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.     

Pressure effect on denaturant-induced unfolding of hen egg white lysozyme.

The influence of hydrostatic pressure (< or =100 MPa) on denaturant-induced unfolding of hen egg white lysozyme was investigated by means of ultraviolet spectroscopy at various temperatures. Assuming a two-state transition model, the dependence of Gibbs free-energy change of unfolding on the denaturant concentration was calculated. Under applied hydrostatic pressure, these data were interpreted as suggesting that a two-state model is not applicable in a restricted temperature range; the dominant effect of hydrostatic pressure is to affect the cooperativity in protein unfolding due to a chemical equilibrium shift in the direction of the reduction in the system volume. The deviation from the two-state transition model appears to be rationalized by assuming that applied pressure induces an intermediate conformation between the native and unfolded states of the protein. The implication of the thermodynamic stability of protein under pressure was discussed.     

Isolation of a New Intermediary Substance Consisting of Colistin Fatty Acid and l-α,δ-Diaminobutyric Acid from Colistin-producing Cells of Bacillus colistinus Koyama

Several saccharides were found to be significantly effective in providing protection against hydrostatic pressure and high temperature damage in the yeast Saccharomyces cerevisiae. The extent of barotolerance and thermotolerance with seven different sugars showed a linear relationship to their mean number of equatorial OH groups. The same linear relatioship is seen when sugars protect protein molecules against elevated temperatures in vitro. Some sugars were more effective in providing protection against hydrostatic pressure nearly a hundred times than high temperature. Pre-heat shock treatment on yeast cells induce various stress tolerances. In this report, pre-heat shocked cells showed potent protection against elevated temperature, but these cells showed faint protection against elevated pressure.

These results suggest that sugars may protect cells against hydrostatic pressure and high temperature in a similar manner, probably by stabilizing the macromolecule(s), and such type of protection may be suited for pressure stress.     

On the effect of hydrostatic pressure on the conformational stability of globular proteins

The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245‐14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure− volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure−volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 711–718, 2015.     

Water Stress and Protein Synthesis: II. Interaction between Water Stress, Hydrostatic Pressure, and Abscisic Acid on the Pattern of Protein Synthesis in Avena Coleoptiles

Water stress causes a reduction in hydrostatic pressure and can cause an increase in abscisic acid in plant tissues. To assess the possible role of abscisic acid and hydrostatic pressure in water stress effects, we have compared the effects of water stress, abscisic acid, and an imposed hydrostatic pressure on the rate and pattern of protein synthesis in Avena coleoptiles. Water stress reduces the rate and changes the pattern of protein synthesis as judged by a double labeling ratio technique, Abscisic acid reduces the rate but does not alter the pattern of protein synthesis. Gibberellic acid reverses the abscisic acid-induced but not the stress-induced inhibition of protein synthesis. The effect of hydrostatic pressure depends on the gas used. With a 19: 1 N₂-air mixture, the rate of protein synthesis is increased in stressed but not in turgid tissues. An imposed hydrostatic pressure alters the pattern of synthesis in stressed tissues, but does not restore the pattern to that found in turgid tissues. Because of the differences in response, we conclude that water stress does not affect protein synthesis via abscisic acid or reduced hydrostatic pressure.     

Specific induction of heat shock protein 90beta by high hydrostatic pressure

In chondrocytes, a low-amplitude intermittent hydrostatic pressure induces production of extracellular matrix molecules, while high hydrostatic pressure inhibits it. High pressure increases cellular heat shock protein 70 level in a number of cell types on account of increased stabilisation of the heat shock protein 70 mRNA. In our experiments, only bovine primary chondrocytes, but not an immortalized chondrocytic cell line, could resist the induction of the stress response in the presence of continuous 30 MPa hydrostatic pressure. We have recently shown that protein synthesis is required for the stabilization. According to two-dimensional gel electrophoresis the synthesis of heat shock protein 90 was also increased in a chondrocytic cell line and in HeLa cells, and mass spectrometric analysis suggested that the induction was rather due to increase in heat shock protein 90beta than in heat shock protein 90alpha. The stress response was rather intense in HeLa cells, therefore, we investigated the effect of continuous 30 MPa hydrostatic pressure on the expression of the two heat shock protein 90 genes in HeLa cells using Northern and Western blot analyses. Heat shock protein 90beta mRNA level increased within 6 hours of exposure to 30 MPa hydrostatic pressure, while hsp90alpha level remained stable. At protein level there was a clear increase in the heat shock protein 90beta/heat shock protein 90alpha ratio, too. These results show a specific regulation of stress proteins in cells exposed to high hydrostatic pressure.     

Piezotolerance of the respiratory terminal oxidase activity of the piezophilic Shewanella violacea DSS12 as compared with non-piezophilic Shewanella species

The facultative piezophile Shewanella violacea DSS12 is known to alter its respiratory components under the influence of hydrostatic pressure during growth, suggesting that it has a respiratory system that functions in adaptation to high pressure. We investigated the pressure- and temperature-dependencies of the respiratory terminal oxidase activity of the membrane of S. violacea relative to non-piezophilic Shewanella species. We observed that the activity in the membrane of S. violacea was more resistant to high pressure than those of non-piezophilic Shewanella even though DSS12 was cultured under atmospheric pressure. On the other hand, the temperature dependency of this activity was almost the same for all of the tested strain regardless of optimal growth temperature. Both high pressure and low temperature are expected to lower protein flexibility, causing a decrease in enzyme activity, but the results of this study suggest that the mechanism maintaining enzyme activity under high hydrostatic pressure is different from that at low temperature. Additionally, the responses of the activity to the pressure- and temperature-changes were independent of membrane lipid composition. Therefore, the piezotolerance of the respiratory terminal oxidases of S. violacea is perhaps dependent on the properties of the protein itself and not on the lipid composition of the membrane. Our observations suggest that S. violacea constitutively express piezotolerant respiratory terminal oxidases that serve adaptation to the deep-sea environment.     

The effects of the deep-sea environment on transmembrane signaling

Membrane-associated processes may be particularly susceptible to perturbation by the high hydrostatic pressures and low temperatures of the deep ocean. Transmembrane signaling by guanyl nucleotide binding protein (G protein) coupled receptors (GPCRs) is affected at a number of steps: (1) agonist activation of the GPCR; (2) the interaction of the receptor with the heterotrimeric G protein; (3) the G protein GTPase cycle; and (4) the activation and function of the effector element, adenylyl cyclase. The effects of low temperature and high hydrostatic pressures on the A₁ adenosine receptor–inhibitory G protein (G_i)–adenylyl cyclase signaling complex were examined in teleost fishes from three families, Scorpaenidae, Macrouridae and Moridae. In a comparison of teleost fishes, rat and chicken, species with body temperatures from 1 to 40 °C, at atmospheric pressure, A₁ adenosine receptor agonist binding is conserved at the body temperature of the species. In the marine teleost fishes examined, increased pressure decreases agonist efficacy. There are differences among species in the effects of increased hydrostatic pressure on G protein interactions with receptors, GTP binding to G protein α subunits and the intrinsic GTPase activity of α subunits. Adenylyl cyclase activity and modulation are affected by increased pressure in all the species examined, except Antimora rostrata which was unaffected by pressure changes. At pressures approximating those which the species experience in situ adenylyl cyclase activity retains its sensitivity to modulators. To understand the physiological consequences of impaired cell signaling several prototypical human diseases are discussed.     

High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures

High hydrostatic pressure is a new technology in the food processing industry, and is used for cold pasteurization of food products. However, the pressure inactivation of food-borne microorganisms requires very high pressures (generally more than 400 MPa) and long pressure holding times (5 min or more). Carrying out pressure processing at low temperatures without freezing can reduce these parameters, which presently limit the application of this technology, in keeping the quality of fresh raw product. The yeast, Saccharomyces cerevisiae and the bacterium, Lactobacillus plantarum were pressurized for 10 min at temperatures between -20 and 25 degrees C and pressure between 100 and 350 MPa. Pressurization at subzero temperatures without freezing significantly enhanced the effect of pressure. For example, at a pressure of 150 MPa, the decrease in temperature from ambient to -20 degrees C allowed an increase in the pressure-induced inactivation from less than 1 log up to 7-8 log for each microorganism studied. However, for comparable inactivation levels, the kinetics of microorganism inactivation did not differ, which suggests identical inactivation mechanisms. Implications of water thermodynamical properties like compression, protein denaturation, as well as membrane phase transitions, are discussed.     

Effects of hydrostatic pressure and temperature on growth and lipid composition of the inner membrane of barotolerant Pseudomonas sp. BT1 isolated from the deep-sea

A barotolerant member of the genus Pseudomonas was isolated from deep-sea sediment obtained from the Japan Trench, at a depth of 4418 m. The growth temperature was found to affect the hydrostatic pressure range in which the bacterium could grow; the optimum hydrostatic pressure for growth shifted to a higher pressure with increasing temperature. We examined the lipid composition of the inner membrane of cells grown at various hydrostatic pressures and temperatures. The fatty acid components of the inner membrane lipids were C16:0, C16:1, C18:0, and C18:1. The phospholipid components of the inner membrane were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and phosphatidylserine. It is evident that the effects of elevated hydrostatic pressure are comparable to the effects of low temperature on both the fatty acid composition of the inner membrane lipids and the phospholipid composition of the inner membrane of this bacterium.     

Biotechnological properties of distillery and laboratory yeasts in response to industrial stresses

The stress sensitivity of different wild-type strains was evaluated, as well as the response of cells arrested at different cell cycle positions to high hydrostatic pressure (HPP). HHP was chosen both for its importance in food decontamination and assessment of its suitability as a model for stress in general and understanding the yeast stress response. Studies were conducted with four industrial strains and four laboratory wild-type yeast strains (two haploid and two diploid) that differed in their backgrounds. Fundamental differences were found between the laboratory and industrial populations. Industrial strains were clearly more sensitive to hydrostatic pressure and ethanol stresses than the laboratory strains. However, ethanol production was higher in industrial strains than laboratory strains. Furthermore, no correlation was observed between ploidy and stress resistance. Yeast cells arrested in the G1 phase led to an enhancement in pressure tolerance compared to unarrested, G2 arrested, and S arrested cells. Moreover, cells arrested in the S phase were more sensitive to hydrostatic pressure than cells arrested in the G2 phase. Again, industrial strains were more sensitive than laboratory strains. HHP responses of industrial yeasts correlated well with both ethanol concentration and temperature stress, which suggests that it would be a useful model stress.     

Hydrostatic pressure-induced changes in cellular protein synthesis

Hydrostatic pressure is a well-known effector of cellular protein synthesis. High continuous hydrostatic pressure inhibits protein synthesis in general. It has been known for a long time that 30S ribosomal subunit is associated with the effects of pressure on protein synthesis in prokaryotes, however, the mechanisms of action are still not completely understood. Our new data suggest that synthesis of eukaryotic elongation factor-2 (eEF-2) is decreased under 30 MPa continuous hydrostatic pressure. Thus, eEF-2 may have a role in the synthesis of pressure-regulated proteins in eukaryotic cells. The presence of pressure-sensitive proteins indicate that hydrostatic pressure can induce very specific responses in stressed cells. Accumulation of heat shock protein 70 and 90 beta occurs under high pressure, independent of the general inhibition of protein synthesis, although this response appears clearly weaker than during heat stress.     

Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia

The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.     

Pressure-induced changes in the secondary structure of the Escherichia coli methionine repressor protein

The effect of hydrostatic pressure on the conformational properties of the E. coli methionine repressor protein in aqueous solution was investigated by infrared spectroscopy. Changes in hydrostatic pressure produce dramatic changes in the spectral region of the conformation-sensitive amide I band. As the pressure is raised up to 18 kbar, the protein undergoes a rearrangement of alpha-helical segments into beta-type structures; after the pressure is released the beta-strands reconvert into less ordered alpha-helical or random segments.     

Leben unter Druck

Life under pressure Pressure is an abiotic factor that has forced organisms to adapt on many levels. Especially in marine environments is hydrostatic pressure one of the main factors for changes in morphology and physiology. On a macroscopic level there is a need for a reduction of weight, the development of means of buoyancy and new hunting strategies to survive under deep sea conditions. On a cellular level high pressure (>10 MPa) inhibits the metabolism due to a deformation of proteins. In consequence high pressure can be utilized at room temperature to sterilize food. On the other hand there are extremophile microbes and higher animals that withstand the strong mechanical impact thanks to compatible solutes for example. Even the development of healthy tissue like joint cartilage and bone is dependent on the application of pressure.     

Induction of barotolerance by heat shock treatment in yeast

In Saccharomyces cerevisiae, heat shock treatment provides protection against subsequent hydrostatic pressure damage. Such an induced hydrostatic pressure resistance (barotolerance) closely resembles the thermotolerance similarly induced by heat shock treatment. The parallel induction of barotolerance and thermotolerance by heat shock suggests that hydrostatic pressure and high temperature effects in yeast may be tightly linked physiologically.     

Glycine Insertion Makes Yellow Fluorescent Protein Sensitive to Hydrostatic Pressure

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.     

Analysis of outer membrane permeability of Pseudomonas aeruginosa and bactericidal activity of endolysins KZ144 and EL188 under high hydrostatic pressure

The parameters influencing outer membrane permeability of Pseudomonas aeruginosa PAO1 under high hydrostatic pressure were quantified and optimized, using fusion between a specific A1gamma peptidoglycan-binding domain and green fluorescent protein (PBD-GFP). Based on the obtained data, optimal conditions were defined to assess the synergistic bactericidal action between high hydrostatic pressure and peptidoglycan hydrolysis by bacteriophage-encoded endolysins KZ144 and EL188. Under high hydrostatic pressure, both endolysins show similar inactivation of P. aeruginosa as the commonly used hen egg white lysozyme or slightly higher inactivation in the case of EL188 at 150 and 200 MPa. The partial contribution of pressure to the bacterial inactivation increases with higher pressure, while the partial contribution of the enzymes is maximal at the onset pressure of outer membrane permeabilization for the PBD-GFP protein (175 MPa). This study's results demonstrate the usefulness of this approach to determine optimal synergy for hurdle technology applications.     

Sedimentation velocity analyses of the effect of hydrostatic pressure on the 30 S microtubule protein oligomer

Increasing hydrostatic pressure in the analytical ultracentrifuge by increasing rotor velocity and overlayering protein samples with oil caused a depolymerization of the 30 S oligomer of microtubule protein. This results indicates that the reaction of 6 S microtubule protein to form the oligomer was accompanied by a positive volume change. The effect of hydrostatic pressure on the 6 S to 30 S transition was employed to demonstrate the presence of a rapidly reversible equilibrium between these components by showing polymerization or depolymerization of the oligomer during the course of ultracentrifugation. The magnitude of the partial specific volume change accompanying this reaction was estimated from mass fraction measurements of microtubule protein solutions at a variety of hydrostatic pressures to be about 9 X 10(-4) ml g-1.     

Effect of annealing and pressure on microstructure of cornstarches with different amylose/amylopectin ratios

This work focuses on the effect of annealing and pressure on microstructures of starch, in particular the crystal structure and crystallinity to further explore the mechanisms of annealing and pressure treatment. Cornstarches with different amylose/amylopectin ratios were used as model materials. Since the samples covered both A-type (high amylopectin starch: waxy and maize) and B-type (high amylose starch: G50 and G80) crystals, the results can be used to clarify some previous confusion. The effect of annealing and pressure on the crystallinity and double helices were investigated by X-ray diffraction (XRD) and ¹³C CP/MAS NMR spectroscopy. The crystal form of various starches remained unchanged after annealing and pressure treatment. XRD detection showed that the relative crystallinity (RC) of high amylopectin starches was increased slightly after annealing, while the RC of high amylose-rich starches remained unchanged. NMR measurement supported the XRD results. The increase can be explained by the chain relaxation. XRD results also indicated that some of the fixed region in crystallinity was susceptible to outside forces. The effect of annealing and pressure on starch gelatinization temperature and enthalpy are used to explore the mechanisms.