[Evaluation of a rapid trehalase test for the identification of Candida glabrata]. / Candida glabrata'nin tanisinda hizli trehalaz testinin degerlendirilmesi.

Candida species which cause local infections, may also lead to fatal systemic infections. The increasing incidence of non-albicans Candida, especially fluconazole susceptible or resistant dose-dependent C. glabrata, increased the importance of rapid and accurate species level identification for Candida. Rapid and correct identification of C. glabrata is essential for the initiation of the appropriate antifungal therapy. This study was conducted to evaluate the performance of the rapid trehalase test in the diagnosis of C. glabrata isolates. A total of 173 Candida strains isolated from various clinical specimens and identified according to germ tube test, growth on cornmeal Tween 80 agar and the colony morphologies on Mast-CHROMagar Candida medium (Mast Diagnostics, UK), were included to the study. The identification of non-albicans Candida species were also confirmed by API 20CAUX (BioMerieux, France) system. Accordingly 86 (50%) of the isolates were identified as C. glabrata, 48 (28%) C. albicans, 17 (10%) C. krusei, 13 (8%) C. tropicalis, 5 (3%) C. parapsilosis, 3 (2%) C. kefyr and 1 (1%) Cutilis. In order to detect the presence of trehalase enzyme in Condida strains, all isolates were grown on Sabouraud dextrose agar containing 4% glucose and then one yeast colony was emulsified in 50 microl of citrate buffer containing 4% (wt/vol) trehalose for 3 h at 37 degrees C. Presence of glucose which emerged after the action of trehalase on trehalose, was detected by a commercial "urinary glucose detection dipstick" (Spinreacta, Spain). All C. glabrata strains yielded positive result by trehalase test. None C. glabrata isolates were found negative by trehalase test except for one strain of C. tropicalis. In this study, the trehalase test allowed identification of C. globrata with 100% sensitivity and 98.9% specificity. It was concluded that trehalase test is a rapid, cost-effective and simple test that can be used for the accurate identification of C. glabrata.

Carbohydrate accumulation and utilization by oocytes of Rhodnius prolixus.

The processes of accumulation and mobilization of carbohydrate stores in eggs of Rhodnius prolixus were analyzed. During oogenesis, the total amounts of glycogen, glucose, and trehalose increased with an accumulation of proteins, especially when oocytes grew from 1.0 to 1.5 mm in length. At 2.0 mm length, when oocytes were ready for oviposition, nutrient reserves did not increase appreciably and trehalose content decreased. Mating did not affect the final content of carbohydrates or proteins in oocytes of mated and virgin females. A trehalase activity was detected in follicles containing vitellogenic oocytes, 1.0 and 1.5 mm length, in both mated and virgin females. This activity was extremely low in chorionated, 2.0-mm oocytes. After oviposition, glycogen content decreased in fertilized eggs, but not in unfertilized ones, and some was present in newly hatched nymphs. Glucose content remained constant in unfertilized eggs, but increased in fertilized ones, while total protein amount was constant in both groups after egg laying.

Content of glycogen and trehalose and activity of alpha-amylase and trehalase in Galleria mellonella larvae infected with entomopathogenic nematodes Steinernema affinis and S. feltiae.

INTRODUCTION: The influence of infection with two species of entomopathogenic nematodes of Steinernematidae family on metabolism of glycogen and trehalose of the host was studied. MATERIAL AND METHODS: Last instar larvae (L7) of Galleria mellonella were experimentally infected with Steinernema affinis and S. feltiae. At 6, 12, 18 and 24 h after infection concentrations of trehalose and glycogen as well as activity of trehalase and alpha-amylase were determined. RESULTS: The content of glycogen was lower in insects infected with S. feltiae than in the controls and animals infected with S. affinis. The content of trehalose was higher in insects from both infected groups than in the controls. Its concentration was slightly higher in larvae infected with S. affinis than in those infected with S. feltiae. The activity of alpha-amylase after infection with S. affinis was low. It was significantly higher in insects infected with S. feltiae. In animals of both infected groups, following a significant reduction at 6 h, the activity of trehalase remained at a similar level, higher than in the controls. In the paper the effects of infection with (i) different species of entomopathogenic nematodes and (ii) the importance of the developmental stage of the insect-host for changes in its metabolism of glycogen and trehalose were discussed.

Absorption of toxic beta-glucosides produced by plants and their effect on tissue trehalases from insects.

Trehalases present in body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor (Coleoptera), Musca domestica (Diptera), Spodoptera frugiperda and Diatraea saccharalis (Lepidoptera) were assayed in the presence and absence of toxic beta-glucosides produced by plants or their aglycones. The glucosides used were phlorizin, amygdalin, prunasin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with and without esculin. More than 60% of total trehalase activity was found in the midgut of these insects. As a rule, trehalases present in each insect were inhibited by at least two of the glucosides. Prunasin was the best inhibitor in tissues with highest trehalase activity. S. frugiperda beta-glucosidases were not able to hydrolyze esculin. Nevertheless, their larval midguts absorb the intact glucoside that is recovered from the fat body, Malpighian tubules and mainly from haemolymph. Mature larvae fed on a diet containing 3 mM (0.1%) esculin have 0.2 mM esculin in their haemolymph, and weigh 60% of control larvae. In vitro, haemolymph trehalase activity is abolished by 0.5 mM esculin. This inhibition may play a role in the decrease of body weight and in animal survival. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activity in tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why the S. frugiperda development is not impaired by 1% dietary amygdalin, in contrast to what is observed when insects are reared in 0.1% esculin. The data suggest that many plant beta-glucosides are toxic because they inhibit trehalase, a key enzyme controlling glucose availability in insects.

Effects of exposure to tobacco smoke in pregnancies complicted by oligohydramnios and premature rupture of the membranes. II. Activity of brush border enzymes in human amniotic fluid.

The aim of the present study was to assess the activity of membrane enzymes: alanine aminopeptidase (AAP), gamma-glutamyltransferase (GGT) and trehalase in amniotic fluid of women who smoke cigarettes during pregnancy complicated by idiopathic oligohydramnios or premature rupture of the membranes (PROM). The enzyme activity was measured between 22 and 31 (group A) and between 32 and 39 (group B) weeks of gestation. In the women of group A with idiopathic oligohydramnios, AAP activity was five times higher than in PROM women. AAP activity was declining with the progression of gestation, and in the B group women with oligohydramnios, it was over eight times lower than in group A. A threefold increase in GGT activity was found in women of group A with oligohydramnios as compared to women of group A with PROM. No statistically significant differences in trehalase activity were found in amniotic fluid of women with oligohydramnios and PROM, AAP, GGT and trehalase activity in women with idiopathic oligohydramnios correlated with the cadmium ion concentration, and AAP and GGT activity with the lead ion concentration in amniotic fluid which confirms toxical properties of these heavy metals present in cigarette smoke. It has already been confirmed that measurements of the brush border enzyme activity in amniotic fluid are very useful in prenatal diagnosis and detection of the placenta disorders.

[The concentration of trehalose and acitvity of trehalase from Galleria mellonella larvae infected by Steinernema affinis, Bovien 1937 (Nematoda: Rhabditida: Steinernematidae)]. / Zawartosc trehalozy i aktywnosc trehalazy u larw Galleria mellonella zarazonych Steinernema affinis, Bovien 1937 (Nematoda: Rhabditida: Steinernematidae).

The experimental studies were conducted on caterpillars of wax moth Galleria mellonella infected with Steinernema affinis larvae. The concentration of trehalose and the activity of trehalase were measured during the invasion lasting 48h. The level of trehalose and activity of enzyme were slightly lower in infected insects in comparison to the control animals.

Carbohydrate metabolism during starvation in the silkworm Bombyx mori.

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.

Biochemical effects of high intensity constant magnetic fields on worker honey bees.

Hemolymph samples from adult bees that had completed their pupal development and emergence in a 7 Tesla field contained a lower percentage of glucose than controls, indicating that trehalase enzyme activity in honey bees is reduced in strong magnetic fields. Significantly more phospholipids were found in the intestines of magnetic field-exposed bees than in controls. No significant differences were found for fatty acids, triacylglycerols, or steroids.

Are intestinal tumours in Apc+/-mice a suitable model of colorectal carcinoma in man?

In snap frozen sections of the duodenum, jejunum, ileum, the right and left colon of APC+/-mice mucosubstances, activities of brush border glycosidases and proteases, immunoreactivity of sucrase and activities of some enzymes of pericellular proteolysis were studied. Multiple adenomas (tubular or tubulovillous) the numbers of which decreased in the aboral direction occurred in the small intestine. Two tubulovillous adenomas with dysplastic nuclei but with no invasion were found in the right colon. The morphological and histochemical findings resembled those of human colorectal tumours. Activities of brush border enzymes and sucrase immunoreactivity were decreased to various extent or were not present at all. The findings fluctuated even within the same section. Activities of enzymes of pericellular proteolysis were slightly increased in comparison with non affected mucosa. This model is suitable and deserves further studies.

Is the brush border membrane of the intestinal mucosa a generator of "chymosomes"?

1. The microvilli of enterocytes in calf intestine demonstrate high levels of vesiculation activity at the top and at the basal region. 2. The morphology of the vesicles associated with microvilli (100-500 nm diameter, unilamellar, few intramembraneous particles, high AP activity) is very similar to the morphology of vesicles found in the chyme. 3. Vesicles can be purified 6-10 fold from chyme of the calf intestine applying a Mg(++)-precipitation method, used for brush border membrane preparation. 4. Specific activities of alkaline phosphatase and disaccharidases were found to be much higher in chyme vesicles than in the mucosa. 5. Phospholipid content and phospholipid composition is in chyme vesicles different from brush border membrane vesicles. 6. The characterized chyme vesicles are referred to as chymosomes. We consider the mucosa as a large-scale generator of chymosomes, i.e. digestive enzymes bearing vesicles.

Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC-PK1 cells.

The porcine kidney epithelial cell line LLC-PK1 expresses a sodium-coupled glucose cotransporter (SGLT1) together with other differentiation markers of renal proximal tubule such as trehalase and gamma-glutamyltranspeptidase. Expression is regulated by cell density and exogenous differentiation inducers such as hexamethylene bisacetamide (HMBA). Northern blot and PCR analysis of clonal cell populations indicated SGLT1 mRNA was not detectable in subconfluent cultures, but 2.2 and 3.9 kb SGLT1 mRNA species appeared after cell confluence, accompanying expression of the transport activity. SGLT1 mRNA levels were significantly increased after treatment of confluent cultures with HMBA, paralleling increases in the transport activity and immunodetectable 75 kD cotransporter subunit. SGLT1 mRNA was also increased after treatment of cultures with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), an inducer of Na+/glucose cotransport activity. The 3.9 kb SGLT1 transcript showed the largest increase after either HMBA or IBMX treatment. HMBA treatment also resulted in increased mRNA levels of two other differentiation markers--trehalase and gamma-glutamyltranspeptidase. By contrast, trehalase and gamma-glutamyltranspeptidase mRNA levels were not increased by IBMX. Regulation of Na+/glucose symporter expression by either cell density, cyclic AMP elevation, or differentiation inducer treatment occurs, at least in part, at the level of SGLT1 mRNA and can be dissociated from regulation of other differentiation markers.

The effect of somatostatin on small intestinal transport in intractable diarrhoea of infancy.

It has been reported that somatostatin may be an effective antisecretory agent in a range of conditions causing severe secretory diarrhoea. In many children, intractable diarrhoeal illnesses result in significant morbidity and mortality. In a group of seven children with secretory diarrhoea, the effect of i.v. infusion of somatostatin (3.5 micrograms/kg stratum plus 3.5 micrograms/kg/h) on the net mucosal flux of salt and water was assessed using an in vivo steady-state perfusion technique. In one of the seven children who had evidence of deranged mucosal secretion and preserved villus function, somatostatin infusion resulted in a moderate reduction in secretion. In the remaining six, it had little or no beneficial effect. Somatostatin did not alter the rate of glucose absorption.

Fetal intestinal and renal origins of trehalase activity in human amniotic fluid.

Intestinal and renal trehalase isozymes have been distinguished in normal human amniotic fluid on the basis of their membrane-bound character and isoelectric point (pI). The intestinal trehalase was mostly membrane bound in amniotic fluid and had a pI around 4.60. In contrast, the renal form of trehalase was soluble and had a pI around 4.37. These pI values were consistent with those found in extracts of fetal intestinal (pI 4.60) and renal (pI 4.24) tissues. The determination of trehalase isozyme composition of amniotic fluid from pathological pregnancies with anal imperforation and polycystic kidney disease confirmed our findings on the origin of amniotic fluid trehalase. In the sample from a fetus with anal imperforation, low or absent intestinal trehalase isozyme was observed whereas a higher than normal level of renal trehalase activity was found in a fetus with polycystic kidney disease.

Disaccharidase activity in the small intestine of susceptible and resistant mice after primary and challenge infections with Giardia muris.

The activities of four disaccharidases were examined in resistant (C57Bl/6) and susceptible (C3H/HeN) mice during the primary infection with Giardia muris and after challenge with either trophozoite extract or cysts. Significant decreases in lactase, sucrase, trehalase, and maltase activities in C57Bl/6 mice and lactase and sucrase activities in C3H/HeN mice in the anterior 25% of the small intestine were observed on day 10 after infection. The activities of maltase, sucrase, trehalase, and lactase in the jejunum of C3H/HeN mice were significantly reduced after challenge with trophozoite extract, when compared with the uninfected or infected, but not challenged animals. Decreases in enzyme activities of C3H/HeN mice were evident as early as 12 hours after challenge with the extract. The resistant C57Bl/6 mice showed little change in disaccharidase activity after challenge with trophozoite extract. On the other hand, challenge with cysts resulted in a few decreases in disaccharidase activities in both strains of mice: C57Bl/6 mice showed decreases in the duodenum, while disaccharidases of C3H/HeN mice had lower activity more posteriorly. Thus, challenge with parasite antigen results in a more severe disaccharidase deficiency in susceptible hosts when compared with resistant ones.

Characterization of different forms of yeast acid trehalase in the secretory pathway.

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.

Discriminant analysis for assessing the value of amniotic fluid microvillar enzymes in the prenatal diagnosis of cystic fibrosis.

We have analysed the sensitivity, specificity, and reliability of biochemical diagnosis based on microvillar membrane enzyme assay and using discriminant analysis in amniotic fluid samples obtained from 54 pregnancies at high risk for cystic fibrosis and 125 normal pregnancies. Our results show that amniotic fluid trehalase, alkaline phosphatase, alkaline phosphatase isoenzymes and gamma-glutamyltransferase enzyme activities measured during 16-20 gestational weeks, in spite of their non-specificity for cystic fibrosis, have a very good predictive value for fetal cystic fibrosis or exclude the possibility of the disease. Overall enzyme activity analysis provided over 90 per cent reliability of the method.

Amniotic fluid microvillar enzyme activity in fetal malformations.

Prenatal diagnosis of cystic fibrosis based on amniotic fluid microvillar enzyme activity assay has become routine practice in the past few years. Normal (median) values of these enzymes were determined in 177 normal healthy pregnancies between 15-20 gestational weeks and were related to enzyme values measured in 50 pregnancies complicated with congenital malformations, 6 monogenic inherited diseases and 4 chromosomal aberrations. It is concluded that increased trehalase activity has diagnostic importance in detecting fetal kidney diseases, and radial-renal syndrome (with elevated GGT activity), while low enzyme activities may indicate chromosomal aberrations (with no signs of intestinal obstruction). With the collection of further data, the analysis of these enzymes might provide an opportunity to set up diagnostic procedures for the detection of other, non-CF-related cases.

Determination of trehalose in biological samples by a simple and stable trehalase preparation.

A three step purification procedure for trehalase from Saccharomyces cerevisiae with a recovery of 76% of the original activity is presented. The enzyme was activated by a heat shock treatment prior to homogenization of the cells. A mutant strain deleted in SUC genes was used to avoid contamination by invertase. The lyophylized enzyme was stable for, at least, 5 months and could be used to determine trehalose in the range 25 to 500 nmol. The preparation was free of inspecific phosphatases allowing for trehalose determinations in yeast cell free extracts and in insect hemolymph.

Differential location of regulatory and nonregulatory trehalases in Candida utilis cells.

The isolation of vacuoles by density gradient centrifugation of protoplast lysates from Candida utilis cells showed a high specific activity for nonregulatory trehalase in vacuoles whereas the regulatory trehalase activatable by phosphorylation behaves as a cytoplasmic enzyme. The vacuolar trehalase is a glycoprotein that can be precipitated by Con A-Sepharose. Treatment of this enzyme with endo H reduced its reactivity with the lectin without loss of enzyme activity and decreased its apparent molecular weight by gel filtration.