Horacio A. Garda

Effect of Temperature and Salinity Stress on Growth and Lipid Composition of Shewanella gelidimarina

ABSTRACT The maximum growth temperature, the optimal growth temperature, and the estimated normal physiological range for growth of Shewanella gelidimarina are functions of water activity (aw), which can be manipulated by changing the concentration of sodium chloride. The growth temperatures at the boundaries of the normal physiological range for growth were characterized by increased variability in fatty acid composition. Under hyper- and hypoosmotic stress conditions at an aw of 0.993 (1.0% [wt/vol] NaCl) and at an aw of 0.977 (4.0% [wt/vol] NaCl) the proportion of certain fatty acids (monounsaturated and branched-chain fatty acids) was highly regulated and was inversely related to the growth rate over the entire temperature range. The physical states of lipids extracted from samples grown at stressful aw values at the boundaries of the normal physiological range exhibited no abrupt gel-liquid phase transitions when the lipids were analyzed as liposomes. Lipid packing and adaptational fatty acid composition responses are clearly influenced by differences in the temperature-salinity regime, which are reflected in overall cell function characteristics, such as the growth rate and the normal physiological range for growth.

Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes

n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.

Conformation of apolipoprotein AI in reconstituted lipoprotein particles and particle–membrane interaction: Effect of cholesterol

Discoidal recombinant high density lipoproteins (rHDL) of apolipoprotein AI (apoAI) and palmitoyloleoylphosphatidylcholine (POPC), with or without cholesterol, were prepared by cholate dialysis. By gel filtration, rHDL containing 2-4 (Lp2, Lp3 and Lp4) apoAI molecules/particle were obtained. The ApoAI conformation in these rHDL was investigated by tryptophan fluorescence, denaturation with guanidine HCl, and immunoreactivity with two monoclonal antibodies recognizing epitopes in the N-terminal and central domains. Data show that apoAI conformation is highly dependent on particle size as well as on cholesterol. The ability of rHDL to interact with lipid bilayer was studied by measuring leakage induction on POPC and POPC/cholesterol vesicles loaded with terbium/dipicolinic acid. Among the cholesterol-free rHDL, the most efficient ones were the smallest Lp2. Leakage induction on POPC vesicles is dramatically decreased by the presence of cholesterol in Lp2 and Lp3. All the rHDL, but specially those containing cholesterol, induced more leakage on the POPC/cholesterol than on the POPC vesicles. These results suggest that in small cholesterol-poor particles, apoAI could have a conformation determining a high affinity for membranes, which could facilitate cholesterol efflux. After cholesterol enrichment, a conformational change in apoAI could decrease the affinity for membranes allowing the lipoprotein release.

Effect of dexamethasone on the fatty acid composition of total liver microsomal lipids and phosphatidylcholine molecular species.

Dexamethasone depresses Δ6 and Δ5 and increases Δ9 desaturase and synthase activities. Therefore, we investigated the effect on the fatty acid composition of microsomal liver lipids and phosphatidylcholine (PtdCho) molecular species. After 15 d of treatment we found a notable decrease in arachidonic acid, a small decrease in stearic acid, and increases of linoleic, oleic, palmitoleic, and palmitic acids in liver microsomal total lipids and PtdCho. The study of the distribution of the PtdCho molecular species indicated that 18∶0/20∶4n−6, 16∶0/20∶4n−6, and 16∶0/18∶2n−6 predominated in the control animals. Dexamethasone, as expected because of its depressing effect on arachidonic acid synthesis and activation of oleic and palmitic acid synthesis, evoked a very significant decrease in 18∶0/20∶4n−6 PtdCho (P<0.001) and an important increase in 16∶0/18∶2n−6. The invariability of 16∶0/20∶4n−6 PtdCho could be related to the antagonistic effect of arachidonic and palmitic acid synthesis. PtdCho species containing oleic acid were not significant. The bulk fluidity and dynamic properties of the microsomal lipid bilayer measured by fluorometry using the probes 1,6-diphenyl-1,3,5-hexatriene and 4-trimethylammonium-phenyl-6-phenyl-1,3,5-hexatriene showed no significant modification, probably owing to a compensatory effect of the different molecular species, but changes of particular domains not detected by this technique are possible. However, the extremely sensitive Laurdan detected increased lipid packing in the less-fluid domains of the polar-nonpolar interphase of the bilayer, possibly evoked by the change of molecular species and cholesterol/phospholipid ratio. The most important effect found is the decrease of arachidonic acid pools in liver phospholipids as one of the corresponding causes of dexamethasone-dependent pharmacological effects.

The central type Y amphipathic α-helices of apolipoprotein AI are involved in the mobilization of intracellular cholesterol depots

We studied the role of a central domain of human apolipoprotein AI (apoAI) in cholesterol mobilization and removal from cells. In order to check different protein conformations, we tested different sized and cholesterol-content reconstituted apoAI particles (rHDL). Meanwhile cholesterol-free discs were active to induce mobilization, only small cholesterol-containing rHDL were active. To test the influence of a central domain in such events, we used two apoAI variants: one, with its central Y helix pair replaced by the C-terminal domain, and the other having a lysine deleted in central region. The helix-swapping variant decrease the cholesterol pool available to acyl-CoA cholesterol acyl transferase and increase mobilization of newly synthesized cholesterol. Instead, the deletion mutant had no effect on both events. We conclude that the central domain of apoAI is involved in cholesterol cell traffic and solubilization, and that a Y-type charge distribution in polar face may be required, as well as a correct helices-polar face orientation.

Membrane organization and regulation of cellular Cholesterol homeostasis

An excess of intracellular free cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor because it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. We hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line overexpressing stearoyl coenzyme A (CoA) desaturase (SCD cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty acids, and determined this effect on membrane properties, cell viability, and Chol homeostasis. PM in SCD cells has a higher ratio of phospholipids to sphingomyelin and is slightly enriched in Chol. These cells showed an increase in the ratio of cholesteryl esters to free Chol; they were more resistant to Chol toxicity, and they exported more caveolin than control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage.

The effect of osmotic stress on the biophysical behavior of the Bacillus subtilis membrane studied by dynamic and steady-state fluorescence anisotropy.

The thermotropic behavior of intact bacterial membranes and vesicles prepared from total and polar lipids isolated from Bacillus subtilis cultures grown at 37 degrees C in normal (LB) and hyperosmotic (LBN) conditions was studied using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), and 2-diethylamino-6-lauroyl-naphthalene (Laurdan) as fluorescent probes. No phase transition of bulk lipids was observed in these preparations at the range of temperature studied. The anisotropy values (r(s)) for DPH and TMA-DPH in purified membranes showed significant differences between the LB and LBN conditions, suggesting that there was an increase in membrane packing during the adaptation to osmotic stress. Furthermore, generalized polarization (GP) parameters for Laurdan indicated small but significant changes in water relaxation at the membrane hydrophobic/hydrophilic interface. Membrane preparations showed r(s) higher values than those of lipid vesicles and a higher temperature dependence of the Laurdan GP parameter. This fact indicates that membrane proteins increase the lipid packing and keep the membrane more sensitive to temperature changes.

Effect of fenitrothion on dipalmitoyl and 1-palmitoyl-2-oleoylphosphatidylcholine bilayers.

The effects of the organophosphorous insecticide fenitrothion (phosphorothioic acid, O,O-dimethyl O-(3-methyl-4-nitrophenyl) ester; FS) on the physical state of pure dipalmitoyl (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes were investigated. FS lowers the phase transition temperature of DPPC. It has no large effects on the DPPC gel phase, but it increases the order of the liquid-crystalline state of DPPC and POPC. FS also decreases 1,6-diphenyl-1,3,5-hexatriene (DPH) lifetime (tau) in the DPPC and POPC liquid-crystalline states. Since a direct quenching of DPH emission by FS was ruled out, tau shortening is assigned to an increased water penetration in the bilayer. The effect of FS is different from most perturbing agents for which an increased order is accompanied by a higher tau. Furthermore, quenching of DPH by KI was increased by FS in POPC liposomes indicating an increased accessibility of the quencher to the hydrophobic core where DPH distributes. The effect of FS on dipole relaxation at the hydrophilic-hydrophobic interface of POPC bilayers was studied with 2-dimethylamino-6-lauroylnaphthalene (Laurdan). FS produces a decrease in Laurdan tau and a narrowing of its emission band. FS significantly increases the generalized polarization values at both emission band ends. These results indicate that FS may allow the coexistence of microdomains that have different physical properties.

Effect of polyunsaturated fatty acid deficiency on dipole relaxation in the membrane interface of rat liver microsomes

The influence of a fat-free diet on the lipid dynamics of rat liver microsomes and liposomes of microsomal lipids was studied by using different fluorescence methods. Lifetime distribution and rotational diffusion of probes with different localization in the lipid bilayer were measured using multifrequency fluorometry. Lateral mobility was studied by measuring excimer formation of pyrenedodecanoic acid. Dipolar relaxation in the interfacial region was studied using 2-dimethyl-amino-6-lauroylnaphthalene (Laurdan). In spite of large changes in the fatty acid composition of microsomal lipids, polyunsaturated fatty acid deficiency showed no effect on the lifetime distribution and rotational mobility of 1,6-diphenyl-1,3,5-hexatriene (DPH). l-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 2- 7- and 12-(9-anthroiloxy)stearic acids. The treatment did not change the lateral diffusion of pyrenedodecanoic acid, either. However, generalized polarization of Laurdan fluorescence was higher in polyunsaturated fatty acid deficient microsomes as compared to the polyunsaturated fatty acid sufficient ones. This effect was also observed in liposomes of the total microsomal lipids, indicating that the changes in fatty acid composition resulting from polyunsaturated fatty acid deficiency produced a small but significant decrease in the rate of dipolar relaxation in the region of the lipid polar groups of the bilayer. The absence of lipid gel phase domains in rat liver microsomes was also indicated by Laurdan fluorescence features.

Dietary cholesterol induces changes in molecular species of hepatic microsomal phosphatidylcholine.

After 21 days on a diet containing 1g% cholesterol and 0.5g% cholic acid, rats had an increased content of cholesterol in liver microsomal lipids. In liver, both cholesterol content and δ9 desaturase activity increased, whereas δ6 and δ5 desaturase activities decreased. These changes correlated with increases in oleic, palmitoleic, and linoleic acids and decreases in arachidonic and docosahexenoic acids in total microsomal lipids. Similar fatty acid changes were found in phosphatidylcholine (PC), the principal lipid of the microsomal membrane. In PC the predominant molecular fatty acid species (67% of the total) in the control rats were 18:0/20:4, 16:0/20:4, and 16:0/18:2; and they mainly determined the contribution of PC to the biophysical and biochemical properties of the phospholipid bilayer. The cholesterol diet decreased specifically the 18:0/20:4 species, and to a lesser extent, 16:0/20:4 and 18:0/22:6. The 18:1-containing species, especially 18:1/18:2 and less so 16:0/18:1 and 18:1/20:4, were increased. A new 18:1/18:1 species appeared. The independent effects of the presence of cholesterol and change of the fatty acid composition of the phospholipid bilayer of liver microsomes on the packing were studied by fluorescence methods using 6-lauroyl-2,4-dimethylaminonaphthalene, 1,6-diphenyl-1,3,5-hexatriene and 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene, which test different parameters and depths of the bilayer. Data showed that the increase of cholesterol in the membrane, and not the change of the fatty acid composition of phospholipids, was the main determinant of the increased bulk packing of the bilayer. The increase of fluid oleic- and linoleic-containing species almost compensated for the drop in 20:4- and 22:6-containing molecules. But the most important effect was that the general drop in essential n-6 and n-3 polyunsaturated fatty acids meant that this endogenous source for the needs of the animal decreased.