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PubMed2000

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae.

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.

Agrobacterium tumefaciens↗
PubMed1990

On the determination of trehalose-6-phosphate synthase in Saccharomyces.

Trehalose-6-phosphate synthase activity was determined by colorimetric, spectrophotometric and trehalose specific assays. All methods gave comparable results thus confirming our previous findings (1) and those reported by Elander (2). Different strains and mutants of Saccharomyces were carefully re-investigated in relation with the recent claim made by Vandercammen et al. (3) that our spectrophotometric assay over-estimated the enzyme activity and that no differences exist between wild type and mutant strains. In this paper we also confirm the de-activation of the trehalose synthase complex in response to a "glucose signal", and present trehalose-6-phosphate synthase and trehalase activities in different strains measured during all phases of growth on glucose.

Colorimetry↗
PubMed1989

Participation of tumor necrosis factor in the antitumor activity of mycobacterial trehalose dimycolate (cord factor).

Trehalose dimycolate, a mycobacterial glycolipid also known as cord factor, retains some of the antitumor properties of the intact BCG. Murine macrophages incubated in vitro in the presence of trehalose dimycolate for 20 h at 37 degrees C released a factor which was cytotoxic for the L929 tumor cell line. Anti-tumor necrosis factor (TNF) blocked the cytotoxicity of trehalose dimycolate macrophage supernatant. Therefore the antitumor activity of trehalose dimycolate can be attributed to TNF released in the in vitro assay.

Animals↗
PubMed2002

A highly efficient method for long-chain cDNA synthesis using trehalose and betaine.

Obtaining full-length cDNA is important for many molecular biology methods like cDNA library construction or RACE-PCR (rapid amplification of cDNA ends). We have found that the inclusion of betaine alone and in combination with trehalose in reverse transcription results in longer cDNA synthesis products. As shown on the 14-kb long clarithrin mRNA with real-time PCR, a combination of 2 M betaine and 0.6 M trehalose leads to almost 9 times more cDNA with a length of 12.5 kb. This is due to the ability of betaine to resolve secondary structures of the RNA, thereby decreasing its melting temperature. The application of betaine in combination with trehalose should prove useful in all laboratory methods relying on full-length cDNA. (USA).

Betaine↗
PubMed1993

A small influence of HSP90 levels on the trehalose and heat shock element inductions of the yeast heat shock response.

Heat shock protein (HSP) induction is only one change elicited in yeast by heat shock. Trehalose is also accumulated, while declining intracellular pH stimulates plasma membrane ATPase activity. Recently the trehalose induction was shown to be regulated by levels of HSP70 and, to a lesser extent, HSP104. Another HSP which might contribute to regulation is HSP90, especially as HSP90 forms complexes with heat shock transcription factor and several of the regulatory proteins of eukaryotic cells. This possibility was investigated using isogenic yeast strains with normal, decreased or elevated HSP90. The results show HSP90 levels having a small negative influence over the heat inductions of trehalose and the heat shock element, a minor effect compared with the major regulation exerted by HSP70.

Genotype↗
PubMed1995

Induction of heat-shock proteins and accumulation of trehalose by TPN in Saccharomyces cerevisiae.

TPN [Tetrachloroisophthalonitrile], a kind of disinfectant, affected growth in Saccharomyces cerevisiae. Exposed to TPN under no lethal conditions, 70-, 90-kDa protein and hsp104 were induced. Each of them was not uniformly induced; namely, the 70-kDa protein was more sensitive to TPN among other proteins. Trehalose was also accumulated depending on the concentration. The degree of thermotolerance in yeast cells pretreated with 1.0 mg/l TPN was about 100-fold greater than in the control. Under this condition, TPN-inducible proteins were synthesized but trehalose was not accumulated. Although TPN-inducible proteins and trehalose were definitely synthesized with 10.0 or 100.0mg/l TPN treatment, thermotolerance was not acquired.

Blotting, Western↗
PubMed1993

Effect of sucrose, trehalose, hypotaurine, taurine, and blood serum on survival of frozen bull sperm.

Factorially arranged experiments were conducted to study the effects of adding sucrose and trehalose, known to have cryoprotective properties, blood serum, and the antioxidants taurine and hypotaurine on sperm motility after freezing and thawing at different rates. At sugar concentrations of 0.1 M, osmolality of the whole milk (WM) or egg yolk-Tris freezing medium (without glycerol) was about 370 mOsmol. Survival following freezing and thawing was reduced unless osmolality was corrected by adding pure water to reduce osmolality to about 280 mOsmol. Then the post-thaw percentages of motile sperm for control WM, and WM with 0.05 or 0.10 M sucrose, or 0.05 or 0.10 M trehalose, respectively, were 62, 55, 61, 57, and 62%. Thawing semen at 5 degrees C, versus 25 degrees C resulted in 64 versus 70% motile sperm (P < 0.05). Post-thaw survival of sperm stored at 25 degrees C for 24 h in trehalose-treated WM was superior to that in WM (P < 0.05). Hypotaurine and taurine had little effect on sperm survival. Up to 10% (v/v) of heated blood serum was generally beneficial, but gave more variable responses with different bulls. Sperm survival after cooling at -25 degrees C/min was slightly superior to that after cooling at -15 degrees C/min to -100 degrees C. The effects of the compounds studied on motility of frozen-thawed sperm were small, but if they protect sperm cell membranes, as reported for other types of membranes, they may assist sperm in surviving in the reproductive tract of the cow prior to fertilization.

Animals↗
PubMed1993

Protection of freeze-dried Escherichia coli by trehalose upon exposure to environmental conditions.

Freeze-dried cultures of wild-type and genetically engineered strains of Escherichia coli lost their colony-forming ability upon exposure to air, visible light, and certain relative humidity levels. Both strains could be maximally protected from these lethal effects with 100 mM trehalose, a concentration calculated to just saturate the interphospholipid spaces in the cell membrane, thus preserving the liquid-crystalline structure. The trehalose protection was observed for at least 96 h. Trehalose increased viability as much as 2000-4000% over nontreated populations. In all cases, exposure to environmental conditions was more damaging to the genetically engineered strain.

Cryoprotective Agents↗
PubMed1997

Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing.

The influence of the presence of trehalose or sucrose, the osmolality of the freezing medium, and the cooling rate on success of cryopreservation of bull sperm were investigated. Bull semen was frozen at four different cooling rates varying from 40 to 300 degrees C/min in standard Tris-egg yolk medium and in hypertonic or isotonic media containing 0.2 M trehalose or sucrose. Sperm motility, H33258 exclusion, and acrosome morphology were evaluated. In the hypertonic media the motility was almost completely inhibited, but could partly be restored by returning to isotonic conditions. Medium composition and cooling rate had significant effects, with a strong interaction of medium x cooling rate. The optimal cooling rate was found to be between 76 and 140 degrees C/min. At the highest cooling rate, both the presence of the sugars and the high osmolality of the hypertonic media conferred significant protection against fast-cooling damage, sucrose being significantly better than trehalose. At all other cooling rates the medium differences were less pronounced, but the isotonic sugar media were generally superior to standard medium as to preservation of acrosome intactness and H33258 exclusion. This indicates that the isotonic sugar media could enable some improvement of cryopreservation of bull semen over the currently used Tris-egg yolk medium. Further improvement of sperm survival at the AI station should be possible by implementation of controlled rate freezing, using a cooling rate in the optimal range between 76 and 140 degrees C/min.

Animals↗
PubMed1997

Fatty acids stimulate trehalose synthesis in trophocytes of the cockroach (Periplaneta americana) fat body.

Trophocytes from the disaggregated fat body of the cockroach (Periplaneta americana) respond to synthetic hypertrehalosemic hormone (HTH) by increasing the rate of trehalose synthesis. The cells give a similar response when incubated with stearic, oleic, linoleic, or arachidonic acid. A maximal increase in trehalose synthesis was obtained with 1-10 microM fatty acids. Synthesis of trehalose by the trophocytes was also increased by 1 microM prostaglandin F2alpha to nearly the same extent as that evoked by HTH. Furthermore, the data show that the trophocytes are capable of converting linoleic acid into arachidonic acid. This suggests that the cells may convert arachidonic acid, formed from the linoleic acid released by the action of HTH, to a prostaglandin which serves as an integral part of the hypertrehalosemic mechanism.

Animals↗
PubMed1996

Preservation of skin free-flap using trehalose.

In this study, we looked for a solution able to preserve traumatically amputated tissues such as nose tip, ear, and scalp for at least 48 hr that was easy to handle and low in price. Trehalose is a nonreducing disaccharide that can protect many prokaryotes, fungi, and yeasts against adverse conditions by stabilizing cell membranes. In order to study its protective effect, 60 skin free-flaps of the ears of rabbits were preserved in Euro-Collins (EC) solution or in modified Euro-Collins solution in which 7% trehalose replaced glucose (EC + 7%T) for 24, 48, and 72 hr at 4 degrees C. After completion of preservation, these flaps were transferred to the other ear with microvascular anastomosis. After 7 days, we obtained a survival rate of 100% of flaps after preservation for 24 hr in both solutions. But, after preservation for 48 hr or longer, this decreased to 60% (6 of 10 preserved flaps survived) in EC solution and 90% (9 of 10 flaps) in EC + 7%T solution. This difference became clearer after 72 hr of preservation, when the survival rate was higher and light microscopic observation showed less damage for flaps preserved in EC + 7%T solution (20% in EC and 70% in EC + 7%T) (P < 0.05). Thus, EC + 7%T solution was superior to EC solution in the preservation of skin free-flaps of the ear of rabbits. Trehalose shows great promise in the preservation of many organs, including storage of traumatically amputated tissues such as nose tip, ear, and scalp.

Adipose Tissue↗
PubMed1992

Levels of trehalose and glycogen in Arthrobacter globiformis under conditions of nutrient starvation and osmotic stress.

Cells of Arthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-Mr carbohydrates (800 micrograms/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-Mr carbohydrate levels increased to 1300 micrograms/mg protein. Low-Mr pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of alpha,alpha-trehalose. Levels of trehalose in Arthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties of Arthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.

Actinomycetales↗
PubMed1975

Trehalose of Culex pipiens fatigans.

Trehalose was found to occur in the mosquito, Culex pipiens fatigans, to the extent of 23 to 49% of total free sugars in different developmental stages of the insect. Induction of stress to the insects led to significant reduction in the trehalose of larvae and glucose of adults. The label from alpha,alpha'-U-14C-trehalose readily incorporated into lipids of the adults in which triglycerides seemed to undergo rapid turnover.

Animals↗
PubMed2002

Low-temperature-induced changes in trehalose, mannitol and arabitol associated with enhanced tolerance to freezing in ectomycorrhizal basidiomycetes (Hebeloma spp.).

Ectomycorrhizal fungi have been shown to survive sub-zero temperatures in axenic culture and in the field. However, the physiological basis for resistance to freezing is poorly understood. In order to survive freezing, mycelia must synthesise compounds that protect the cells from frost damage, and certain fungal-specific soluble carbohydrates have been implicated in this role. Tissue concentrations of arabitol, mannitol and trehalose were measured in axenic cultures of eight Hebeloma strains of arctic and temperate origin grown at 22, 12, 6 and 2 degrees C. In a separate experiment, mycelia were frozen to -5 degrees C after pre-conditioning at either 2 degrees C or 22 degrees C. For some, especially temperate strains, there was a clear increase in specific soluble carbohydrates at lower growth temperatures. Trehalose and mannitol were present in all strains and the highest concentrations (close to 2.5% and 0.5% dry wt.) were recorded only after a cold period. Arabitol was found in four strains only when grown at low temperature. Cold pre-conditioning enhanced recovery of mycelia following freezing. In four out of eight strains, this was paralleled by increases in mannitol and trehalose concentration at low temperature that presumably contribute towards cryoprotection. The results are discussed in an ecological context with regard to mycelial overwintering in soil.

Agaricales↗
PubMed2007

Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis for sugars monitoring.

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection.

Archaeal Proteins↗
PubMed1998

Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch.

Enzymes that convert starch and dextrins to alpha,alpha-trehalose and glucose were found in cell homogenates of the hyperthermophilic acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from alpha-1,4 to alpha-1,1. The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the alpha-1,4 linkage adjacent to the alpha-1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively, and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70 degrees C for SsTDFE and 85 degrees C for SsTFE. The third enzyme identified was an alpha-glycosidase (Ss alpha Gly), which catalyzed the hydrolysis of the alpha-1,4 glucosidic linkages in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85 degrees C.

Glucose↗
PubMed2006

Combined effect of betaine and trehalose on osmotic tolerance of Escherichia coli in mineral salts medium.

In mineral salts medium, supplementing with betaine in combination with increased production of endogenous osmoprotectant from a second copy of the trehalose biosynthetic genes (otsBA) improved growth of E. coli and increased the MIC for xylose, glucose, sodium lactate and NaCl. With these compounds, this combination was more effective than either betaine or trehalose alone. With succinate, this combination was no more effective than betaine alone. Neither approach improved tolerance to ethanol. A combination of betaine and increased trehalose may improve strain productivity for many bioproducts by promoting growth in the presence of high sugar concentrations.

Betaine↗
PubMed2005

Comparative investigation by two analytical approaches of enthalpy relaxation for glassy glucose, sucrose, maltose, and trehalose.

PURPOSE: In an effort to understand the stability of glassy sugars such as glucose, sucrose, maltose, and trehalose, the molecular mobility below the glass transition temperature (Tg) was investigated by an enthalpy relaxation measurement with differential scanning calorimetry (DSC). METHODS: The glassy sample was aged over several days at (Tg - 10) K to (Tg - 30) K, before a DSC heating scan was taken. The relaxed enthalpy (deltaHrelax) was estimated from the endothermic peak area. The enthalpy relaxation time was analyzed from the time course of deltaHrelax using two different approaches; Kohlrausch-Williams-Watts (KWW) and extended Adam-Gibbs (exAG). RESULTS: TauKWW, which is defined as the mean average enthalpy relaxation time in a distribution, and tau(eff)0 and tau(eff)infinity, which correspond to the enthalpy relaxation time of the initial minimum and final maximum cooperative rearrangement region, were estimated by KWW and exAG, respectively. And three activation energies for enthalpy relaxation were calculated from the Arrhenius plot. CONCLUSIONS: Although these deltaEs originated from different theoretical backgrounds, almost the same trend was observed for a comparison of the values of the four sugars. The finding that the deltaEs of glassy trehalose were the largest among the four sugars may support the reason that glassy trehalose is an effective stabilizer.

Calorimetry, Differential Scanning↗