Rapid compressions in a captive bubble apparatus are isothermal.

Captive bubbles are commonly used to determine how interfacial films of pulmonary surfactant respond to changes in surface area, achieved by varying hydrostatic pressure. Although assumed to be isothermal, the gas phase temperature (Tg) would increase by >100 degrees C during compression from 1 to 3 atm if the process were adiabatic. To determine the actual change in temperature, we monitored pressure (P) and volume (V) during compressions lasting <1 s for bubbles with and without interfacial films and used P x V to evaluate Tg. P x V fell during and after the rapid compressions, consistent with reductions in n, the moles of gas phase molecules, because of increasing solubility in the subphase at higher P. As expected for a process with first-order kinetics, during 1 h after the rapid compression P x V decreased along a simple exponential curve. The temporal variation of n moles of gas was determined from P x V >10 min after the compression when the two phases should be isothermal. Back extrapolation of n then allowed calculation of Tg from P x V immediately after the compression. Our results indicate that for bubbles with or without interfacial films compressed to >3 atm within 1 s, the change in Tg is <2 degrees C.     

Kinetics of Initiation of Germination of Bacillus pumilus Spores by Hydrostatic Pressure

The kinetics of initiation of germination and inactivation by hydrostatic pressure of phosphate-buffered Bacillus pumilus spores is shown to be a consecutive first-order process at 25 C. The effect of increasing pressure at constant temperature was studied, and rate constants were derived by using the criteria of heat resistance, refractility, and stainability. The calculated volume change of activation (DeltaVdouble dagger) was -139 +/- 6 cm(3)/mole for loss of heat resistance, -158 +/- 8 cm(3)/mole for the loss of refractility, and -153 +/- 4 cm(3)/mole for the change in permeability to dilute stains for the pressure range 800 to 1,010 atm at 25 C. It is suggested that the spore exists as a Donnan phase and that pressure triggers germination by influencing the equilibrium.     

Effects of hydrostatic pressure on the molecular structure and endothermic phase transitions of phosphatidylcholine bilayers: a Raman scattering study

The temperature dependences of the Raman spectra of aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were monitored at different but constant pressures between 1 and 1210 bar. The changes observed in these Raman spectra are discussed in terms of the effects of high pressure on the phase state and molecular structure of lipid bilayers. It is demonstrated that the temperature of the endothermic gel to liquid-crystal phase transition, as well as the temperature of the pretransition, increases linearly with increasing hydrostatic pressure. The dTm/dP values obtained from a wide range of pressures are 20.8 degrees C X kbar-1 for DPPC and 20.1 degrees C X kbar-1 for DMPC. The dTp/dP value for DPPC is 16.2 degrees C X kbar-1. It is also shown that the volume change that occurs at the gel to liquid-crystal transition is not constant; i.e., d delta Vm/dP decreases by 6.2% (DPPC) or 6.3% (DMPC) per kilobar pressure. The volume change at the pretransition is also pressure dependent; the d delta Vp/dP value of DPPC decreases by 4.7% per kilobar pressure.     

Entrainment of the circatidal swimming activity rhythm in the cumacean Dimorphostylis asiatica (Crustacea) to 12.5-hour hydrostatic pressure cycles

The cumacean Dimorphostylis asiatica (Crustacea) exhibits a circatidal swimming activity rhythm. The animals were exposed to a 12.5 hr sinusoidal change of hydrostatic pressure of 0.3 atm amplitude in the laboratory. Under constant dark conditions, most of the specimens were entrained to a daily bimodal swimming activity rhythm by the hydrostatic pressure cycle. A small number of individuals exhibited a unimodal daily rhythm, with no apparent entraining from the administered cycles. A marked feature was a flexible phase relationship between the entrained daily bimodal rhythm and the hydrostatic pressure cycles: the swimming activity of most of the specimens occurred around the pressure-decreasing phase, but for a small number of individuals it coincided with the pressure-increasing phase. Such flexibility suggests a weak entraining effect of hydrostatic pressure on the circatidal rhythm of this species. When exposed to 24 hr light-dark cycles and a hydrostatic pressure cycle simultaneously, the specimens exhibited a rhythmic activity entrained by the hydrostatic pressure cycle during the dark period, which closely resembles the temporal activity pattern of this species in the field. The light cycles entrained the swimming activity via direct inhibition and induction of activity (i.e., masking). Under light-dark conditions, the specimens exhibited activity on the pressure-increasing phase more frequently compared with specimens kept in constant darkness.     

Passive shedding of erythrocyte antigens induced by membrane rigidification

Rigidification of the cell membrane lipid bilayer can lead to an increase in the degree of exposure of membrane proteins to either side of the membrane. It is shown in this study that excess increase of the membrane lipid microviscosity (‘hyper-rigidification’) in intact human erythrocytes can cause the release of Rh₀(D) and A blood group antigens from the cell surface which can then be collected from the supernatant by affinity chromatography. The most efficient antigen shedding was achieved upon incorporation of cholesteryl hemisuccinate (CHS) (incubation for 2 h at 37 °C in a mixture of 200 μg/ml CHS, 3.5% polyvinylpyrrolidone 1% bovine serum albumin, 0.5% glucose in phosphate-buffered saline) followed by application of hydrostatic pressure (1 500 atm, 5 min) which increases the lipid microviscosity by about 2-fold. This technique can be of general application for isolation of membrane proteins without disruption of the cells or the use of detergents.     

Molecular mobility in the monolayers of foam films stabilized by porcine lung surfactant.

Certain physical properties of a range of foam film types that are believed to exist in vivo in the lung have been investigated. The contribution of different lung surfactant components found in porcine lung surfactant to molecular surface diffusion in the plane of foam films has been investigated for the first time. The influence of the type and thickness of black foam films, temperature, electrolyte concentration, and extract composition on surface diffusion has been studied using the fluorescence recovery after photobleaching technique. Fluorescent phospholipid probe molecules in foam films stabilized by porcine lung surfactant samples or their hydrophobic extracts consisting of surfactant lipids and hydrophobic lung surfactant proteins, SP-B and SP-C, exhibited more rapid diffusion than observed in films of its principal lipid component alone, L-alpha-phosphatidylcholine dipalmitoyl. This effect appears to be due to contributions from minor lipid components present in the total surfactant lipid extracts. The minor lipid components influence the surface diffusion in foam films both by their negative charge and by lowering the phase transition temperature of lung surfactant samples. In contrast, the presence of high concentrations of the hydrophillic surfactant protein A (SP-A) and non-lung-surfactant proteins in the sample reduced the diffusion coefficient (D) of the lipid analog in the adsorbed layer of the films. Hysteresis behavior of D was observed during temperature cycling, with the cooling curve lying above the heating curve. However, in cases where some surface molecular aggregation and surface heterogeneity were observed during cooling, the films became more rigid and molecules at the interfaces became immobilized. The thickness, size, capillary pressure, configuration, and composition of foam films of lung surfactant prepared in vitro support their investigation as realistic structural analogs of the surface films that exist in vivo in the lung. Compared to other models currently in use, foam films provide new opportunities for studying the properties and function of physiologically important alveolar surface films.     

Pressure effects on the composition and thermal behavior of lipids from the deep-sea thermophile Methanococcus jannaschii.

The deep-sea archaeon Methanococcus jannaschii was grown at 86 degrees C and under 8, 250, and 500 atm (1 atm = 101.29 kPa) of hyperbaric pressure in a high-pressure, high-temperature bioreactor. The core lipid composition of cultures grown at 250 or 500 atm, as analyzed by supercritical fluid chromatography, exhibited an increased proportion of macrocyclic archaeol and corresponding reductions in aracheol and caldarchaeol compared with the 8-atm cultures. Thermal analysis of a model core-lipid system (23% archaeol, 37% macrocyclic archaeol, and 40% caldarchaeol) using differential scanning calorimetry revealed no well-defined phase transition in the temperature range of 20 to 120 degrees C. Complementary studies of spin-labeled samples under 10 and 500 atm in a special high-pressure, high-temperature electron paramagnetic resonance spectroscopy cell supported the differential scanning calorimetry phase transition data and established that pressure has a lipid-ordering effect over the full range of M. jannaschii's growth temperatures. Specifically, pressure shifted the temperature dependence of lipid fluidity by ca. 10 degrees C/500 atm.     

Pressurization facilitates adenovirus-mediated gene transfer into vein graft

We investigated whether application of non-distending hydrostatic pressure facilitates gene transfer into vein grafts. An external jugular vein was placed in a chamber with 100 microl adenovirus solution at a titer of 10(10) pfu/ml and was pressurized to up to 8 atm above ambient pressure for 10 min. Histochemical analysis demonstrated a positive transgene expression in all layers of the vessel wall. Gene transfer with 8 atm pressurization resulted in an approximately 50 times higher transgene expression than that without pressurization. Under 8 atm pressurization, the efficiency of gene transfer reached a plateau at 7.5 min. The application of hydrostatic pressure may improve the effectiveness of intraoperative genetic engineering of vein grafts.     

Initiation of Germination and Inactivation of Bacillus pumilus Spores by Hydrostatic Pressure

The effect of hydrostatic pressures as high as 1,700 atm at 25 C on the heat and radiation resistance of Bacillus pumilus spores was studied. Phosphate-buffered spores were more sensitive to compression than spores suspended in distilled water. Measurements of the turbidity of suspensions, the viability, refractility, stainability, dry weight, and respiratory activity of spores, and calcium and dipicolinic acid release were made for different pressures and times. Initiation of germination occurred at pressures exceeding 500 atm and was the prerequisite for inactivation by compression. The rate of initiation increased with increasing pressure at constant temperature. This result is interpreted as a net decrease in the volume of the system during initiation as a result of increased solvation of the spore components.     

Destabilization of a lipid non-bilayer phase by high pressure

Pressure is found to destabilize the non-bilayer phase with respect to the bilayer in a model lipid system. The lamellar to inverted hexagonal (H₁₁) phase transition of aqueous egg phosphatidylethanolamine is shifted to higher temperatures by hydrostatic pressure. The slope of the increase in transition temperature is constant to beyond 300 bar, and is greater than that seen for other lipid phase transitions. This behavior is consistent with the hypothesis that increasing chain disorder drives the conversion from the bilayer into the hexagonal phase. If this non-bilayer lipid phase is an intermediate in membrane fusion, then pressure should inhibit the process. This may explain the inhibition of chemical transmission at neural synapses by pressure.     

Redetermination of the pressure dependence of the lipid bilayer phase transition.

The effect of pressure on the phase transition temperature for the dipalmitoyllecithin bilayer was redetermined by following the volume change accompanying the transition. These measurements were carried out isothermally with the transition from the ordered to the disordered phase induced by decreasing the pressure. This contrasts with our previous measurements which were carried out at constant pressure and increasing temperature. The transition at every temperature was sharp and confirmed our previous observation that the volume change associated with the transition (0.033 mL g-1) is invariant with pressure. However, our present measurements, in contrast to our previous results, indicate that dP m/dTm at all pressures is in agreement with the 1 atm value of delta H/Tm delta V within experimental error where Tm and Pm are the temperature and pressure of the phase transition, respectively. These results, which are now in agreement with all other known pressure data, indicate that the entropy change associated with the transition is invariant with pressure.     

Biological limits of temperature and pressure

Most biologists do not take into account that the greatest portion of today's biosphere is in the realm of environmental extremes, most of it being cold and under pressure. Since bacteria have the ability to adapt to environmental extremes, a close examination for the presence and/or growth of bacteria at high and low temperatures, low temperature and reduced pressure (less than 1 atm), low temperature and increased hydrostatic pressure should be made. It is also within the realm of possibility that life may have arisen in an environmental extreme on the primordial earth and then evolved over time to live under moderate temperatures and 1 atm. Microbial life has been demonstrated at temperatures slightly greater than 90°C, below 0°C, at hydrostatic pressures of 1100 atm, and possibly at cold temperatures in the atmosphere (less than 1 atm). Laboratory experiments have shown that certain enzyme reactions can occur above 100°C under hydrostatic pressure, at –26°C and at 5°C under hydrostatic pressure.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements.

The microviscosity of the lipid phase of bovine serum high density lipoprotein was determined by fluorescence polarization measurements on a lipophilic probe (1,6-diphenyl-1,3,5-hexatriene) dissolved in the lipoprotein. At 25°C the average microviscosity was 6.1 ± 0.5 poise, and the activation energy calculated from a plot of log η versus $\text{1}\text{T}$ was 13±3Kcal/mole. A constant slope for the Arrhenius plot from 0 to 46°C indicated no apparent phase transitions in this temperature range.Comparison of the present results with reported microviscosity values for rat lymphocyte membranes and liposomes [Shinitzky and Inbar (1974) $\text{J. Mol. Biol. 85}$, 603] indicates a more rigid environment of the probe in the high density lipoprotein system fluidity of the lipid appears to be considerably decreased in the lipoprotein relative to organic solvent or oil solutions of lipids, probably as a result of the anisotropic environment of the probe, high total cholesterol, and presence of protein in these particles.     

The effect of helium and of hydrogen at high pressure on the cell division of Tetrahymena pyriformis W.

The rate of cell division of Tetrahymena growing in an observational high pressure vessel was measured at selected pressures of helium, hydrogen and at high hydrostatic pressure. Pressures greater than 100 atm reduced the rate of division, but the gases inhibited division to a lesser degree than pure hydrostatic pressure. Hydrogen's effect was distinguishable from that of hydrostatic pressure at 130 atm or more, while helium's effect appeared at 175 atm. These inert gases probably counteract the action of pressure by stabilising apolar pressure-labile targets.     

Glycerol-induced baroprotection in erythrocyte membranes

A system for applying hydrostatic pressures up to 10,000 atm upon cell suspensions for time intervals from a few seconds to several minutes is described. The K+ content of toad red blood cells was used as an indication of the degree of membrane injury induced by the hyperbaric condition. It is practically not affected for pressures up to 2000 atm in experiments lasting 3 or 10 min. falling markedly for pressures of 5000 or 8000 atm. The duration of the applied pressure and its intensity are additive regarding the magnitude of the baroinjury. Glycerol, a cryoprotective agent. at 4.0 M, confers partial but significant baroprotection, which is characterized by a smaller decline of the cell K+ content of the glycerol-treated cells in comparison to the untreated cells, submitted to the same conditions of pressure and time. Baroinjury is compatible with a reversible mechanism. However, irreversible membrane damage occurs for a pressure of 8000 atm applied for 10 min. Baroinjury is discussed in terms of alterations of the lipid leaflet or of membrane proteins, and the mechanism of baroprotection in terms of stabilization of membrane components, under the effect of high pressure, by the association of glycerol with the proteins or the phosphate head groups of phospholipids.     

The role of the gel <=> liquid-crystalline phase transition in the lung surfactant cycle

Lipid polymorphism plays an important role in the lung surfactant cycle. A better understanding of the influence of phase transitions on the formation of a lipid film from dispersions of vesicles will help to describe the mechanism of action of lung surfactant. The surface pressure (or tension) of dispersions of DPPC, DMPC, and DPPE unilamellar vesicles was studied as a function of temperature. These aggregates rapidly fuse with a clean air-water interface when the system is at their phase transition temperature (Tm), showing a direct correlation between phase transition and film formation. Based on these results, an explanation on how fluid aggregates in the alveolar subphase can form a rigid monolayer at the alveolar interface is proposed.     

Hydrostatic Pressure Effects on Protein Synthesis

The effects of high hydrostatic pressure on several phases of cell-free protein synthesis have been examined. The initial rate of polyuridylic acid (poly U)-directed synthesis of polyphenylalanine showed an apparent increase at 100 atm, above which the synthetic rate was reduced sharply with increased pressure up to 640 atm where 95% inhibition was observed. The magnitude of the inhibition of polyphenylalanine synthesis with increased pressure depended strongly on the magnesium salt concentration in the reaction system. Misreading of the poly U message, as measured by insertion of leucine in place of phenylalanine, dropped rapidly with increased pressure from 1 to 350 atm, above which the amount of misreading increased. Enzymatic activation of transfer RNAs (tRNAs) was reduced by increased pressure in the range 100-640 atm, where the rate of tRNA aminoacylation was 80% inhibited. Both nonenzymatic attachment of phenylalanyl-tRNA (phe-tRNA) to the poly U-ribosome complex and stability of the phe-tRNA-poly U-ribosome complex were decreased at high pressures (100-900 atm). The results of the action of pressure on the various phases of cell-free protein synthesis suggest that the major pressure-sensitive element in the protein synthetic machinery is the ribosome.     

Quantification of lipid bilayer effective microviscosity and fluidity effect induced by propofol

Electron spin resonance (ESR) spectroscopy with nitroxide spin probes was used as a method to probe the liposome microenvironments. The effective microviscosities have been determined from the calibration of the ESR spectra of the probes in solvent mixtures of known viscosities. In the first time, by measuring ESR order parameter (S) and correlation time (tau(c)) of stearic spin probes, we have been able to quantify the value of effective microviscosity at different depths inside the liposome membrane. At room temperature, local microviscosities measured in dimyristoyl-l-alpha phosphatidylcholine (DMPC) liposome membrane at the different depths of 7.8, 16.95, and 27.7 A were 222.53, 64.09, and 62.56 cP, respectively. In the gel state (10 degrees C), those microviscosity values increased to 472.56, 370.61, and 243.37 cP. In a second time, we have applied this technique to determine the modifications in membrane microviscosity induced by 2,6-diisopropyl phenol (propofol; PPF), an anaesthetic agent extensively used in clinical practice. Propofol is characterized by a unique phenolic structure, absent in the other conventional anaesthetics. Indeed, given its lipophilic property, propofol is presumed to penetrate into and interact with membrane lipids and hence to induce changes in membrane fluidity. Incorporation of propofol into dimyristoyl-l-alpha phosphatidylcholine liposomes above the phase-transition temperature (23.9 degrees C) did not change microviscosity. At 10 degrees C, an increase of propofol concentration from 0 to 1.0 x 10(-2) M for a constant lipid concentration mainly induced a decrease in microviscosity. This fluidity effect of propofol has been qualitatively confirmed using merocyanine 540 (MC540) as lipid packing probe. Above 10(-2) M propofol, no further decrease in microviscosity was observed, and the microviscosity at the studied depths (7.8, 16.95, and 27.7 A) amounted 260.21, 123.87, and 102.27 cP, respectively. The concentration 10(-2) M was identified as the saturation limit of propofol in dimyristoyl-l-alpha phosphatidylcholine liposomes.     

Behavioral thermoregulation in mice subjected to high pressure

Mice exposed to normoxic He and Ne at increased pressure and allowed to choose between a neutral and a cool environment showed a preference for the cooler environment. This behavior was apparent at 5.7 but not at 2.5 atm He. At 11.3 atm He and Ne, the behavior was associated with a similar reduction in the deep body temperature to a new steady level. The reduction in body temperature increased with pressure, up to 35 atm He, the maximum studied. Since the heat transfer of the He and Ne gas mixtures is different and both gases exert negligible anesthetic effects, the hydrostatic pressure most likely affects behavioral thermoregulation by affecting neuronal function.     

Effects of High Hydrostatic Pressure on Morphogenesis in Naegleria gruberi (Schardinger)*

SYNOPSIS. The ameboid phase of Naegleria gruberi can be activated to transform to the flagellate phase, and cysts to excyst and transform to the flagellate phase, by a limited treatment with high hydrostatic pressure followed by release. The most effective treatment at 21 G is 45 min at 3500 psi (238 atm), which leads to almost 100% transformation. Following this dose of high pressure, 50% of amebae transform within 55–70 min after release of pressure, and nearly all within 75–120 min. Nearly all cysts hatch and transform within 200–240 min after release. Pressures of 4000 psi (272 atm) and above, and of 1000 psi (68 atm) and below, were ineffective at any duration of treatment.