Structural change in lipid bilayers and water penetration induced by shock waves: molecular dynamics simulations

The structural change of a phospholipid bilayer in water under the action of a shock wave is numerically studied with unsteady nonequilibrium molecular dynamics simulations. The action of shock waves is modeled by the momentum change of water molecules, and thereby we demonstrate that the resulting collapse and rebound of the bilayer are followed by the penetration of water molecules into the hydrophobic region of the bilayer. The high-speed phenomenon that occurs during the collapse and rebound of the bilayer is analyzed in detail, particularly focusing on the change of bilayer thickness, the acyl chain bend angles, the lateral fluidity of lipid molecules, and the penetration rate of water molecules. The result shows that the high-speed phenomenon can be divided into two stages: in the first stage the thickness of bilayer and the order parameter are rapidly reduced, and then in the second stage they are recovered relatively slowly. It is in the second stage that water molecules are steadily introduced into the hydrophobic region. The penetration of water molecules is enhanced by the shock wave impulse and this qualitatively agrees with a recent experimental result.     

Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure

By use of neutron diffraction, the structural parameters of oriented multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine with deuteriocarbon chains/cholesterol (molar ratio 70:30), multilamellar lipid vesicles composed of pure lipids and lipid/cholesterol mixtures, and crystalline purple membrane patches from Halobacterium halobium have been measured at pressures up to 2 kbar. Pressurization of the oriented 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/cholesterol multilayers results in an in-plane compression with the mean deuteriocarbon chain spacing of 4.44 A obtained under ambient conditions decreasing by 3-7% at 1.9 kbar. The thickness for this bilayer increases by approximately equal to 1.5 A, but the net bilayer volume decreases and the isothermal compressibility is estimated to be in the range (-0.1 to -0.6) X 10(-4)/bar at 19.0 degrees C. The d spacings for multilamellar vesicles composed of lipids in the liquid crystalline state and lipid/cholesterol mixtures increase linearly as a function of pressure, suggesting that these bilayers are also compressed in the membrane plane. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine MLVs in the gel state, the d spacing decreases with pressure. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, the hexagonally packed chains are anisotropically compressed in the bilayer plane, resulting in a pseudohexagonal chain packing at 1.9 kbar. The bilayer compressibility is (-0.4 or -0.5) X 10(-4)/bar depending on whether the chain tilt increases with pressure or terminal methyl groups of apposing lipid monolayers approach each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hydrostatic pressure on bilayer phase behavior and dynamics of dilauroylphosphatidylcholine.

Deuterium nuclear magnetic resonance spectroscopy was used to study the thermotropic phase behavior of dilauroylphosphatidylcholine (DLPC) bilayers at pressures up to 221 MPa. Pressure was found to separate the liquid crystal to gel transition from the gel to ordered crystalline phase transition. The jump in chain order observed on cooling through the transition into the gel phase was found to be small and thus consistent with the trend in longer chain saturated diacyl phosphatidylcholines. On cooling, DLPC was observed to enter an unusual state above the transition into the gel phase. This unusual state displayed fluid-like conformational order but short transverse relaxation times. It was found to be much better pronounced and to span a broader temperature range at elevated pressure than at lower pressures. Transverse relaxation measurements of deuterons on the chain alpha-carbons revealed a substantial slowing of molecular motions within the temperature range of the unusual fluid phase. The observation of such a phase at high pressure appears to be consistent with recent reports of an unusual fluid phase, Lx, in DLPC at ambient pressure.     

The effects of pressure and cholesterol on rotational motions of perylene in lipid bilayers

Using steady-state fluorescence polarization measurements, an isothermal pressure-induced phase transition was observed in dimyristoyl-L-alpha-phosphatidylcholine multilamellar vesicles containing perylene. The temperature-to-pressure equivalence, dT/dP, estimated from the phase transition pressure, P1/2, is about 22 K X kbar-1, which is comparable to values determined from diphenylhexatriene polarization (Chong, P.L.-G. and Weber, G. (1983) Biochemistry 22, 5544-5550). In addition, we have employed a new method, introduced in this paper, to calculate the rate of in-plane rotation (Rip) and the rate of out-of-plane rotation (Rop) of perylene in lipid bilayers. The effects of pressure and cholesterol on the rotational rates of perylene in two lipid bilayer systems have been examined. They are 1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) multilamellar vesicles (MLV) and 50 mol% cholesterol in POPC (MLV). Rop is smaller than Rip due to the fact that the out-of-plane rotation requires a larger volume change than the in-plane rotation. Cholesterol seems not to affect Rop significantly, but pressure causes a decrease in Rop by about a factor of three. In contrast, the effects of pressure and cholesterol on Rip are less straightforward. At 1 atm cholesterol increases Rip by a factor of about two. Similarly, in the absence of cholesterol 1.5 kbar pressure essentially triples Rip. However, if both cholesterol is added and pressure is applied, Rip decreases sharply. The possible interactions between cholesterol and perylene are discussed.     

Effect of hydrostatic pressure on translational fidelity

We have used the application of hydrostatic pressure to modify the misreading of polyuridylate template. Pressure was used to test ribosomes isolated from Escherichia coli strains containing mutations in the S12 ribosomal protein which lead to streptomycin-resistance and -dependence. The incorporation of phenylalanine into polypeptide, at a given pressure, was found to vary with the source of ribosomes and was found to correlate with S12-dependent changes in rates of incorporation suggesting a role of the S12 ribosomal protein in the pressure effect. Streptomycin partially alleviated the increased pressure-resistance in those cases where control rates of incorporation were found to be stimulated by the addition of streptomycin. In contrast, the misincorporation of isoleucine was substantially more sensitive to pressure application, regardless of ribosome source or the presence of streptomycin. These results suggest that the application of hydrostatic pressure affects at least two distinct ribosomal reactions important to the discrimination of these two amino acids.     

Effect of hydrostatic pressure on starch gelatinisation

Samples of wheat and potato starches, mixed with water to four concentrations were subjected to preselected hydrostatic pressures (in the range 200–1500 MPa) and temperatures. Subsequent examination in a polarising microscope revealed that the effect of high hydrostatic pressure was to lower the gelatinisation temperature. With the exception of the low water content samples, the samples did not appear to be greatly affected in any other way by hydrostatic pressure (as evidenced by staining behaviour, appearance in the polarising microscope and subsequent gelatinisation behaviour at ambient pressure). Reduction in gelatinisation temperature was a non-linear function of pressure, being greatest at high pressure. The effect was also more pronounced at the higher water contents. The significance of these results with respect to thermodynamic models of starch gelatinisation is discussed.     

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure

Differential polarized phase fluorometry was used to quantify the rotational rate ($\text{R}$) and limiting anisotropy ($\text{r}{}_{\mathrm{\infty }}$) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by $\text{r}{}_{\mathrm{\infty }}$. In addition to the well-known elevation of the transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with $\text{r}{}_{\mathrm{\infty }}$. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.     

Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers: a FT-IR study

The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers have been studied by Fourier-transform infrared spectroscopy (FT-IR) over the pressure range of 0.001-25 kbar. The results derived from the PRODAN C = O stretching band, the correlation field splitting of the methylene scissoring mode, and the methylene symmetric stretching mode as well as the absorption of the naphthalene ring show that in the sample of 4% (w/w) PRODAN in dimyristoyl-L-alpha-phosphatidylcholine (DMPC) at pH 6.8, most of the PRODAN molecules are embedded in the bilayers. In contrast, at pH 3.0, PRODAN was found to reside either on the membrane surface or dispersed in water. Compared to DMPC, egg yolk phosphatidylcholine (egg PC), which contains a substantial amount of unsaturated fatty acyl chains, is more susceptible to PRODAN permeation. The present study shows that the pressure dependence of the location of PRODAN in lipid membranes is different from that of tetracaine, a local anesthetic, in lipid bilayers. The model regarding the PRODAN location in lipid bilayers derived from the present infrared data has been compared with that obtained with previous fluorescence studies.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

Lipid peroxidation and water penetration in lipid bilayers: A W-band EPR study

Lipid peroxidation plays a key role in the alteration of cell membrane's properties. Here we used as model systems multilamellar vesicles (MLVs) made of the first two products in the oxidative cascade of linoleoyl lecithin, namely 1-palmitoyl-2-(13-hydroperoxy-9,11-octadecanedienoyl)-lecithin (HpPLPC) and 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-lecithin (OHPLPC), exhibiting a hydroperoxide or a hydroxy group at position 13, respectively. The two oxidized lipids were used either pure or in a 1:1 molar ratio mixture with untreated 1-palmitoyl-2-linoleoyl-lecithin (PLPC). The model membranes were doped with spin-labeled lipids to study bilayer alterations by electron paramagnetic resonance (EPR) spectroscopy. Two different spin-labeled lipids were used, bearing the doxyl ring at position (n) 5 or 16: γ-palmitoyl-β-(n-doxylstearoyl)-lecithin (n-DSPPC) and n-doxylstearic acid (n-DSA).Small changes in the acyl chain order in the sub-polar region and at the methyl-terminal induced by lipid peroxidation were detected by X-band EPR. Concomitantly, the polarity and proticity of the membrane bilayer in those regions were investigated at W band in frozen samples. Analysis of the g_xx and A_zz parameters revealed that OHPLPC, but mostly HpPLPC, induced a measurable increase in polarity and H-bonding propensity in the central region of the bilayer. Molecular dynamics simulation performed on 16-DSA in the PLPC–HpPLPC bilayer revealed that water molecules are statistically favored with respect to the hydroperoxide groups to interact with the nitroxide at the methyl-terminal, confirming that the H-bonds experimentally observed are due to increased water penetration in the bilayer. The EPR and MD data on model membranes demonstrate that cell membrane damage by oxidative stress cause alteration of water penetration in the bilayer.     

Orientation and rotational freedom of fluorescent probes in lecithin bilayers.

The fluorescence polarization properties of lecithin bilayers stained with 2,6-MANS and 1,8 ANS under applied potential steps have been studied. The fluorescence signal components of both dyes were found to have different sign and relative amplitude, suggesting that 1,8-ANS and 2,6-MANS behave differently when bound to black lipid membranes. In order to determine the location and the extent of rotational brownian motions of the bound chromophores, the experimental data were analyzed by using a simplified physico-mathematical model. According to it 2,6-MANS appears to have a ratio rho/tau higher than 1.8-ANS (rho being the rotational relaxation of in plane rotations and tau the lifetime of the excited singlet state of the bound molecules), suggesting that the former chromophore is more tightly held inside the bilayers. Furthermore, 2,6-MANS is found to possess the absorption and emission oscillators more closely oriented to the normal of membrane surface, while 1,8-ANS has both oscillators almost near the plane of the bilayers. The results furnish also a fair estimate of the random molecular motion own by the phospholipid molecules at room temperature. The comparison of the present data with those obtained from squid axon membranes confirms the validity of the proposed physical model, yielding a rough estimate of the axon membrane-area covered by integral protein macromolecules. These preliminary results derived from lecithin model membranes suggest that fluorescence polarization techniques can provide valuable informations if applied to study the macromolecular organization of in vitro reconstituted membranes.     

Effect of hydrostatic pressure on the mitochondrial ATP synthase

The effects of hydrostatic pressure on three different preparations of mitochondrial H+-ATPase were investigated by studies of the hydrolytic activity, of the spectral shift and quantum yield of the intrinsic protein fluorescence, and of filtration chromatography. Both membrane-bound and detergent-solubilized forms of the mitochondrial F0-F1 complex were reversibly inactivated in the pressure range of 600-1800 bar, whereas with soluble F1-ATPase the inactivation was irreversible. Pressure inactivation of soluble F1-ATPase was facilitated by decreasing the protein concentration, indicating that dissociation is an important factor. In the presence of 30% glycerol, soluble F1-ATPase becomes inactivated by pressure in a reversible fashion, recovering the original activity. ATPase activity measured in an aqueous medium returns to the original values when incubated under high pressure in a glycerol-containing medium without substrate and is even enhanced when Mg-ATP is present. ATP hydrolysis returns to 80% of its original value in the case of the F0-F1 complex. Fluorescence studies under pressure revealed a red shift in the spectral distribution of the emission of tyrosine fluorescence of soluble F1-ATPase. A decrease in the quantum yield of intrinsic fluorescence was also observed upon subjection to pressure. The fluorescence intensity decreased monotonically as a function of pressure when the sample was in an aqueous medium, whereas it presented a biphasic behavior in a 30% glycerol medium. Gel filtration studies demonstrated that the hydrodynamic properties of the F1-ATPase are preserved if the enzyme is subjected to pressure in the presence of glycerol but they are modified when the same procedure is performed in an aqueous medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of high hydrostatic pressure on hydration and activity of ribozymes

Formation and stabilization of RNA structure in the cell depends on its interaction with solvent and metal ions. High hydrostatic pressure (HHP) is a convenient tool in an analysis of the role of small molecules in the structure stabilization of biological macromolecules. Analysis of HHP effect and various concentrations of ions showed that water induce formation of the active ribozyme structure. So, it is clear that water is the driving force of conformational changes of nucleic acid.     

Effect of high hydrostatic pressure on the BK channel in bovine chromaffin cells.

The activity of the BK channel of bovine chromaffin cells was studied at high hydrostatic pressure, using inside-out patches in symmetrical KCl solution, Ca2+-free and at V(H) = -60 to -40 mV. Pressure increased the probability of channels being open (900 atm increasing the probability 30-fold), and it increased the minimum number of channels apparent in the patches. The pressure activation of the channel was reversed on decompression. Channel conductance was unaffected. It was shown that pressure did not act by raising the temperature, or by affecting [Ca] or pH, or the order of the membrane bilayer, and it was concluded that pressure most likely acted directly on the channel proteins and/or their modulating reactions.     

Effect of high hydrostatic pressure on murine norovirus in Manila clams

Aims: Eating raw or insufficiently cooked bivalve molluscs contaminated with human noroviruses (NVs) can result in acute cases of gastroenteritis in humans. Manila clams (Ruditapes philippinarum) are particularly prone to exposure to NVs due to the brackish environment in which they are farmed which is known to be susceptible to human faecal contamination. High hydrostatic pressure processing (HHP) is a food treatment technique that has been shown to inactivate NV. Methods and results: In this study we investigated the ability of HHP to inactivate murine norovirus (MNV‐1), a recognised surrogate for NV, in experimentally contaminated manila clams. Pools of contaminated live clams were subjected to hydrostatic pressure ranging from 300 to 500 MPa for different time intervals of between one and 10 min. The trial was repeated three times, at monthly intervals. Conclusions: Virus vitality post‐treatment was assessed and the data obtained indicates that the use of high hydrostatic pressures of at least 500 MPa for 1 min was effective in inactivating MNV‐1. Significance and Impact of the Study: HHP results to be an effective technique that could be applied to industrial process to obtain safe Manila clams ready to eat.     

The effect of hydrostatic pressure and heavy water upon regeneration in Blepharisma

• 1.1. Hydrostatic pressure ruptures postperistomal pieces of Blepharisma very rapidly at 15,000 pounds per square inch (psi) and more slowly at lower pressures.
• 2.2. At 4000 and 3000 psi blepharismas do not cytolyze but they fail to regenerate while under pressure, although they regenerate normally when brought to atmospheric pressure.
• 3.3. Regeneration occurs with considerable delay at 2500 and 2000 psi; at 1500 to 1000 psi regeneration occurs with relatively little delay.
• 4.4. Brief exposure of 10 to 15 sec to 15,000 psi damages postperistomal pieces of Blepharisma to some extent, inasmuch as regeneration at atmospheric pressure thereafter is slightly delayed, the time for 50 per cent regeneration being increased by 20 to 40 per cent.
• 5.5. Whole blepharismas are about as sensitive as postperistomal pieces: death occurs rapidly at 15,000 psi.
• 6.6. Brief exposure to 15,000 psi, insufficient to cause disintegration, alters the whole blepharismas to some extent inasmuch as they shorten and become larger in circumference. However, the division rate of such individuals implanted into culture medium is comparable to that of controls.
• 7.7. Under high hydrostatic pressure (3000–4000 psi), which just prevents regeneration, incorporation of ¹⁴C-uridine continues at a somewhat lower rate than in the control.
• 8.8. Pressure which stops regeneration also considerably delays the macronuclear changes which occur during regeneration. As soon as pressure is released the macronucleus continues its changes in shape, and regeneration is completed.

     

Effect of hydrostatic pressure on halothane-induced depression of mitochondrial respiration

Anesthetized animals are awakened when subjected to increased atmospheric pressure. Whether all phenomena associated with the anesthetic state are similarly reversed is not known. Since the anesthetic halothane produces a dose-related reversible depression of rat liver mitochondrial respiration, the effect of 51 atmospheres of pressure on the drug's action was evaluated. It is concluded that application of pressure does not antagonize the inhibition produced by this anesthetic.