Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.     

Regulation of soluble and membrane-bound trehalase activity and expression of the enzyme in the larval midgut of the bamboo borer Omphisa fuscidentalis

Diapausing larvae of Omphisa fuscidentalis contain soluble and membrane-bound trehalase in the midgut. Soluble trehalase activity accounts for three-fourths of the total trehalase activity in midgut homogenates. The exposure of diapausing larvae to juvenile hormone analog (JHA) induced pupation, accompanied by an increase in soluble trehalase activity at the beginning of the prepupal period. Injection of 20-hydroxyecdysone (20E) increased the level of soluble trehalase activity 5 days postinjection in a dose-dependent manner. In contrast, no increase in membrane-bound trehalase activity was observed under the same conditions. We cloned the cDNAs that encode the soluble and membrane-bound forms of trehalase in O. fuscidentalis trehalase-1 (OfTreh-1) and trehalase-2 (OfTreh-2), respectively. Treh-1 encodes a 581-aa protein while Treh-2 encodes a 648-aa protein with one putative transmembrane domain near the C-terminus. The mRNA expression level of Treh-1 was 27-fold higher than that of Treh-2 in diapausing larval midgut. Following the exposure of diapausing larvae to JHA, Treh-1 mRNA expression increased gradually until the prepupal period whereupon it increased dramatically; in contrast, the mRNA expression of Treh-2 remained at its initial level. Similarly, 20E upregulated Treh-1 expression but had no effect on Treh-2 expression. Taken together, these results suggest that an increase in the soluble trehalase activity at pupation is caused by upregulation of Treh-1 gene. Moreover, membrane-bound trehalase does not appear to be involved in the dynamic changes in the hemolymph trehalose concentration that occur during the larval-pupal transformation.     

高温对家蚕三品系血淋巴中糖水平的影响（英文）

家蚕Bombyx mori的两个二化性品系热耐受型NB4D2和热敏感型CSR2均适合于温带气候,而多化性的PM（Pure Mysore) 品系适合于热带气候,将这3种品系5龄幼虫分别置于32℃和36℃的高温下,观察高温对其5龄幼虫至蛹期血淋巴中糖含量及海藻糖酶活性的影响。结果表明： PM幼虫和蛹的死亡率均小于NB4D2和CSR2。在蜕皮期间血淋巴海藻糖水平较高,而葡萄糖水平及海藻糖酶活性较低。32℃和36℃的高温下,幼虫蜕皮期间血淋巴中糖含量及海藻糖酶活性仅在其各自的水平上表现为小幅度的增加。蜕皮后幼虫血淋巴中海藻糖含量显著下降,而葡萄糖含量和海藻糖酶活性显著上升。在较高温度下,蜕皮后幼虫血淋巴中海藻糖含量下降幅度更大,而葡萄糖含量及海藻糖酶活性上升水平也更加显著。25±1℃下取食幼虫血淋巴中葡萄糖含量显著下降,海藻糖含量显著上升;3℃和36℃下PM 和NB4D2取食幼虫血淋巴葡萄糖和海藻糖含量以及海藻糖酶活性增加,而CSR2均减少或降低。吐丝幼虫血淋巴中葡萄糖含量及海藻糖酶活性显著下降,海藻糖小幅度下降。而在较高温度下,耐热型PM 和NB4D2吐丝家蚕血淋巴糖含量含量和海藻糖酶活性明显增加,而热敏感型CSR2的则明显下降。这3种品系蛹发育期的血淋巴糖含量及海藻糖酶活性均下降。在两较高温度下,PM蛹期血淋巴糖和海藻糖酶活性增加,而NB4D2 36℃时增加幅度小于32℃时。对于CSR2,32℃时观察到其血淋巴葡萄糖含量增加,但当环境温度增加到36℃时其血淋巴葡萄糖含量降至正常水平下。然而,当CSR2的蛹置于32℃和36℃时血淋巴海藻糖含量及其酶活性下降,且36℃时下降幅度更大。因此,桑蚕对高温的适应取决于家蚕的品系及发育阶段,并可通过其血淋巴糖及海藻糖酶活性水平进行验证。     

Regulation of trehalase activity during the cell cycle of Saccharomyces cerevisiae

Synchronous cultures of Saccharomyces cerevisiae prepared by selection of small unbudded cells from an elutriating rotor were used to measure trehalase activity during the cell cycle. After the small cells had been removed from the rotor, the remainder was used to prepare asynchronous control cultures. Both synchronous and control cultures were studied for two cell cycles. In asynchronous cultures the trehalase activity of crude cell lysates rose continuously. In synchronized populations trehalase activity increased from the beginning of budding onwards. However, around the period of cell division the enzyme activity dropped rapidly but transiently by more than 5-fold. The same changes were found during the second budding cycle. Measurements of invertase and glucose-6-phosphate dehydrogenase activities in the same synchronous and asynchronous cultures revealed a continuous increase for both enzymes. Incubation of cell lysates with cAMP-dependent protein kinase before assaying for trehalase resulted in a 2-fold enhancement of enzyme activity in asynchronous control cultures. In synchronized cells this treatment also led to a significant stimulation of trehalase activity, and largely abolished the cell-cycle-dependent oscillatory pattern of enzyme activity. These results suggest that the activity of trehalase during the cell cycle is regulated, presumably at the post-translational level, by a phosphorylation-dephosphorylation mechanism.     

Properties of a germination mutant of Phycomyces blakesleeanus

Spores of the Phycomyces blakesleeanus strain S440 germinated only for some 4 to 7% when activated with a heat treatment or with ammonium acetate. Contrary to wild type spores, they showed no increase in trehalase activity during or after the activating treatment. This was not due to a variant trehalase or a defective protein kinase but rather to the absence of an increase in cellular cyclic AMP which normally occurs in the wild type. Phosphodiesterase activity in the mutant was comparable to wild type activity and in both strains phosphodiesterase was inactivated by a heat treatment. The phosphodiesterase inhibitor 1-isobutyl, 3-methyl xanthine caused germination and trehalase activation in the wild type but not in the mutant. The results corroborate the importance of cyclic AMP in the breaking of dormancy and the activation of trehalase in this fungus.     

Transport of yeast vacuolar trehalase to the vacuole

We have tested yeast secretory mutants, which define different stages of the secretory pathway, for their levels of vacuolar trehalase activity. Mutations that cause accumulation of secretory proteins in the endoplasmic reticulum or in the Golgi body lead to diminished vacuolar trehalase activity. Mutations that cause accumulation of secretory vesicles have no effect on vacuolar trehalase activity. None of the mutations affects cytoplasmic trehalase activity. These results provide further evidence for the existence of a compartmentalized trehalase in yeast, and demonstrate that the enzyme enters the secretory pathway.     

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger

Abstract Resting cells of the fission yeast Schizosaccharomyces pombe , suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen iannophilus , the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.     

Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores

Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity.     

Influence of octopamine or trehalase activity in muscle and hemolymph of the American cockroach, Periplaneta americana L.

Injection of adult male cockroaches (Periplaneta americana) with 10 μl 1 μM octopamine causes elevated activity of trehalase (α,α-trehalose glucohydrolase; EC 3.2.1.28) in hemolymph and muscle but not in gut. Tyramine, dopamine and glutamate, at the same concentration, failed to elicit any effect on trehalase activity. Determination of some kinetic parameters for muscle and hemolymph trehalase reveal that octopamine causes an increase in V_max without any significant alteration in the K_m of the enzyme for trehalose. The results are discussed in terms of the physiological significance of octopamine-mediated activation of tissue trehalase.     

A stalk-specific localization of trehalase activity in Dictyostelium discoideum.

By utilizing ultra-microtechniques, trehalase activity was followed in specific cell types during the differentiation cycle of Dictyostelium discoideum. When whole organisms were assayed, trehalase activity was found to be high in the early stages of differentiation, decreased to its lowest point at 14 h, and then increased at the end of the cycle. By microdissection of freeze-dried individuals, the activity of trehalase could be followed during the migration of pre-stalk and pre-spore cells. No activity was observed at any stage of spore cell development, whereas stalk cells showed a rapid increase in activity upon maturation. An increasing gradient of activity was found from the apex of the stalk toward the base. This localization of trehalase in stalk cells resolves some contradictory results in the literature concerning the role of the enzyme during differentiation.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATalpha and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae

Various microorganisms produce the disaccharide trehalose during their symbiotic and pathogenic interactions with plants. Trehalose has strong effects on plant metabolism and growth; therefore, we became interested to study its possible role in the interaction of Arabidopsis thaliana with Plasmodiophora brassicae, the causal agent of clubroot disease. We found that trehalose accumulated strongly in the infected organs (i.e., the roots and hypocotyls) and, to a lesser extent, in the leaves and stems of infected plants. This accumulation pattern of trehalose correlated with the expression of a putative trehalose-6-phosphate synthase (EC 2.4.1.15) gene from P. brassicae, PbTPS1. Clubroot formation also resulted in an induction of the Arabidopsis trehalase gene, ATTRE1, and in a concomitant increase in trehalase (EC 3.2.1.28) activity in the roots and hypocotyls, but not in the leaves and stems of infected plants. Thus, induction of ATTRE1 expression was probably responsible for the increased trehalase activity. Trehalase activity increased before trehalose accumulated; therefore, it is unlikely that trehalase was induced by its substrate. The induction of trehalase may be part of the plant's defense response and may prevent excess accumulation of trehalose in the plant cells, where it could interfere with the regulation of carbon metabolism.     

Inducible trehalase enzyme activity of Morchella conica Persoon mycelium

Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.     

Reversibility characteristics of glucose-induced trehalase activation associated with the breaking of dormancy in yeast ascospores

The breaking of dormancy in yeast ascospores by addition of glucose is associated with a sudden tenfold increase in the activity of trehalase. The rapid activation of trehalase is followed by a slower inactivation process which is greatly retarded in the presence of nitrogen sources and cycloheximide. When glucose is washed away from the spores after some time and the spores resuspended in glucose-free medium, the trehalase activity decreases sharply. Subsequent addition of new glucose partially reactivates the enzyme. The extent of reactivation decreases further with each subsequent activation/inactivation step. Changing the duration of the inactivation periods has no effect on this diminution of the reversibility. However, prolonging the duration of the activation step speeds up the loss of reversibility. On the other hand, addition of a nitrogen source or cycloheximide completely prevents the loss of reversibility. The results of the reversibility studies are in agreement with the phosphorylation mechanism which has been proposed for the underlying molecular process of trehalase activation. Apparently, they are also in agreement with proteolytic breakdown being responsible for the inactivation of trehalase after its initial activation. However, the effect of cycloheximide and nitrogen sources, at least in ascospores, does not appear to be due to inhibition or repression of protease synthesis, respectively, since the addition in the presence of glucose of a nitrogen source after trehalase inactivation immediately reactivates the enzyme completely.     

Trehalase activity in dormant and activated spores of Phycomyces blakesleeanus

Summary Heat treatment of Phycomyces sporangiospores, which breaks dormancy, causes a very rapid 10- to 15fold increase in trehalase activity; soon after the heat shock the enzyme activity decays. This phenomenon can be repeated several times by repeating the heat shocks. Prolonging the heat treatment over the minimum required time delays the decay of enzyme activity. Cycloheximide does not prevent the rise in enzyme activity. It is suggested that heat treatment converts temporarily an inactive form of trehalase into an active one. Optimal enzyme activity is obtained at pH 7.5 and the enzyme requires metal ions for maximal activity. The possible role of trehalase in the spore-activation process is discussed.     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

Characterization of putative soluble and membrane-bound trehalases in a hemipteran insect, Nilaparvata lugens

Trehalose is the main blood sugar of insects, and the enzyme trehalase is involved in energy metabolism and controlling trehalose levels in cells. Two forms (soluble and membrane-bound) of trehalase and the corresponding genes (NlTre-1 and NlTre-2) were identified from the brown planthopper, Nilaparvata lugens. Both NlTre-1 and NlTre-2 contain trehalase signature motifs, and NlTre-2 contains a putative transmembrane domain. Comparison of trehalase activity and gene mRNA level at different developmental stages, or following application of 20-hydroxyecdysone (20E), suggests that NlTre-1 and NlTre-2 encode a soluble trehalase and a membrane-bound trehalase respectively. Soluble trehalase activity accounted for the majority of total trehalase activity in N. lugens. Only soluble trehalase activity and NlTre-1 mRNA level could be induced by 20E. Additionally, only soluble trehalase activity was significantly higher in macropterous individuals than in brachypterous morphs. These results indicate that only soluble trehalase is differentially expressed between macropterous and brachypterous individuals and is more responsive to hormone stimulus.     

CONTROL OF TREHALASE SYNTHESIS IN NEUROSPORA CRASSA

When an aconidial strain (STL6A) of Neurospora crassa is grown on carbon sources such as glucose, maltose, sucrose, etc., trehalase activity per unit weight of mycelium is very low. By contrast, media containing arabinose, glutamic acid, glycine, etc., which support growth only poorly, produce mycelium with very high trehalase activity. Retarding growth limiting the supply of a necessary nutrient, altering the pH and temperature, or adding toxic substances, however, does not derepress trehalase activity. Repression and derepression of trehalase was found to be reversible through the transfer of cultures to appropriate media. It is likely that the increase in trehalase activity results from de novo synthesis because labeled enzyme can be isolated from acrylamide gels after isolation from medium containing C¹⁴-labeled leucine and after purification by other means. These experiments are interpreted in terms of catabolite repression which may be correlated with events during growth and conidiation.     

Methods for the Rapid Purification of Trehalase from Dictyostelium Discoideum

A rapid and reliable method for the preparation of homogeneous trehalase from the cellular slime mold, Dictyostelium discoideum for usage in enzyme characterization studies and trehalose assays was developed. This procedure takes advantage of the fact that trehalase activity is secreted by Dictyostelium during the course of development, the major fraction being released late in fruiting body formation. Purification of trehalase to electrophoretic homogeneity was accomplished utilizing the techniques of ultrafiltration, streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel chromatography and preparative disc gel electrophoresis. Analysis of the purified enzyme by analytical polyacrylamide disc gel electrophoresis demonstrated the presence of a single protein band which was stainable with Coomassie blue. Assay of trehalase activity in eluates from segments of a companion gel indicated that all of the recovered trehalase activity was associated with this band of protein. Examination of the substrate specificity of the purified enzyme indicated absolute specificity for trehalose.     

Activation of neutral trehalase by fermentable sugars and cAMP in the fission yeast Schizosaccharomyces pombe

Abstract Derepressed cells of Schizosaccharomyces pombe 972 h⁻ suspended in the presence of glucose or other fermentable sugars displayed a transient activation of trehalase which was not blocked by cycloheximide. Repressed cells were unable to show glucose-induced trehalase stimulation. Nitrogen sources, protonophores or uncouplers failed to produce direct trehalase activation but increased the activity of the enzyme in the presence of glucose. Exogenous cAMP induced a rapid and pronounced stimulation of trehalase in both repressed and derepressed cells suggesting that the response to glucose includes activation of adenylate cyclase as part of a cAMP signalling pathway that increases the catalytic activity of trehalase by enzyme modification.