Hypoxia Effects on Trypanosoma cruzi Epimastigotes Proliferation, Differentiation, and Energy Metabolism.

Trypanosoma cruzi, the causative agent of Chagas disease, faces changes in redox status and nutritional availability during its life cycle. However, the influence of oxygen fluctuation upon the biology of T. cruzi is unclear. The present work investigated the response of T. cruzi epimastigotes to hypoxia. The parasites showed an adaptation to the hypoxic condition, presenting an increase in proliferation and a reduction in metacyclogenesis. Additionally, parasites cultured in hypoxia produced more reactive oxygen species (ROS) compared to parasites cultured in normoxia. The analyses of the mitochondrial physiology demonstrated that hypoxic condition induced a decrease in both oxidative phosphorylation and mitochondrial membrane potential (ΔΨm) in epimastigotes. In spite of that, ATP levels of parasites cultivated in hypoxia increased. The hypoxic condition also increased the expression of the hexokinase and NADH fumarate reductase genes and reduced NAD(P)H, suggesting that this increase in ATP levels of hypoxia-challenged parasites was a consequence of increased glycolysis and fermentation pathways. Taken together, our results suggest that decreased oxygen levels trigger a shift in the bioenergetic metabolism of T. cruzi epimastigotes, favoring ROS production and fermentation to sustain ATP production, allowing the parasite to survive and proliferate in the insect vector.

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

Hydrogen Peroxide Generation as an Underlying Response to High Extracellular Inorganic Phosphate (Pi) in Breast Cancer Cells.

According to the growth rate hypothesis (GRH), tumour cells have high inorganic phosphate (Pi) demands due to accelerated proliferation. Compared to healthy individuals, cancer patients present with a nearly 2.5-fold higher Pi serum concentration. In this work, we show that an increasing concentration of Pi had the opposite effect on Pi-transporters only in MDA-MB-231 when compared to other breast cell lines: MCF-7 or MCF10-A (non-tumoural breast cell line). Here, we show for the first time that high extracellular Pi concentration mediates ROS production in TNBC (MDA-MB-231). After a short-time exposure (1 h), Pi hyperpolarizes the mitochondrial membrane, increases mitochondrial ROS generation, impairs oxygen (O2) consumption and increases PKC activity. However, after 24 h Pi-exposure, the source of H2O2 seems to shift from mitochondria to an NADPH oxidase enzyme (NOX), through activation of PKC by H2O2. Exogenous-added H2O2 modulated Pi-transporters the same way as extracellular high Pi, which could be reversed by the addition of the antioxidant N-acetylcysteine (NAC). NAC was also able to abolish Pi-induced Epithelial-mesenchymal transition (EMT), migration and adhesion of MDA-MB-231. We believe that Pi transporters support part of the energy required for the metastatic processes stimulated by Pi and trigger Pi-induced H2O2 production as a signalling response to promote cell migration and adhesion.

3-Bromopyruvate: A new strategy for inhibition of glycolytic enzymes in Leishmania amazonensis.

The compound 3-bromopyruvate (3-BrPA) is well-known and studies from several researchers have demonstrated its involvement in tumorigenesis. It is an analogue of pyruvic acid that inhibits ATP synthesis by inhibiting enzymes from the glycolytic pathway and oxidative phosphorylation. In this work, we investigated the effect of 3-BrPA on energy metabolism of L. amazonensis. In order to verify the effect of 3-BrPA on L. amazonensis glycolysis, we measured the activity level of three glycolytic enzymes located at different points of the pathway: (i) glucose kinases, step 1, (ii) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), step 6, and (iii) enolase, step 9. 3-BrPA, in a dose-dependent manner, significantly reduced the activity levels of all the enzymes. In addition, 3-BrPA treatment led to a reduction in the levels of phosphofruto-1-kinase (PFK) protein, suggesting that the mode of action of 3-BrPA involves the downregulation of some glycolytic enzymes. Measurement of ATP levels in promastigotes of L. amazonensis showed a significant reduction in ATP generation. The O2 consumption was also significantly inhibited in promastigotes, confirming the energy depletion effect of 3-BrPA. When 3-BrPA was added to the cells at the beginning of growth cycle, it significantly inhibited L. amazonensis proliferation in a dose-dependent manner. Furthermore, the ability to infect macrophages was reduced by approximately 50% when promastigotes were treated with 3-BrPA. Taken together, these studies corroborate with previous reports which suggest 3-BrPA as a potential drug against pathogenic microorganisms that are reliant on glucose catabolism for ATP supply.

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment.

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ±â€¯0.1674 mM Pi and Vmax = 198.6 ±â€¯10.23 Pi × h-1 × mg protein-1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.

Characterization of inorganic phosphate transport in the triple-negative breast cancer cell line, MDA-MB-231.

BACKGROUND: Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaPi-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (Pi) transporter in this cancer type remains elusive. METHODS: In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated Pi transport with cell migration and adhesion. RESULTS: We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in Pi transport. We observed that the inorganic phosphate is dependent on sodium transport (K0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of Pi, following the Michaelis-Menten kinetics (K0,5 = 0.08 mM Pi). PFA, monensin, furosemide and ouabain inhibited Pi transport, cell migration and adhesion. CONCLUSIONS: Taken together, these results showed that the uptake of Pi in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.

Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2) generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 µM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 µM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 µM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

Rhodnius prolixus: modulation of antioxidant defenses by Trypanosoma rangeli.

Trypanosoma rangeli is a protozoan parasite of insects and mammals that is challenged by the constant action of reactive oxygen species, generated either by its own metabolism or through the host immune response. The aim of this work was to investigate whether T. rangeli is able to modify the redox state of its insect vector, Rhodnius prolixus, through the modulation of such antioxidant enzymes as superoxide dismutase (SOD), catalase, and GPx present in the midgut of the insect. We verified that in R. prolixus fed with blood infected with T. rangeli there is an increase in SOD activity in the anterior and posterior midguts. However, the activities of enzymes related to hydrogen peroxide and hydroperoxides metabolism, such as catalase and GPx, were decreased in relation to the insect control group, which was only fed blood. These changes in the redox state of the vector led to an increase in lipid peroxidation and thiol oxidation levels in the anterior and posterior midgut tissues. We also verified that the addition of 1 mM GSH in the blood meal of the infected insects increased the proliferation of these parasites by 50%. These results suggest that there is an increase in oxidative stress in the insect gut during T. rangeli infection, and this condition could contribute to the control of the proliferation of these parasites.

Nox4: a hydrogen peroxide-generating oxygen sensor.

Nox4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for Nox4 require upstream activators, either calcium or organizer/activator subunits (p47(phox), NOXO1/p67(phox), and NOXA1). Nox4 may also be unusual as it reportedly releases hydrogen peroxide (H2O2) in contrast to Nox1-Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection. Our studies were undertaken (1) to identify the Nox4 ROS product using a membrane-free, partially purified preparation of Nox4 and (2) to test the hypothesis that Nox4 activity is acutely regulated not by activator proteins or calcium, but by cellular pO2, allowing it to function as an O2 sensor, the output of which is signaling H2O2. We find that approximately 90% of the electron flux through isolated Nox4 produces H2O2 and 10% forms superoxide. The kinetic mechanism of H2O2 formation is consistent with a mechanism involving binding of one oxygen molecule, which is then sequentially reduced by the heme in two one-electron reduction steps first to form a bound superoxide intermediate and then H2O2; kinetics are not consistent with a previously proposed internal superoxide dismutation mechanism involving two oxygen binding/reduction steps for each H2O2 formed. Critically, Nox4 has an unusually high Km for oxygen (∼18%), similar to the values of known oxygen-sensing enzymes, compared with a Km of 2-3% for Nox2, the phagocyte NADPH oxidase. This allows Nox4 to generate H2O2 as a function of oxygen concentration throughout a physiological range of pO2 values and to respond rapidly to changes in pO2.

Identification of uncoupling protein 4 from the blood-sucking insect Rhodnius prolixus and its possible role on protection against oxidative stress.

Uncoupling proteins (UCPs) play a critical role in the control of the mitochondrial membrane potential (ΔΨm) due to their ability to dissipate the proton gradient, which results in the uncoupling of mitochondrial respiration from ATP production. Most reactive oxygen species generation in mitochondria occurs in complex III, due to an increase of semiquinone (Q(-)) half-life. When active, UCPs can account as a potential antioxidant system by decreasing ΔΨm and increasing mitochondrial respiration, thus reducing Q(-) life time. The hematophagous insect Rhodnius prolixus, a vector of Chagas disease, is exposed to a huge increase in oxidative stress after a blood meal because of the hydrolysis of hemoglobin and the release of the cytotoxic heme molecule. Although some protective mechanisms were already described for this insect and other hematophagous arthropods, the putative role of UCP proteins as antioxidants in this context has not been explored. In this report, two genes encoding UCP proteins (RpUcp4 and RpUcp5) were identified in the R. prolixus genome. RpUcp4 is the predominant transcript in most analyzed organs, and both mRNA and protein expression are upregulated (13- and 3-fold increase, respectively) in enterocytes the first day after the blood feeding. The increase in UCP4 expression is coincident with the decrease in hydrogen peroxide (H2O2) generation by midgut cells. Furthermore, in mitochondria isolated from enterocytes, the modulation of UCP activity by palmitic acid and GDP resulted in altered ΔΨm, as well as modulation of H2O2 generation rates. These results indicate that R. prolixus UCP4 may function in an antioxidation mechanism to protect the midgut cells against oxidative damage caused by blood digestion.

Inhibition of ecto-phosphatase activity in conidia reduces adhesion and virulence of Metarhizium anisopliae on the host insect Dysdercus peruvianus.

Metarhizium anisopliae is an entomopathogenic fungus with the ability to infect a broad range of arthropods, and have evolved distinct strategies for their attachment to hosts. Here, we describe the characterisation of ecto-phosphatase activity on the conidia surface of M. anisopliae and its relevance in the host interaction process. Ecto-phosphatase activity was linear for 60 min and during this time, was linear with the increase of cell density. The optimum pH was in the acidic range and some divalent metals, such as Cu(2+), Cd(2+) and Zn(2+), inhibited ecto-phosphatase activity. The activity was also reduced by phosphatase inhibitors. Importantly, the inhibition of phosphatase activity in conidia reduced the adhesion to Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) integument and, consequently and indirectly, M. anisopliae infection. The results herein presented show, for the first time, the importance of ecto-phosphatase activity in M. anisopliae conidia and provide the first evidence of its direct involvement in adhesion and host infection.

Cell signaling through protein kinase C oxidation and activation.

Due to the growing importance of cellular signaling mediated by reactive oxygen species (ROS), proteins that are reversibly modulated by these reactant molecules are of high interest. In this context, protein kinases and phosphatases, which act coordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. The major mechanism by which these proteins may be modified by oxidation involves the presence of key redox-sensitive cysteine residues. Protein kinase C (PKC) is involved in a variety of cellular signaling pathways. These proteins have been shown to contain a unique structural feature that is susceptible to oxidative modification. A large number of scientific studies have highlighted the importance of ROS as a second messenger in numerous cellular processes, including cell proliferation, gene expression, adhesion, differentiation, senescence, and apoptosis. In this context, the goal of this review is to discuss the mechanisms by which PKCs are modulated by ROS and how these processes are involved in the cellular response.

Leishmania amazonensis: inhibition of 3'-nucleotidase activity by Cu2+ ions.

Free Cu(2+) is toxic due to the capacity of free copper ions to catalyze the production of reactive oxygen species (ROS) that can modify the structure and/or function of biomolecules. In addition, non-specific binding to enzymes, which modifies their catalytic activities, can occur. In this work, the mechanisms underlying the ability of copper to inhibit 3'-nucleotidase from Leishmania amazonensis (La3'-nucleotidase) were investigated. To that end, La3'-nucleotidase activity was assayed with CuCl(2) in the presence of ascorbate or hydrogen peroxide to discriminate non-specific binding effects from pro-oxidant effects of copper. Copper inhibitory effects were greater at more acidic pH than at alkaline pH. The addition of enzyme substrate, adenosine 3'-monophosphate (3'AMP), prevented the inhibition of enzyme activity by copper. Thiol-containing compounds were able to protect the enzyme activity against inhibition due to copper. The specific copper chelating agent bathocuproine sulphonate (BCS) restored enzyme activity after pre-treatment of the enzyme with copper. La3'-nucleotidase activity was found to be resistant to ROS generated during oxidation reactions of ascorbate and hydrogen peroxide catalyzed by copper. Our results suggest that Cu(2+) ions exert their inhibitory effects by binding to specific motifs of the 3'-nucleotidase protein and that the enzyme appears to be extremely resistant to ROS.

The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi.

Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS) which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.

Characterization of an ecto-5'-nucleotidase activity present on the cell surface of Tritrichomonas foetus.

Tritrichomonas foetus is the causative agent of sexually transmitted trichomoniasis in cattle. In females, the infection can be associated with infertility, vaginitis, endometritis, abortion or pyometra, leading to significant economic losses in cattle raising. T. foetus is devoid of the ability to synthesize purine nucleotides de novo, depending instead on salvaging purines from the host environment. Ecto-5'-nucleotidase catalyzes the final step of extracellular nucleotide degradation, the hydrolysis of nucleoside 5'-monophosphates to the corresponding nucleosides and Pi. In this work we show that living, intact cells of T. foetus were able to hydrolyze 5'AMP at a rate of 12.57 ± 1.23 nmol Pi × h(-1) × 10(-7) cells at pH 7.2 and the 5'AMP hydrolysis is due to a plasma membrane-bound ecto-enzyme activity. The apparent K(m) for 5'AMP was 0.49 ± 0.06 mM. In addition to 5'AMP, the enzyme hydrolyzed all substrate monophosphates tested except 3'AMP. No divalent metals or metal chelators were able to modulate enzyme activity. Phosphatase inhibitors did not have an effect on ecto-5'-nucleotidase activity while ammonium molybdate did inhibit the activity in a dose dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. These results indicate the existence of an ecto-5'-nucleotidase that may play a role in the salvage of purines.

Ecto-phosphatases in protozoan parasites: possible roles in nutrition, growth and ROS sensing.

The cellular plasma membrane contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells. Ecto-phosphatases are ecto-enzymes that presumably hydrolyze extracellular phosphorylated substrates, releasing free inorganic phosphate. Although, several alternative functions have been suggested for these enzymes, such as participation in proliferation, differentiation, adhesion, virulence, and infection, little is known about the physiological roles of these enzymes in protozoa parasites. In this review, we discuss the principal features of ecto-phosphatases in protozoan parasites that are causative agents of important diseases such as Chagas' disease, leishmaniasis, amoebiasis, giardiasis, trichomoniasis and, sleeping sickness.

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction.

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.

Giardia duodenalis: biochemical characterization of an ecto-5'-nucleotidase activity.

In this work, we biochemically characterized the ecto-5'-nucleotidase activity present on the surface of the living trophozoites of Giardia duodenalis. Two sequences of the 5'-nucleotidase family protein were identified in the Giardia genome. Anti-mouse CD73 showed a high reaction with the cell surface of parasites. At pH 7.2, intact cells were able to hydrolyze 5'-AMP at a rate of 10.66 ± 0.92 nmol Pi/h/10(7) cells. AMP is the best substrate for this enzyme, and the optimum pH lies in the acidic range. No divalent cations had an effect on the ecto-5'-nucleotidase activity, and the same was seen for NaF, an acid phosphatase inhibitor. Ammonium molybdate, a potent inhibitor of nucleotidases, inhibited the enzyme activity in a dose-dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. The results indicate the existence of an ecto-5'-nucleotidase that could play a role in the salvage of purines.

Biochemical characterization of an ecto-ATP diphosphohydrolase activity in Candida parapsilosis and its possible role in adenosine acquisition and pathogenesis.

In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.