Differential temperature acclimatization responses in the membrane phospholipids of Posthodiplostomum minimum and its second intermediate host, Lepomis macrochirus

The effects of temperature change on phospholipid content in metacercariae of Posthodiplostomum minimum and their second intermediate hosts, Lepomis macrochirus, were examined to gauge similarities in the homeoviscous adaptation of host and parasite membranes to environmental thermal change. Heart, liver, and muscle tissues from individual L. macrochirus responded to environmental temperature declines with a decrease in the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Increases in membrane PE concentration increase membrane fluidity, maintaining fish membrane function as environmental temperature declines. However, the metacercariae of P. minimum exhibit changes in cholesterol levels, total lipid levels, and lipid composition (PE/PC) that contrast the normal changes for homeoviscous membrane adaptation exhibited by their fish intermediate hosts. The parasites seem to rely on their hosts for homeoviscous adaptation within normal developmental temperature ranges, pooling both cholesterol and PE as energetic stores for development and ontological transitions signaled by elevated temperatures.     

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

Effect of acidic conditions on fatty acid composition and membrane fluidity of<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from Crescenza cheese

The effect of low pH on minimum pH, growth kinetics and adaptive response to acidity conditions were studied on nineEscherichia coli strains having food origin and two collection strains. Moreover, the effects of low pH on the modification of fatty acid (FA) composition and membrane fluidity of the strains were studied. Also the effects of a pre-adaptation to pH 5 on acidic resistance and FA composition were considered. The results showed the crucial role of unsaturated FA in acid adaptation ofE. coli. Cyclization and isomerization of membrane FA represented the most interesting response to acidic growth conditions in theE. coli strains considered. In particularcis/trans isomerization was shown to play an important role in modulating membrane composition and fluidity under acidic conditions. Only one strain was able to adapt to acidic stress increasing its ability to grow at low pH. It responded to pH decrease, conversely to each other strain, by increasing its membrane fluidity at the lowest pH values considered. The results obtained showed that the determination of membrane fluidity in intact whole cells by fluorescence polarisation can represent a useful tool to study instantaneous stress-induced membrane fluidity modifications.     

Regulation of membrane lipid bilayer structure during salinity adaptation: a study with the gill epithelial cell membranes of Oreochromis niloticus

A significant variation in the membrane fluidity (as assessed by DPH-fluorescence polarisation) and membrane lipid bilayer composition is noticed in the subcellular membranes of the gill epithelial cells of Oreochromis niloticus due to exposure of the fish to 1% saline water for 1 month. Also, a 70% enhanced activity of Na(+)-K(+)-ATPase in plasma membranes and a 2.5-fold increase of glucose-6-phosphate dehydrogenase in microsomal membranes are recorded in the treated fish. The changed membrane structure and fluidity along with the changed enzymatic activity of Na(+)-K(+)-ATPase help the influx the Na(+) rather than the efflux of K(+) through the gill epithelial cells during salinity adaptation.     

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.     

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.     

Changes of the fluidity of mitochondrial membranes induced by the permeability transition.

The dynamic properties of protein and lipid regions of mitochondrial membranes during the permeability transition (PT) process were studied by following the anisotropy changes of hematoporphyrin (HP) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. We show that opening of the PT pore is accompanied by a remarkable increase of mitochondrial membrane fluidity which is specifically localized to protein sites, while lipid domains are unaffected. The increased membrane fluidity is not related to the collapse of transmembrane potential that follows the PT, as demonstrated by a comparison between the anisotropy properties of permeabilized mitochondria and impermeable, depolarized organelles. Parameters such as osmotic swelling and temperature, which are shown to affect the mitochondrial membrane dynamics in the absence of permeability transition, cannot alone account for the pore dynamical properties. We suggest that the observed increase in fluidity is mainly due to a conformational change of pore-forming protein(s) during the "assembly" of the PT pore.     

Adaptation of biological membranes to temperature. The lack of homeoviscous adaptation in the sarcoplasmic reticulum

Temperature adaptation of biological membranes was examined by comparing the fragmented sarcoplasmic reticulum preparation of goldfish acclimated to different temperatures. Membrane fluidity was estimated using the fluorescence polarization technique. There was considerable variation between preparations, but no consistent differences in fluidity were observed between 5- and 25°C-acclimated goldfish, fish species adapted over an evolutionary period to arctic or desert temperatures, and rat. The fatty acid composition of the sarcoplasmic reticulum preparations of differently acclimated goldfish showed differences in the proportion of mono- and polyunsaturated fatty acids while the proportion of saturated fatty acids remained relatively constant. However, the fatty acid composition of sarcoplasmic reticulum phosphoglycerides became more unsaturated in the order rat, desert pupfish, arctic sculpin, which correlates with their respective environmental or body temperature. It is concluded that differences in membrane components other than fatty acids are important in determining membrane dynamic structure. The inability to demonstrate homeoviscous adaptation in sarcoplasmic reticulum is supported by other evidence suggesting that functions of the sarcoplasmic reticulum that are measured in vitro are not affected by such modifications of their phosphoglyceride fatty acid composition as occur during thermal acclimation.     

Fluorescence polarization studies and biochemical properties of membranes exfoliated from the cell surface of rabbit thymocytes in situ

In the course of our work on membrane phenomena related to the differentiation of lymphocytes in the rabbit thymus, we isolated membranous material from the extracellular compartment of this organ. With respect to their ultra-structural appearance, enzyme activity, lipid composition (cholesterol/phospholipid molar ratio, fatty acid composition of total phospholipids, phospholipid composition) and lipid fluidity, these membranes were shown to exhibit characteristics similar to those of purified plasma membranes isolated from disrupted thymocytes. Moreover, their antigenic specificity as determined in a cytotoxicity adsorption test was identical. From our experiments, we hypothesize that the extracellular membrane fragments found in the rabbit thymus are derived mainly from material shed by immature thymocytes.     

The influence of microtubule integrity on plasma membrane fluidity in L929 cells

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after approximately 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.     

The influence of microtubule integrity on plasma membrane fluidity in L929 cells

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after ~ 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.     

Lipid fluidity and composition of intestinal microvillus membranes isolated from rats of different ages

The lipid composition and fluidity of microvillus (luminal) membranes isolated from the small intestines of Fisher 344 rats aged 6, 17, and 117 weeks were compared. Lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, was significantly greater in rats aged 6 weeks as compared to 17 or 117 weeks. A lipid thermotropic transition was observed at 17.5 +/- 1.3 degrees C in the membranes of the youngest group, approx. 5-6 degrees C lower than that of the older animals. The differences in lipid composition which account for the higher fluidity of the youngest preparations include a decreased cholesterol/phospholipid molar ratio in both the proximal and distal halves of the small intestine and, in the proximal half alone, increases in the lipid/protein ratio and double bond index. The foregoing reduction in cholesterol/phospholipid ratio derives mainly from a higher content of total phospholipid, and the increment in double bond index results from an increase in arachidonic acid residues. The results demonstrate an age-dependent decrease in fluidity of intestinal microvillus membranes in the early post-weaning period in the rat. This pattern was unlike that of the microvillus membrane p-nitrophenylphosphatase, whose specific activity declined progressively in the older age groups.     

Abnormal fluidity state in membranes of malignant hyperthermia pig skeletal muscle

The fluidity state was analyzed on sarcoplasmic reticulum membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the 5-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hypothesis for this syndrome.     

Effect of fatty acid deficiency on microsomal membrane fluidity and cooperativity of the UDP-glucuronyltransferase

The effect of fat deprivation on microsomal membrane fluidity of guinea-pig livers and the kinetic cooperativity of UDP-glucuronyl transferase towards its natural substrate, the UDP-glucuronic acid, were studied. Fat deprivation in the diet of weanling guinea-pigs evoked a typical essential fatty-acid-deficient pattern in the composition of the microsomal membrane. The unsaturated:saturated fatty acid ratio progressively declined in the membrane during the 21-day period tested. This decline determined a gradual increase in the fluorescence anisotropy (rs) of the membrane labeled with diphenylhexatriene and the apparent microviscosity of the lipid bilayer calculated from these values increased from 1.1 to 1.8 poise. In addition, when the infinitely slow decaying fluorescence anisotropy ((r infinity), which is proportional to the square of the lipid order parameter, was calculated from rs data, a significant increase in these parameters was also obtained. Furthermore, this decrease of the double bond index:saturated acid ratio of the membrane was associated with a parallel increase in Hill coefficients of the UDP-glucuronyl transferase that gradually lost the negative homotropic effect and cooperativity of UDP-glucuronic acid. The Hill coefficient varied from 0.39 to 0.98 during the 21-day period studied. Our observations indicate on one side that changes in the fat composition of the diet are accompanied by modifications in the lipid composition and fluidity of the microsomal membrane, and the apparent cooperativity of the enzyme. On the other side, they suggest that the evaluation of Hill coefficients of UDP-glucuronyl transferase might be used as a sensitive test to investigate conformational changes in the microsomal membrane of the liver.     

Fluidity of rat liver Golgi membranes in streptozotocin diabetes. A spin label study

The mobility of 5-doxylstearic acid spin label (5-SASL) in the intact rat liver Golgi membranes of streptozotocin diabetes was studied as a function of free blood sugar level and temperature. During development of diabetes, indicated by the increase of the free blood sugar level, the membrane fluidity measured in the physiological temperature range (1) does not change in comparison with control in light diabetes, (2) decreases significantly in advanced diabetes and (3) again increases to the control level in heavy diabetes (the free blood sugar levels being 200-250 mg/100 ml, 250-350 mg/100 ml and greater than 350 mg/100 ml, respectively). The development of streptozotocin diabetes is accompanied by significant changes in lipid composition of liver Golgi membranes as also shown in our previous observations. The measurements of motion of 5-SASL in Golgi membranes as well as in vesicles, made from commercially available lipids of composition close to the liver Golgi membranes, show that a decrease of cholesterol contents is the main factor which induces the increase membrane fluidity. We suggest that in the heavy diabetes the hemostatic regulation in the lipid composition leads to minimization of alterations in membrane fluidity to obtain comparatively normal activity of certain membrane enzymes.     

Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins

The effect of different dietary fats on peritoneal macrophage plasma membrane fluidity, intracellular cyclic AMP (cAMP) production, GTP hydrolysis and TNF binding and TNF-induced IL1 and IL6 production was investigated. After a four week period, fluidity, as determined by both fluorescence recovery after photobleaching (FRAP) and anisotropy was lowest and highest in animals fed corn and fish oil respectively. After eight weeks feeding, lateral membrane movements were decreased substantially in fish, olive and coconut oil fed dietary groups, whereas an increase in the corn oil fed group was observed, no effect was observed in macrophages from the butter fed group. However, an increase in the packing was observed in macrophages from all dietary groups except in the olive oil fed group. GTPase values for the coconut oil and butter groups were higher than in any other dietary group. After receiving the diet for 8 weeks these differences between the groups were no longer apparent. Exposure of macrophages to TNF had no effect on the rate of GTP hydrolysis. A major enhancement of cAMP production became apparent between weeks 4 and 8 of dietary treatment. After 4 weeks on the diet, values were significantly higher from cells of animals fed corn and olive oils than from animals fed fish oil. After 8 weeks, while there was a general enhancement of production, further differences became apparent. Feeding corn and coconut oils resulted in the highest values and olive oil and chow in the lowest. It is proposed that fats rich in n-3 fatty acids (fish oils) alter membrane fluidity, decrease TNF binding affinity, GTPase activity and cAMP production which appears not to modify cytokine production after short term dietary supplementation. However, after long term feeding it appears that increases in the sensitivity of the TNF receptors plays a major role in modifying cytokine production. (Mol Cell Biochem 166: 135-143, 1997)     

Changes of erythrocyte membrane fluidity associated with childhood obesity: a molecular study using fluorescence spectroscopy

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene has been used to investigate the physical state of erythrocyte membranes from 36 obese children in absence of endocrine and metabolic disorders involving alterations of blood lipid pattern. A decrease of membrane fluidity, as shown by an increase of fluorescence polarization, was evident in obese subjects in comparison with the controls. The study of membrane composition has revealed an increase of cholesterol content and of cholesterol to phospholipid molar ratio, both being positively correlated with the observed changes of membrane fluidity. Moreover a correlation of membrane fluidity is also observed with body-mass index and plasma cholesterol levels. The results are discussed in the light of the possible use of erythrocyte membrane as a model to detect early alterations in the exchanges between erythrocyte membrane and lipoproteins in obesity.     

Glycolipid transfer protein interaction with bilayer vesicles: modulation by changing lipid composition

Glycosphingolipids (GSLs) are important constituents of lipid rafts and caveolae, are essential for the normal development of cells, and are adhesion sites for various infectious agents. One strategy for modulating GSL composition in lipid rafts is to selectively transfer GSL to or from these putative membrane microdomains. Glycolipid transfer protein (GLTP) catalyzes selective intermembrane transfer of GSLs. To enable effective use of GLTP as a tool to modify the glycolipid content of membranes, it is imperative to understand how the membrane regulates GLTP action. In this study, GLTP partitioning to membranes was analyzed by monitoring the fluorescence resonance energy transfer from tryptophans and tyrosines of GLTP to N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine present in bilayer vesicles. GLTP partitioned to POPC vesicles even when no GSL was present. GLTP interaction with model membranes was nonpenetrating, as assessed by protein-induced changes in lipid monolayer surface pressure, and nonperturbing in that neither membrane fluidity nor order were affected, as monitored by anisotropy of 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, even though the tryptophan anisotropy of GLTP increased in the presence of vesicles. Ionic strength, vesicle packing, and vesicle lipid composition affected GLTP partitioning to the membrane and led to the following conclusion: Conditions that increase the ratio of bound/unbound GLTP do not guarantee increased transfer activity, but conditions that decrease the ratio of bound/unbound GLTP always diminish transfer. A model of GLTP interaction with the membrane, based on the partitioning equilibrium data and consistent with the kinetics of GSL transfer, is presented and solved mathematically.