This study aimed to detect intracellular trehalose in boar sperm that were cryopreserved with liposomes and conduct an analysis of its effects on some characteristics of thawed sperm, including rheological properties. First, soybean lecithin cholesterol-based liposomes were produced and characterized in the presence of 300 mM trehalose. Next, semen samples were frozen in two freezing media: a control medium with 300 mM trehalose and an experimental medium supplemented with 300 mM trehalose and 10% liposomes, both of which were thawed and then studied to ascertain their integrity, motility, rheological response, and trehalose quantities by testing two methods of spermatic lysis via high-performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD). The results found spherical liposomes measuring 357 nm that were relatively stable in an aqueous medium and had an entrapment efficiency of 73%. An analysis of the cryopreserved ejaculates showed that their viability and motility did not significantly differ between groups (P > 0.05). The viscous response of the samples was influenced by the extracellular medium rather than by the freezing–thawing process, which resulted in a loss of interaction between the cells and cryoprotectants. Finally, intracellular trehalose levels were determined using HPLC-ELSD, with no differences observed (P > 0.05) when comparing both sperm lysis methods. The use of liposomes with trehalose appears to be a promising option for boar semen cryopreservation, with a marked effect on rheological properties. The proposed HPLC-ELSD method was effective for measuring trehalose in cryopreserved cell samples.

The impact of slowly digestible sugars in reducing the risk of developing obesity and related metabolic disorders remains unclear. We hypothesised that such carbohydrates (CHO), resulting in a lower glycaemic and insulinaemic response, may lead to greater postprandial fat oxidation rates in subjects with impaired glucose tolerance (IGT). The present study intends to compare the postprandial metabolic responses to the ingestion of glucose (GLUC) v. trehalose (TRE) and sucrose (SUC) v. isomaltulose (IMU). In a randomised, single-blind, cross-over design, ten overweight IGT subjects were studied four times, following ingestion of different CHO drinks either at breakfast or in combination with a mixed meal at lunch. Before and 3 h after CHO ingestion, energy expenditure, substrate utilisation and circulating metabolite concentrations were determined. Ingestion of CHO drinks with a meal resulted in an attenuated rise in GLUC ( − 33 %) and insulin ( − 14 %) concentrations following TRE when compared with GLUC and following IMU, an attenuation of 43 and 34 % when compared with SUC ingestion, respectively. Additionally, there was less inhibition of the rise in NEFA concentrations and less decline in postprandial fat oxidation (22 %) after IMU when compared with SUC, whereas TRE did not differ from GLUC. The attenuated rise in GLUC and insulin concentrations following IMU ingestion attenuated the postprandial inhibition of fat oxidation compared with SUC when co-ingested with a meal. This suggests that exchange of SUC in the diet for IMU may result in a more favourable metabolic response and may help to reduce the risks associated with obesity and type 2 diabetes.

The proposed impact of slowly digestible sources of dietary carbohydrate in reducing the risk of developing obesity and related metabolic disorders remains unclear. The aim of the present study was to compare the postprandial metabolic response to the ingestion of glucose v. trehalose. We hypothesised that the reduced digestion and absorption rate of trehalose is accompanied by an attenuated glycaemic and insulinaemic response, leading to a less inhibited postprandial fat oxidation rate. In a randomised, single-blind, cross-over study, ten overweight subjects ingested two carbohydrate drinks (75 g carbohydrate equivalents of trehalose or glucose) following an overnight fast (08.40 hours) and together with a standardised mixed meal (12.30 hours; 25 % total energy content was provided as either glucose or trehalose). Blood samples were collected before ingestion and every 30 min thereafter for a period of 3 h; substrate use was assessed by indirect calorimetry and expired breath samples were collected. Ingestion of carbohydrates with a mixed meal resulted in a lower peak glucose response and a lower change in area under the curve (ΔAUC) following trehalose when compared with glucose. Differences in peak insulin response and ΔAUC were observed with trehalose when compared with glucose during the morning and afternoon. These differences were accompanied with a reduced carbohydrate oxidation after trehalose when ingested as a drink, whilst no significant differences in fat oxidation between drink were observed.

A study was conducted to quantify the ability of entrapped, monoxenically produced spores of an arbuscular mycorrhizal fungus to germinate and reproduce the fungal life cycle after cryopreservation. No germination was obtained after incubation of entrapped spores in glycerol and mannitol and subsequent cryopreservation at −70 °C, regardless of the concentration of cryoprotectants and duration of incubation. Incubation for 1 d in 0.5 M sucrose, and for 1 and 2 d in 0.5 M trehalose, led to spore germination after cryopreservation at −70 °C. Lower cryopreservation temperatures were tested with entrapped spores incubated for 1 d in 0.5 M trehalose. The highest germination rate, estimated by the percentage of potentially infective beads (%PIB), was obtained at −100 °C. A %PIB of 95% (water agar medium) to 100% (Strullu–Romand medium) was obtained at this temperature. Thereafter, %PIB rapidly decreased at −140 and −180 °C. Heavy sporulation and high internal root colonization were obtained after re-association of the entrapped spores, incubated for 1 d in 0.5 M trehalose and subsequently cryopreserved at −100 °C, with transformed carrot roots. This demonstrates the ability of entrapped spores to reproduce the fungal life cycle following cold treatment.

The non-reducing disaccharide trehalose is a major storage compound in most fungi. For a better understanding of carbon metabolism in the ectomycorrhizal-forming basidiomycete Amanita muscaria, trehalase activity was analysed from mycelium growing in liquid culture or on agar plates, and from mycorrhizas. Trehalase activity was found in both the culture medium and in mycelial extracts. The excreted trehalase was purified to apparent homogeneity by anion-exchange chromatography, gel filtration and preparative gel electrophoresis. The identified enzyme belongs to the group of acid trehalases. The apparent molecular mass of the native enzyme was estimated to be 165 kDa by gel filtration and 135 kDa by SDS–PAGE. Isoelectrofocusing indicated an isoelectric point (pI) of approx. 3.7, which is typical for acid trehalases. The enzyme is highly specific for its substrate trehalose, with an apparent Km of 0.38 mM. Validamycin and sucrose act as competitive inhibitors with Ki values of 45 nM and 15 mM, respectively. The activity of acid trehalase excreted into the growth medium is independent of the carbon source (glucose or trehalose), revealing that the enzyme is not regulated by its substrate trehalose. Nevertheless, fungal hyphae grown in the absence of an external carbon source showed enhanced enzyme excretion. The biochemical characteristics of the trehalase activity measured in the mycelium are in the same range as those determined for the excreted enzyme. The enzyme is localized in the cell wall and its activity is strongly decreased in ectomycorrhizas.

Metacestodes of Hymenolepis diminuta affect several aspects of female reproductive physiology in Tenebrio molitor and such effects are mediated via the endocrine system. The effects on male reproduction are less well known and were studied with respect to the Bean-Shaped Accessory Glands (BAGs). The size and wet and dry weight of BAGs from infected and uninfected beetles were compared and rose to a plateau from 0–6 days post-emergence in uninfected beetles but in infected individuals continued to increase in both size and weight. These effects were density independent. Glands from both infected and uninfected beetles were assayed for trehalase activity measured by its ability to convert the sugar trehalose to glucose. The activity of this enzyme, per mg wet weight, was not affected by the parasite. However, total activity per gland increased in infected males. Total protein content and electrophoretic profiles of BAGs from infected and uninfected individuals showed no change in profile but showed an increase in all protein subunits per gland over a broad molecular weight range.

To study the response of non-mycorrhizal and mycorrhizal maize plants to drought, the changes in the pools of non-structural carbohydrates and amino acids were analysed in leaves and roots of two maize cvs. Plants well colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) (60% of root length infected) and comparable non-mycorrhizal plants were subjected to moderate drought stress by reducing the water supply. This stress induced a conspicuous increase in the trehalose pool in the mycorrhizal roots, probably because it was accumulated by the fungal symbiont. Furthermore, glucose and fructose were accumulated in leaves and roots of non-mycorrhizal plants but not in the mycorrhizal ones. Starch disappeared completely from the leaves of both mycorrhizal and non-mycorrhizal plants in response to drought. Activities of soluble acid invertase and trehalase were also measured. Acid invertase activity increased during drought in the leaves of both non-mycorrhizal and mycorrhizal plants whilst in the roots it was unaffected in non-mycorrhizal plants and decreased in the mycorrhizal ones. Without drought stress, trehalase activity was considerably higher in the leaves and roots of mycorrhizal plants than in those of non-mycorrhizal plants. It increased conspicuously during drought, primarily in the leaves of non-mycorrhizal plants. A drought-induced accumulation of amino acids as well as imino acids was found in roots and leaves of both mycorrhizal and non-mycorrhizal plants; leaves of mycorrhizal plants accumulated more imino acids than those of non-mycorrhizal ones. Our results show that drought stress and the presence of a mycorrhizal fungus have a considerable effect on carbon partitioning, imino acid and amino acid accumulation in maize plants.

The amounts of cyclic AMP (cAMP), fructose-2,6-bisphosphate (F26BP), trehalose and glycogen were determined in cell suspension cultures of the ectomycorrhiza-forming fungus Amanita muscaria (L. ex Fr.) Hooker. For the assay of cAMP a protocol was developed that enabled the detection of as little as 50 fmol of this secondary messenger by an enzyme-linked immuno assay (EIA). Values varied from <1 and up to 5 pmol cAMP mg−1 d. wt according to the age of the fungal culture. Typically, a transient increase in cAMP occurred after c. 4 d of culture of the fungus on glucose-containing medium. This increase (up to 100%) was followed by the start of the logarithmic growth phase, and by a more persistent increase in F26BP. In parallel, glucose in the medium started to decrease, whilst the amounts of fungal carbohydrates, especially the disaccharide trehalose, increased. From these data we assume that a high initial rate of glucose uptake caused an increase in the fungal pools of storage carbohydrates and, via activation of an adenylate cyclase, of cAMP. According to data reported for yeast cells this should enhance the formation of F26BP by phosphorylation of relevant enzymes. In animal and yeast cells an increase in the concentration of F26BP stimulates glycolysis by activation of the ATP-dependent phosphofructokinase (PFK). A. muscaria also possesses an F26BP activated PFK and, under conditions of symbiosis, host-derived carbohydrates are supplied mainly in the form of glucose. The implications of these findings to the regulation of carbohydrate metabolism of symbiotic plant root/fungus structures (ectomycorrhiza) are discussed.

Several examples are presented that show the successful application of uranyl acetate and ammonium molybdate negative staining in the presence of trehalose for TEM studies of filamentous and tubular structures. The principal benefit to be gained from the inclusion of trehalose stems from the considerably reduced flattening of the large tubular structures and the greater orientational freedom of single molecules due to an increased depth of the negative stain in the presence of trehalose. Trehalose is likely to provide considerable protection to protein molecules and their assemblies during the drying of negatively stained specimens. Some reduction in the excessive density imparted by uranyl acetate around large assemblies is also achieved. Nevertheless, in the presence of 1% (w/v) trehalose, it is desirable to increase the concentration of negative stain to 5% (w/v) for ammonium molybdate and to 4% for uranyl acetate to produce satisfactory image contrast. In general, the ammonium molybdate-trehalose negative stain is more satisfactory than the uranyl acetate-trehalose combination, because of the greater electron beam sensitivity of the uranyl negative stain. Reassembled taxol-stabilized pig brain microtubules, together with collagen fibrils, sperm tails, helical filaments, and reassociated hemocyanin (KLH2), all from the giant keyhole limpet Megathura crenulata, have been studied by negative staining in the presence of trehalose. In all cases satisfactory TEM imaging conditions were readily obtained on the specimens, as long as regions of excessively deep stain were avoided.

Polyphenism is a common feature in the mustard aphid, Lipaphis erysimi (Kalt.) in tropical countries. The function related changes in basic biochemical pool are reported during development of polyphenic forms. A gradual decrease in the protein content in this aphid was recorded, as the nymphs advanced in their instar number and reached adult stage. However, the protein content was significantly lower in the apterous forms as compared to alate. The number, density and mobility of protein bands varied as the nymphs passed through different instars and the number of bands was more in alate forms. The glycogen and trehalose contents showed a decrease upto the third instar and showed an increase in the fourth instar, but again declined in the adults of both the phases.

Decrease in trehalose (40–60%) and total sugar (58–68%) content was observed in both sexes of ageing Callosobruchus maculatus. The variations in the level of trehalose and total sugar with age, suggest that these serve as energy sources responsible for the maintenance of metabolism in the process of ageing in bruchids.