Distribution of 5-doxylstearic acid in the membranes of mammalian cells

Concentration-dependent spin broadening of ESR spectra of the nitroxide 5-doxylstearic acid has been used to evaluate the distribution of 5-doxylstearic acid in the membranes of intact mouse thymus-bone marrow (TB) and Chinese hamster ovary (CHO) cells. TB cells, CHO cells, erythrocytes, and isolated plasma membranes from CHO cells were labelled with 5-doxylstearic acid and the peak to peak linewidths of the central line of the resulting ESR spectra were measured. The measured line widths were linearly dependent on the amount of 5-doxylstearic acid incorporated into the sample over the range of 0-0.18 mol nitroxide per mol lipid. In erythrocytes, the relationship between linewidths approximated a linear function at lower concentrations of 5-doxylstearic acid, up to 0.07 mol nitroxide per mol lipid. The amount of broadening of the central line for a given amount of 5-doxylstearic acid was far less for intact cells than for either erythrocytes or plasma membrane, indicating that the 5-doxylstearic acid samples a much larger lipid pool in the intact cells. With the broad assumption that the mobility of the 5-doxylstearic acid is similar in different membranes, the size of the lipid pool sampled by 5-doxylstearic acid is approximately equal to the total cellular lipid in intact cells. If a given concentration of 5-doxylstearic acid sampled only the plasma membrane of TB or CHO cells, we would expect to see a linewidth corresponding to a 12-20-fold greater local concentration of 5-doxylstearic acid than was observed, since the plasma membranes of CHO and TB cells represent only 5-8 percent of the total cellular lipid. Therefore, the 5-doxylstearic acid must distribute into most or all cellular membranes of intact cells and is not localized in the plasma membrane alone.     

Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis–trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis–trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.     

Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus

Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

Induced Heat Sensitivity of Wheat Roots and Protecting Effect of Ethanol and Kinetin

Wheat seedlings were subjected to heat shock for 2 min at 45°C. The seedlings were then incubated at 25°C or higher temperatures (usually 35°C). At 25°C the root tips survived the heat shock, but not at temperatures above 34°C, unless they had been pretreated with ethanol or kinetin, After 1 h in ethanol and after more than 15 h in kinetin the root meristem survived a high incubation temperature after the heat shock. Immediately after heat treatment the glyceride content in treated root tips was higher than in untreated roots. The same was observed after heat treatment of root tips pretreated in ethanol and kinetin. The content of ether extractable lipids was not changed by the heat shock.     

Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C₁₂E₈) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C₁₂E₈ DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C₁₂E₈ DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C₁₂E₈ treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C₁₂E₈ to the study of DRMs.     

HSP16.6 Is Involved in the Development of Thermotolerance and Thylakoid Stability in the Unicellular Cyanobacterium, Synechocystis sp. PCC 6803

The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47°C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43°C for 1 h and subsequent challenge exposure at 49°C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress. Received: 14 October 1999 / Accepted: 16 November 1999     

Changes in cell morphology and actin organization during heat shock in Dictyostelium discoideum: does HSP70 play a role in acquired thermotolerance?

In response to heat shock (34°C, 30 min), cell morphology and actin organization in Dictyostelium discoideum are drastically changed. Loss of pseudopodia and disappearance of F-actin-containing structures were observed by using fluorescence microscopy. These changes were paralleled by a rapid decrease of the F-actin content measured by a TRITC-phalloidin binding assay. The effects of heat shock on cell morphology and actin organization are transient: After heat shock (34°C) or during a long-term heat treatment (30°C), cell morphology, F-actin patterns and F-actin content recovered/adapted to a state which is characteristic for untreated cells. Because F-actin may be stabilized by increased amounts of heat shock proteins, their response and interaction with F-actin was analyzed. After a 1 h heat treatment (34°C), the major heat shock protein of D. discoideum (HSP70) showed maximally increased synthesis rates and levels. During recovery from a 34°C shock or during a continuous heat treatment at 30°C, the HSP70 content first increased and then declined slowly toward normal levels. Pre-treatment of cells with a short heat shock of 30 min at 34°C stabilized the F-actin content when the cells were exposed to a second heat shock. Furthermore, a transient colocalization of HSP70 and actin was observed at the beginning of heat treatment (30°C) using immunological detection of HSP70 in the cytoskeletal actin fraction.     

Effect of heat shock on protein synthesis in Locusta migratoria epidermis

Heat shock induced by an increase in temperature from 30°C to 47°C led to changes in protein synthesis in wing pads of the fifth larval instar of Locusta migratoria. Synthesis of heat shock proteins in the molecular weight range of 85,000, 70,000 and 18,000–22,000 was first detected at a threshold temperature of 45°C and was found to be highest at 47°C. A marked decline in the synthesis of many other proteins was also evident at 47°C. Recovery of general protein synthesis was observed when wing pads were shifted back to 30°C after a 2-h heat shock at 47°C. Heat shock protein patterns in Locusta and Drosophila were compared.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Structural reorganizations of the lipids and proteins of erythrocyte membranes under the action of low temperatures

The reversible structural rearrangement of lipids and protein oligomerization has been shown to occur during cooling in membranes of model systems (liposome, erythrocyte shadows) and native erythrocytes. Analysing the dependence of Azz in membrane probes (5- or 15-doxylstearic acids) in the Arrhenius plots a conclusion on the structural changes at 13-19 degrees C and within the range of interior water freezing from -17 up to -19 degrees C has been drawn, the last transition is smoothed out in the presence of glycerin. Using diamide joining spectrin and electrophoresis in polyacrylamide gel it has been determined that the low temperatures cause the spatial approach of proteins of spectrin-actinic complex and formation connections between the erythrocyte membrane proteins which aren't destroyed by dodecylsulfate.     

Mechanism of Saccharomyces cerevisiae yeast cell death induced by heat shock. Effect of cycloheximide on thermotolerance

The mechanism of yeast cell death induced by heat shock was found to be dependent on the intensity of heat exposure. Moderate (45°C) heat shock strongly increased the generation of reactive oxygen species (ROS) and cell death. Pretreatment with cycloheximide (at 30°C) suppressed cell death, but produced no effect on ROS production. The protective effect was absent if cycloheximide was added immediately before heat exposure and the cells were incubated with the drug during the heat treatment and recovery period. The rate of ROS production and protective effect of cycloheximide on viability were significantly decreased in the case of severe (50°C) heat shock. Treatment with cycloheximide at 39°C inhibited the induction of Hsp104 synthesis and suppressed the development of induced thermotolerance to severe shock (50°C), but it had no effect on induced thermotolerance to moderate (45°C) heat shock. At the same time, Hsp104 effectively protected cells from death independently of the intensity of heat exposure. These data indicate that moderate heat shock induced programmed cell death in the yeast cells, and cycloheximide suppressed this process by inhibiting general synthesis of proteins.     

Heat stress affects the organization of microtubules and cell division in Nicotiana tabacum cells

To investigate the effects of heat stress on the plant cytoskeleton, the structure of microtubule arrays in N. tabacum suspension cells incubated at 38 or 42°C was analysed. Whilst incubation at 42 °C resulted in the disruption of the majority of cellular microtubules after 30 min, in cells exposed to 38 °C all the microtubule arrays were preserved even after 12 h of incubation, although their organization was altered. The most susceptible were the microtubules of the mitotic spindle and the phragmoplast. Several abnormalities were observed: (i) splitting of the spindle into several parts; (ii) elongation of the spindles; (iii) formation of microtubule asters in mitotic cells, and (iv) elongation of phragmoplast microtubules. Exposure of cells to 38 °C caused a decrease in the mitotic index but an accumulation of telophase cells. The recovery of normal microtubule organization occurred after 12 h. Treatment of the cells subjected to heat stress conditions with an inhibitor of protein synthesis, cycloheximide, did not prevent either the alterations of microtubule organization or accumulation of cells containing phragmoplasts. Therefore, heat shock proteins do not seem to be directly responsible for the microtubule disorganization induced by heat stress.     

Low-pH association of proteins with the membranes of intact red blood cells. I. Exogenous glycophorin and the CD4 molecule

Glycophorin and CD4 proteins are tightly associated with intact human erythrocyte membranes after a short-time incubation at low pH (1-2 min, pH lower than 5, 37 degrees C). Flow cytometry and epifluorescence microscope observations showed that after incubation of red cells with fluorescein isothiocyanate (FITC) labeled glycophorin at pH values lower than 5, the erythrocyte membrane and subsequently formed ghost membranes were fluorescent. Unlabeled glycophorin was reacted with mouse erythrocytes using the same low-pH conditions. Flow cytometry and fluorescence microscopy showed that anti-glycophorin monoclonal antibodies were able to recognize the epitopes of glycophorin associated with the mouse erythrocytes. Kinetic experiments showed that the interaction of FITC-glycophorin with red cell membranes can be monitored by a decrease in the fluorescence intensity. Erythrocyte associated glycophorin was not removed from the membranes after 24 h incubation in human plasma (in vitro, 39 degrees C). A glycoprotein extract containing CD4 was isolated from a T4-lymphoma cell line (CEM). This protein extract was incubated with erythrocytes using the same low-pH conditions. Fluorescently labeled monoclonal antibodies against CD4 stained the red cells after association of CD4 with the membranes. Electron microscopy showed 10 nm immunoglobulin G-coated gold beads associated with CD4-bearing erythrocyte membranes after incubation with anti-CD4 antibodies and then with the gold beads. The potential use of the CD4-erythrocyte complex as a therapeutical agent against acquired immune deficiency syndrome (AIDS) is suggested.     

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells.     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Chilling-induced changes in membrane fluidity and antioxidant enzyme activities in Coffea arabica L. roots

Exposure of coffee to low temperatures caused growth inhibition, changes in metabolic rates, and membrane alterations. Root tissue exposed to 10 °C evolved significantly lower rates of metabolic heat compared with controls grown at 25 °C, and the values were closely associated with the observed root growth inhibition. Electron paramagnetic resonance spectra of intact tissues showed that the spin probe 5-doxylstearic acid was capable to intercalate within the cellular membrane lipids. Indeed, at the depth of the 5th carbon atoms of the alkyl chains, the nitroxide radical detected more rigid membranes in seedlings exposed to 10 °C compared with 25 °C treated samples. Ascorbate peroxidase and catalase activities did not show appreciable changes under chilling conditions, while guaiacol peroxidase activity increased 55 % compared to the control. On the other hand, glutathione reductase activity decreased, in parallel to a significant decline in the capacity to reduce triphenyl-tetrazolium. Our results showed a marked correlation between lipid peroxidation and root tissue damage, which seemed to be associated with increased membrane rigidity. This revised version was published online in July 2006 with corrections to the Cover Date.