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PubMed1993

Assay of trehalose with acid trehalase purified from Saccharomyces cerevisiae.

An enzymatic end-point assay of trehalose using acid trehalase from yeast is described. After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system. Pre-existing glucose is determined in a control reaction from which acid trehalase is omitted. When intact cells are analysed for trehalose, pre-existing glucose can be washed out with ice-cold water without reducing the trehalose content of the cells. A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95 degrees C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid trehalase preparation used. As described previously (Mittenbühler, K. and Holzer, H., 1988, J. Biol. Chem. 263, 8537-8543; Mittenbühler, K., 1988, Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose-6-phosphate, glucose-1-phosphate, galactose. The application of the new trehalose assay to yeast cells grown to different growth stages and at various temperatures is presented.

Biological Assay↗
PubMed1994

Do trehalose and dimethyl sulfoxide affect intermembrane forces?

The sugar trehalose is produced in some organisms that survive dehydration and desiccation, and it preserves the integrity of membranes in model systems exposed to dehydration and freezing. Dimethyl sulfoxide, a solute which permeates membranes, is added to cell suspensions in many protocols for cryopreservation. Using a surface forces apparatus, we measured the very large, short-range repulsion between phosphatidylcholine bilayers in water and in solutions of trehalose, sorbitol, and dimethyl-sulfoxide. To the resolution of the technique, the force-distance curves between bilayers are unchanged by the addition of trehalose or sorbitol in concentrations exceeding 1 kmol.m-3. A relatively small increase in adhesion in the presence of trehalose and sorbitol solutions may be explained by their osmotic effects. The partitioning of trehalose between aqueous solutions and lamellar phases of dioleylphosphatidylcholine was measured gravimetrically. The amount of trehalose that preferentially adsorbs near membrane surfaces is at most small. The presence of dimethyl sulfoxide in water (1:2 by volume) makes very little difference to the short-range interaction between deposited bilayers, but it sometimes perturbs them in ways that vary among experiments: free bilayers and/or fusion of the deposited bilayers were each observed in about one-third of the experiments.

Adhesiveness↗
PubMed1989

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans.

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose) and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures - highly homogeneous with regard to the respective cell form produced - contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5-2% to the biomass of M. rouxii and 1.5-6% to that of C. albicans; the values for trehalose ranged from 0.2-11% in the former and 1-3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions - regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.

Candida albicans↗
PubMed2002

Intracellular delivery of trehalose into mammalian cells by electropermeabilization.

The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.

Animals↗
PubMed2002

Fed-batch cultivation of baker's yeast followed by nitrogen or carbon starvation: effects on fermentative capacity and content of trehalose and glycogen.

An industrial strain of Saccharomyces cerevisiae (DGI 342) was cultivated in fed-batch cultivations at a specific growth rate of 0.2 h(-1). The yeast was then exposed to carbon or nitrogen starvation for up to 8 h, to study the effect of starvation on fermentative capacity and content of protein, trehalose and glycogen. Nitrogen starvation triggered the accumulation of trehalose and glycogen. After 8 h of starvation, the content of trehalose and glycogen was increased 4-fold and 2-fold, respectively. Carbon starvation resulted in a partial conversion of glycogen into trehalose. The trehalose content increased from 45 to 64 mg (g dry-weight)(-1), whereas the glycogen content in the same period was reduced from 55 to 5 mg (g dry-weight)(-1). Glycogen was consumed faster than trehalose during storage of the starved yeast for 1 month. Nitrogen starvation resulted in a decrease in the protein content of the yeast cells, and the fermentative capacity per gram dry-weight decreased by 40%. The protein content in the carbon-starved yeast increased as a result of starvation due to the fact that the content of glycogen was reduced. The fermentative capacity per gram dry-weight was, however, unaltered.

Biomass↗
PubMed2004

Effect of nitrogen limitation and surplus upon trehalose metabolism in wine yeast.

Trehalose metabolism in yeast has been related to stress and could be used as a stress indicator. Winemaking conditions are stressful for yeast and understanding trehalose metabolism under these conditions could be useful for controlling alcoholic fermentation. In this study, we analysed trehalose metabolism of a commercial wine yeast strain during alcoholic fermentation by varying the nitrogen levels from low (below adequate) to high (excess). We determined trehalose, nitrogen, sugar consumption and expression of NTH1, NTH2 and TPS1. Our results show that trehalose metabolism is slightly affected by nitrogen availability and that the main consumption of nitrogen occurs in the first 24 h. After this period, nitrogen is hardly taken up by the yeast cells. Although nitrogen and sugar are still available, no further growth is observed in high concentrations of nitrogen. Increased expression of genes involved in trehalose metabolism occurs mainly at the end of the growth period. This could be related to an adaptive mechanism for fine tuning of glycolysis during alcoholic tumultuous fermentation, as both anabolic and catabolic pathways are affected by such expression.

Fermentation↗
PubMed1999

Trehalose phosphorylase activity and carbohydrate levels during axenic fruiting in three Agaricus bisporus strains.

Three strains of Agaricus bisporus (B430, 116, and 155.8), which share the ability to form hyphal aggregates on solid media under axenic conditions, were investigated with respect to carbohydrate levels and activities of enzymes involved in their carbon metabolism. The size and macroscopic appearance of the aggregates, when grown on diluted medium, suggest that substrate limitation plays a role in the process of fruiting body development in A. bisporus. The enzymes trehalose phosphorylase (TP), mannitol dehydrogenase (MD), and glucose-6-phosphate dehydrogenase (G6PD) seem to be developmentally regulated, in contrast to hexokinase (HK). Activities of TP (measured in the direction of trehalose degradation), MD, and G6PD were higher in the hyphal aggregates compared with the mycelium, whereas HK activity varied little. In the period preceding the axenic formation of hyphal aggregates, synthesis of trehalose by TP approximately doubled in the mycelium. The carbohydrate levels, which were measured by HPLC, varied in a way similar to their corresponding enzymes. The results indicate synthesis of trehalose in the mycelium of A. bisporus before the hyphal aggregates arise. Subsequently, translocation of the trehalose takes place from the mycelium to the emerging aggregates. In these small aggregates the trehalose is rapidly broken down to yield glucose and glucose-1-phosphate, serving as carbon and energy sources for further growth of the aggregates and for the synthesis of the osmolyte mannitol.

Agaricus↗
PubMed2004

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress.

The comparative analysis of growth, intracellular content of Na+ and K+, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na+ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.

Ascomycota↗
PubMed2003

An in vitro evaluation of the glycation potential of a natural disaccharide, trehalose.

BACKGROUND: Glucose, an osmotic agent generally used in continuous ambulatory peritoneal dialysis (CAPD) dialysate, has a critical characteristic of forming advanced glycation endproducts (AGEs). We undertook this study to investigate whether a possible osmotic agent, trehalose, formed fewer AGEs than glucose. METHODS: Hemoglobin (Hb), a counter-protein of AGE, was incubated in four kinds of medium; glucose-phosphate buffered saline (PBS), autoclaved glucose-PBS, trehalose-PBS, and autoclaved trehalose-PBS, for 3, 7, 14, and 30 days, respectively. Polymerization of the Hb molecule was detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and carboxymethylated Hb was detected by Western blotting, using specific mono-clonal antibody for carboxymethylated N-terminal valine-Hb (CMV-Hb). RESULTS: PBS containing glucose showed bands of polymerized Hb molecule, a phenomenon which was markedly exaggerated by autoclaving. Likely, PBS containing glucose showed the formation of CMV-Hb in the long incubation of 30 days, and PBS containing autoclaved glucose showed accelerated formation of CMV-Hb in an incubation as short as 3 days. By contrast, PBS containing trehalose showed much less increase in a band of 30 k Dalton and in CMV-Hb formation even in autoclaved medium. CONCLUSIONS: Our present in vitro study clearly showed the superior characteristic of trehalose to produce fewer AGEs. Based upon the results of this study, we propose that the application of trehalose should be considered for CAPD solution.

Animals↗
PubMed2005

Microneedles coated with porous calcium phosphate ceramics: effective vehicles for transdermal delivery of solid trehalose.

Trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) is recognized as a promising fast-dissolving solid reservoir capable of stabilizing the native structure of proteins and suitable for loading with a wide variety of bioactive substances. Currently, there is a growing interest in developing cost-effective methods for immobilizing solid trehalose on arrays of microneedles for delivering protein-based and DNA-based vaccine to the epidermis. In the present work, micro-porous calcium phosphate coatings were used to provide a biocompatible interface with a large surface area for the effective immobilization of trehalose on microneedles. Calcium phosphate coatings with varying degrees of porosity were electrochemically synthesized on the tips of stainless steel acupuncture needles and loaded with solid trehalose. Skin experiments were designed to determine the ability of micro-porous calcium phosphate coatings to deliver solid trehalose into epidermis without breaking during insertion. The mechanical performance of the coatings was assessed by inserting the tips of the coated needles into human skin to an average depth of 100-300 microm and then removing them for analysis by scanning electron microscopy. Microporous calcium phosphate coatings loaded with trehalose effectively breached the stratum corneum and allowed direct access to the epidermis without breaking and without stimulating nerves in deeper tissues.

Administration, Cutaneous↗
PubMed1993

Properties of gel phase lipid-trehalose bilayers upon rehydration.

When dipalmitoylphosphatidylcholine bilayers dried under vacuum in different concentrations of trehalose are rehydrated in buffer without the sugar they show different physicochemical properties in the gel state in comparison to the normal gel state. Dry DPPC/trehalose mixtures are readily dispersed in buffer below the phase transition showing by electron microscopy a morphology similar to liposomes prepared by dispersing the lipids in buffer above the phase transition temperature. In these conditions, an increase in the peak at 570 nm of merocyanine after the dehydration-rehydration process in the presence of the sugar is observed and the water permeation increases to values comparable to those found in the fluid state as indicated by the activation energy values and the osmotic volume. The trehalose-dried liposomes rehydrated in buffer show a similar osmotic response to hypertonic gradient as DPPC liposomes without sugar near the phase transition temperature. In accordance with this behavior the trehalose-dried liposomes are lysed below the phase transition temperature by lysoderivatives. These modifications of the gel state of hydrated phospholipids by trehalose can only be achieved if a drastic dehydration is performed in the presence of the sugar. After rehydration the changes in the gel state can be detected after dyalizing the rehydrated membranes in media without trehalose during at least 24 h. These results suggest that trehalose is still intercalated between the phospholipids after restoring water to the dried liposomes either at temperatures below or above the phase transition.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Trehalose lowers membrane phase transitions in dry yeast cells.

Recent work has clearly demonstrated a direct correlation between the amount of trehalose present in the yeast Saccharomyces cerevisiae and its ability to tolerate dehydration, but has failed to elucidate the specific role played by trehalose. By using Fourier transform infrared spectroscopy we measured the transition temperature of phospholipids in both intact S. cerevisiae and isolated plasma membranes dried in the presence and absence of trehalose. Our results show that trehalose lowers the temperature of the dry gel to liquid crystal phase transition in yeast from around 60 degrees C to about 40 degrees C, thus allowing yeast rehydrated above 40 degrees C to avoid the damaging effects of passing through a phase transition. These results explain both the need for trehalose and the observation that yeast must be rehydrated with warm water if they are to remain viable. Only when trehalose is present is the dry transition within a physiologically tolerable range and only when the cells are rehydrated above 40 degrees C will they avoid passing through a phase transition.

Cell Membrane↗
PubMed1992

Cryoprotective activity of synthetic glycophospholipids and their interactions with trehalose.

A series of glycophospholipids have been synthesized by conjugating mono-, di- and trisaccharides with dioleoylphosphatidylethanolamine (DOPE) by reductive amination. These synthetic glycophospholipids were incorporated into egg phosphatidylcholine (PC) vesicles and tested for the release of vesicle-entrapped calcein after five cycles of freeze and thaw. N-isomaltotriosyl-DOPE and N-(N-acetylneuramin-lactosyl)-DOPE had approximately equal activity in cryoprotection; whereas N-lactosyl-DOPE and N-galactosyl-DOPE were without effect. However, the cryoprotective effect of trehalose was significantly reduced with the incorporation of N-lactosyl-DOPE and N-galactosyl-DOPE, but not with the trisaccharide-DOPE conjugates. FTIR studies of mixtures of trehalose and glycophospholipid revealed that trehalose forms H-bonding with the phosphodiester group of N-(N-acetylneuramin-lactosyl)-DOPE but not that of N-galactosyl-DOPE. The presence of interaction of trehalose with the trisaccharide-DOPE conjugate and the absence of interaction with the monosaccharide-DOPE conjugate were also demonstrated by the change of the HII phase transition temperature of dielaidoyl phosphatidylethanolamine as measured by the differential scanning calorimetry. The ability of trehalose to restore the membrane order of the rigid dipalmitoyl phosphatidylcholine bilayer perturbed by the presence of glycophospholipids is also correlated with the ability of trehalose to interact with trisaccharide-DOPE conjugate but not with the monosaccharide-DOPE conjugate. These observations have shed some light on the mechanism of the cryoprotective activity of the membrane bound carbohydrates.

Calorimetry, Differential Scanning↗
PubMed1988

Dependence of trehalose protective action on the initial phase state of dipalmitoylphosphatidylcholine bilayers.

Dipalmitoylphosphatidylcholine (DPPC) bilayers hydrated in the presence of trehalose were equilibrated at various temperatures (4, 20, and 60 degrees C) corresponding to the crystalline Lc, gel L beta', and liquid-crystalline L alpha phases, respectively, and then desiccated at these temperatures or freeze-dried at -80 degrees C to ca. DPPC dihydrate. The thermotropic behavior of the resulting DPPC/trehalose mixtures was investigated by differential scanning calorimetry and found to be dependent not only on the trehalose concentration but also on the phase state of the hydrated bilayers prior to their drying. Trehalose was most effective when the desiccation was carried out from the L alpha phase at 60 degrees C. In this case, one trehalose molecule per two DPPC molecules was sufficient to depress the melting temperature from values typical of DPPC dihydrate to 45 degrees C. Trehalose's influence decreased when dried from the L beta' phase and was significantly less pronounced when dried from the Lc phase. These data show that trehalose's protective influence depends on the initial phase state of the lipid bilayer and reaches its maximum in the liquid-crystalline state. The possible role of this effect in anhydrobiosis is pointed out.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana.

Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, contain large amounts of trehalose which they use as a major substrate for energy metabolism and biosynthesis for development under aerobic conditions at 25 degrees C. When cysts are placed at 42 degrees C (heat shock) these pathways stop, and the cysts re-synthesize the trehalose that was utilized during the previous incubation at 25 degrees C. Glycogen and glycerol, produced from trehalose at 25 degrees C, appear to be substrates for trehalose synthesis during heat shock. Anoxia prevents trehalose synthesis in cysts undergoing heat shock. These results are consistent with the view that trehalose may play a protective role in cells exposed to heat shock, and other environmental insults, in addition to being a storage form of energy and organic carbon for development.

Animals↗
PubMed1993

Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases.

Trehalose-6-phosphate (P) competitively inhibited the hexokinases from Saccharomyces cerevisiae. The strongest inhibition was observed upon hexokinase II, with a Ki of 40 microM, while in the case of hexokinase I the Ki was 200 microM. Glucokinase was not inhibited by trehalose-6-P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose-6-P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia lipolytica, was particularly sensitive to the inhibition by trehalose-6-P: when assayed with 2 mM fructose an apparent Ki of 5 microM was calculated. Two S. cerevisiae mutants with abnormal levels of trehalose-6-P exhibited defects in glucose metabolism. It is concluded that trehalose-6-P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.

Binding, Competitive↗
PubMed1992

Construction of Saccharomyces cerevisiae strains that accumulate relatively low concentrations of trehalose, and their application in testing the contribution of the disaccharide to stress tolerance.

Genetically related diploid strains of Saccharomyces cerevisiae that accumulate varied amounts of trehalose during starvation for nitrogen have been constructed. Strains that produced greater than 5% trehalose (dry cell weight) were more tolerant of thermal, or freeze-thaw stresses than strains that produced less than 4% trehalose. Thus trehalose appears to play a role in stress tolerance of yeast. The significance of these results is that, for the first time, a series of related, unmutated strains have been used to test the effect of trehalose on thermotolerance. Previous studies employed either heat shock treatment, or mutated strains to provide trehalose variations, and as such the contribution of the disaccharide to stress tolerance could not necessarily be separated from other factors such as heat shock proteins.

Hot Temperature↗
PubMed1994

Intracellular transduction of trehalose synthesis by hypertrehalosemic hormone in the fat body of the tropical cockroach, Blaberus discoidalis.

Fat body of adult male Blaberus discoidalis cockroaches exposed to B. discoidalis hypertrehalosemic hormone (HTH) in vitro showed a decline in tissue glycogen as carbohydrate increased in the medium. In vivo HTH injections increased hemolymph carbohydrate and fat body glycogen phosphorylase activity > 2-fold compared to controls. In vivo trehalose synthesis was unaffected by agents that enhance intracellular levels of cyclic nucleotides including: dibutyrl cAMP, dibutyryl cGMP, forskolin (adenylyl cyclase activator) and isobutyl methylxanthine (IBMX) or theophylline (cAMP phosphodiesterase inhibitors). DbcAMP+IBMX stimulated trehalose biosynthesis of fat body in vitro and had additive effects with a minimally stimulatory HTH concentration. However, adenylyl cyclase activity was unaffected by HTH either with isolated fat body or fat body membrane preparations. We conclude that cAMP is not a second messenger for HTH, but cAMP can stimulate trehalose production independent of HTH through actions on common regulatory events related to trehalose biosynthesis. Dibutyryl cGMP and phorbol esters and diacylglycerol (activators of protein kinase C) also failed to stimulate trehalose biosynthesis in vitro. Extracellular Ca2+ enhanced HTH-dependent phosphorylase activity, glycogenolysis and hypertrehalosemia and maintained basal levels of phosphorylase a at twice those observed in the absence of Ca2+. However, Ca2+ entry by Ca2+ ionophore (A23187) did not mimic HTH effects. Results of these studies demonstrated that extracellular Ca2+ is important for HTH-dependent trehalose biosynthesis but cAMP and cGMP are not involved.

3',5'-Cyclic-AMP Phosphodiesterases↗