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PubMed2005

High-resolution TOF-SIMS imaging of eukaryotic cells preserved in a trehalose matrix.

A novel, trehalose-glycerol matrix was utilized to generate high-resolution, TOF-SIMS images of macrophages and glial cells. Viable cells incubated in 50 mM trehalose, then lyophilized in a 50 mM trehalose, 10-15% (w/w) glycerol rinse, are preserved and chemically profiled. These experiments demonstrate the utility of the disaccharide matrix as an efficient, cost-effective alternative to cryogenics for SIMS and other ultrahigh-vacuum (UHV) analyses of biological species. Cellular processes on oligodendrocytes and astrocytes, 1-3 mum in width, were well resolved for cells in the trehalose-glycerol matrix. The viscous cell matrixes were fractured and analyzed at room temperature and maintained their three-dimensional integrity under UHV. Images have been generated with a Au primary ion source near the static limit of 10(12) ions/cm2. Though these nucleated cells do not remain viable after desiccation, TOF-SIMS imaging and subsequent rehydration reveals structural and morphological preservation. Eliminating the inherent obstacles associated with cryogenic analysis opens the door to greater utility of SIMS as a bioanalytical tool, such as lipid mapping of single cells in the nervous system.

Animals↗
PubMed1997

Trehalose prevents myoglobin collapse and preserves its internal mobility.

A quantitative model, which involves diffusion on a temperature-dependent potential, is utilized to analyze the time-dependence of geminate CO recombination to sperm whale myoglobin in a trehalose glass and the accompanying spectral shifts. Most of the recombination is inhomogeneous. This is due to higher geminate reactivity rather than slower protein relaxation. A fraction of the hemes undergoes relaxation with a concomitant increase in the barrier height for recombination. The activation energy for conformational diffusion (relaxation) is considerably lower than in glycerol/water. "Protein collapse", manifested in glycerol/water by a decrease in the equilibrium conformational separation between the bound and deoxy states, is completely prevented in trehalose. We postulate that the high internal viscosity in glycerol/water is due to dehydration of the heme pocket. Trehalose prevents the escape of the few vital internal water molecules and thus preserves the internal lability of the protein. This might be important in understanding the ability of trehalose to protect against the adverse effects of dehydration.

Animals↗
PubMed1999

Enhancement of the foaming properties of protein dried in the presence of trehalose.

Surface tension, foamability, and foam stability kinetics have been measured for the pure proteins bovine serum albumin (BSA) and beta-lactoglobulin, before and after aqueous solutions of the proteins had been subjected to different drying conditions, and also for whey protein concentrate (WPC). Pure proteins were air-dried, at 78 or 88 degrees C, in the presence and absence of sucrose or trehalose, at a mass ratio of 5:1 sugar/protein. WPC was spray-dried in the presence of various sugars: trehalose, sucrose, lactose, and lactitol. Spray-drying WPC without sugars resulted in a dramatic decrease in the foam stability, whereas drying in the presence of sugars gave better retention of the original foaming properties. Trehalose in particular resulted in almost complete retention of the foam stability observed for the nondried WPC. Pure beta-lactoglobulin showed similar behavior, but trehalose did not seem to afford the same protection to BSA.

Animals↗
PubMed2002

Effects of sucrose and trehalose on the preservation of the native structure of spray-dried lysozyme.

PURPOSE: To investigate the effects of sucrose, trehalose, sucrose/ dextran mixtures, and sucrose/trehalose mixtures on the preservation of the native structure of spray-dried lysozyme in the solid state. METHODS: The intensity of the alpha-helical band and the melting enthalpies (deltaH(m)) of spray-dried lysozyme in the dried form and in aqueous solution were obtained using second derivative FTIR and differential scanning calorimetry (DSC) respectively. RESULTS: The intensity of the alpha-helical band and the deltaH(m) of spray-dried lysozyme obtained were linearly correlated and both suggest that the stabilization of lysozyme in the dried form was excipient concentration-dependent with a close to maximum stabilization being conferred by sucrose or trehalose at a mass ratio 1-2 (sugar:enzyme). Sucrose appeared to be more effective than trehalose on a weight by weight basis whilst stabilizing effects of dextran/sucrose or trehalose/ sucrose mixtures were found to be additive. CONCLUSION: Dehydration during spray drying was considered the main stress to the denaturation of lysozyme. A major effect of the sugars in protecting lysozyme against dehydration was attributable to hydrogen bonding between the sugar and protein molecules, which lead to an increase in the change in the negative value of the free energy between native and denatured states.

Chemistry, Pharmaceutical↗
PubMed2004

Effect of aging on the physical properties of amorphous trehalose.

PURPOSE: The purpose of this investigation was i) to study the effect of physical aging on crystallization and water vapor sorption behavior of amorphous anhydrous trehalose prepared by freeze-drying, and ii) to determine the effects of water sorption on the relaxation state of the aged material. METHODS: Freeze-dried trehalose was aged at 100 dgrees C for varying time periods to obtain samples with different degrees of relaxation. The glass transition temperature (Tg) and enthalpic relaxation were determined by differential scanning calorimetry, and the rate and extent of water uptake at different relative humidity values were quantified using an automated vapor sorption balance. RESULTS: Annealing below the Tg caused nucleation in the amorphous trehalose samples, which decreased the crystallization onset temperature on subsequent heating. However, no crystallization was observed below the Tg even after prolonged annealing. Physical aging caused a decrease in the rate and extent of water vapor sorption at low relative humidity values. Moreover, the water sorption removed the effects of physical aging, thus effectively causing enthalpic recovery in the aged samples. This recovery occurred gradually in the glassy phase and was not associated with a glass to rubber transition. We believe this aging reversal to be due to volume expansion during water sorption in the amorphous structure. CONCLUSIONS: Thermal history of amorphous materials is a crucial determinant of their physical properties. Aging of amorphous trehalose led to nucleation below the Tg, and decrease in rate and extent of water sorption. Sorption of water resulted in irreversible changes in the relaxation state of the aged material.

Absorption↗
PubMed2000

Trehalose expression confers desiccation tolerance on human cells.

Many organisms that withstand desiccation express the disaccharide trehalose. We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector. Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI). Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days. Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water. This study shows that mammalian cells can be engineered to retain viability in the absence of water.

Bacterial Proteins↗
PubMed1997

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae.

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52 degrees C for 4.5 min), ethanol (20% v/v for 30 min), H2O2 (0.3 M for 60 min), rapid freezing (-196 degrees C for 20 min, cooling rate 200 degrees C min-1), slow freezing (-20 degrees C for 24 h, cooling rate 3 degrees C min(-1)), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H2O2 stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H2O2 and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H2O2 tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast.

Acetic Acid↗
PubMed1997

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose.

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.

Amino Acid Sequence↗
PubMed1995

Trehalose as a possible precursor of the sulfated L-galactan in the ascidian tunic.

Among sulfated polysaccharides, those in the tunic of ascidians are unique: their major constituent sugar is galactose, which occurs exclusively in the L-enantiomeric form. Incorporation of D-[14C]glucose into tunic slices in vitro revealed that the cells epimerize D-glucose into L-galactose during biosynthesis of the sulfated polysaccharides. The interconversion of these two sugars involves exchange of hydrogen atoms at the epimerization sites with protons of the medium. Tunic cells also synthesize trehalose, although this disaccharide is not a prominent constituent of the tissue. Pulse-chase experiments using D-[14C]glucose reveal that incorporation of label into trehalose precedes the synthesis of the sulfated L-galactan. In addition, the loss of label from trehalose coincides with the appearance of label in the sulfated L-galactan. Based on these results, we speculate that trehalose in the ascidian tunic may be a precursor of the sulfated L-galactan.

Animals↗
PubMed2005

Role of trehalose transport and utilization in Sinorhizobium meliloti--alfalfa interactions.

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rml021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.

Bacterial Proteins↗
PubMed1991

Trehalose-6-phosphate synthase/phosphatase complex from bakers' yeast: purification of a proteolytically activated form.

A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers' yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4.5-fold), and increased TPS activity at 5 mM-UDP-glucose/10 mM-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (Ki about 5 mM), but increased TPP activity about six-fold (Ka about 4 mM). Phosphate (6 mM) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.

Enzyme Activation↗
PubMed2003

Different roles for the stress-activated protein kinase pathway in the regulation of trehalose metabolism in Schizosaccharomyces pombe.

The Wis1p-Sty1p mitogen-activated protein kinase cascade is a major signalling system in the fission yeast Schizosaccharomyces pombe for a wide range of stress responses. It is known that trehalose functions as a protective metabolite to counteract deleterious effects of environmental stresses. Herein it is reported that the expression of genes related to trehalose metabolism in S. pombe, ntp1(+) (neutral trehalase) and tps1(+) [trehalose-6-phosphate (T6P) synthase], is partially regulated by the Sty1p kinase under salt-induced osmotic stress and conditions of slight oxidative stress and is fully dependent on this kinase under severe oxidative stress. This control is carried out through transcription factors Atf1p/Pcr1p during osmotic stress and through Pap1p during exposure to low levels of oxidative stress. However, all three transcription factors are needed for gene expression under conditions of extreme oxidative stress. In addition, a role for Sty1p in the modulation of post-transcriptional activation of trehalase mediated by Pka1p/Sck1p kinases, as well as in the activity of T6P synthase under such stressful conditions has been demonstrated. These results reveal a novel dual action of the Wis1p-Sty1p pathway in the regulation of trehalose metabolism in fission yeast.

Activating Transcription Factors↗
PubMed1976

[Separation of structural studies of the molecular species of monomycolates and dimycolates of alpha-D-trehalose present in Mycobacterium phlei (author's transl)].

Mixtures of dimycolates of alpha-D-trehalose (cord factor) and monomycolates have been isolated from Mycobacterium phlei and separated as trimethylsilyl derivatives according to the polarity of the fatty acid residues. The free glycolipids can be recovered by mild hydrolysis. Silylation and disilylation reactions did not induce any isomerisation. The structure of these trehalose esters has been determined by using a series of reactions elaborated on synthetic acyl-sugar models. Free hydroxyl groups are transformed into tetrahydropyran ethers, deacylated by dimsyl sodium, methylated and the sugar derivatives are hydrolyzed. The O-methyl-sugars obtained contain a methyl ether group located at the position where the acyl group was present initially. Identification by gas chromatography of the O-methyl-sugars thus allows the location of the fatty acid residues in the glycolipid. It has been demonstrated that no migration occurs. Two types of 6-monomycoloyl-alpha-D-trehalose have been isolated, differing by the nature of the mycolic acid residues. One of them, called MA, contains "alpha-phlei mycolic acid". The other one, called MB, contains "gamma-phlei mycolic acid" which is the ester of 2-eicosanol with the omega-carboxyl function of a dicarboxylic mycolic acid. Three types of 6,6'-dimycoloyl-alpha-D-trehalose (cord factor) have been obtained. Two of them are symmetrical diesters, containing either two residues of alpha-mycolic acid, or two residues of gamma-mycolic acid. The last one is an unsymmetrical diester, the hydrolysis of which gives one mole of alpha-mycolic acid and one mole of gamma-mycolic acid. The ratio of the different diesters in the cord factor fraction might be explained by some equilibrium between the different 6-ester groups or by a transformation in situ of one species into another one by biochemical modification of the mycolic acid residues.

Chromatography, Gas↗
PubMed1995

A novel approach for investigating reaction mechanisms in cells. Mechanism of deoxy-trehalose synthesis in Saccharomyces cerevisiae studied by 1H-NMR spectroscopy.

A new approach is proposed for investigating the mechanism of metabolite synthesis in cells. This method, based on the competition between various substrates, allows the flux along a pathway, which is normally independent of the concentration of the corresponding precursor in the external medium, to be divided into partial fluxes. In particular, the mechanism deoxy-trehalose synthesis in glucose-grown repressed Saccharomyces cerevisiae was studied, by 1H-NMR spectroscopy, using the competition between 2-deoxy-D-glucose (dGlc) and 2-fluoro-deoxy-D-glucose (FdGlc) with respect to hexokinase. S. cerevisiae cells, suspended in a standard pyrophosphate medium containing about 5 x 10(7) cells/ml, were incubated with 30 mM glucose and various concentrations of dGlc and FdGlc. Apart from dGlc6P and FdGlc6P, trehalose and the dissacharides relative to dGlc, i.e. dideoxy-trehalose (dGlc-dGlc) and deoxytrehalose (dGlc-Glc), are observed while their analogues relative to FdGlc (FdGlc-FdGlc, FdGlc-Glc) are surprisingly absent. For the same external concentration of dGlc and FdGlc, the internal concentration of FdGlc6P is about three times larger than that of dGlc6P. The ratio of the FdGlc6P and dGlc6P concentrations is independent of the incubation times and proportional to the FdGlc and dGlc concentrations in the suspension. The dGlc6P concentration can thus be reduced at will by increasing the [FdGlc]/[dGlc] ratio. Under these conditions, the dGlc-Glc concentration was found to vary linearly with that of dGlc6P. The present data clearly show that deoxy-trehalose is not synthesized from UDP-dGlc and Glc6P but from UDP-Glc and dGlc6P. This conclusion was also confirmed by an experiment in which S. cerevisiae cells were previously charged with dGlc6P and then incubated with glucose.

Binding, Competitive↗
PubMed1999

Weak organic acid treatment causes a trehalose accumulation in low-pH cultures of Saccharomyces cerevisiae, not displayed by the more preservative-resistant Zygosaccharomyces bailii.

Weak organic acid food preservatives exert pronounced culture pH-dependent effects on both the heat-shock response and the thermotolerance of Saccharomyces cerevisiae. In low-pH cultures, they inhibit this stress response and cause strong induction of respiratory-deficient petites amongst the survivors of lethal heat treatment. In higher pH cultures, 25 degrees C sorbic acid treatment causes a strong induction of thermotolerance without inducing the heat-shock response. In this study we show that trehalose, a major stress protectant, accumulates rapidly in S. cerevisiae exposed to sorbate at low pH. In pH 3.5 cultures, a 25 degrees C sorbate treatment is as effective as a 39 degrees C heat shock in inducing trehalose. This weak-acid-induced trehalose accumulation is enhanced in the pfk1 S. cerevisiae mutant, indicating that it arises through inhibition of glycolysis at the phosphofructokinase step. The more preservative-resistant food spoilage yeast Zygosaccharomyces bailii differs from S. cerevisiae in that: (1) its basal thermotolerance is not strongly affected by culture pH; (2) it does not display trehalose accumulation in response to 25 degrees C sorbate treatment at low pH; and (3) there is no induction of respiratory-deficient petites during lethal heating with sorbate. This probably reflects Z. bailii being both petite-negative and better equipped for maintenance of homeostasis during weak-acid, pH or high-temperature stress.

Food Preservatives↗
PubMed2001

Changes in glycogen and trehalose content of Streptomyces brasiliensis hyphae during growth in liquid cultures under sporulating and non-sporulating conditions.

Streptomyces brasiliensis ATCC 23727 showed extensive sporulation when cultured in a liquid medium containing galactose and glutamic acid as carbon and nitrogen sources. Under such conditions, glycogen and trehalose are accumulated in the hyphae coinciding with spore formation. The results reported here suggest that glycogen accumulated in sporogenic hyphae is converted into trehalose during the final period of spore maturation. Glycogen is also accumulated in the hyphae when S. brasiliensis is cultured under conditions which did not support sporulation. Under such conditions, however, glycogen degradation is not accompanied by accumulation of trehalose. This suggest that the conversion of glycogen into trehalose might be a sporulation-specific event in streptomycetes.

Glycogen↗
PubMed2005

Autonomous oscillations in Saccharomyces cerevisiae during batch cultures on trehalose.

We report that autonomous oscillations, which usually happen in aerobic glucose-limited continuous cultures of yeast at low dilution rate, were also observed in trehalose discontinuous cultures of Saccharomyces cerevisiae. This unexpected oscillatory behaviour was therefore examined using fast Fourier transformation of online gas measurements. This robust mathematical analysis underlined the existence of two types of oscillation. The first was found to be linked to the cell cycle because (a) the periodicity corresponded to a fraction of the generation time and (b) the oscillations were accompanied by a transient increase in the budding index, mobilization of storage carbohydrates, and fermentative activity. Moreover, these oscillations occurred in a range of specific growth rates between 0.04 and 0.15 h(-1). All these criteria were consistent with the cell-cycle-related metabolic oscillations observed in the same range of growth rates in glucose-limited continuous cultures. The second type were short-period respiratory oscillations, independent of the specific growth rate. Both types of oscillation were found to take place consecutively and/or simultaneously during batch culture on trehalose. In addition, mobilization of intracellular trehalose emerged as a key parameter for the sustainability of these autonomous oscillations as they were no longer observed in a mutant defective in neutral trehalase activity. We propose that batch culture on trehalose may be an excellent device for further investigation of the molecular mechanisms that underlie autonomous oscillations in yeast.

Cell Cycle↗
PubMed2000

Molecular identification of a taste receptor gene for trehalose in Drosophila.

The molecular nature of sweet taste receptors has not been fully explored. Employing a differential screening strategy, we identified a taste receptor gene, Tre1, that controls the taste sensitivity to trehalose in Drosophila melanogaster. The Tre1 gene encodes a novel protein with similarity to G protein-coupled seven-transmembrane receptors. Disruption of the Tre1 gene lowered the taste sensitivity to trehalose, whereas sensitivities to other sugars were unaltered. Overexpression of the Tre1 gene restored the taste sensitivity to trehalose in the Tre1 deletion mutant. The Tre1 gene is expressed in taste sensory cells. These results provide direct evidence that Tre1 encodes a putative taste receptor for trehalose in Drosophila.

Animals↗