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PubMed1994

Heat-shock response in a yeast tps1 mutant deficient in trehalose synthesis.

Exponential cells of the Saccharomyces cerevisiae tps1 mutant underwent a rapid loss of viability upon a non-lethal heat exposure (from 28 to 42 degrees C). However, a further more severe heat stress (52.5 degrees C 5 min) induced an increase in the fraction of viable cells. This mutant can not synthesize trehalose either at 28 degrees C or at 42 degrees C due to the lack of a functional trehalose-6P synthase complex. In control experiments carried out with the wild-type W303-1B, heat-stressed exponential phase cultures grown on YPgal at 28 degrees C acquired thermotolerance to a higher extent than identical cultures grown on YPD, although in both cultures the level of stored trehalose was negligible. These data suggest that the bulk of trehalose accumulated in yeast upon mild heat treatments is not sufficient to account for the acquisition of thermotolerance.

Genes↗
PubMed1996

Effects of temperature shifts on the activities of Neurospora crassa glycogen synthase, glycogen phosphorylase and trehalose-6-phosphate synthase.

Conidiospore germlings of Neurospora crassa submitted to a heat shock at 45 degrees C accumulate trehalose and degrade glycogen. The opposite occurs upon reincubation at a physiologic temperature (30 degrees C). These observations suggest a temperature-dependent mechanism for the preferential synthesis of one or the other sugar reserve. Here we show that concomitant with these shifts of temperature, occurred reversible changes in the activities of glycogen synthase and phosphorylase. Glycogen synthase was inactivated at 45 degrees C while phosphorylase was activated. The reverse was true when the cells were shifted back to 30 degrees C. Addition of cycloheximide did not prevent the reversible enzymatic changes, which remained stable after gel filtration. Apparently, the effects of temperature shifts occurred at the level of reversible covalent enzymatic modifications. Trehalose-6-phosphate synthase properties were also affected by temperature. For instance, the enzyme was less sensitive to in vitro inhibition by inorganic phosphate at 50 degrees C than at 30 degrees C. Fructose-6-phosphate partially relieved the inhibitory effect of phosphate at 30 degrees C but not at 50 degrees C. These effects of the assay temperature, inorganic phosphate, and fructose-6-phosphate, on trehalose-6-phosphate synthase activity, were more evident for crude extracts obtained from heat-shocked cells. Altogether, these results may contribute to explain the preferential accumulation of trehalose 45 degrees C, or that of glycogen at 30 degrees C.

Cycloheximide↗
PubMed2005

Trehalose uptake through P2X7 purinergic channels provides dehydration protection.

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.

Adenosine Triphosphate↗
PubMed2005

Effects of process variables on the powder yield of spray-dried trehalose on a laboratory spray-dryer.

A systematic examination is presented of the effects of process variables on the powder yield of amorphous trehalose obtained from the Buchi Model 191 laboratory-scale mini spray dryer. By using a specially made, narrow cyclone the powder yield could be greatly improved at all process temperatures examined. Calculations of the separation efficiencies of the improved cyclone and the manufacturer's standard cyclone are given, which show that the former's higher tangential particle velocity at the radius of the exit duct is responsible for the improved performance. The powder yield increases with higher process temperatures, owing to improved droplet drying and reduced droplet/particle deposition on the walls of the drying chamber. A maximum in the powder yield is reached, however, after which it decreases sharply. This is caused by heating of the cyclone wall to >10 degrees C above the so-called 'sticky point' of the trehalose, causing increased particle deposits on the walls of the tower and cyclone. Increasing liquid feed flow rate or decreasing atomizing air flow rate too extensively were both detrimental to powder yield. The drying air flow rate should be as high as possible to ensure sufficient enthalpy throughput to dry the trehalose adequately to give a high powder yield. The enthalpy balance calculation for drying trehalose with the new cyclone was used successfully to interpret the results obtained. Some recommendations for optimizing powder yield of an amorphous material are given.

Powders↗
PubMed2005

Phase behavior and rheological properties of enzymatically synthesized trehalose decanoate aqueous solutions.

Surface tension properties of an enzymatically synthesized equimolar mixture of trehalose mono- and didecanoate in aqueous solutions have been determined. At 20 degrees C a critical micellar concentration (CMC) of 50 micromol/l and a minimal surface tension of 28 mN/m have been obtained. Above the CMC, it has been shown that up to a concentration of 42 wt%, and in a 20-60 degrees C temperature range the sugar ester aqueous solutions do not form any crystalline structure, nor present any phase transition, and the trehalose decanoate molecules form an isotropic worm-like micellar phase. The rheological properties indicate however a more complicated picture in the same concentration and temperature ranges. In steady shear, the viscosity of the trehalose decanoate solutions do not exhibit any shear rate dependence from 1 to 100 s(-1) for concentrations up to 42 wt%. Below 0.8 wt%, the viscosity remains constant and close to that of water; then, between 0.8 and 23 wt%, the viscosity shows a quadratic increase with surfactant concentration. For higher concentrations, up to 42 wt%, no further significant increase in viscosity is observed. In oscillatory shear experiments, the solutions exhibit viscoelastic properties. The observed rheological behavior as a function of concentration and temperature may be due to a progressive evolution of the trehalose decanoate molecular associations: as the concentration increases, the system evolves towards an entangled and/or partially branched or cross-linked micellar network, and eventually a multiconnected network of cross-linked micelles.

Cross-Linking Reagents↗
PubMed2004

Effects of the supplementation of trehalose extender containing egg yolk with sodium dodecyl sulfate on the freezability of goat spermatozoa.

Two experiments were conducted to determine the effect of sodium dodecyl sulfate (SDS) added to a trehalose-egg yolk extender on the cryopreservation of goat spermatozoa. In Experiment 1, semen from four goats was frozen in trehalose extender (osmolality = 370, pH = 7) containing 4 and 20% (v/v) glycerol and egg yolk, respectively, and 0.035-0.2% SDS. After thawing, sperm motility and acrosome integrity were assessed using a computer-assisted sperm analysis (CASA) system and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Both motility and progressive motility were improved (P < 0.05) by increasing the concentration of SDS in the trehalose-egg yolk extender, with the best results obtained with SDS at 0.1% (80.0 +/- 1.5% and 65.0 +/- 1.7%, respectively). There were no significant differences in path velocity when spermatozoa were frozen in a diluent containing 0.035, 0.05, 0.075, or 0.1% SDS, but path velocity decreased significantly with 0.2% SDS. The percentage of acrosome-intact sperm were highest (P < 0.05) when 0.05% (74.0 +/- 1.1) and 0.075% (70.0 +/- 1.2) SDS were used. In Experiment 2, the effect of diluent storage time (6, 24, or 48 h) before freezing on the cryoprotective effect of SDS was investigated. Prolonged storage of the diluent had slight cryoprotective effects when 0.2% SDS is used, while motility and the acrosome integrity of the cryopreserved spermatozoa improved slightly when the extender was stored for 48 h at 5 degrees C before use. In conclusion, goat sperm freezability was significantly improved when sperm were frozen in a trehalose-egg yolk extender containing an adequate concentration of SDS.

Acrosome↗
PubMed2001

Trehalose effect on low temperature protein dynamics: fluctuation and relaxation phenomena.

We performed spectral diffusion experiments in trehalose-enriched glycerol/buffer-glass on horseradish peroxidase where the heme was replaced by metal-free mesoporphyrin IX, and compared them with the respective behavior in a pure glycerol/buffer-glass (Schlichter et al., J. Chem. Phys. 2000, 112:3045-3050). Trehalose has a significant influence: spectral diffusion broadening speeds up compared to the trehalose-free glass. This speeding up is attributed to a shortening of the correlation time of the frequency fluctuations most probably by preventing water molecules from leaving the protein interior. Superimposed to the frequency fluctuation dynamics is a relaxation dynamics that manifests itself as an aging process in the spectral diffusion broadening. Although the trehalose environment speeds up the fluctuations, it does not have any influence on the relaxation. Both relaxation and fluctuations are governed by power laws in time. The respective exponents do not seem to change with the protein environment. From the spectral dynamics, the mean square displacement in conformation space can be determined. It is governed by anomalous diffusion. The associated frequency correlation time is incredibly long, demonstrating that proteins at low temperatures are truly nonergodic systems.

Biochemical Phenomena↗
PubMed2002

An anhydrous polymorphic form of trehalose.

An anhydrous polymorphic form of alpha,alpha-trehalose was prepared from trehalose dihydrate by two different drying methods: (1) heating under vacuum; and (2) heating in hot air. Preparation of this anhydrous form by vacuum heating showed good reproducibility. This form was characterized by X-ray powder diffraction analysis and differential scanning calorimetry. This anhydrous form was converted to an amorphous phase at 127 degrees C and was found to be hygroscopic. At 43% relative humidity at 25 degrees C, this form rapidly reverted to dihydrate, while the amorphous phase remained unchanged. When an amorphous phase coexisted with this form, the rate of water adsorption to the amorphous phase was slower than that to the amorphous phase alone. These properties of this anhydrous form of alpha,alpha-trehalose may explain the effects of trehalose in dehydration tolerance of plants and insects in the desert.

Calorimetry, Differential Scanning↗
PubMed2003

Stabilization of membranes in human platelets freeze-dried with trehalose.

Human blood platelets are normally stored in blood banks for 3-5 days, after which they are discarded. We have launched an effort at developing means for preserving the platelets for long term storage. In previous studies we have shown that trehalose can be used to preserve biological membranes and proteins during drying and have provided evidence concerning the mechanism. A myth has grown up about special properties of trehalose, which we discuss here and clarify some of what is fact and what is misconception. We have found a simple way of introducing this sugar into the cytoplasm of platelets and have successfully freeze-dried the trehalose-loaded platelets, with very promising results. We present evidence that membrane microdomains are maintained intact in the platelets freeze-dried with trehalose. Finally, we propose a possible mechanism by which the microdomains are preserved.

Blood Platelets↗
PubMed2000

Intracellular trehalose improves osmotolerance but not desiccation tolerance in mammalian cells.

Trehalose has been shown to play a role in osmotolerance or desiccation tolerance in some microorganisms, anhydrobiotic invertebrates and resurrection plants. To test whether trehalose could improve stress responses of higher eukaryotes, a mouse cell line was genetically engineered to express bacterial trehalose synthase genes. We report that the resulting levels of intracellular trehalose ( approximately 80 mM) are able to confer increased resistance to the partial dehydration resulting from hypertonic stress, but do not enable survival of complete desiccation due to air drying.

Animals↗
PubMed2003

Anhydrobiosis without trehalose in bdelloid rotifers.

Eukaryotes able to withstand desiccation enter a state of suspended animation known as anhydrobiosis, which is thought to require accumulation of the non-reducing disaccharides trehalose (animals, fungi) and sucrose (plants), acting as water replacement molecules and vitrifying agents. We now show that clonal populations of bdelloid rotifers Philodina roseola and Adineta vaga exhibit excellent desiccation tolerance, but that trehalose and other disaccharides are absent from carbohydrate extracts of dried animals. Furthermore, trehalose synthase genes (tps) were not found in rotifer genomes. This first observation of animal anhydrobiosis without trehalose challenges our current understanding of the phenomenon and calls for a re-evaluation of existing models.

Acclimatization↗
PubMed1997

Effect of temperature on the role of Hsp104 and trehalose in barotolerance of Saccharomyces cerevisiae.

We have studied the effect of temperature on the contribution of Hsp104 and trehalose to barotolerance using mutants deficient in Hsp104 and trehalose synthesis. When compared with a corresponding wild type strain, mutants of Hsp104 did not show temperature dependent barotolerance when the incubation temperature during the hydrostatic pressure treatment was increased. However, a mutant deficient in trehalose synthesis showed features similar to a wild type strain. Furthermore, the Hsp104 level was low in the insoluble fraction of the wild type strain after pressure treatment at 35 degrees C but not at 4 degrees C, and the protein profiles in the insoluble fraction were different between 35 degrees C and 4 degrees C. In contrast to the Hsp104 deficient mutants, the protein profile of the wild type after pressure treatment at 35 degrees C favors the role of Hsp104 as a disaggregator of proteins during hydrostatic pressure stress. These results suggest that the role of Hsp104 in barotolerance is temperature dependent in contrast to trehalose.

Adaptation, Physiological↗
PubMed2002

Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations.

We evaluated freeze-thawing tolerance of heterospermic ram spermatozoa (Pampinta breed) in a base diluent (Tris, citric acid, fructose, egg yolk, glycerol) with the addition of different trehalose concentrations (0-400 mOsm). We chose sperm motility, acrosome integrity and hypo-osmotic swelling test as parameters to evaluate cryopreservation capacity. We obtained the best results for 50 and 100 mOsm trehalose-supplemented extenders, with values (referred to fresh semen values) of 65% for motility, 75% for acrosome integrity and 50% for hypo-osmotic swelling test, while freeze-thawing tolerance diminished significantly for 200 and 400 mOsm of the disaccharide. Fertility values measured at lambing were 47.1 and 44.6% (2 consecutive years), using semen cryopreserved in 100 mOsm trehalose-containing diluent, which is 2.5 times greater than those obtained with the base diluent (18.5 and 14.5%). We conclude that the membrane-protecting disaccharide trehalose confers a greater cryoprotective capacity to the base extender, when added up to 100 mOsm. This action is reflected in the different sperm membranes, the motile activity and in vivo fertility.

Acrosome↗
PubMed1998

Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose.

Trehalose, a sugar produced by a wide variety of organisms, has long been known for its role in protecting certain organisms from desiccation. Recent work in yeast indicates that trehalose also promotes survival under conditions of extreme heat, by enabling proteins to retain their native conformation at elevated temperatures and suppressing the aggregation of denatured proteins. The latter property, however, seems to impair the recovery of cells from heat shock if they fail to degrade trehalose after the stress has passed. These multiple effects of trehalose on protein stability and folding suggest a host of promising applications.

Animals↗
PubMed2000

Stabilisation of Salmonella vaccine vectors by the induction of trehalose biosynthesis.

The growth of an aroA mutant of Salmonella typhimurium (SL3261) in minimal medium containing 0.5 M NaCl resulted in the intracellular accumulation of 2.2 micromol trehalose/mg total protein. The vacuum drying of these bacteria in the presence of trehalose allowed the recovery of 35% of the viable cells that were present before drying. In contrast, bacteria cultured in control medium accumulated 0.4 micromol trehalose/mg total protein and only 5% of the viable cells were recovered after vacuum drying with trehalose. Similar results were obtained when S. typhimurium SL3261, expressing the vaccine antigen (F1-antigen) of Yersinia pestis, was cultured in minimal medium with or without 0.5 M NaCl and dried in the presence of trehalose. Although these results indicate the potential for trehalose stabilisation of vaccine strains of S. typhimiurium, growth in minimal medium containing 0.5 M NaCl resulted in the loss of invasion competence of the bacteria.

Animals↗
PubMed2002

Role of trehalose biosynthesis in environmental survival and virulence of Salmonella enterica serovar typhimurium.

The otsA and otsB genes, encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase respectively, have been isolated from Salmonella enterica serovar typhimurium and nucleotide-sequenced. Induction of trehalose biosynthesis by exposure of bacteria to high osmotic strength resulted in the intracellular accumulation of trehalose. An otsA mutant of S. enterica serovar typhimurium was more susceptible to killing by heat, and grew poorly under conditions of high osmolarity. The wild-type and otsA mutant strains showed similar abilities to colonise spleen tissues after oral dosing of mice. These findings suggest that the otsBA gene products play a role in environmental survival, but not in virulence, of S. enterica serovar typhimurium.

Animals↗
PubMed2002

Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA.

Trehalose is a nonreducing disaccharide that plays a major role in many organisms, most notably in survival and stress responses. In Mycobacterium tuberculosis, it plays a central role as the carbohydrate core of numerous immunogenic glycolipids including "cord factor" (trehalose 6,6'-dimycolate). The classical pathway for trehalose synthesis involves the condensation of UDP-glucose and glucose-6-phosphate to afford trehalose-6-phosphate, catalyzed by the retaining glycosyltransferase OtsA. The configurations of two anomeric positions are set simultaneously, resulting in the formation of a double glycoside. The three-dimensional structure of the Escherichia coli OtsA, in complex with both UDP and glucose-6-phosphate, reveals the active site at the interface of two beta/alpha/beta domains. The overall structure and the intimate details of the catalytic machinery reveal a striking similarity to glycogen phosphorylase, indicating a strong evolutionary link and suggesting a common catalytic mechanism.

Amino Acid Sequence↗
PubMed2004

Yeast adapt to near-freezing temperatures by STRE/Msn2,4-dependent induction of trehalose synthesis and certain molecular chaperones.

Virtually nothing is known about the biochemical adaptations in eukaryotic cells that may enhance survival at low temperatures or upon freezing. Here we demonstrate an adaptive response in yeast that is activated below 10 degrees C and increases tolerance to low temperatures and freezing. This response involves a dramatic accumulation of the chemical chaperone trehalose and induction of trehalose-synthesizing enzymes (Tps1, Tps2) and certain heat shock proteins (Hsp104, Hsp42, Hsp12, Ssa4). mRNAs for these proteins increase dramatically below 10 degrees C and even at 0 degrees C. Their expression requires Msn2,4 transcription factors but also involves marked mRNA stabilization. Upon return to 30 degrees C, TPS1, TPS2, and HSP104 mRNAs, trehalose levels and tolerance to freezing fall dramatically within minutes. Mutants lacking trehalose or Msn2,4 die more rapidly at 0 degrees C and upon freezing. Thus, below 10 degrees C, yeast show an adaptive response that sustains viability at low or freezing temperatures, which are commonly encountered in natural environments and laboratory refrigerators.

Adaptation, Physiological↗