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PubMedDate not supplied

Secretion of sucrase by Leishmania donovani.

Leishmania donovani promastigotes were collected, washed, resuspended in buffer, and assayed for sucrase activity. No activity was observed in the intact washed cells, but activity was measurable when the cells were permeabilized with Triton X-100. Intracellular sucrase activity was highest in promastigotes grown at pH 7.4, somewhat lower in promastigotes grown at pH 5.5, and significantly lower in "amastigotes" grown at pH 5.5. No trehalase, lactase, or maltase activities were observed. Assay of the medium in which the cells had grown showed that most the sucrase activity was extracellular, i.e. was secreted into the medium during growth.

Animals↗
PubMed1998

The treA gene of Bacillus subtilis is a suitable reporter gene for the archaeon Methanococcus voltae.

The similarity of the transcriptional apparatus of Archaea with that of Eucarya makes studies of their transcriptional regulation especially interesting. Such investigations are greatly facilitated by reporter genes. The concomitant analysis of several promoters for investigations of regulatory patterns requires different reporter genes. The archaeon Methanococcus voltae is a moderately halophilic mesophile. The treA gene from Bacillus subtilis appeared to be a good candidate for a reporter, since its product trehalase is salt-resistant. We show that it is indeed expressed under the control of a M. voltae promoter and that the enzyme is easily testable in cell lysates.

Bacillus subtilis↗
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↗
PubMed1990

The effect on intestinal disaccharidase activity of feeding galactose to growing rats.

The effect of supplementation of the diet with galactose on the age-related decline of intestinal lactase activity was investigated in 108 growing rats. Starting from 14 days of age, the rats were divided into two groups and fed with chow, and with fluid either as tap water or 5% galactose solution. At 14 days the specific lactase activity was 112.8 +/- 3.2 mumol min-1 (g protein)-1, which decreased to less than 10% of this value at maturity. Galactose supplementation did not prevent the decline. The increase of maltase, sucrase and trehalase was also unaffected. The result suggests that galactose plays no significant role in the regulation of disaccharidase activities in the rat.

Animals↗
PubMed1987

Rat jejunal disaccharidase activity increases biphasically during early post-natal development.

1. Injection of hydrocortisone into 9-day-old rats induces the early appearance of sucrase in jejunal homogenates, the time course of the subsequent increase and magnitude of the final effect being similar to that seen to start on day 16 during normal development. 2. Cytochemical comparison of the effect of hydrocortisone and normal development on the appearance of a mixture of sucrase, maltase, isomaltase and trehalase disaccharidases (alpha-glucosidase activity) shows this enzyme to appear first in enterocytes at the base of the villus. Enzyme activity then increases and spreads along the whole villus during the next 96 h. 3. The rate at which enterocytes migrate along the villus after hydrocortisone injection is not significantly different from that measured during the early phase of normal development. The later phase of normal development is associated with a threefold increase in cell migration rate and a twofold increase in crypt depth. 4. The rate at which alpha-glucosidase activity increases in enterocytes at the base of the villus during early normal development is similar to that determined after hydrocortisone injection into younger animals. This rate of appearance increases eight to tenfold during normal development, shortly after the appearance of solid food in the stomach of normal control animals. 5. Injection of steroid hormones into young rats is generally supposed to mimic events taking place normally at weaning. Present results show alpha-glucosidase induction during normal development to be under more complicated control than had been previously suspected.

Aging↗
PubMed1981

Multiple drug resistance in Mycobacterium avium: is the wall architecture responsible for exclusion of antimicrobial agents?

Whole cells of Mycobacterium avium, characterized by their negative response in the nine biochemical tests used for mycobacterial identification in our laboratory, turned positive for nitrate reductase, Tween-80 hydrolysis, beta-glucosidase, acid phosphatase, alkaline phosphatase, penicillinase, and trehalase after their wall portion was removed to yield spheroplasts. This suggested that the negative results in most of the biochemical procedures were caused by the exclusion mechanism at the wall level. Preliminary transmission and scanning electron microscopic studies showed differences at wall level between laboratory-maintained opaque, dome-shaped (SmD) and host-recycled smooth, transparent (SmT) colony type variants of M. avium and suggested the presence of an outer regularly structured layer in SmT variants. Comparative ultrastructural studies utilizing different polysaccharide coloration methods confirmed the presence of an outer polysaccharide layer in SmT variants which was probably related to their enhanced pathogenicity for experimental animals and drug resistance as compared to that of SmD variants. These findings are discussed with respect to multiple drug resistance, virulence, and gene expression of M. avium.

Anti-Bacterial Agents↗
PubMed1996

Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications.

The accumulation of trehalose is a critical determinant of stress resistance in the yeast Saccharomyces cerevisiae. We have constructed a yeast strain in which the activity of the trehalose-hydrolyzing enzyme, acid trehalase (ATH), has been abolished. Loss of ATH activity was accomplished by disrupting the ATH1 gene, which is essential for ATH activity. The delta ath1 strain accumulated greater levels of cellular trehalose and grew to a higher cell density than the isogenic wild-type strain. In addition, the elevated levels of trehalose in the delta ath1 strain correlated with increased tolerance to dehydration, freezing, and toxic levels of ethanol. The improved resistance to stress conditions exhibited by the delta ath1 strain may make this strain useful in commercial applications, including baking and brewing.

Arabidopsis Proteins↗
PubMed1999

Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.

Journal Article↗
PubMed1978

Intestinal enzyme activities in germfree, conventional, and gnotobiotic rats associated with indigenous microorganisms.

Various enzyme activities involved in the active transport system, glycolysis, and digestion were assayed in various parts of the gastrointestinal tracts of germfree, conventional, and gnotobiotic rats associated with indigenous bacteria. The activity levels of alkaline phosphatase, glucose 6-phosphatase, adenosine triphosphatase, and disaccharidases in the upper small intestine were highest in all parts of the gastrointestinal tracts of various kinds of gnotobiotic, conventional, and germfree rats. Alkaline phosphatase, glucose 6-phosphatase, and adenosine triphosphatase activities in the upper small intestine of germfree rats were, respectively, 2.3-, 2.9-, and 1.7-fold higher than those in conventional rats. Similar to the results of these enzymes, sucrase, maltase, trehalase, and lactase activities in the upper small intestine of germfree rats were, respectively, 1.6-, 1.5-, 2.3-, and 1.8-fold higher than those in conventional rats. In various gnotobiotic rats, enzyme activity levels were intermediate between those in germfree and conventional rats. These findings suggest that those enzymatic activities are strongly depressed by the association with the indigenous microorganisms in the epithelial mucosa of the upper small intestine of rats. The levels of pyruvate kinase, hexokinase, and lactate dehydrogenase activities were highest, respectively, in the stomach, cecum, and the upper small intestine and cecum in all parts of the gastrointestinal tracts in various kinds of gnotobiotic, conventional, and germfree rats. It was also shown that six kinds of gastrointestinal bacteria, including lactobacilli, significantly depressed the enzyme activity levels to levels between those of the germfree and conventional rats in the upper small intestine of gnotobiotic rats.

Adenosine Triphosphatases↗
PubMed1973

Trehalose metabolism in germinating spores of Streptomyces hygroscopicus.

Evidence is presented which indicates that utilization of trehalose is an early event in spore germination in Streptomyces hygroscopicus. Early in spore germination and before any increase in cell mass, the activity of trehalase increased more than 15-fold, while the intracellular content of trehalose fell to very low levels. On the other hand, increased activity of the trehalose phosphate synthetase or disappearance of glycogen did not occur until later in germination, during which time cell mass was also increasing.

Disaccharides↗
PubMed1974

Trehalose metabolism by Bacillus popilliae.

Trehalose was found to be utilized more readily than glucose for the growth of Bacillus popilliae NRRL B-2309MC. The pathway of degradation of trehalose was elucidated and found to differ from that reported for other organisms. Trehalase and trehalose phosphorylase activities could not be detected. Rather, trehalose was found to undergo phosphoenolpyruvate (PEP)-dependent phosphorylation, and the resulting trehalose 6-phosphate was cleaved by a phosphotrehalase to equimolar amounts of glucose and glucose 6-phosphate. The phosphotrehalase was purified 34-fold and shown to have a pH optimum of 6.5 to 7.0 and a K(m) for trehalose 6-phosphate of 1.8 mM. A mutant missing the phosphotrehalase failed to grow on trehalose but grew normally on other sugars. The mutant accumulated [(14)C]trehalose as [(14)C]trehalose 6-phosphate. Phosphorylation of trehalose by dialyzed extracts was at least 25 times faster with PEP than with adenosine 5'-triphosphate, and the phosphorylation activity was associated primarily with the particulate fraction. These data and the results of studies of [(14)C]trehalose uptake suggest that trehalose is transported into the cell as trehalose 6-phosphate by a PEP:sugar phosphotransferase system. Cell extracts of other strains of B. popilliae were also found to produce [(14)C]sugar phosphate from [(14)C]trehalose and to have phosphotrehalase activity.

Bacillus↗
PubMed1989

Trehalose accumulation in vegetative cells and spores of Myxococcus xanthus.

The disaccharide trehalose is found in the spores and cysts of a variety of organisms. We analyzed developing cells of Myxococcus xanthus for trehalose accumulation. Vegetative cells grown in media with low osmotic strengths contained less than 5 micrograms of trehalose per mg of protein. Spores formed in fruiting bodies accumulated up to 1,100 micrograms of trehalose per mg of protein. Spores formed in liquid culture following the addition of glycerol contained up to 300 micrograms of trehalose per mg of protein. The trehalose contents of both spore types decreased rapidly during the early stages of germination. Trehalase activity was not detected in extracts of dormant or germinating spores. Trehalose accumulation in M. xanthus was also associated with elevated osmotic strength. Vegetative cells accumulated up to 214 micrograms of trehalose per mg of protein when grown in media containing elevated levels of solutes.

Disaccharides↗
PubMed1992

Glucose-induced regulatory defects in the Saccharomyces cerevisiae byp1 growth initiation mutant and identification of MIG1 as a partial suppressor.

Saccharomyces cerevisiae byp1-3 mutants displayed a long lag phase when shifted from a nonfermentable carbon source to a medium containing glucose. The byp1-3 mutation also caused several defects in regulatory phenomena which occur during the transition from the derepressed state to the repressed state. As opposed to wild-type cells, the addition of glucose to cells of the byp1-3 mutant grown on nonfermentable carbon sources did not induce a cyclic AMP signal. Fructose-2,6-bisphosphate formation and inactivation of fructose-1,6-bisphosphatase were severely delayed, but trehalase activation was not affected. In addition, the induction of pyruvate decarboxylase both at the level of activity and that of transcription was very slow compared with that in wild-type cells. These pleotropic defects in glucose-induced regulatory phenomena might be responsible for the very long lag phase of byp1-3 cells and the inability of ascospores to initiate growth after germination on glucose media. Screening of a yeast gene library for clones complementing the byp1-3 phenotype resulted in the isolation of a truncated form of the previously described zinc finger transcription repressor MIG1. The entire MIG1 gene and the truncated form suppressed even on a single-copy vector the growth initiation defect but not the regulatory abnormalities of the byp1-3 mutant. MIG1 is not allelic to byp1-3.

Antimycin A↗
PubMed1967

Constitutive and induced trehalose transport mechanisms in spores of the fungus Myrothecium verrucaria.

Trehalose is absorbed by two distinct systems-one constitutive, the other induced by turanose and to a lesser extent by nigerose but not by trehalose. The constitutive system is apparently mediated by a surface trehalase; the induced system has the characteristics of a permease. The specificity of the induced system is apparently limited to the alpha glucosyl-glucose or glucosyl-fructose linkage, because absorption of kojibiose, nigerose, maltose, isomaltose, turanose, sucrose, and melezitose, in addition to that of trehalose, was increased. Absorption of beta-linked or of galactose-containing disaccharides was not increased. The constitutive and induced trehalose-absorbing systems differ in their activity, specificity, lability to acid treatment, effects of substrate concentration, and pH optima. Both systems require oxygen, and no marked differential effects of inhibitors were observed. The activity of the induced system is proportional to log turanose concentration (from about 1 to 300 mug/ml), and is an approximate linear function of time of exposure (from about 1 to 50 min). Accumulation of trehalose occurred against a concentration gradient in both systems but particularly in the induced. No leakage was observed. The activity of the induced system declined slowly upon removal of the inducer. Accumulated trehalose is metabolized after activation by azide as are the endogenous trehalose reserves. The accumulated trehalose appears to enter the endogenous trehalose pool found in these spores, although some data suggest it may be more accessible. Respiratory data indicate that absorbed trehalose is available for metabolism while in transit from the external membrane to the internal pool.

Chromatography, Paper↗
PubMed1979

Enzyme activities and morphological appearance in functioning and excluded segments of the small intestine after shunt operation for obesity.

Five patients in whom small-intestinal bypass was performed for severe obesity had a second operation 11-19 months later because of insufficient weight loss. Mucosal enzyme activities and histological appearance were investigated in biopsies from different parts of the functioning and excluded small intestine. These were compared with biopsies form corresponding sites obtained at the first operation. In addition to a prominent increase in length, circumference, and mucosal thickness in the functioning shunt, the disaccharidases and two intracellular beta-galactosidases increased in specific activity,, especially in the distal ileal part of the shunt. In the excluded segment of the small intestine different enzymes showed a different response: trehalase increased and alkaline phosphate decreased significantly. Other enzymes that were measured showed a varied pattern. The results indicated that not only the luminal content but also other, presumably hormonal, factors regulated the enzyme activities, and that different regulating factors influenced the various enzymes differently. The marked adaptive increase in mucosal surface of the functioning shunt could be one factor in explaining the weight stabilisation and, in some cases, weight increase after the initial rapid weight loss after the operation for small-intestinal bypass. The increase in specific enzyme activities would further increase the digestive capacity of the shunt.

Adult↗
PubMedDate not supplied

Effect of hydrocortisone on the maturation of human foetal kidney explants in serum-free organ culture.

The effect of hydrocortisone on the in vitro maturation of human foetal kidney was investigated. Following legal therapeutic abortions, explants of renal cortex from foetuses aged 13-18 weeks were cultured for 5 days in serum-free Leibovitz's L-15 medium at 37 degrees C in a mixture of 95% air - 5% CO2, without hormone (controls) or with hydrocortisone at concentrations of 12.5, 25, or 50 ng/mL, which are the levels representative of different gestational periods. During the studied period of culture, the overall architecture of the renal structures was preserved without any evident signs of nephrogenesis induced by hydrocortisone. DNA synthesis was measured by incorporation of [3H]thymidine and was stimulated on day 5 by 80% with the addition of hydrocortisone at 12.5 ng/mL, and by 131% with 50 ng/mL. In autoradiograms, the sites of [3H]thymidine incorporation were the same after hydrocortisone addition, but the number of labelled nuclei was higher in 5-day explants supplemented with hydrocortisone at 50 ng/mL. The activities of some brush border enzymes (leucylnaphthylamidase, maltase, and alkaline phosphatase) were not influenced by hydrocortisone when compared with controls. Trehalase activity was decreased on day 5 with 12.5 and 50 ng/mL. A concentration of 12.5 ng/mL diminished gamma-glutamyltransferase activity by 29% on day 5. The incorporation of [3H]leucine into proteins was not influenced by any concentration of the glucocorticoid hormone. This study indicates that hydrocortisone directly influences cell proliferation and certain brush border enzymic activities in human developing kidney maintained in organ culture.

Blood↗
PubMed2005

Immediate early genes of glucocorticoid action on the developing intestine.

Prior studies have demonstrated that glucocorticoid hormones elicit functional maturation of the small intestine as evidenced by their ability to induce increases in the expression of various digestive hydrolases, such as sucrase-isomaltase and trehalase. However, these increases have a lag time of approximately 24 h, suggesting that they are secondary effects of hormone action. To identify candidate primary response genes, we performed microarray analysis on pooled RNA from jejunums of untreated postnatal day 8 mouse pups and from littermates who earlier received dexamethasone 2 h. Fluorescent dye-labeled samples were hybridized in quadruplicate to glass-spotted cDNA microarrays containing 15,000 cDNA clones from the National Institute of Aging cDNA clone set. Analysis of the resulting signals using relatively stringent criteria identified 66 transcripts upregulated and 36 downregulated by 2 h of glucocorticoid treatment. Among the upregulated transcripts, the magnitude of the increase detected by microarray ranged from 1.4- to 16-fold. Selected mRNAs from throughout the range were subsequently analyzed by Northern blot analysis. Of 11 mRNAs chosen all were confirmed, and there was a strong correlation between the magnitude of the increase observed from the microarray analysis and from Northern blot analysis. Additional time points showed that these transcripts peaked between 2 and 6 h and had returned to baseline by 24 h. Gene ontology analysis showed pleiotropic effects of dexamethasone on the developing intestine and pointed to genes in the development category as being likely candidates for mediation of glucocorticoid-induced maturation of intestinal function.

Animals↗
PubMed1980

Effect of genetic diabetes on enzymes of mouse intestinal brush-border membrane.

Activities of intestinal enzymes were measured in genetically diabetic mouse of strain C57BL/KsJ-dbm to determine the long-term effects of genetic and uncontrolled diabetes on intestinal digestive function. Specific activities of enzymes were measured in intestinal homogenates, brush-border membrane fractions, and everted sacs from diabetic mice and their littermate controls. Sucrase, maltase, trehalase, alkaline phosphatase, and leucylnaphthylamidase activities were elevated in diabetes; lactase did not show any changes. The increases in disaccharidase activities in diabetes were in homogenates from both proximal and distal intestine but the increases in distal were more pronounced than in the proximal. Measurement of enzyme activities in brush-border membrane fractions showed a pattern similar to that observed in homogenates. Hydrolysis of sucrose and trehalose by everted sacs was markedly higher in the diabetic mice. It is therefore concluded that in genetic diabetes the digestive function of the intestine is stimulated, that the increased enzymes were incorporated into the brush-border membrane, and that the additional enzymes are accessible to the substrates in the intestinal lumen and so of physiological significance.

Alkaline Phosphatase↗