linoleic (C18 : 2 delta 9, 12) and gamma-linolenic (gamma-C18 : 3) acids in the 1- and 2-positions, respectively. Phosphatidylethanolamine has been subfractionated into seven different diglyceride species. In 15 degrees C cells, the amounts of fractions 2 (1-linolenoyl-2-linoleoyl) and 3 (1-linolenoyl-2-palmitoleoyl, 1-linolenoyl-2-oleoyl) increased while there was a great decrease in subfraction 7 (1-myristoyl-2-palmitoleoyl, 1-palmitoyl-2-palmitoleoyl). Since subfractions 1 and 2 contain over 70% linoleic (C18 : 2) and linolenic (C18 : 3) acids, these fractions might be composed mainly of 1-linolenoyl-2-linolenoyl and 1-linolenoyl-2-linoleoyl molecular species at 15 degrees C. These data support evidence that phosphatidylethanolamine would play a principal role as an acceptor of acyl chains for temperature acclimation.     

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°C, 5–25°C and –5–15°C for cultures grown at 38, 28 and 18°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.     

Thermoregulation in the cold after physical training at different ambient air temperatures

Since human thermoregulation at rest is altered by cold exposure, it was hypothesized that physical training under cold conditions would alter thermoregulation. Three groups (n = 8) of male subjects (mean age 24.3 +/- 0.9 years) were evaluated: group T (interval training at 21 degrees C), group CT (interval training at 1 degrees C), and group C (no training, equivalent exposure to 1 degrees C). Each group was submitted, before and after 4 weeks of interval training (5 d/week), to a cold air test at rest (SCAT) (dry bulb temperature (Tdb) = 1 degrees C) for a 2-h period for evaluation of the thermoregulatory responses. During SCAT, after the training/acclimation period, group T exhibited a higher rectal temperature (Tre) (P < 0.05) without significant change in mean skin temperature (Tsk) whereas metabolic heat production (M) was higher at the beginning of the SCAT (P < 0.05). For group CT, no thermoregulatory change was observed. Group C showed a lower Tre (P < 0.05) without significant change in either Tsk or in M, suggesting the development of a hypothermic general cold adaptation. This study showed, first, that the cold thermoregulatory responses induced by an interval training differed following the climatic conditions of the training and, second, that this training performed in the cold prevented the development of a general cold adaptation.     

Interaction of melittin with glycosphingolipids and phospholipids in mixed monolayers at different temperatures. Effect of the lipid physical state

The influence of the liquid-expanded or liquid-condensed state of the lipid interface induced by changes of temperature on the lipid-protein interactions and their two-dimensional miscibility was studied for mixtures of melittin with different phospholipids (DPPC, DMPC, DOPC egg PC) and gangliosides (G_M1, G_D1a) in mixed monolayers at the air/145 mM NaCl interface. The critical amount of melittin at which a phase separation takes place in the mixed film increases as the glycosphingolipid or phospholipid is more liquid-expanded. The lipid-protein interaction increases the stability of both melittin and the lipid. The interaction of melittin with gangliosides is thermodynamically more favorable as these are more liquid-expanded. The interaction of melittin with phospholipids, on the other hand, is more favorable when the lipids are in the liquid-condensed state even if these films show lateral immiscibility at a lower proportion of protein compared to lipids in the liquid-expanded state. Hydration-dehydration effects in the polar head group region are likely to participate in these lipid-protein interactions.     

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.     

Effects of species of VA-mycorrhizal fungi on growth and mineral uptake of sorghum at different temperatures

Sorghum [Sorghum bicolor (L.) Moench] plants were grown in growth chambers at 20, 25 and 30°C in a low P Typic Argiudoll (3.65 µg P g^–1 soil, pH 8.3) inoculated with Glomus fasciculatum, Glomus intraradices, and Glomus macrocarpum to determine effects of vesicular-arbuscular mycorrhizal fungi (VAMF) species on plant growth and mineral nutrient uptake. Sorghum root colonization by VAMF and plant responses to Glomus species were temperature dependent. G. macrocarpum colonized sorghum roots best and enhanced plant growth and mineral uptake considerably more than the other VAMF species, especially at 30°C. G. fasciculatum enhanced shoot growth at 20 and 25°C, and mineral uptake only at 20°C. G. intraradices depressed shoot growth and mineral uptake at 30°C. G. macrocarpum enhanced shoot P, K, and Zn at all temperatures, and Fe at 25 and 30°C above that which could be accounted for by increased biomass. Sorghum plant growth responses to colonization by VAMF species may need to be evaluated at different temperatures to optimize beneficial effects.     

Effects of zinc on lipids of erythrocytes from carp (Cyprinus carpio L.) acclimated to different temperatures

We compared the effect of zinc (0.01, 0.1, 0.5 and 1 mM) at two temperatures (5 and 20 degrees C) on erythrocytes from summer and winter acclimatised carp. An increase in temperature from 5 to 20 degrees C increased the unsaturation index (UI) and relative proportion (UI/SFA) of unsaturated to saturated fatty acids in total lipids of the red cells. At 5 degrees C, the unsaturation index of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) decreased (30-40%) in the presence of 1 mM zinc. The change in unsaturation of phospholipids in the presence of zinc at 5 degrees C is probably responsible for the alteration in structural integrity of erythrocyte membrane as observed by hemolysis and the decreased thiol group content in the erythrocytes. In light of this result, zinc may be considered an environmental hazard for these fish at low temperatures.     

Composition and physical properties of lipids from plasma membranes of dog kidney

Lipid composition, physical state of major phospholipid classes and transbilayer migration of phosphatidylcholine have been determined in plasma membranes of the dog kidney. The lipid composition of brush-border membranes markedly differs from that of antiluminal membranes with respect to: (a) the total phospholipid content; (b) the cholesterol to phospholipid ratio (C/P); (c) the distribution of the major phospholipid classes. Sphingomyelin present in large amounts in both luminal and antiluminal membranes extracts exhibits a transition of phase between 20 and 44 degrees C approximately. In the range of temperature studied (5-55 degrees C) no phase transitions were detected for the other phospholipid species. Our data suggest that: (1) at physiological temperature the higher C/P ratio of brush-border membranes is in large part responsible for their lower fluidity; (2) both the relatively low cholesterol and high sphingomyelin contents contribute to the thermotropic transitions observed in intact membranes. Finally transbilayer migration of phosphatidylcholine in brush-border membranes is a very slow process with a half time of 6.5 h at 37 degrees C which compares with that of other biological membranes.     

Effects of K+-depolarization on the physical state of membrane lipids in brain synaptosomes of the rat

The microstructure of lipid bilayer in synaptosomes from rat brain upon K+-depolarization (30 mM) was studied using the inductive resonance energy transfer (IRET) from proteins to the fluorescent probes, pyrene and DMC (4-dimethylaminochalcone). The effectiveness of IRET was not changed by the K+-depolarization. The monomer-to-eximer ration (Fm285/Fe285) of pyrene fluorescence intensities in IRET was 1.5 times lower upon depolarization than in controls. This suggested a decreased microviscosity of the lipid bilayer in immediate environment to proteins of the synaptosomal membrane. The Fm338/Fee338 ratio as well as polarization of DMC fluorescence indicative of the bulk lipid phase were not altered under these conditions. Neither cytochalasin B not colchicine had any effect on fluorescence polarization of DMC both in control and depolarized synaptosomes. It is suggested that the increased lateral mobility of protein-associated lipid molecules found in depolarized synaptosomes may be caused by alterations in the activity of ion channels and ion pumps or by restructuring of the cytoskeletal network.     

An X-ray diffraction study on phase transition temperatures of various membranes isolated from Tetrahymena pyriformis cells grown at different temperatures

Mitochondrial, microsomal and pellicular membranes were isolated from Tetrahymena cells grown at 39°C or 15°C, and phospholipids, in turn, were separated from total lipids extracted from these membranes. The effect of growth temperature on their solid-to-fluid phase transition temperature was examined by wide-angle X-ray diffraction. The transition temperatures of phospholipids from mitochondria, microsomes and pellicles were 21, 19 and 26°C for cells grown at 39°C and ?8, ?3 and 6°C for cells grown at 15°C, respectively. All phospholipids were found in a completely fluid state at these growth temperatures. From a comparison between the phospholipids and total lipids from pellicles of cells grown at 39°C, a triterpenoid alcohol, tetrahymanol, caused the transition temperature to increase. The alignment of tetrahymanol in membranes was examined with pellicle's total lipid oriented in a sample holder.