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PubMed1998

Evaluation of new improved solution containing trehalose in free skin flap storage.

BACKGROUND DATA: In this study, we evaluated a new intracellular type (IT-K) solution containing trehalose in a rabbit free skin flap storage model. Trehalose is a nonreducing disaccharide that can stabilise cell membranes under various stressful conditions. MATERIAL AND METHOD: Seventy-two free skin flaps of the ear of rabbits were preserved in Euro-Collins (EC) solution or in IT-K solution for 24, 48, and 72 h at 4 degrees C. After completion of preservation, these flaps were replanted to the other ear by microsurgical techniques. Viability study and photo documentation were performed daily for 7 days. Tissue specimens were taken 24 h after vascular anastomosis, fixed in 10% formaldehyde and stained with haematoxylin and eosin (HE). Survival rates were analysed by Fisher's exact test for comparison of the two experimental groups. Values of P < 0.05 were considered to be statistically significant. RESULTS: After 7 days, a survival rate of 100% of flaps were observed in both solutions after 24 h of preservation. After preservation for 48 h in IT-K solution the survival rate was 100%. However, in EC solution survival decreased to 75% (9 of 12 preserved flaps survived). This difference increased to 33.3% (4 of 12 flaps) in EC solution and 91.6% (11 of 12 flaps) (P < 0.01) in IT-K solution when the flaps were stored for 72 h. Light microscopic examination also showed less damage in flaps preserved in IT-K solution than in these preserved in EC solution. CONCLUSION: IT-K solution was superior to EC solution in the preservation of free skin flaps on rabbit ears when stored for 48 and 72 h.

Animals↗
PubMed2003

Drosophila Gr5a encodes a taste receptor tuned to trehalose.

Recent studies have suggested that Drosophila taste receptors are encoded by a family of G protein-coupled receptor genes comprising at least 56 members. One of these genes, Gr5a, has been shown by genetic analysis to be required by the fly for behavioral and sensory responses to a sugar, trehalose. Here, we show that Gr5a is expressed in neurons of taste sensilla located on the labellum and legs. Expression is observed in most if not all labellar sensilla and suggests that many taste neurons express more than one receptor. We demonstrate by heterologous expression in a Drosophila S2 cell line that Gr5a encodes a receptor tuned to trehalose. This is the first functional expression of an invertebrate taste receptor.

Animals↗
PubMed1982

Radiolysis of alpha, alpha-trehalose in concentrated aqueous solution; the effect of co-irradiated proteins and lipids.

gamma-Radiolysis (dose-rate: 0 X 89 Gy/s) or electron (e)-radiolysis (dose-rate: 5 X 10(7) Gy/s) of unbuffered aqueous solutions of alpha, alpha-trehalose (concentration: 60 mg/ml, radiation dose: 20 kGy) at 0 degree C yielded glucose (Ggamma = 1 X 7; Ge = 0 X 63) and 5-deoxyxylohexodialdose (Ggamma = 0 X 21; Ge = 0 X 05). Buffering at pH-values of 5 X 0 or 5 X 5 and irradiation caused increased formation of these monomeric products, particularly of the deoxy-compound. On addition of increasing amounts of bovine serum albumin or ovalbumin (10-30 mg/ml) and irradiation the yields of products were markedly reduced. The decrease in glucose formation was less pronounced when sperm whale myoglobin was present during gamma- or electron-irradiation. The G-values of 5-deoxyxylohexodialdose, however, were increased by 45 per cent (gamma-irradiation) and 70 per cent (electron-irradiation) at approximately 10 mg/ml of admixed myoglobin. Further increase in myoglobin concentration led to a gradual decrease in the yields of the deoxy-product. The observed effects are explained by scavenging of water radicals and by interactions of the added substrates with sugar radicals. Emulsified lipids (palmitic acid methylester or trilinoleic glycerol) did not affect the radiation-induced formation of products from trehalose.

Deoxy Sugars↗
PubMed2004

Freeze-drying of mannitol-trehalose-sodium chloride-based formulations: the impact of annealing on dry layer resistance to mass transfer and cake structure.

The objective of this article was to study the mechanism by which annealing increases the primary drying time in mannitol-trehalose-sodium chloride-based formulations. The thermal events occurring during annealing and the glass transition of the frozen solutions were monitored with differential scanning calorimetry (DSC). Manometric temperature measurement was used to evaluate the dry layer resistances during primary drying. The morphologies of the freeze-dried cakes were examined by scanning electron microscopy (SEM). The degrees of crystallinity of mannitol and sodium chloride (NaCl) in freeze-dried cakes were determined by powder X-ray diffraction (XRD). DSC results indicated that annealing during freezing did not increase the glass transition temperature (Tg') significantly, but there was a distinct decrease of deltaCp at Tg' with annealing, suggesting a decrease in amorphous content. SEM revealed that most mannitol crystallized as the delta-form during annealing at -23 degrees C, and further crystallized as the alpha-form, together with NaCl crystallization, during subsequent annealing at -33 degrees C. The powder XRD results demonstrated that annealing caused crystal growth of mannitol and NaCl, and thus prevented the partial collapse observed without annealing. However, the highly crystallized mannitol blocked the pathways for water vapor escape, contributing to the increase in the dry layer resistance and thus the longer times for primary drying. Freeze-dried cakes without annealing had lower dry layer resistances because partial collapse created larger channels for water vapor escape. Therefore, two-step annealing in freezing makes mannitol-trehalose-sodium chloride-based formulations robust in freeze-drying, but annealing increases the dry layer resistances, thereby extending primary drying.

Chemistry, Pharmaceutical↗
PubMed2003

Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells.

Our previous studies had shown that a combination of the bio-antioxidant catalase and the membrane stabilizer trehalose in the conventional freezing mixture affords better cryoprotection to hematopoietic cells as judged by clonogenic assays. In the present investigation, we extended these studies using several parameters like responsiveness to growth factors, expression of growth factor receptors, adhesion assays, adhesion molecule expression, and long-term culture-forming ability. Cells were frozen with (test cells) or without additives (control cells) in the conventional medium containing 10% dimethylsulfoxide (DMSO). Experiments were done on mononuclear cells (MNC) from cord blood/fetal liver hematopoietic cells (CB/FL) and CD34(+) cells isolated from frozen MNC. Our results showed that the responsiveness of test cells to the two early-acting cytokines, viz. interleukin-3 (IL-3) and stem cell factor (SCF) in CFU assays was better than control cells as seen by higher colony formation at limiting concentrations of these cytokines. We, therefore, analyzed the expression of these two growth factor receptors by flow cytometry. We found that in cryopreserved test MNC, as well as CD34(+) cells isolated from them, the expression of both cytokine receptors was two- to three-fold higher than control MNC and CD34(+) cells isolated from them. Adhesion assays carried out with CB/FL-derived CD34(+) cells and KG1a cells showed significantly higher adherence of test cells to M210B4 than respective control cells. Cryopreserved test MNC as well as CD34(+) cells isolated from them showed increased expression of adhesion molecules like CD43, CD44, CD49d, and CD49e. On isolated CD34(+) cells and KG1a cells, there was a two- to three-fold increase in a double-positive population expressing CD34/L-selectin in test cells as compared to control cells. Long-term cultures (LTC) were set up with frozen MNC as well as with CD34(+) cells. Clonogenic cells from LTC were enumerated at the end of the fifth week. There was a significantly increased formation of CFU from test cells than from control cells, indicating better preservation of early progenitors in test cells. Our results suggest that use of a combination of catalase and trehalose as a supplement in the conventional freezing medium results in better protection of growth factor receptors, adhesion molecules, and functionality of hematopoietic cells, yielding a better graft quality.

Antigens, CD34↗
PubMed2001

Trehalose becomes the most abundant non-structural carbohydrate during senescence of soybean nodules.

Carbohydrate metabolism and symbiont survival were studied in nodules of soybean (G. max [L.] Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101), induced to senesce simultaneously by application of the photosynthesis inhibitor dichloromethyl urea (DCMU). The plant-borne carbohydrates sucrose and starch started to decline after 2 d and reached background levels after 8 d, in parallel with the decline of nitrogenase. However, the microsymbiont-borne disaccharide trehalose declined only by about 40% and subsequently remained at a constant level of c. 6 mg x g(-1) dry weight up to 14 d, when nodules softened and decayed. The number of re-isolated viable bacteria was not significantly decreased in senescent nodules as compared to control nodules. These results indicate that during terminal senescence of nodules an appreciable part of the bacteria conserve their trehalose pools and survive.

Bradyrhizobium↗
PubMed1986

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus.

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.

Disaccharides↗
PubMed2005

A combination of catalase and trehalose as additives to conventional freezing medium results in improved cryoprotection of human hematopoietic cells with reference to in vitro migration and adhesion properties.

BACKGROUND: Cryopreservation of hematopoietic cells from cord blood is an essential component in unrelated transplant settings. Cell damage during freezing is caused by multiple factors, of which membrane damage and oxygen free radical generation are two major factors. It was reported earlier that a combination of catalase and trehalose as additives in freezing medium affords better cryoprotection in terms of long-term culture assays. STUDY DESIGN AND METHODS: Mononuclear and CD34+ cells isolated from cord blood were used as a source of hematopoietic cells. KG1a cell line was used as a model system in adhesion assays. The cells were frozen in a programmable freezer and stored at -196 degrees C. Various homing-related assays were carried out on the frozen cells. RESULTS: Herein it is reported that these two additives afford better preservation of adhesion- and migration-related properties of the frozen cells. The test cells frozen with additives resulted in improved migration toward stromal-derived factor-1alpha and showed higher expression of its receptor CXCR4. Colony-forming unit assay of migrated test cells showed that these cells are early progenitors having capacity to give rise to all types of myeloid colonies. Test cells also show increased expression of FLT3R and improved responsiveness to FLT3 ligand, the homing-related cytokine. Adhesion to stroma and extracellular matrix was better in test cells as compared to control cells. CONCLUSION: The present data provide evidence that addition of catalase and trehalose to the conventional freezing medium preserves migration- and adhesion-related properties of the hematopoietic graft.

Antibodies↗
PubMed1976

Transglycosylic reactions of nucleotides of deoxy sugars. Biosynthesis of deoxy analogues of alpha,alpha'-trehalose.

The biosynthesis of alpha,alpha'-trehalose was investigated in a reaction catalyzed with yeast UDP-Glc :glucose-6-phosphate 1-glucosyl transferase. It was revealed that the hydroxyls at positions C-2, C-3, C-4 and C-6 are not essential for preserving its substrate properties in this reaction. The affinity of the enzyme and the rate of hexose incorporation into trehalose decreases in following sequence: UDP-glucose, UDP-2-deoxy-D-glucose, UDP-6-deoxy-d-glucose, UDP-4-deoxy-D-glucose and UDP-3-deoxy-D-glucose.

Deoxy Sugars↗
PubMed1979

Property and activity of mycoloyl esters of methyl glucoside and trehalose. Effect on mitochondrial oxidative phosphorylation related to organization of suspensions and to acyl-chain structures.

Methyl alpha-D-glucoside and alpha, alpha'-D-trehalose acylated on their primary hydroxyl group by alpha-alkyl-beta-hydroxy acids (mycolic acids) with 18, 32 and more than 80 carbon atoms were studied. These cord factors and analogues when dispersed in water gave hydrated and organized phases which bore resemblances and differences to phospholipid liposomes. Phase transition temperature was determined by fluorescence for the C32 derivatives and by differential scanning calorimetry for the C80 derivatives. A narrow temperature interval between the gel and the fluid states was observed, indicating that this transition was a strongly cooperative phenomenon. The activity on mitochondria of these diacyl trehaloses was studied with succinate as substrate. The fluid suspensions were more active than the rigid ones. There was no influence of the relative configuration of the mycoloyl residue. Acyl chains with 18, 32 and more than 80 carbon atoms had rather similar activities on phosphorylation, but differences appeared when considering effects on respiration. Electron microscopy suggested that cord factor weakened the peripheral part of the inner membrane, but cristae looked unchanged. These observations, together with the results of our previous monolayer studies of these glycolipids, led us to propose a model for interaction of cord factor with mitochondria.

Animals↗
PubMed1976

Correlation of the characters fermentation of trehalose, non-transmissible resistance to tetracycline, and relatively long flagellar wavelength in Proteus morganii.

An investigation of strains of Proteus morganii isolated from patients in Odense, Denmark, and of 21 serotype strains revealed a close correlation between the fermentation of trehalose and a non-transmissible resistance to tetracycline. The trehalose fermenting, tetracycline resistant strains had a longer flagellar wavelength than the non-fermenting, sensitive strains. Resistance to chloramphenicol occurred only in strains of the former group.

Chloramphenicol↗
PubMed1980

The occurrence of the trehalose fermenting, tetracycline and polymyxin resistant phenotype among the Enterobacteriaceae.

In Proteus morganii, P. mirabilis, and Providencia stuartii the ability to ferment trehalose and resistance to tetracycline were associated in 90%-97% of the strains. The same was true of at least 78% of the strains of Serratia marcescens. Proteus vulgaris showed a more quantitative association of the two traits. As the characters occur independently in 3-10% of the strains, the association is considered to be due to simultaneous selection in some natural niche. The trehalose fermenting, tetracycline and polymyxin resistant species ferment few other carbohydrates, fewer than the remainder of the Serratia species.

Drug Resistance, Microbial↗
PubMed1961

Blood trehalose and flight metabolism in the blowfly.

The concentration of trehalose in the blood of Phormia regina was found to determine the rate of energy expenditure during flight as reflected in measurements of the wing-beat frequency. Fat body was found to be the source of blood trehalose; either endogenous or exogenous substrates are used for its synthesis.

Adipose Tissue↗
PubMed1996

Disruption of the SNF1 gene abolishes trehalose utilization in the pathogenic yeast Candida glabrata.

The SNF1 gene product, a serine/threonine protein kinase, is a global regulatory protein which has been isolated from several organisms. In Saccharomyces cerevisiae the SNF1 gene product is essential for the derepression of glucose repression since snf1 strains are unable to utilize sucrose, galactose, maltose, melibiose, or nonfermentable carbohydrates. Moreover, the SNF1 gene product was suggested to interact with additional regulatory pathways and to affect the expression of multiple target genes as reflected by the pleiotropic nature of the snf1 mutation. Here we report the characterization of the SNF1 homolog of Candida glabrata, a pathogenic yeast phylogenetically related to S. cerevisiae. The carbon utilization spectrum of C. glabrata is considerably narrower than that of other pathogenic yeasts, and the majority of the strains utilize solely glucose and trehalose from among 20 of the most commonly tested carbohydrates. Disruption of the C. glabrata SNF1 homolog resulted in the loss of the ability to utilize trehalose, indicating that even in an organism with such a limited carbon utilization spectrum, the regulatory mechanism governing catabolic repression is preserved.

Amino Acid Sequence↗
PubMed2000

In vivo administration of mycobacterial cord factor (Trehalose 6, 6'-dimycolate) can induce lung and liver granulomas and thymic atrophy in rabbits.

Trehalose 6,6'-dimycolate (TDM) is a cell surface molecule of Mycobacterium tuberculosis. TDM induced a loss of body weight and prominent granulomas in the liver and lungs by the intravenous injection of TDM into rabbits. TDM also induced atrophy of the thymus and spleen due to apoptosis. By contrast, sulfolipid (2,3,6, 6'-tetraacyl trehalose 2'-sulfate) induced neither toxicity, nor granuloma formation, nor atrophy of the thymus and spleen. In rabbits the histopathological changes were more dramatic than in mice. The rabbit model may be more sensitive and may provide more information on the beneficial or pathological effects of TDM.

Animals↗
PubMed1987

Location of acyl groups of trehalose-containing lipooligosaccharides of mycobacteria.

A variant of a Mycobacterium sp. originating in a patient with Crohn's disease, but not necessarily implicated in the disease, provided a simple version of a newer class of species-specific surface glycolipids, the trehalose-containing lipooligosaccharides. A combination of high-resolution 1H nuclear magnetic resonance, methylation, ethylation, and absolute configurational analysis established the structure of the oligosaccharide unit as beta-D-Glcp(1----3)-alpha-L-Rhap(1----3)-alpha-D-Glcp(1----1)-alph a-D-Glcp (where Glc is glucose, Rha is rhamnose, and p is pyranosyl), and gas chromatography-electron impact mass spectrometry allowed identification of the fatty acyl esters as primarily 2,4-dimethyltetradecanoate. The relative simplicity of the glycolipid combined with the application of a mild methylation procedure and californium-252 plasma desorption mass spectrometry allowed recognition of three such acyl residues on the 3-, 4-, and 6-hydroxyl positions of the terminal glucosyl residue of the trehalose unit. Thus, the glycolipid is decidedly amphipathic yet is clearly not membranous. This observation leads to speculation about the role of these novel lipooligosaccharides in contributing to the outer segment of the hydrophobic barrier of the cell wall of certain mycobacteria.

Chemical Phenomena↗
PubMed1988

Germination of Saccharomyces cerevisiae ascospores without trehalose mobilization as revealed by in vivo 13C nuclear magnetic resonance spectroscopy.

Saccharomyces cerevisiae ascospores germinate in the presence of acetate without any detectable trehalose degradation, as revealed by high-resolution nuclear magnetic resonance spectroscopy and by a standard colorimetric assay. The results presented here substantiate the hypothesis that in S. cerevisiae trehalose supplies energy during dormancy of the spores and not during the germination process.

Acetates↗
PubMed1995

Molecular analysis of treB encoding the Escherichia coli enzyme II specific for trehalose.

A gene bank of partially Sau3A-digested Escherichia coli DNA ligated in plasmid pBR322 was screened for the ability to complement a mutant unable to metabolize trehalose at low osmolarity. The resulting plasmid was shown to contain the genes encoding transport (treB) and metabolic (treC) functions. The complementing DNA region was sequenced and shown to contain an operon of two genes, with treB as the promoter proximal gene and with treC as the promoter distal gene. The transcriptional start point was determined, and one major transcript was detected. The control region of the operon was found to contain consensus binding motifs for the cyclic AMP-catabolite activator protein complex and for a specific repressor protein whose gene, treR, is located immediately upstream of treB, being transcribed in the same direction as treB treC. The products of both genes could be expressed in minicells in which TreB revealed itself as a protein with an apparent molecular weight of 42,000. The gene product of treB consists of 485 amino acids with a calculated molecular weight of 52,308. It showed high homology to enzymes IIScr of enteric bacteria specific for the uptake of sucrose and encoded by plasmid pUR400 of enteric bacteria. Like enzyme IIScr, enzyme IITre belongs to the EIIBC domain type and lacks a covalently bound EIIA domain. Instead, enzyme IITre-mediated phosphorylation of trehalose requires the activity of enzyme IIAGlc, a component of the major glucose transport system.

Adaptation, Physiological↗