Nucleotidase Enzyme Research Articles

Efn1 and Efn2 are extracellular 5'-nucleotidases induced during the fission yeast response to phosphate starvation.

Schizosaccharomyces pombe adapts to phosphate starvation by upregulating the expression of a cell surface acid phosphatase that mobilizes inorganic phosphate from the extracellular milieu, as well as transmembrane transporters that take up inorganic phosphate and glycerophosphocholine. This study identifies two paralogous extracellular 5'-nucleotidase enzymes, Efn1 and Efn2, encoded by genes that are highly transcriptionally induced during acute phosphate starvation, as major proteins secreted into the medium by phosphate-starved fission yeast cells. Secreted Efn1 and Efn2 catalyze the release of inorganic phosphate from all ribonucleoside monophosphates, with a preference for CMP. Secretion of Efn1 and Efn2 enables phosphate-starved fission yeast to thrive by using extracellular CMP as a source of inorganic phosphate. The starvation-induced production of extracellular 5'-nucleotidases adds a new layer of pro-adaptive function during phosphate limitation.

Partial Purification of 5'-Nucleotidase Enzyme in Patients with Renal Failure and Kinetic Study of the Purified Enzyme in Salah al-Din

This study involved separating and partially purifying the 5'-Nucleotidase enzyme isoforms from the sera of patients with renal failure. The purification process consisted of several steps, beginning with precipitation using 30% ammonium sulfate, resulting in a purification fold of 1.2625 and an enzyme yield of 65.63%. This was followed by membrane filtration using a buffer solution, achieving a purification fold of 1.651 and an enzyme yield of 49%. The protein fraction separated from the dialysis bag was then passed through a gel filtration column, reaching a purification fold of 51.88 and an enzyme yield of 43.1%. The enzyme isoforms from the separated protein fraction were further separated using gel filtration, employing ion-exchange chromatography techniques. This resulted in three isoforms of the 5'-Nucleotidase enzyme with varying degrees of purity, the highest for isoform. at 2.297-fold. The study also included kinetic studies of the enzyme purified by gel filtration, which involved determining the Km value under the influence of varying concentrations of the enzyme's substrate, 5'-AMP, for the purified enzyme from the sera of patients with renal failure, which was found to be 12.4. The effects of temperature, pH, and reaction time on enzyme activity were also studied.

Evaluation of the level of 5'-nucleotidase and some biochemical parameters in the sera of beta-thalassemia patients

One of the hydrolytic enzymes that catalyzes the hydrolysis of 5' -nucleotides into ribonucleosides and phosphate is the 5'nucleotidase, which is found in several organs. Oxidative stress shows depletion of antioxidants, such as glutathione (GSH), and the increased concentration of product of lipid peroxidation, such as MDA. The study comprised a total of 54 subjects including patients with beta thalassemia (n=27) and healthy volunteers (n=27) matched by age and gender. Hematology (Hb and % PCV), 5'-nucleotidase, Ferritin, GSH and MDA were measured using different methods and devices. The 5'nucleotidase, Ferritin, and MDA levels were significantly increased (p<0.001) in beta-thalassemia patients compared to the controls. As opposed to that, the Hb, % PCV and GSH levels were significantly decreased (p<0.001) in patients with beta-thalassemia. There was a negative significant correlation (r = -0.146, -0.245, p<0.05) between Hb and 5'- NT enzyme and PCV% with 5'- NT enzyme, respectively. While Hb had a high significant correlation (r = 0.956, p<0.05) with PCV%. In addition, 5'- NT enzyme had a positive significant correlation with GSH, MDA and Ferritin (r = 0.584, 0.442, 0.735, p<0.05), respectively. The present findings indicated an increase in the level of effectiveness of the 5'nucleotidase enzyme, ferritin and GSH in β thalassemia patients while decreasing in MDA.

Abstract 3960: Therapeutic targeting of HIF-1 alpha induced CD73 expression in experimental esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) is one of the most aggressive human cancers with poor prognosis, and the overall 5-year survival rate is less than 20 percent. Prognosis for EAC remains poor even with modern combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches for EAC treatment improvements are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to cancer aggressiveness and poor clinical prognosis. Overexpression of hypoxia-inducible factor 1-alpha (HIF-1 alpha) and immunosuppressive CD73, an ecto-5’-nucleotidase enzyme in cancer can give rise to tumor progression with drug resistance. CD73 has never been proposed as a therapeutic target in EAC and its relationship with hypoxia or HIF-1 alpha has not also been investigated in EAC. In this study, we therefore investigated the therapeutic targeting of HIF-1 alpha and CD73 by acriflavine in experimental EAC. Methods: Hypoxia in EAC cells were induced by 3D culture and hypoxic exposure. NanoCulture® plates and dishes were used for 3D cultures. For hypoxic exposure, cells were placed in a sealed modular incubator chamber flushed with a gas mixture containing 1% O2, 5% CO2 and 94% N2. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Nanoparticle albumin-bound paclitaxel (NPT) was used as chemotherapeutic agent, whereas acriflavine was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 and Cell Titer-Glo (CTG) luminescent assays, in vivo tumor growth was detected by measuring subcutaneous xenografts, apoptosis was detected by cleaved caspase 3/PARP expressions and hypoxia-targeting was detected by HIF-1 alpha/CD73 expressions. Results: We observed overexpression of both HIF-1 alpha and CD73 in 3D culture and hypoxic exposure of EAC cells. Interestingly acriflavine treatment drastically inhibited both HIF-1 alpha and CD73 expression in EAC 3D culture and hypoxic exposure. 3D culture was more resistant to antiproliferative effect of chemotherapeutic agent NPT over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavine showed stronger antiproliferative effects in 3D culture than in 2D culture. We also observed hypoxia inside the 3D culture spheroids. In addition, acriflavine showed significant in vivo antitumor efficacy both as monotherapy and in combination with NPT. In subcutaneous xenografts using OE19 EAC cells, acriflavine monotherapy exhibited a significant decrease in relative tumor volume to 55.02% compared to control (p=0.04) and addition of NPT with acriflavine also showed a significant enhancement effect of tumor regression as tumor size decreased to 32.70% compared to control (p=0.002). Conclusion: These results support the potential of acriflavine as HIF-1 alpha and CD73 targeting and its combination with chemotherapy NPT as an effective option for EAC therapy. Citation Format: Md Sazzad Hassan, Aktar Ali, Saisantosh Ponna, Dimitri Scofield, Niranjan Awasthi, Mark Jantz, Urs von Holzen. Therapeutic targeting of HIF-1 alpha induced CD73 expression in experimental esophageal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3960.

Ecto-5'-nucleotidase (Nt5e/CD73)-mediated adenosine signaling attenuates TGFβ-2 induced elastin and cellular contraction.

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor β (TGFβ) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFβ regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFβ signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFβ-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFβ signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFβ signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.

Senna petersiana (Bolle) leaf extract modulates glycemic homeostasis and improves dysregulated enzyme activities in fructose-fed streptozotocin-induced diabetic rats

Ethnopharmacological relevanceSenna petersiana (Bolle) is a native South African medicinal shrub combined locally with other plant products to manage diabetes or used as a single therapy for several other ailing conditions. Aim of the studyThis study evaluated the antidiabetic and antilipidemic effects of S. petersiana leaf ethanol extract and its modulatory effects on dysregulated enzyme activities in fructose-fed streptozotocin-induced diabetic rats. Materials and methodsSix groups of 6-weeks old male Sprague Dawley rats were used in this study. Diabetes was induced in four of the groups by injecting (i.p.) 40 mg/kg of streptozotocin after a two-weeks feeding of 10% fructose via drinking water, while animals in the two normal groups were given similar volume of vehicle buffer and normal drinking water, respectively. After the confirmation of diabetes, treatment with 150 and 300 mg/kg body weight of the ethanolic leaf extract of S. petersiana proceeded for a period of 6 weeks. ResultsOral administration of S. petersiana leaf extract significantly lowered blood glucose, food and liquid intake, glycosylhaemoglobin in blood, liver and cardiac biomarkers, and lipid profile in serum and atherogenic index (AIP) in both the low and high-dose treated animal groups. This was accompanied by a simultaneous increase in Homeostatic Model Assessment-beta (HOMA-β) score, serum high-density lipoproteins cholesterol (HDL-c), and insulin levels. It also improved pancreatic and serum-reduced glutathione (GSH) levels, catalase, and superoxide dismutase (SOD) enzymes activities with a simultaneous reduction in malondialdehyde (MDA) and nitric oxide (NO) concentrations. Moreover, the extract modulated dysregulated α-amylase, lipase, cholinesterase, and 5′ nucleotidase enzyme activities in pancreatic tissue as well as glycogen metabolism in the liver. Analysis of the phytochemicals in the S. petersiana extract showed the presence of phytol, 4a,7,7,10a-tetramethyldodecahydrobenzo[f]-chromen-3-ol, phytol acetate, solasodine glucoside, cassine, veratramine and solasodine acetate. Amongst these compounds, solasodine glucoside had the best binding energy (ΔG) with the selected diabetes-linked enzymes via molecular docking simulation. ConclusionData from this study demonstrate the antidiabetic effects of S. petersiana leaf extract via the modulation of the dysregulated indices involved in type 2 diabetes and its associated complications. Although it has been shown safe in animals, further toxicological studies are required to ensure its safety for diabetes management in humans.

CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities

Background aimsExtracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors compared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lymphocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immunomodulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay. MethodsThe bona fide MSC antigen CD73 possesses ecto-5’-nucleotidase activity that cleaves pro-inflammatory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5’-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5’-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity. ResultsThe authors showed that the ecto-5’-nucleotidase activity of the MSC-EV preparations did not correlate with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5’-nucleotidase activity was resistant to EV-destroying detergent treatment. ConclusionsEcto-5’-nucleotidase activity neither reflects the potency of the authors’ MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5’-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors’ MSC-EV products. The development of appropriate potency assays for MSC-EV products remains challenging.

Regulation of Microglial Functions by Purinergic Mechanisms in the Healthy and Diseased CNS.

Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood–brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5′-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation.

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5'-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.

Активность фермента 5`-нуклеотидазы у больных хроническим гепатитом С с обострением естественного течения болезни и его терапевтическая коррекция

Активность фермента 5`-нуклеотидазы у больных хроническим гепатитом С с обострением естественного течения болезни и его терапевтическая коррекция

Estrogen receptors modulate ectonucleotidases activity in hippocampal synaptosomes of male rats

Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and adenosine, are among least investigated signaling factors that participate in 17β-estradiol (E2)-mediated synaptic rearrangements in rodent hippocampus. Their levels in the extrasynaptic space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases1-3 (NTPDase1-3)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, the aim of the present study was to get closer insight in the E2-induced decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors (ERs i.e. ERα, ERβ or GPER1) responsible for the observed effects of E2. In this study we show indiscriminate participation of estradiol receptor α (ERα), -β (ERβ) and G- protein coupled estrogen receptor 1 (GPER1) in the mediation of E2 actions in hippocampal synaptosomes of male rats. Synaptic NTPDase1-3 activities are modulated only through activation of ERβ, while activation of ERα, -β and/or non-classical GPER1 decreases synaptic eN activity. Since both ATP and adenosine function as neuromodulators in the hippocampal networks, influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance.

Abstract 3871: Monitoring CD73 and CD39 ectonucleotidase activities and their modulation by antibodies and small molecules inhibitors

Abstract Modern immunotherapy approaches focusing on bolstering T cell responses and augmenting cell mediated immunity with final tumor destruction have ushered a new era and a turning point in cancer treatment. This was demonstrated by recent successes of several immunotherapeutic regimes, such as monoclonal antibody blocking of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD1), which boosted the development of this treatment modality. Although much success have been generated with multiple tumors at different stages, tumors can deploy multiple mechanism to allow their survival by avoiding immunesurveillance. Thus, identification of alternate immunosuppressive pathways that can be targeted to enhance tumor clearance are disparately needed. It is well known that adenosine produced in the tumor microenvironment (TME) hampers the immune reaction towards cancer cells by disabling the cytotoxic anti-tumor immune response while enhancing the proliferation and polarization of immunosuppressive cells and the neovascularization that support tumor growth. It is also known that modulation of adenosine levels in the TME limits tumor growth and improves anti-tumor immune activity. Therefore, targeting the enzymes that produce adenosine and adenosine receptors as potential biomarkers for treatment response and tumor progression can provide a novel therapeutic strategy that synergize with other check point immunoblockade antibodies. Towards this goal we have developed a novel assay to monitor the activity of the two enzymes responsible for generating extracellular adenosine in the TME, namely, ectonucleotidase triphosphate diphosphohydrolase 1 (CD39) and ecto 5'-nucleotidase (CD73). Since adenosine monophosphate (AMP) is generated from adenosine triphosphate (ATP) and adenosine diphosphate (ADP) by CD 39 followed by generation of adenosine (Ado) from AMP by CD73, we monitored the activity of both enzymes in different tumor cell lines with varied levels of both enzymes. We were able to correlate the activity of the ecto enzymes with their protein level on the cell surface and carried out enzyme characterization using both antibodies and small molecules selective for their targets. Our results clearly show that small molecule inhibitors of CD73 provide a better strategy to combat adenosine levels while small molecules towards CD39 were shown to be promiscuous since they inhibit other enzymes such as kinases. For targeting CD39, a better strategy might be the use of antibodies since they are selective to their cognate enzyme protein. Because the therapeutic potential of targeting CD39 and CD73 is not dependent solely on their nucleotidase enzyme activity but also due to their non-enzymatic role in tumor progression, antibodies might provide an alternative approach for targeting these enzymes. Citation Format: Said A. Goueli, Kevin Hsiao. Monitoring CD73 and CD39 ectonucleotidase activities and their modulation by antibodies and small molecules inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3871.

Erratum to: 17β-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes.

17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24 h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.

17β-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes.

17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.

NTPDase and 5'-nucleotidase as inflammatory markers in cattle naturally infected by Eurytrema coelomaticum.

The aim of this study was to evaluate seric NTPDase and 5'nucleotidase activities of cattle naturally infected by Eurytrema coelomanticum, as well as to correlate them to histopathological lesions in the pancreas and the degree of parasitism. Blood samples and pancreas of 51 bovines were collected on a slaughterhouse in Southern Brazil: 33 from cattle naturally infected by E. coelomanticum (the Group A), and 18 from uninfected animals (the Group B). Infected animals showed an average of 532 parasites per pancreas. In the pancreatic histology, ducts displayed hyperplasia, stenosis, proliferation of fibrous tissue, and interstitial inflammatory infiltration of lymphocytes. The serum from infected animals showed an increase in NTPDase activity when ATP was used as substrate (P<0.001). For the ADP substrate, there was no difference between groups regarding NTPDase activity (P=0.37), as well as 5'-nucleotidase activity (P=0.27). Correlating NTPDase activity (ATP substrate) with the degree of histopathological lesions (rho=0.66, P<0.001) and the parasitic load on the pancreas (rho=0.65, P<0.001), a positive correlation was observed. Similar results were found between the degree of histopathological lesions and NTPDase activity (ADP substrate; rho=0.29, P=0.03), and 5'nucleotidase activity (rho=0.35, P=0.01). Based on the results of NTPDase and 5'nucleotidase enzymes in cattle naturally infected by E. coleomanticum, it is possible to suggest that these enzymes are involved in the modulation of inflammation, and they can act as markers of inflammatory response.

Characterization of extracellular nucleotide metabolism in Candida albicans.

Candida albicans is the most frequent agent of human disseminated fungal infection. Ectophosphatase and ectonucleotidase activities are known to influence the infectious potential of several microbes, including other non-albicans species of Candida. With the present work we aim to characterize these ecto-enzymatic activities in C. albicans. We found that C. albicans does not have a classical ecto-5'-nucleotidase enzyme and 5'AMP is cleaved by a phosphatase instead of exclusively by a nucleotidase that also can use 3'AMP as a substrate. Moreover, these enzymatic activities are not dependent on secreted soluble enzymes and change when the yeast cells are under infection conditions, including low pH, and higher temperature and CO2 content.

Abstract 16348: Ecto-Nucleotidase-Derived Adenosine Promotes Mineralization Through A2a Receptor in Calcified Aortic Valves Disease

Background: Mineralization plays a crucial role in the development of calcific aortic valve disease (CAVD). The expression of ecto-nucleotidases may promote the mineralization of valve interstitial cells (VICs). We hypothesized that the expression of NPP1, which generates adenosine monophosphate (AMP), and 5’nucleotidase, an enzyme using AMP as a substrate to produce adenosine, may co-regulate the mineralization of the aortic valve. Methods: We have investigated the expression of NPP1 and 5’nucleotidase in CAVD tissues and determined the role of these ecto-nucleotidases on the mineralization of isolated VICs. The signaling pathway involving adenosine receptors was also studied. Results: In CAVD tissues, we documented that NPP1 and 5’nucleotidase enzyme activities were increased. NPP1 and 5’nucleotidase were co-expressed by VICs. In cell culture we found that mineralization induced by ATP was decreased by silencing NPP1 and 5’nucleotidase, suggesting a role for adenosine. Adenosine and specific A2a receptor agonist increased the calcification of VICs. Silencing of A2a receptor and the use of A2a-/- receptor mouse VICs abrogated adenosine-induced mineralization. Also, A2a receptor-mediated mineralization of VICs was negated by the transfection of a mutant dominant negative Gαs vector. Inhibition of the protein kinase A (PKA) pathway prevented adenosine-induced mineralization of VICs and expression of NPP1. We next showed that activation of PKA promoted in luciferase assay the activity of the NPP1 promoter. By using chromatin immunoprecipitation assay (ChIP) we documented that the cAMP response element binding protein (CREB), downstream of PKA, physically interacted with the NPP1 promoter region. Furthermore, the transfection of a mutant dominant active CREBDIEDML in isolated VICs increased the NPP1 promoter activity by 6.5-fold. Conclusion: The overexpression of NPP1 and 5’nucleotidase in CAVD promotes the mineralization of the aortic valve through A2a adenosine receptor, which signals through Gαs and the cAMP/PKA/CREB pathway. CREB is a positive regulator of NPP1 promoter activity in a positive feedback loop. The ecto-nucleotidases and/or A2a adenosine receptor could represent potential novel pharmaceutical targets in CAVD.

In vitro Anti-Snake Venom Properties of Carisssa spinarum Linn Leaf Extracts

In vitro inhibitory activity of Carissa spinarum leaf extracts against Bungarus caeruleus and Vipera russelli toxic snake venom enzymes was determined. The leaf extracts in non-polar and polar solvents were concentrated and were used for inhibitory activity determination. Methanol extracts (100 μg.mL−1) inhibited acetylcholinesterase, phospholipase A2, hyaluronidase, phosphomonoesterase, phosphodiesterase, 5′ nucleotidase enzymes of B. caeruleus and V. russelli venoms.

QUINAZOLIN-4-PIPERIDIN-4-METHYL SULFAMIDE COMPOUNDS ARE SPECIFIC INHIBITORS OF HUMAN NPP1 AND PREVENT CALCIFICATION OF VALVE INTERSTITIAL CELLS

s S245 promote the mineralization of valve interstitial cells (VICs), the main cellular component of the aortic valve. We hypothesized that the expression of NPP1, which generates adenosine monophosphate (AMP), and 5’nucleotidase, an enzyme using AMP as a substrate to produce adenosine, may co-regulate the mineralization of the aortic valve. METHODS: We have investigated the expression of NPP1 and 5’nucleotidase in CAVD tissues and determined the role of these ecto-nucleotidases on the mineralization of isolated VICs. RESULTS: In CAVD tissues, we documented that NPP1 and 5’nucleotidase enzyme activities were increased. NPP1 and 5’nucleotidase were co-expressed by VICs. In cell culture we found that mineralization induced by ATP was decreased by silencing NPP1 and 5’nucleotidase, suggesting a role for adenosine. Adenosine and specific A2a receptor agonist increased the calcification of VICs. Silencing of A2a receptor and the use of A2a-/receptor mouse VICs abrogated adenosine-induced mineralization. Also, A2a receptor-mediated mineralization of VICs was negated by the transfection of a mutant dominant negative Gas vector. Inhibition of the protein kinase A (PKA) pathway prevented adenosine-induced mineralization of VICs and expression of NPP1. We next showed that activation of PKA promoted in luciferase assay the activity of the NPP1 promoter. By using chromatin immunoprecipitation assay (ChIP) we documented that the cAMP response element binding protein (CREB), downstream of PKA, physically interacted with the NPP1 promoter region. Furthermore, the transfection of a mutant dominant active CREBDIEDML in isolated cells increased the NPP1 promoter activity by 6.5-fold. CONCLUSION: The overexpression of NPP1 and 5’nucleotidase in CAVD promotes the mineralization of the aortic valve through A2a adenosine receptor, which signals through Gas and the cAMP/PKA/CREB pathway. CREB is a positive regulator of NPP1 promoter activity in a positive feedback loop. The ecto-nucleotidases and/or A2a adenosine receptor could represent potential novel pharmaceutical targets in CAVD. 372 ROLE OF LONG NON-CODING RNAS IN THE REGULATION OF THE NOTCH PATHWAY: IMPLICATION FOR CALCIFIC AORTIC VALVE STENOSIS F Hadji, M Boulanger, S Guauque-Olarte, R Bouchareb, Y Bosse, P Mathieu

NPP1 AND 5’NUCLEOTIDASE PROMOTE THE MINERALIZATION OF THE AORTIC VALVE: ROLE OF A2A RECEPTOR AND CREB

Mineralization of the aortic valve is the major culprit in the development of calcific aortic valve disease (CAVD). The expression of ecto-nucleotidases, which exert a control over extracellular nucleotides/nucleosides, may promote the mineralization of valve interstitial cells (VICs), the main cellular component of the aortic valve. We hypothesized that the expression of NPP1, which generates adenosine monophosphate (AMP), and 5’nucleotidase, an enzyme using AMP as a substrate to produce adenosine, may co-regulate the mineralization of the aortic valve. We have investigated the expression of NPP1 and 5’nucleotidase in CAVD tissues and determined the role of these ecto-nucleotidases on the mineralization of isolated VICs. In CAVD tissues, we documented that NPP1 and 5’nucleotidase enzyme activities were increased. NPP1 and 5’nucleotidase were co-expressed by VICs. In cell culture we found that mineralization induced by ATP was decreased by silencing NPP1 and 5’nucleotidase, suggesting a role for adenosine. Adenosine and specific A2a receptor agonist increased the calcification of VICs. Silencing of A2a receptor and the use of A2a-/- receptor mouse VICs abrogated adenosine-induced mineralization. Also, A2a receptor-mediated mineralization of VICs was negated by the transfection of a mutant dominant negative Gαs vector. Inhibition of the protein kinase A (PKA) pathway prevented adenosine-induced mineralization of VICs and expression of NPP1. We next showed that activation of PKA promoted in luciferase assay the activity of the NPP1 promoter. By using chromatin immunoprecipitation assay (ChIP) we documented that the cAMP response element binding protein (CREB), downstream of PKA, physically interacted with the NPP1 promoter region. Furthermore, the transfection of a mutant dominant active CREBDIEDML in isolated cells increased the NPP1 promoter activity by 6.5-fold. The overexpression of NPP1 and 5’nucleotidase in CAVD promotes the mineralization of the aortic valve through A2a adenosine receptor, which signals through Gαs and the cAMP/PKA/CREB pathway. CREB is a positive regulator of NPP1 promoter activity in a positive feedback loop. The ecto-nucleotidases and/or A2a adenosine receptor could represent potential novel pharmaceutical targets in CAVD.