Activities of ectonucleotidases and adenosine deaminase in platelets of cattle experimentally infected by Fasciola hepatica.

The enzymatic activities of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) are important in regulating the concentration of adenine nucleotides, molecules known to be involved on platelet aggregation. Fasciolosis causes coagulation disorders that have not been completely elucidated. Taking into consideration the association between the purinergic system and hemostasis, this study aimed to evaluate the enzymatic activities of NTPDase (hydrolyze ATP and ADP), 5'-nucleotidase (hydrolyze AMP) and ADA (deamination of adenosine) in platelets from cattle experimentally infected by Fasciola hepatica on days 20, 40, 60 and 80 post-infection (PI). For this study, 10 healthy Friesian steers were separated into two groups: the group A (n = 5) was used as uninfected control, and the group B was composed of steers experimentally infected by F. hepatica (n = 5). The number of platelets did not differ between groups in the periods evaluated. Reduction of NTPDase (p < 0.05) hydrolysing ATP (days 20, 40 and 60 PI), and ADP (days 40, 60 and 80 PI), and on 5'-nucleotidase hydrolyzing AMP (days 40 and 60 PI) was observed. A reduction (p < 0.05) in ADA activity on day 20 PI, as well as an increase (p < 0.05) in ADA activity on days 40 and 60 PI was observed when compared to the control. Based on these results, we can conclude that ATP, ADP and AMP hydrolysis and adenosine deamination were altered in platelets of cattle infected by F. hepatica. Considering the importance of the purinergic system in hemostasis, it is believed that those changes may contribute to the coagulation impairment observed in acute fasciolosis described in the literature.

Toxoplasmosis treatment with diphenyl diselenide in infected mice modulates the activity of purinergic enzymes and reduces inflammation in spleen.

Toxoplasma gondii, an intracellular protozoan, may cause chronic infection in the brain tissue of the host inducing a systemic pro-inflammatory profile. Chronic infections can induce numerous physiological changes, such as alterations in the immune and oxidative profiles. Diphenyl diselenide (PhSe)2, an organoselenium compound, has shown antioxidant and immunomodulatory activities in recent studies. So, the aim of this study was to investigate the activity of purinergic enzymes and reactive oxygen species (ROS) in serum and spleen of mice chronically infected by T. gondii, untreated and treated with (PhSe)2. For this experiment, were divided into four groups: Group A (healthy mice), Group B (healthy mice treated with (PhSe)2), Group C (infected mice) and Group D (infected mice treated with (PhSe)2). Group C and group D were infected via oral route with ME49 Toxoplasma gondii strain. Groups B and D were treated subcutaneously with 5 µmol kg-1 of (PhSe)2. Chronic T. gondii infection induced splenomegaly and physiological changes in the spleen and raised histologic inflammatory markers, ROS levels and the activity of purinergic enzymes activity such as NTPDase, 5´nucleotidase and ADA. In serum, the infection increased 5´nucleotidase and ADA activities. (PhSe)2per se has managed to decrease ROS levels and ADA activity and increase NTPDase and 5´nucleotidase in spleen. In infected mice, treatment with (PhSe)2 reversed splenomegaly, reduced histological inflammatory markers, ROS levels and ADA activity in the spleen. Our results prove that chronic toxoplasmosis can induce splenomegaly, heightens ROS levels and purinergic enzyme activity in mice. These results suggest that (PhSe)2 is a potential therapy for the alterations found in the spleen in chronic T. gondii infection.

Loss of ectonucleotidases from the coronary vascular bed after ischemia-reperfusion in isolated rat heart.

BACKGROUND: Ectonucleotidase plays an important role in the regulation of cardiac function by controlling extracellular levels of adenine nucleotides and adenosine. To determine the influence of ischemia-reperfusion injury on ectonucleotidase activity in coronary vascular bed, we compared the metabolic profile of adenine nucleotides during the coronary circulation in pre- and post-ischemic heart. METHODS: Langendorff-perfused rat hearts were used to assess the intracoronary metabolism of adenine nucleotides. The effects of ischemia on the adenine nucleotide metabolism were examined after 30 min of ischemia and 30 min of reperfusion. Adenine nucleotide metabolites were measured by high performance liquid chromatography. RESULTS: ATP, ADP and AMP were rapidly metabolized to adenosine and inosine during the coronary circulation. After ischemia, ectonucleotidase activity of the coronary vascular bed was significantly decreased. In addition, the perfusate from the ischemic heart contained a considerable amount of enzymes degrading ATP, AMP and adenosine. Immunoblot analysis revealed that the perfusate from the ischemic heart dominantly contained ectonucleoside triphosphate diphosphohydrolase 1, and, to a lesser extent, ecto-5'-nucleotidase. The leakage of nucleotide metabolizing enzymes from the coronary vascular bed by ischemia-reperfusion was more remarkable in aged rats, in which post-ischemic cardiac dysfunction was more serious. CONCLUSION: Ectonucleotidases were liberated from the coronary vascular bed by ischemia-reperfusion, resulting in an overall decrease in ectonucleotidase activity in the post-ischemic coronary vascular bed. These results suggest that decreased ectonucleotidase activity by ischemia may exacerbate subsequent reperfusion injury, and that levels of circulating ectonucleotidase may reflect the severity of ischemic vascular injury.

Activities of ectonucleotidases and adenosine deaminase in platelets of dogs experimentally infected with Rangelia vitalii.

Rangeliosis is a disease which affects dogs in Brazil, caused by a piroplasm known as Rangelia vitalii. This disease causes a lot of clinico-pathological features, including the coagulation disorders associated with bleeding. The cause of these changes has not yet been determined. Considering the association of purinergic system and hemostasis this study aimed to evaluate the activity of enzymes that hydrolyze ATP, ADP and AMP; and deamination of adenosine in platelets from dogs experimentally infected with R. vitalii. For this study, 12 healthy young dogs (females) were used, separated in two groups. Group A (n=5) were uninfected controls, and group B were experimentally infected with R. vitalii (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite after five days post-inoculation (PI). Blood samples were collected to quantitate and separate platelets (Day 0, 12 and 21 PI) and to measure the enzymatic activities (Day 12 and 21 PI). The activity of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) was measured in platelets. A reduction (P<0.01) in the number of platelets was observed in R. vitalii-infected blood at Days 12 and 21 PI. At Day 12 PI, a reduction (P<0.01) in the hydrolysis of ATP, ADP and AMP, and deamination of adenosine was observed in dogs infected with R. vitalii. At Day 21 PI the ADA activity remained decreased, unlike the activity of NTPDase which increased (P<0.05). Based on these results we can conclude that ATP, ADP and AMP hydrolysis and adenosine deamination were altered in platelets of R. vitalii-infected dogs. Considering the importance of the purinergic system in hemostasis, it is believed that those changes contribute to the coagulation disorders and bleeding observed in R. vitalii-infected dogs and discussed in this manuscript.

Potential roles of serum ATPase and AMPase in predicting diagnosis of colorectal cancer patients.

Colorectal cancer (CRC) is a common gastrointestinal cancer with high incidence and mortality rates. CRC may be associated with regulation of circulating nucleotides. This study aimed to evaluate the serum levels of nucleotide-metabolizing enzymes (ATPase and AMPase) in patients with CRC and to explore the clinical diagnostic value of these enzymes. The gene set variation analysis (GSVA) score of the ATP-adenosine signature was calculated using tumor samples from The Cancer Genome Atlas (TCGA). ATP-adenosine signaling plays a central role in CRC progression. A total of 135 subjects, including 87 patients with CRC and 48 healthy controls, were included. The serum levels of ATPase and AMPase in the CRC group were significantly higher than those in the control group (P < 0.05). Furthermore, ATP and AMP hydrolysis levels significantly increased in the advanced CRC group (P < 0.05). ATP and AMP hydrolysis was decreased by the ENTPDase inhibitors (POM-1 and ARL67156) and CD73 inhibitor (APCP). The sensitivities of ATPase and AMPase were 95.4% and 75.9%, respectively, which were higher than those of CEA (67.8%) and CA19-9 (72.4%). The specificities of ATPase and AMPase were 69.9% and 73.9%, respectively, which were higher than that of CA19-9 (47.8%). The combination of CEA, ATPase, and AMPase demonstrated high sensitivity (92.0%) and specificity (87.0%). Collectively, ATPase and AMPase activities are upregulated in CRC with considerable diagnostic significance. The combination of CEA, ATPase, and AMPase may provide a novel approach for CRC screening.

Lymphocyte ecto-5'-nucleotidase deficiency in agammaglobulinemia.

Fresh peripheral blood lymphocytes from eight patients with congenital agammaglobulinemia demonstrate reduced ecto-5'-nucleotidase activity when compared to the mean activity of normal subjects and patients with other forms of immunoglobulin deficiency. A specific defect of ecto-5'-nucleotidase is further suggested by normal values for lymphocyte ecto-adenosinetriphosphatase and ecto-nonspecific phosphatase. The data provide evidence for an enzyme deficiency in this X-linked, B lymphocyte deficiency syndrome.

Ecto-enzyme of granulocytes: 5'-nucleotidase.

The 5'-nucleotidase of guinea pig polymorphonuclear leukocytes is localized exclusively on the plasma membrane of intact cells. The active site of this enzyme faces the external medium, not the cytoplasm.

Detection of DNA 3'-phosphatase activity based on exonuclease III-assisted cascade recycling amplification reaction.

DNA 3'-phosphatase is an essential enzyme, which plays a pivotal role in repairing DNA damage. The peculiar activity of DNA 3'-phosphatase has been proved to associate with a variety of human pathologies. Therefore, sensitive determination of DNA 3'-phosphatase is necessary for clinical diagnosis and therapy. Here, we develop a simple, sensitive, and specific fluorescent biosensor including three DNA chains of hairpin DNA1, hairpin DNA2 and fluorescence probe DNA (FP) for detecting the activity of DNA 3'-phosphatase. First, biotin-modified hairpin DNA1 binds with streptavidin-modified magnetic beads (MB) to get MB-DNA1. DNA 3'-phosphatase can hydrolyze phosphate groups on MB-DNA1 to form hydroxyl groups, which leads to the polymerization extension and nicking endonuclease cleavage reaction to obtain the trigger DNA1 fragment (tDNA1). Next, two cyclic amplification reactions are designed. In cycle I, the tDNA1 hybridizes with the hairpin DNA2, which leads the hairpin structure of DNA2 opened and the fluorescence signal of 6-carboxy-fluorescein (FAM) labeled on hairpin DNA2 turned on. This cyclic reaction is amplified by exonuclease III (Exo III). At the same time, the trigger DNA2 fragment (tDNA2) is obtained. In cycle II, similarly, the tDNA2 hybridizes with FP. Thus, the fluorescence signal of FAM labeled on FP released, which multiplies with the fluorescence signal from cycle I. Finally, this strategy is applied to determine two typical DNA 3'-phosphatases including T4 polynucleotide kinase (T4 PNK) and alkaline phosphatase (ALP) with the detection limit (LOD) of 0.0033 and 0.00037 U/mL, respectively. The method provides a promising platform to evaluate the DNA 3'-phosphatase activity in the complicated biological samples and can be potentially applied in the relevant fields such as biomedical research, drug discovery and clinical diagnosis.

Heterogeneity of 5'-nucleotidase activity in lymphocytes in chronic lymphocytic leukemia.

The specific activity of 5'-nucleotidase was determined in lymphocyte plasma membranes from 14 normal subjects and 10 patients with chronic lymphocytic leukemia (CLL). Whereas the enzyme was present in the preparation from normal lymphocytes, in 7 out of 10 CLL patients the membranes had markedly decreased or no detectable 5'-nucleotidase activity. The lack of this activity from the lymphocytes of most patients with CLL constitutes an alteration in a plasma membrane enzyme from the normal cell. The presence of the enzyme in the lymphocytes of some patients with CLL and its decrease in others provide further evidence for biochemical heterogeneity among patients with this disorder.

Lead poisoning. Further observations on erythrocyte pyrimidine-nucleotidase deficiency and intracellular accumulation of pyrimidine nucleotides.

Pyrimidine nucleotides, detectable in normal erythrocytes only in trace quantities if at all, were found to comprise 7-80% of the intracellular nucleotide pools in nine subjects with severe lead over-burden. Blood lead concentrations ranged from approximately equal to 200- to 400-microgram/dl packed cells, and the greatest accumulations of pyrimidine-containing nucleotides occurred in the two subjects with the highest blood lead levels. Most of the patients had mild or moderate anemia and moderate basophilic stippling evident in Wright's-stained peripheral smears. Pyrimidine nucleotidase activities were inhibited to 13-28% of the mean activity in normal control erythrocytes and even more so (5-15%) when compared to specimens with increased reticulocytes and young cells. Reticulocytosis was absent in two subjects and modest to moderate in the remainder, but erythrocyte assays revealed the substantial elevations in populations of young mean cell age. Inappropriately low reticulocyttial elevations in glucose-6-phosphate dehydrogenase expected in populations of young mean cell age. Inappropriately low reticulocyte responses may reflect hematopoietic suppressive effects of lead at a variety of metabolic loci.

Human platelet collagenase.

The presence of proteolytic enzymes such as cathepsin and elastase in platelets and the important role of collagen in platelet aggregation suggested that collagenase might be present in platelets. Epinephrine, ADP, and collagen liberate collagenase from platelets in plasma as measured by the hydrolysis of [(14)C]glycine-labeled collagen fibrils. The collagenase activity appeared in an early phase of platelet aggregation and was not a part of the release reaction. However, only 50% of the total collagenase could be liberated by the aggregating agents used. Sucrose density gradient analysis of platelet homogenates using appropriate sub-cellular markers indicated that collagenase appeared in both the granule and membrane fractions. Gel-filtered platelets failed to show collagenase activity before exposure to aggregating agents but released more collagenolytic activity than was found in platelet-rich plasma. This observation was explained by the finding that collagenolytic activity was inhibited by normal human plasma. One of the inhibitors is alpha(1)-antitrypsin as demonstrated by decreased inhibition in plasma from a patient with homozygous alpha(1)-antitrypsin deficiency. Platelet collagenase activity could also be demonstrated by its ability to decrease the viscosity of collagen solutions and to produce collagen fragments similar to those produced by other mammalian collagenases on disk gel electrophoresis. The observation that partially purified platelet collagenase could destroy the platelet-aggregating activity of collagen suggests that the enzyme might function in a negative feedback mechanism limiting thrombus formation.

Lipopolysaccharide alters nucleotidase activities from lymphocytes and serum of rats.

ATP exerts a proinflammatory role and induces cytokine release by acting at P2X(7) receptors. The product of ATP hydrolysis is the nucleoside adenosine, an important immunomodulator. The main source of extracellular adenosine is the hydrolysis of extracellular ATP by a group of ecto-enzymes: ENTPDase family, NPP family and ecto-5'-nucleotidase. Considering the role of ATP and adenosine in inflammatory processes, we investigated the effect of lipopolysaccharide on ectonucleotidases activities and expression in lymphocytes from mesenteric lymph nodes and serum of rats, in order to better understand the involvement of extracellular nucleotide hydrolysis in an endotoxemia model. We observed significant changes on nucleotidase activities from lymphocytes and serum of rats after in vitro and in vivo exposure to LPS. In vitro results have shown an increase on nucleotide hydrolysis in lymphocytes and a decrease on the enzyme activity of NPP in blood serum. In vivo, we observed an increase on nucleotide hydrolysis in lymphocytes and a decrease in the hydrolysis of all nucleotides tested in blood serum. After 24 and 48 h of LPS treatment, there was a reduction in NTPDase1, 2, 3 and ecto-5'-nucleotidase transcripts. These results suggest that there is a time-dependent enhancement of extracellular nucleotides metabolism in lymphocytes and blood serum after the induction of an endotoxemic model. The changes observed suggest that these enzymes can act in the regulation of extracellular nucleosides and nucleotides in a model able to trigger inflammatory process.

Serum sialyltransferase and 5'-nucleotidase as reliable biomarkers in women with breast cancer.

Sialyltransferase and 5'-nucleotidase were measured in the sera of 135 women with breast cancer: 53 undergoing mastectomy for primary cancer and 83 receiving different modalities of palliative therapy for metastatic disease. The objective of this study was to determine whether these enzyme levels were associated with the extent of the disease and whether changes in these enzyme levels could be correlated with success or failure of treatment. Mastectomy caused a rapid fall of elevated enzyme levels to within the normal range in all patients with stage I breast cancer but not in those with stage II or III disease. In women with metastatic disease, elevated enzyme levels fell only in patients responding to treatment. Thus serum sialyltransferase and 5'-nucleotidase activities are reliable biomarkers of breast cancer activity, and serial measurement of these enzyme activities provides a useful tool for the monitoring of disease activity and success or failure of the treatment.

Enzyme markers in acute leukemias: advances during the last decade.

Besides the recent advances in immunology that provided important information for the characterization of leukemia cells, enzyme marker analysis is another area of progress in leukemia research. Assays of enzyme activities, both quantitative enzyme levels and qualitative isozyme changes, are of value in the classification of leukemias. The interest in the study of enzyme markers is due not only to technical improvements and new biochemical possibilities, but also to the involvement of enzyme marker analysis in the so-called "multiple marker analysis," which combines traditional and recently developed techniques in leukemia research. The aims of this review are to describe well-known and potential enzyme markers as well as to stress the relevance of enzyme marker analysis combined with multiple marker analysis for leukemia subclassification and the understanding of normal hematopoietic cell differentiation.

Alterations in serum glycosyltransferases and 5'-nucleotidase in breast cancer patients.

We have measured sialyltransferase, galactosyltransferase, and fucosyltransferase as sell as 5'-nucleotidase in the serum of breast cancer patients. Serum sialyltransferase values in 65 normal healthy females ranged from 2.6 to 8.5 units, with a mean of 5.4. In 25 women with operable primary breast cancer, serum sialyltransferase levels were found to be between 6.2 and 15.4 units. Marked elevation of this enzyme level (range, 8.8 to 36 units) was observed in 48 patients with metastatic breast cancer. Galactosyltransferase and fucosyltransferase measurements, however, showed considerable overlap between the controls and the cancer patients. On the other hand serum 5'-nucleotidase and sialyltransferase in breast cancer patients showed very similar patterns. Thus, serum 5'-nucleotidase values in 44 normal females ranged from 11.4 to 23.2 units, whereas the levels found in 30 patients with metastasis were between 25 and 71.8 units. The tissue origin of abnormal levels of serum glycosyltransferases and 5'-nucleotidase was discussed in relation to their physiological significance as well as their role as markers for diagnosing early malignant breast neoplasm and for monitoring the extent of metastasis.

Leucine aminopeptidase as an echo-enzyme of polymorphonuclear neutrophils.

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5'-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5'-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.

Canine neutrophil plasma membrane markers.

The purpose of this investigation was to determine which enzyme activities are true canine neutrophil plasma membrane markers. Three enzymes thought to be present on plasma membranes were chosen for study: 5'-nucleotidase, magnesium-dependent adenosine triphosphatase (Mg2+-ATPase), and leucine aminopeptidase. Both 5'-nucleotidase and Mg2+-ATPase were found to be ectoenzymes in the canine neutrophil but additional Mg2+-ATPase activity was located intracellularly. An endogenous inhibitor of 5'-nucleotidase was found in the cytosol of canine neutrophils. The specific 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate also inhibited the canine enzyme in intact cells. Leucine aminopeptidase was located solely in the myeloperoxidase-containing granules of the canine neutrophil. Plasma membrane, as identified by the presence of Mg2+-ATPase and 5'-nucleotidase activities, was separated from other cell organelles by Percoll-density gradient centrifugation of a 10 000 X g supernatant of nitrogen cavitated neutrophils.

Absence of 5'-nucleotidase in porcine polymorphonuclear leucocyte membranes.

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5'-nucleotidase inhibitor alpha, beta-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5'-nucleotidase. An antibody against mouse liver 5'-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5'-nucleotidase and the necessity for distinguishing between true 5'-nucleotidase and non-specific phosphatase activity is discussed.

Isolation of human neutrophil plasma membranes employing neutrophil cytoplasts and changes in the cell-surface proteins upon cell activation.

A plasma membrane fraction, highly enriched in 5'-nucleotidase activity, was prepared from human neutrophils by disruption of previously formed neutrophil cytoplasts (enucleated neutrophils), which were devoid of intracellular organelles. This plasma membrane fraction shows an extremely low contamination by specific and azurophilic granule markers as compared to previous reported preparations. Nevertheless, a novel tertiary granule (Mollinedo, F. and Schneider, D.L. (1984) J. Biol. Chem. 259, 7143-7150), unlike specific and azurophilic granules, fuses partially with the cell surface under the experimental conditions used for cytoplast preparation. Comparison between the external cell-surface proteins in resting neutrophils and neutrophil cytoplasts by lactoperoxidase-catalyzed iodination showed some differences both in deletion and in addition of proteins. In resting cells, iodine was incorporated into at least 13 proteins ranging in size from over 200 to 30 kDa. A 140 kDa polypeptide, representing the major labeled surface component in resting neutrophils, was absent from cytoplasts. Furthermore, high-molecular-weight proteins (110 and over 160 kDa were more exposed to iodination after cytoplast preparation. Activation of human neutrophils by N-formylmethionylleucylphenylalanine induced some alterations in the pattern of labeled cell-surface proteins, which correlated to a certain degree with those observed during cytoplast preparation.

Preparation of a highly purified surface membrane fraction from rabbit polymorphonuclear leucocytes by high-voltage free-flow electrophoresis.

A surface membrane fraction of high purity and good yield has been prepared from homogenates of rabbit peritoneal polymorphonuclear leucocytes, using a preliminary sorbitol density gradient sedimentation followed by preparative high voltage electrophoresis in a thin flowing buffer film. Enrichment values for the plasma membrane marker enzyme 5'-nucleotidase and 125I-labelled Lens culinaris lectin, after the latter had been applied at the whole cell level, were 18-fold and 6-fold, respectively. Contamination of the surface membrane fraction by other organelles was negligible and approximately 1 mg of surface membrane protein can be obtained from 2 . 10(9) leucocytes. A triacylglycerol-rich, protein-poor fraction that lacks any definable structure in electron microscopy separates discretely from the surface membrane vesicles during electrophoresis. It is considered that this may be a contaminant not previously recognized as present in membrane fractions prepared by more conventional procedures.