Critical damage to the cellular organelles of Saccharomyces cerevisiae under sublethal conditions upon high pressure carbon dioxide treatment

ABSTRACT

High pressure carbon dioxide treatment is a non-thermal pasteurization technique. However, critical damage, resulting from the treatment, to microbial cells has not been observed directly, and the detailed mechanism of the microbicidal activity is not understood. In this study, we analyzed the damage to Saccharomyces cerevisiae organelles, which were visualized using organelle-specific GFP fusion proteins. Yeast strains were subjected to high pressure carbon dioxide treatments at 30°C and 4.0?MPa for 2–8?h. Reduction in yeast cell viability was accompanied by the disruption of endoplasmic reticulum, nuclear membrane, Golgi body, and nucleolus. However, visible damage to the cell membrane was not observed. Fluorescence microscopy was utilized to confirm that high pressure carbon dioxide treatment damaged membranes of major organelles, but not the cell membrane.     

Identification of a regulatory element for yeast tryptophan permease Tat2 ubiquitination using high hydrostatic pressure

ABSTRACT

Tryptophan uptake in the yeast Saccharomyces cerevisiae is extremely sensitive to high pressure; therefore, the growth of tryptophan auxotrophic strains is impaired. Degradation of tryptophan permease Tat2 is enhanced at 25?MPa, depending on Rsp5 ubiquitin ligase. Any defect in Tat2 ubiquitination confers high pressure growth capacity, which is a luminous phenotype of the yeast used to explore the mechanism by which Rsp5 mediates Tat2 ubiquitination. Here we show that the N-terminal four (K17, K20, K29, and K31) among five lysines are required for efficient Tat2 degradation under high pressure. We found that a domain spanning D70 to S76 is also critical for Tat2 degradation at 25?MPa probably because of the recognition by Bul1, an adaptor protein of Rsp5. Defects in Tat2 ubiquitination do not produce any remarkable mutant phenotype at 0.1?MPa. Therefore, we suggest that high pressure is a unique and advanced tool to explore ubiquitination-dependent Tat2 regulation.     

The role of proteasome in yeast Saccharomyces cerevisiae response to sub-lethal high pressure treatment

Hydrostatic pressure is a physical factor that can induce stress in organisms. This stress leads to growth inhibition, cellular arrest, and cellular death, and these effects depend on the degree of pressure, temperature, and sensitivity of the organisms to hydrostatic pressure. Genomics studies of yeast cells under conditions recovering from high pressure-induced cellular damage showed evidence that multiprotein complexes or membrane proteins, and not soluble proteins, are the critical targets. We performed a metabolomic analysis. The metabolomics results suggested that membrane-spanning proteins broke down after high pressure treatment and recovery conditions. We also found 13 genes that were common to essential and pressure-induced gene groups. Among these 13 genes, more than 10 were associated with proteasome structure and functions. This suggests that proteasome structure or functions can be the critical target or a highly important factor. This hypothesis is supported by the fact that yeast cells are sensitive to the proteasome inhibitor MG132 after high pressure treatment.     

Possible mechanism of high pressure inactivation of microorganisms

Abstract

We have formulated the hypothesis that the ATPase bound to the cell membrane is strongly involved in high pressure inactivation. The stability of the membrane bound ATPase under pressure would in turn be dependent on the fluidity of the membrane. It has been postulated that cells with a more fluid membrane would be more resistant to pressure. We have confirmed this by comparing cells of Lactobacillus plantarum which differed in fluidity of the membrane. Damage of the membrane by pressure was shown by staining with propidium iodide. Leakage of ATP from the cells was observed when the cells were subjected to pressure, which was also an indication of membrane damage. ATP could not be formed after severe pressure treatment, but could after mild pressure treatment. This is also in line with the above mentioned hypothesis.     

The YPR153W gene is essential for the pressure tolerance of tryptophan permease Tat2 in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, hydrostatic pressure at 25?MPa is known to be nonlethal but significantly impairs the uptake of tryptophan by the permease Tat2, thereby inhibiting the growth of strains that require tryptophan from the medium. Here, we found that the lack of the YPR153W gene, so far poorly characterized for its role in yeast, caused a serious adverse effect on the growth at 10–25?MPa in the strain that required tryptophan. Deletion for YPR153W resulted in an increased rate of pressure-induced degradation of Tat2, suggesting that Tat2 is destabilized in the YPR153W deletion mutant at 25?MPa. Overexpression of the TAT2 gene enabled the deletion mutant to grow at 25?MPa. These results suggest that Ypr153w is essential for the stability and proper transport function of Tat2 under pressure at 10–25?MPa.

ABBREVIATIONS: Trp⁺: the phenotype of tryptophan prototrophic strains that synthesize tryptophan de novo; Trp^?: the phenotype of tryptophan auxotrophic strains that require tryptophan from the medium; YPR153W: an yeast poorly characterized gene; Ypr153w: a protein encoded by the YPR153W gene; ypr153wΔ: a deletion mutant for the YPR153W gene; Tat2: the high-affinity tryptophan permease; MCT10: a human aromatic amino acid transporter     

Effects of high hydrostatic pressure on β-glucan content,swelling power,starch damage,and pasting properties of high-β-glucan barley flour

ABSTRACT

The effects of high hydrostatic pressure (HHP) on the physicochemical properties of high-β-glucan barley flour were investigated in the present study. Dough samples were made from two types of barley flour with low and high β-glucan content, respectively, and treated with HHP (200–600?MPa) for 10?min. Although the elevation of pasting properties for the samples treated at 600?MPa was reduced to the same extent as that in wheat flour at normal atmospheric pressure, β-glucan content was maintained regardless of the pressure applied. The significant increase in starch damage of the dough samples at 550 and 600?MPa was confirmed by the results of microscopic observation, which revealed that elliptical starch granules were cracked and damaged in samples with low β-glucan at 600?MPa, and in samples with high β-glucan content at 400?MPa or more. X-ray diffraction patterns of the samples treated at 600?MPa indicated the formation of amylose-lipid complexes that were considered to inhibit the elevation of viscosity.     

High hydrostatic pressure pasteurization of a draft sake brewed using a Niigata-sake yeast

ABSTRACT

Draft (non-pasteurized) sake typically has a fresh flavor, but usually has to be consumed as soon as possible because of its short shelf life due to the potential for over-fermentation by residual yeast. In contrast, thermally pasteurized sake has a long shelf life, but the fresh flavor is lost during heat sterilization. High hydrostatic pressure (HHP) treatment can be used as a non-thermal pasteurization process while maintaining the characteristics of a draft sake. We evaluated the HHP inactivation behavior of sake yeast for the application of HHP pasteurization as an alternative to thermal pasteurization. The results showed complete pasteurization of sake yeast in the draft sake by HHP treatment at 400?MPa for 10?min. The viable cell count in HHP-pasteurized sake was below the detection limit during storage at 10°C for 3 months. Thus, we have established a HHP pasteurization technology to prevent over-fermentation and succeeded in producing a prototype of HHP-sparkling-cloudy sake, which we have designated AWANAMA.     

Enrichment of free amino acid content and reduction of astringent taste compounds in soybean by high hydrostatic pressure

ABSTRACT

We investigated the effect of high hydrostatic pressure (HHP) on the distribution of free amino acids and isoflavones in soybean immediately after pressure treatment (100–600?MPa, 10–60?min). HHP-treatment at 200 and 300?MPa resulted in high accumulation of free amino acids. Additionally, Gly, Val, and Pro levels increased even after HHP of 400?MPa. The application of HHP-treatment to soybean decreased sources of an astringent taste such as genistein-, daizein-, and glycitein-type molecules under all pressure conditions over 200?MPa. High accumulation of free amino acids with specific pressure–time conditions would be caused by the acceleration of proteolysis and specific amino acid metabolism. We concluded that HHP alone with no subsequent storage enabled the enrichment of specific amino acids such as GABA, Glu, Lys, and Pro in soybean. Soybean with enrichment of these amino acids and improved taste should be an innovative component of functional foods.     

Conformational fluctuations in a green fluorescent protein-like Akane family protein: a high-pressure fluorescence study at 0.1–700 MPa

ABSTRACT

We have investigated conformational fluctuations in a new green fluorescent protein(GFP)-like protein rb-Akane found in a red-brown-colored octocoral, Scleronephthya gracillima (Kuekenthal)), with high pressure fluorescence spectroscopy at 0.1–700?MPa. Besides the green fluorescence at 510?nm, two red fluorescence peaks are observed at 590 and 629?nm, the relative intensity of which varies reversibly with pressure. The phenomenon is interpreted as representing the cis–trans isomerization of the chromophore accompanied by the conformational transition between two sub-states of the red fluorescence form of rb-Akane. The two sub-states are separated only marginally in free energy (ΔG⁰?=?1.9?±?0.4?kJ?mol^?1), but significantly in partial molar volume (ΔV⁰?=??19.8?±?1.4?ml?mol^?1) at 0.1?MPa (pH 7.5, 25°C). Above 500?MPa, the fluorescence at λ_max 629?nm undergoes another reversible change with pressure, showing the onset of unfolding.     

Microwave heating in high pressure reaction vessels

Abstract

We report the use of microwave -hydrothermal processing to synthesize various ceramic powders. Microwave-hydrothermal processing is compared with conventional hydrothermal processing in the crystallisation of MoO₂. The presence of microwave field leads to accelerated kinetics of crystallization of the finely divided molybdenum dioxide particles. Existing microwave heated pressure vessels for chemical synthesis cannot be used above 250 MPa and 270°C because they contain parts made of polymeric materials. The objective of this work is to associate a microwave source to a high pressure vessel in a way such that it might be used to carry out reactions in aqueous media at pressures around 100 MPa and temperatures above the critical point of water.     

A damage model based on failure threshold weakening

A variety of studies have modeled the physics of material deformation and damage as examples of generalized phase transitions, involving either critical phenomena or spinodal nucleation. Here we study a model for frictional sliding with long-range interactions and recurrent damage that is parameterized by a process of damage and partial healing during sliding. We introduce a failure threshold weakening parameter into the cellular automaton slider-block model which allows blocks to fail at a reduced failure threshold for all subsequent failures during an event. We show that a critical point is reached beyond which the probability of a system-wide event scales with this weakening parameter. We provide a mapping to the percolation transition, and show that the values of the scaling exponents approach the values for mean-field percolation (spinodal nucleation) as lattice size L is increased for fixed R. We also examine the effect of the weakening parameter on the frequency-magnitude scaling relationship and the ergodic behavior of the model.     

Quantitative analysis of dissociation of LDH by high pressure native PAGE

ABSTRACT

High pressure native polyacrylamide gel electrophoresis has been designed to visualize the dissociation/association process of protein complexes. This paper reports this methodology in more quantitative way by inspecting pressure dissociation of pig heart lactate dehydrogenase, a tetrameric protein, which was extensively investigated in spectroscopic methods. We observed the change of electrophoresis pattern with pressure up to 150?MPa. By optimizing the buffer system and careful image analysis of the stained gels, we quantified all the dissociates in the process of pressurization. We discussed the characteristics of our methodology by comparing the result with the previously reported.     

Effect of high pressure on growth and bacteriocin production of Pediococcus acidilactici HA-6111-2

This study was aimed to investigate the effect of high pressure processing (HPP, 200–600?MPa) on the (i) survival of Listeria innocua and Pediococcus acidilactici HA-6111-2; (ii) production of bacteriocin bacHA-6111-2 and (iii) activity of bacteriocin against untreated and pressure-treated L. innocua cells. Inactivation of P. acidilactici was observed for pressures of >300?MPa. However, at this pressure level, L. innocua was more sensitive. Bacteriocin crude extract was pressure stable, with a decrease for pressures of ≥400 MPa. Pressures of ≤200?MPa did not affect bacteriocin production when compared with non-pressure-treated cells, whereas higher pressures caused a 2- to 4-fold decrease on the maximum level of bacteriocin production. Growth curves of P. acidilactici were fitted with the modified Gompertz model. The lag phase period depended on the magnitude of the pressure applied: there was a delay in the exponential phase as pressure increased and, as a consequence, in the beginning of bacteriocin production. Since P. acidilactici HA-6111-2 and its bacteriocin have shown resistance to pressures up to 300–400 MPa, they could be used in combination with HPP in order to improve food safety.     

Effect of high hydrostatic pressure and reducing sodium chloride and phosphate on physicochemical properties of beef gels

ABSTRACT

The effect of high hydrostatic pressure (HHP) treatment (100–200?MPa, 10?min, 20°C) combined with sodium chloride and sodium phosphate on the physicochemical properties of beef gels was investigated. The water content, cooking losses, color, protein composition by SDS-PAGE analysis and texture parameters of beef gels were determined. The beef gels treated with high pressure at 150?MPa showed a synergistic effect in the increased water content and the decreased cooking losses compared with the unpressurized gels. The L*, a* and b* color values of beef gels were slightly decreased under HHP treatment at 100–200?MPa. In the SDS-PAGE analysis, the staining intensity of the α-actinin protein band was decreased in pressurized samples. The cohesiveness, adhesiveness, gel strength and modulus of elasticity were improved after HHP treatment. Application of high pressure treatment (150–200?MPa) before heat treatment would be beneficial for the manufacturing of low salt and/or low phosphate meat products for a healthy diet.     

Pressure tolerance of Artemia cysts compressed in water medium

ABSTRACT

The high pressure tolerance of cysts of Artemia salina was investigated up to several GPa in water. No survival was observed after exposure to 1.0?GPa for 15?min. After exposure to 2.0?GPa for the same time duration, the hatching rate had recovered to 33%, but decreased to 8% following compression at 7.5?GPa. This contrasts with results using Fluorinert? as the pressure-transmitting medium where 80–88% recovery was observed. The lower survival rate in water is accompanied by swelling of the eggs, indicating that liquid H₂O close to the ice-VI crystallization pressure penetrated inside the eggs. This pressure exceeds the stability limit for proteins and other key biomolecules components within the embryos that could not be resuscitated. Rehydration takes several minutes and so was not completed for all samples compressed to higher pressures, prior to ice-VI formation, resulting in renewed survival. However H₂O penetration inside the shell resulted in increased mortality.     

The impact of scaling on single event upset in 6T and 12T SRAMs from 130 to 22 nm CMOS technology

ABSTRACT

As transistor sizes scale down to nanometres dimensions, CMOS circuits become more sensitive to radiation. High-performance static random access memory (SRAM) cells are prone to radiation-induced single event upsets (SEU) which come from the natural space environment. The SEU generates a soft error in the transistor due to the strike of an ionizing particle. Thus, this paper compares the endurance of 12T SRAM and 6T SRAM circuit on 130 up to 22?nm CMOS technology towards SEU. Besides that, this paper discusses the trend of critical linear energy transfer (LET) and collected charge due to technology scaling for the respective circuit. The critical LET (LETcrit) and critical charge (Qcrit) of 6T are approximately 50% lower compared with 12T SRAMs.     

Review: high pressure generation techniques beyond the limit of conventional diamond anvils

ABSTRACT

Current anvil designs and problems associated with various efforts to generate static high pressures beyond the limit of conventional diamond anvil cells (DACs) (～400?GPa) are reviewed. Pressures of up to 1?TPa have been reported by one research group using the double-stage DAC (ds-DAC) technique, but no other research group has successfully reproduced this high pressure result. Some research groups have used toroidal anvils, achieving pressures of >400?GPa. We have conducted numerous ds-DAC experiments and investigated the problems associated with such experiments. They include problems associated with various pressure scales in the multi-megabar region, difficulties in obtaining reliable X-ray diffraction patterns from micron-sized samples, and physical property measurements of tiny materials that may be harder than diamond. Each of these problems is discussed, following the summary of various experiments.     

Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp^?) strains under pressures of 15–25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp^? cells to grow at 15–25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.     

Moderate hydrostatic pressure–temperature combinations for inactivation of Bacillus subtilis spores

We report the effect of using moderate hydrostatic pressure, 40–140?MPa, at moderate temperature (38–58°C) to inactivate Bacillus subtilis spores in McIlvaine's citric phosphate buffer at pH 6. We have investigated several parameters: pressure applied, holding time, pressure cycling, and temperature. The kinetics of spore inactivation is reported. The results show that spore inactivation is exponentially proportional to the time the sample is exposed to pressure. Spore germination and inactivation occur at the hydrostatic pressures/temperature combinations we explored. Cycling the pressure while keeping the total time at high pressure constant does not significantly increase spore inactivation. We show that temperature increases spore inactivation at two different rates; a slow rate below 33°C, and at a more rapid rate at higher temperatures. Increasing pressure leads to an increase in spore inactivation below 95?MPa; however, further increases in pressure give a similar rate kill. The time dependence of the effect of pressure is consistent with the first-order model (R²?>?0.9). The thermal resistance values (Z_T) of B. subtilis spores are 30°C, 37°C, and 40°C at 60, 80, 100?MPa. The increase in Z_T value at higher pressures indicates lower temperature sensitivity. The pressure resistance values (Z_P) are 125, 125 and 143?MPa at 38°C, 48°C, and 58°C. These Z_P values are lower than those reported for B. subtilis spores in the literature, which indicates higher sensitivity at pressures less than about 140?MPa. We show that at temperatures <60°C, B. subtilis spores are inactivated at pressures below 100?MPa. This finding could have implications for the design of the sterilization equipment.     

Dissociation of the light-harvesting membrane protein complex I from Rhodobacter sphaeroides under high hydrostatic pressure

The light-harvesting complex 1 (LH1) from Rhodobacter sphaeroides is an excellent model system for investigating the stability of oligomeric membrane proteins under high hydrostatic pressure. The currently investigated LH1 forms a 16-meric ring structure of B825 subunits. B825 is a heterodimer of transmembrane α- and β-polypeptide chains, which non-covalently binds two bacteriochlorophyll a molecules. These pigment molecules were used as intrinsic spectroscopic sensors to follow the dissociation reaction. Our results demonstrate that the LH1 dissociates into B825 subunits through an intermediary tetrameric unit B845. The dissociation mechanism depends on pressure. At ～200–500?MPa the dissociation corresponds to a pseudo-first-order reaction, characterised by the apparent reaction rate at atmospheric pressure k₀?=?3·10^?5?s^?1, activation volume ΔV^??=??4?mL/mol, and free energy of activation ΔG^??=?26?kJ/mol. Below 200?MPa and above 500?MPa, the reaction is more complex, including further dissociation of B825 into monomers B777.