Controlling the membrane fluidity of yeasts during coupled thermal and osmotic treatments

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Previous work by our team has allowed us to relate the mortality of cells exposed to a combination of thermal and osmotic treatments to leakage of cellular components through an unstable membrane when lipid phase transition occurs. In this study, yeast viability was measured after numerous osmotic and thermal treatments. In addition, the fluidity of yeast membranes was assessed according to a(w) and temperature by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy measurement. The results show that there is a negative correlation between the overall fluidity variation undergone by membranes during treatments and yeast survival. Using a diagram of membrane fluidity according to a(w) and temperature, we defined dehydration and rehydration methods that minimize fluidity fluctuations, permitting significantly increased yeast survival. Thus, such membrane fluidity diagram should be a potential tool for controlling membrane state during dehydration and rehydration and improve yeast survival. Overall fluidity measurements should now be completed by accurate structural analysis of membranes to better understand the plasma membrane changes occurring during dehydration and rehydration.     

The response of bacterial spores to vacuum treatments. II. Germination and viability studies.

The viability of Bacillus megaterium spores has been determined after exposure to vacuum dehydration at temperatures between 0 and 65 °C, for periods up to 24 hr. A curvilinear relationship has been demonstrated between viability and drying temperature, with minimum viability occuring around 15 °C and increases in viability being shown above 35 °C. In contrast to vegetative bacteria, reequilibration of the dried spores to 2 × 10^?3 or 10 Torr aqueous vapor pressure, and/or subsequent exposure to oxygen had no effect on viability. Dehydration, rehydration and oxygen treatments had no effect on the time for outgrowth of the spores or on the growth rate of the resultant vegetative cells. Physical loss of spores from samples was not demonstrated during any of these treatments. Evidence has been presented for a novel type of spore activation, which occurs during vacuum dehydration at high temperatures, to an extent that is dependent upon drying time. The mechanism of this activation is unlike that of conventional heat or chemical activation but is oxygen independent and unaffected by reequilibration to 2 × 10^?3 or 10 Torr.     

Viability of Escherichia coli after combined osmotic and thermal treatment: a plasma membrane implication

This study investigates the influence of temperature (T) and osmotic pressure (Pi) on the viability of Escherichia coli K12 during an osmotic treatment. Osmotic shock (dehydration and rehydration within 1 s) in liquid media at different temperatures (4, 10, 30 and 37 degrees C) and different levels of osmotic pressure (26, 30, 35, 40, 82 and 133 MPa) were realized.Results show that a sudden dehydration, below 40 MPa, destroyed up to 80% of the bacterial population for each tested temperature, whereas viability was greater than 90% for an osmotic pressure less than 26 MPa. The influence of T and Pi on the membrane's physical structure is finally considered to explain the results in light of FTIR and electron microscopy study of the influence of temperature and osmotic pressure on E. coli membrane phospholipids conformation.     

Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperatures above 0 degrees C

Cell injury due to osmotic dehydration, which is regarded as a major cause of injury during freeze-thaw processes, was examined closely using a perfusion microscope. Human prostatic adenocarcinoma cells (PC-3), which were put in a chamber, were subjected to hyperosmotic stresses by perfusing NaCl solutions of varying concentrations into the chamber. Cells were exposed to 2.5 and 4.5M NaCl solutions for 1-60 min by changing the concentrations at 0.2, 1, and 10 M/min. Decrease in cell viability was biphasic: the viability decreased first after the increase in NaCl concentration due to dehydration and then after return to isotonic conditions due to rehydration. Rehydration was substantially more responsible for cell injury than dehydration, which was marked at lower NaCl concentrations and lower temperatures. Injury resulting from contraction was negligible at the 2.5 M NaCl solution. While the hypertonic cell survival, which was determined without a return to isotonic conditions, was almost independent of time of exposure to hyperosmotic concentrations, the post-hypertonic survival after returning to isotonic conditions decreased with increasing exposure time, suggesting that the rehydration-induced injury was a consequence of time-dependent alteration of the plasma membrane. The post-hypertonic survival was lower for higher NaCl concentrations and higher temperatures, which was qualitatively consistent with previous studies. Effects of the rate of concentration change on the post-hypertonic cell survival were observed at 4.5 M; the highest rate of survival was obtained by slower increase and faster decrease in the NaCl concentration. However, the effect was negligible at 2.5 M.     

Effect of xylitol and trehalose on dry resistance of yeasts

The effects of dehydration/rehydration on two strains of Saccharomyces cerevisiae: S600, a metabolically engineered xylose-utilising strain, and H158, the non-xylose-utilising host strain; and on the naturally xylose-utilising yeast Pachysolen tannophilus CBS 4044, were compared after glucose and xylose utilisation respectively. The yeast strains differed in their ability to excrete and accumulate intracellular xylitol. A high intracellular xylitol content before and after dehydration coincided with a higher viability after a dehydration/rehydration cycle. The intracellular trehalose content increased during dehydration in all three yeast strains, but this did not correspond to enhanced cell viability after dehydration/rehydration. The results are discussed in relation to the ability of xylitol and trehalose to structure water. Received: 9 July 1996 / Received revision: 29 October 1996 / Accepted: 2 November 1996     

Anhydrobiosis in yeast: is it possible to reach anhydrobiosis for yeast grown in conditions with severe oxygen limitation?

The yeast Saccharomyces cerevisiae was shown to be extremely sensitive to dehydration–rehydration treatments when stationary phase cells were subjected to conditions of severe oxygen limitation, unlike the same cells grown in aerobic conditions. The viability of dehydrated anaerobically grown yeast cells never exceeded 2 %. It was not possible to increase this viability using gradual rehydration of dry cells in water vapour, which usually strongly reduces damage to intracellular membranes. Specific pre-dehydration treatments significantly increased the resistance of anaerobic yeast to drying. Thus, incubation of cells with trehalose (100 mM), increased the viability of dehydrated cells after slow rehydration in water vapour to 30 %. Similarly, pre-incubation of cells in 1 M xylitol or glycerol enabled up to 50–60 % of cells to successfully enter a viable state of anhydrobiosis after subsequent rehydration. We presume that trehalose and sugar alcohols function mainly according to a water replacement hypothesis, as well as initiating various protective intracellular reactions.     

Dehydration,rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, <Emphasis Type="Italic">Belgica antarctica</Emphasis>

We investigated molecular responses elicited by three types of dehydration (fast, slow and cryoprotective), rehydration and overhydration in larvae of the Antarctic midge, Belgica antarctica. The larvae spend most the year encased in ice but during the austral summer are vulnerable to summer storms, osmotic stress from ocean spray and drying conditions due to wind and intense sunlight. Using suppressive subtractive hybridization (SSH), we obtained clones that were potentially responsive to dehydration and then used northern blots to evaluate the gene’s responsiveness to different dehydration rates and hydration states. Among the genes most responsive to changes in the hydration state were those encoding heat shock proteins (smHsp, Hsp70, Hsp90), antioxidants (superoxide dismutase, catalase), detoxification (metallothionein, cytochrome p450), genes involved in altering cell membranes (fatty acid desaturase, phospholipase A2 activating protein, fatty acyl CoA desaturase) and the cytoskeleton (actin, muscle-specific actin), and several additional genes including a zinc-finger protein, pacifastin and VATPase. Among the three types of dehydration evaluated, fast dehydration elicited the strongest response (more genes, higher expression), followed by cryoprotective dehydration and slow dehydration. During rehydration most, but not all, genes that were expressed during dehydration continued to be expressed; fatty acid desaturase was the only gene to be uniquely upregulated in response to rehydration. All genes examined, except VATPase, were upregulated in response to overhydration. The midge larvae are thus responding quickly to water loss and gain by expressing genes that encode proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in this Antarctic environment.     

再水合溶液能有效改善急性脱水女举重运动员的运动表现

本研究旨在探讨女子举重运动员急性脱水后口服4种不同组成的溶液,对液体存留率、胃肠道舒适度、血液值和无氧动力的影响。本研究以12位大学生女子举重运动员为对象,采交叉、平衡次序设计;研究参与者于脱水2%体重后分别补充相当于1.5倍脱水量的低渗透压电解质液(HES)、等渗透压电解质液(IES)、运动饮料(SB)或纯水(W),补充时间为脱水后立即、再水合期的30 min、60 min及90 min。再水合期间记录胃肠舒适分数、测量体重(计算液体存留率),并采集静脉血以测量血液渗透压、葡萄糖及钠、钾与氯离子,于脱水前与再水合期120 min时进行Wingate无氧动力测验。研究结果显示:补充HES、IES及运动饮料(SB)于再水合期90 min时的胃肠道舒适分数显著低于补充纯水(W)、而补充3种溶液于再水合期60 min时的血糖值则显著高于补充纯水(W),补充HES于再水合期120 min时的血钾值显著高于补充运动饮料(SB)及纯水(W);但补充4种溶液后的无氧动力与液体存留率并无显著差异。本研究证实举重运动员急性脱水后补充4种不同口服再水合溶液并不会影响再水合后的无氧动力,但补充含有糖类及电解质的溶液能有较佳的胃肠道舒适度,并血糖与血液电解质有维持的效果。     

Critical water contents at leaf,stem and root level leading to irreversible drought-induced damage in two woody and one herbaceous species

Plant water content is a simple and promising parameter for monitoring drought-driven plant mortality risk. However, critical water content thresholds leading to cell damage and plant failure are still unknown. Moreover, it is unclear whether whole-plant or a specific organ water content is the most reliable indicator of mortality risk. We assessed differences in dehydration thresholds in leaf, stem and root samples, hampering the organ-specific rehydration capacity and increasing the mortality risk. We also tested eventual differences between a fast experimental dehydration of uprooted plants, compared to long-term water stress induced by withholding irrigation in potted plants. We investigated three species with different growth forms and leaf habits i.e., Helianthus annuus (herbaceous), Populus nigra (deciduous tree) and Quercus ilex (evergreen tree). Results obtained by the two dehydration treatments largely overlapped, thus validating bench dehydration as a fast but reliable method to assess species-specific critical water content thresholds. Regardless of the organ considered, a relative water content value of 60% induced significant cell membrane damage and loss of rehydration capacity, thus leading to irreversible plant failure and death.     

Fluid balance in exercise dehydration and rehydration with different glucose-electrolyte drinks

After exercise dehydration (3% of body weight) the restoration of water and electrolyte balance was followed in 6 male subjects. During a 2 h rest period after exercise, a drink of one of four solutions was given as 9 X 300 ml portions at 15 min intervals: control (C-drink), high potassium (K-drink), high sodium (Na-drink) or high sugar (S-drink). An exercise test (submaximal and supramaximal work) was performed before dehydration and after rehydration. Dehydration reduced plasma volume by 16%, a process reversed on resting even before fluid ingestion began, due to release of water accumulated in the muscles during exercise. After 2 h rehydration, plasma volume was above the initial resting value with all 4 drinks. The final plasma volumes after the Na-drink (+14%) and C-drink (+9%) were significantly higher than after the K- and S-drinks. The Na-drink favoured filling of the extracellular compartment, whereas the K- and S-drinks favoured intracellular rehydration. In spite of the higher than normal plasma volume after rehydration, mean heart rate during the submaximal test was 10 bpm higher after rest and rehydration than in the initial test, and was not different between the drinks. The amount of work which could be performed in the supramaximal test (105% VO2max) was 20% less after exercise dehydration and subsequent rest and rehydration than before. This reduction was similar for all drinks, and may be due to a decreased muscle glycogen content (70% of initial) at the time of the second test.     

Fate of Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage

Total plate counts were determined on boneless cooked, cubed chicken meat obtained from a commercial processor. Survival of the natural flora was determined after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and 50, 85, and 100 C for 10 min. Total counts of bacteria in the rehydrated samples were determined during storage of the meat at 4, 22, and 37 C until spoilage odor was detectable. Meat samples were inoculated with Staphylococcus aureus, then dried, rehydrated, and stored at the same temperatures. Numbers of surviving organisms in the inoculated samples were determined with use of both selective and nonselective media. Representative genera surviving the various rehydration treatments were determined. Approximately 32% of the bacteria in the meat survived during dehydration and rehydration at room temperature. Many numbers and types of vegetative bacteria also survived rehydration at 50 C. When meat was rehydrated at 85 or 100 C, the initial count was less than one per gram. The only organisms isolated from samples rehydrated at 85 or 100 C were of the genus Bacillus. S. aureus in inoculated samples survived dehydration and rehydration at 60 C. Storage of all rehydrated samples at 4 C gave a good shelf life (18 or more days). The study indicates that freeze-dehydrated meat should be produced with adequate microbiological control and that such meat should be rehydrated in very hot water.     

The response of bacterial spores to vacuum treatments. I. Design and characterization of the vacuum apparatus

A vacuum apparatus has been described that has enabled samples of bacterial spore suspensions to be dehydrated at defined temperatures between 0 and 65 °C, with facilities for reequilibration of the dried samples to aqueous vapor pressures between 5 × 10^?4 and 10 torr and subsequent exposure to dry gases. The apparatus has been characterized using sample temperature/drying-time profiles, and drying rate/ drying-time curves, and the reproducibility of the dehydration and rehydration techniques has been established. Biological data have confirmed the suitability of the apparatus since no loss of spores from samples has been observed during any of these experimental treatments. On the basis of measurements recorded during rehydration of dried spores, it is suggested that dehydration occurs at specific sites in the spore which are of two types, 1) reversibly dehydrated (rehydratable) and 2) irreversibly dehydrated (nonrehydratable).     

Effects of Dehydration and Rehydration on Photosynthesis of Detached Leaves of the Resurrective Plant Boea hygrometrica

Fluorescence induction kinetics was used to investigate the effects of dehydration and rehydration on photosynthesis of detached leaves of the desiccation-tolerant, resurrective plant Boea hygrometrica (Bunge) R. Br. In comparison with the desiccation-intolerant plant Chirita heterotricha Merr., the PSⅡphotochemical activity of Boea hygrometrica was characterized by a faster decline during dehydration and a much higher capacity of recovery during rehydration. By means of native PAGE, it was further shown that the thylakoid pigment-protein complexes of Boea hygrometrica were highly stable during dehydration and rehydration. These features may contribute to the extreme desiccation resistance of photosynthesis apparatus of resurrective plant Boea hygrometrica.     

Increased activities of superoxide dismutase and catalase are not the mechanism of desiccation tolerance induced by hardening in the moss Atrichum androgynum

Abstract

The effects of treatments that increase desiccation tolerance were tested on the activity of the enzymes superoxide dismutase (SOD) and catalase (CAT) in the moss Atrichum androgynum subjected to a drying/wetting cycle. Hardening by both abscisic acid (ABA) pretreatment and partial dehydration significantly increased the rate of recovery of photosynthesis during rehydration following desiccation. Hardening treatments had little effect on SOD activity. In non-hardened plants, SOD activity increased three-fold during desiccation for 32 h at 52% rh, but hardened material tended to display smaller increases in activity. During rehydration, SOD activities rapidly declined to their initial values in all treatments. Hardening by partial dehydration, but not ABA, reduced CAT activity. After desiccation for 32 h, material from all treatments displayed about half the initial CAT activity, and activity did not change during subsequent rehydration. Results show that, while the induction of SOD appears to play a role in desiccation tolerance, a similar induction occurred in both hardened and non-hardened mosses. Induction of greater activities of enzymes that scavenge reactive oxygen species is not responsible for the added tolerance induced by hardening treatments.     

Osmoregulation in Onymacris rugatipennis, a free-ranging tenebrionid beetle from the Namib Desert.

Haemolymph levels of organic and inorganic constituents were investigated in the tenebrionid Onymacris rugatipennis during dehydration and rehydration. The major osmolar effectors are sodium (26%), chloride (24%), amino acids (18%), and sugars (11%); regulation of haemolymph osmotic pressure (OP) during dehydration is effected largely by a reduction in the haemolymph content of these constituents. Changes in amino acid levels are not the result of interchanges with soluble protein. During rehydration, the main contributors to osmoregulation are sodium (26%), chloride (24%), and an increase in haemolymph solute(s) not measured in this study (31%). Of the sodium removed from the haemolymph during dehydration, 21.2% was excreted. Faecal losses of potassium during dehydration far exceeded the amounts removed from the haemolymph; however, haemolymph potassium levels were strongly regulated during rehydration. Regulatory efficiency increases as desiccation proceeds, and is greatest only when this species is severely challenged.     

Relationship between the fatty acid composition of lipids and the viability of dried yeast Saccharomyces cerevisiae

Summary The fatty acid composition of the total lipids and phospholipids of intact and convectively dried yeast has been studied and an increase has been detected in the degree of unsaturation of fatty acids during dehydration. The validity of the inverse relation between the viability of the yeast population during dehydration and rehydration and the degree of unsaturation of the fatty acids has been demonstrated by regression analysis. The results may help to predict the viability of dried yeast.     

Water balance and osmoregulation in Stenocara gracilipes,a wax-blooming tenebrionid beetle from the Namib Desert

Dehydration (10 days at 27 degrees C) of the Namib tenebrionid Stenocara gracilipes resulted in a rapid weight loss (17.5%), and a substantial decline in haemolymph volume (72%). Although the lipid content decreased significantly, metabolic water production was insufficient to maintain total body water (TBW). Rehydration (no food) resulted in increases in haemolymph volume, body weight (sub-normal), and TBW to normality. Haemolymph osmolality, sodium, potassium, chloride, amino acids, and sugars (trehalose and glucose), were all subject to osmoregulatory control during both dehydration and rehydration. Major osmolar effectors in this species are sodium, chloride, and amino acids, with most of the contribution to regulation of haemolymph osmolality coming from changes in the levels of these constituents. Changes in amino acid levels are not the result of interchange with soluble protein during dehydration (the possibility exists during extended rehydration, however). Despite faecal losses of sodium being low (8.2% of that removed from the haemolymph during dehydration), sodium concentrations do not return to normal during rehydration. Chloride concentrations increase supra-normally when access to water is allowed, and remain elevated throughout the rehydration period. Although faecal loss of potassium greatly exceeded the amount removed from the haemolymph (by approximately 1.8 times), haemolymph potassium levels were strongly regulated during rehydration. S. gracilipes demonstrates an exquisite capacity to regulate haemolymph osmolality under conditions of both acute water-shortage and -abundance. Together with an efficient water economy (drinking when fog-water is available, and a superb water conservation mechanism in the form of wax-bloom production), this must serve to contribute to long-term survival of this species in an otherwise harsh abode.     

Characterization of Solute Efflux from Dehydration Injured Soybean (Glycine max L. Merr) Seeds

Soybean (Glycine max L. Merr) seeds lose their tolerance of dehydration between 6 and 36 hours of imbibition. Soybean axes and cotyledons were excised 6 hours (tolerant of dehydration) and 36 hours (susceptible) after commencing imbibition and subsequently dehydrated to 10% moisture. Kinetics of the efflux of potassium, phosphate, amino acid, sugar, protein, and total electrolytes were compared in the four treatments during rehydration. Only slight differences were observed in the kinetics of solute efflux between the two cotyledon treatments dehydrated at 6 and 36 hours suggesting that the cotyledons may retain their tolerance of dehydration at this stage of germination. Several symptoms of injury were observed in the axes dehydrated at 36 hours. An increase in the initial leakage of solutes during rehydration, as quantified by the y-intercept of the linear regression line for solute efflux between 2 and 8 hours suggests an increased incidence of cell rupture. An increase in the rate of solute efflux (slope of regression line between 2 and 8 hours) from fully rehydrated axes was observed in comparison to axes dehydrated at 6 hours. The Arrhenius activation energy for potassium, phosphate, and amino acid efflux decreased and for protein remained unchanged. Both observations indicate an increase in membrane permeability in dehydration-injured tissue. Increasing the H⁺ concentration of the external solution increased K⁺ efflux from both control and dehydrated/rehydrated samples, increased sugar efflux from axes at 6 hours imbibition but decreased sugar efflux from axes at 36 hours imbibition, indicating changes in membrane properties during germination. The dehydration treatment did not alter the pattern of the pH response of axes dehydrated at 6 or 36 hours but did increase the quantity of potassium and sugar efflux from dehydration injured axes. These results are interpreted as indicating that dehydration of soybean axes at 36 hours of imbibition increased both the incidence of cell rupture during rehydration and altered membrane permeability of the rehydrated tissue.