Biomedical subjects

José Roberto Meyer-Fernandes

Publications and source records attributed to José Roberto Meyer-Fernandes.

17 recordsLinked to original sources

PubMed2006

Trypanosoma brucei brucei: biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities.

In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2006

Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions.

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.

Animals↗

PubMed2006

Iron modulates ecto-phosphohydrolase activities in pathogenic trichomonads.

The presence of iron in the extracellular medium is essential for both in vivo and in vitro survival of pathogenic microorganisms, including Trichomonas vaginalis and Tritrichomonas foetus. In these parasites, iron is directly involved in the proliferation, protein expression and activation of critical enzymes. The purpose of this study was to investigate the role of iron in ecto-ATPase, ecto-phophatase and secreted phosphatase activities of these trichomonads. We observed that trichomonads grown in iron-depleted medium exhibited a remarkable decrease in both ecto-ATPase and ecto-phosphatase activities, when compared to those cultivated under control conditions (iron-rich medium). Furthermore, parasites grown in iron-depleted medium restored their enzyme activities when they were re-inoculated into fresh iron-rich medium. We demonstrated that modulation of ecto-phosphohydrolase activities is due neither to enzyme-iron nor to substrate-iron complex formation, since iron addition directly to the medium where the enzymatic reactions occurred did not alter their activities. Previously, we had reported that a fresh clinical isolate of T. vaginalis was much more cytotoxic to epithelial cell monolayers than a long-term cultured one. In this study we witnessed that the fresh isolate of T. vaginalis presented higher activities to all herein investigated enzymes than the long-term cultured one. Altogether, our data clearly point out that iron has a pivotal role in the expression of phosphohydrolases in both trichomonads.

Adenosine Triphosphatases↗

PubMed2006

Effect of extracellular ATP on the human leukaemic cell line K562 and its multidrug counterpart.

Extracellular ATP (ATPo) is capable of inducing different events on cells through receptor activation. The effect produced by ATPo was studied in the cell line K562 and its multidrug resistant (MDR) counterpart, Lucena 1. Lower ATPo concentrations (1 mM and 2.5 mM) led to high (3)H-thymidine incorporation but no increase in cell number. Similarly, the cell cycle profile indicated an increase of cells in S phase and a decrease in G1 and G2, suggesting that the cells did not duplicate their DNA content. Higher doses of ATP (5 mM and 10 mM), as well as UTP (5 mM) and the P2X(7) agonist BzATP, were cytotoxic. However, no expression of P2X(7) receptors could be detected by Western Blot nor were the cells permeabilised by ATP, suggesting that pore formation was not involved in cell death. Both ecto-ATPase and ecto-5'-nucleotidase activity could be demonstrated at the surfaces of K562 and Lucena 1 cells, the latter presenting a higher ecto-5'-nucleotidase activity. Adenosine induced cell death at lower concentrations (2.5 mM) on both cell lines. Furthermore, an increased number of dead cells could be observed when 5 mM Adenosine was used compared to the same concentrations of ATPo. It still remains to be elucidated the nature of the receptors involved in the induction of cell death in these cells.

5'-Nucleotidase↗

PubMed2006

Leishmania amazonensis: characterization of an ecto-phosphatase activity.

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.

Animals↗

PubMed2006

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi.

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 84.6 +/- 11.3 nmol Pi h(-1) mg(-1) mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 +/- 27.6 nmol Pi h(-1) mg(-1)) in the presence of 5 mM MgCl2, with values of Vmax and apparent Km for Mg-ATP(2-) corresponding to 541.9 +/- 48.6 nmol Pi h(-1) mg(-1) cellular dry weight and 1.9 +/- 0.2 mM, respectively. The Mg2+-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1(V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg2+ -stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P2 purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.

Adenosine Triphosphatases↗

PubMed2006

An ectophosphatase activity in Cryptococcus neoformans.

There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.

Cryptococcus neoformans↗

PubMed2006

The occurrence of free D-alanine and an alanine racemase activity in Leishmania amazonensis.

Free D-amino acids are implicated in several biological functions. This study examined the presence of D-alanine in Leishmania amazonensis. Measuring chiral amino acid content by high-performance liquid chromatography we detected a significant amount of free D-alanine in promastigotes of these parasites. D-alanine accounts for 8.9% of total free alanine and is found primarily in the soluble fraction. Specific racemization of L-alanine to D-alanine was detected in cell lysates and this enzyme activity was inhibited by D-cycloserine, an alanine racemase inhibitor. Furthermore, we were able to decrease this pool of D-amino acid by treating our cultures with D-cycloserine. We demonstrate for the first time the existence of a significant amount of free D-alanine in L. amazonensis and an alanine racemase activity present in cell lysates. The restriction of D-alanine to bacteria, some fungi and now in L. amazonensis opens a new perspective on treatment of diseases caused by these microorganisms.

Alanine↗

PubMed2005

Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity.

In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2005

Characterization of an ecto-ATPase activity in Cryptococcus neoformans.

Cryptococcus neoformans is the causative agent of pulmonary cryptococcosis and cryptococcal meningoencephalitis, which are major clinical manifestations in immunosuppressed patients. In the present study, a surface ATPase (ecto-ATPase) was identified in C. neoformans yeast cells. Intact yeasts hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 29.36+/-3.36nmol Pi/hx10(8) cells. In the presence of 5 mM MgCl(2), this activity was enhanced around 70 times, and an apparent K(m) for Mg-ATP corresponding to 0.61mM was determined. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases, V-ATPases, Na(+)-ATPases or P-ATPases had no effect on the cryptococcal ATPase, but extracellular impermeant compounds reduced enzyme activity in living cells. ATP was the best substrate for the cryptococcal ecto-enzyme, but it also efficiently hydrolyzed inosine 5'-triphosphate (ITP), cytidine 5'-triphosphate (CTP), guanosine 5'-triphosphate (GTP) and uridine-5'-triphosphate (UTP). In the presence of ATP, C. neoformans became less susceptible to the antifungal action of fluconazole. Our results are indicative of the occurrence of a C. neoformans ecto-ATPase that may have a role in fungal physiology.

Adenosine Triphosphatases↗

PubMed2004

A Mg-dependent ecto-ATPase is increased in the infective stages of Trypanosoma cruzi.

In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42 +/- 0.31 nmol Pi/h x 10(8) cells). ATP hydrolysis was stimulated by MgCl2, and the Mg-dependent ecto-ATPase activity was 27.15 +/- 2.91 nmol Pi/h x 10(8) cells. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2 or SrCl2. The apparent Km for Mg-ATP2- was 0.61 mM, and free Mg2+ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'.diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg2+-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.

Adenosine Triphosphatases↗

PubMed2003

A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms.

In this work, we describe how living cells of Trypanosoma brucei procyclic forms were able to hydrolyze extracellular p-nitrophenylphosphate (pNPP). These intact parasites, which had their viability determined by motility and the Trypan blue method, presented a low level of pNPP hydrolysis in the absence of any divalent metal (0.72+/-0.07 nmol pNP/mg min). Interestingly, in the presence of 5mM MgCl(2), ectophosphatase activity of 1.91+/-0.21 nmol pNP/mg min was observed. The ectophosphatase activity was also stimulated by MnCl(2), CoCl(2) and CuCl(2) but not by CaCl(2) and CdCl(2) and was inhibited by ZnCl(2). The addition of Mg(2+), Mn(2+), Co(2+) and Cu(2+) to extracellular medium increased the ectophosphatase activity in a dose-dependent manner. At 5mM pNPP, half-maximal stimulation of pNPP hydrolysis was obtained with 0.39+/-0.05 mM MgCl(2), 0.33+/-0.03 mM MnCl(2), 1.63+/-0.12 mM CoCl(2) and 2.04+/-0.33 mM CuCl(2). In the absence of any divalent metal (basal activity) the apparent K(m) for pNPP was 0.66+/-0.09 mM, while at saturating MgCl(2) concentrations the corresponding apparent K(m) for pNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.27+/-0.03 mM. The Mg(2+)-stimulated pNPP hydrolysis was strongly inhibited by ZnCl(2) and vanadate, while the metal-independent basal phosphatase activity was less inhibited by these phosphotyrosyl phosphatase inhibitors.

Animals↗

PubMed2003

Ecto-ATPase activity on the surface of Trypanosoma cruzi and its possible role in the parasite-host cell interaction.

This study describes the possible role of Mg(2+)-dependent ecto-ATPase activity on the Trypanosoma cruzi-host cell interaction. Mg(2+)-dependent ecto-ATPase activity is observed on the cell body and flagellar membranes of the parasite and is about 20 times greater in trypomastigotes, as compared with epimastigotes. Suramin (a competitive antagonist of P2 receptors) and the impermeant agent 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS), both inhibitors of ecto-ATPases, strongly inhibited ATPase activity and the adhesion and internalization of both evolutive forms by mouse resident macrophages. Suramin inhibited the growth of epimastigotes, suggesting a direct participation of ecto-ATPase activity in this process. To overcome the presence of suramin in the culture medium during the time of growth, Mg(2+) ecto-ATPase activity was enhanced 4-fold, as compared with control parasites. The over-expression in enzyme activity was followed by a dramatic increase in the adhesion of epimastigotes to resident macrophages above the level observed for non-treated parasites.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2003

Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei.

Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [(32)P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3-5 nmol pNP min(-1) mg(-1), linearly for up to at least 30 min. The activity was not significantly increased by Mg(2+), Mn(2+), Ca(2+) and Co(2+), but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn(2+) and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5-5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min(-1) mg(-1)) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a K(m) value of 0.29 mM with pNPP and was insensitive to the Fe(2+)/H(2)O(2)/ascorbate system.

Animals↗

PubMed2002

Ecto-ATPases in protozoa parasites: looking for a function.

The plasma membrane of cells 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. Cell membrane ecto-ATPases are integral membrane glycoproteins that are millimolar divalent cation-dependent, low specificity enzymes that hydrolyze all nucleoside triphosphates. Their physiological role is still unknown. However, several hypotheses have been suggested such as; (i). protection from cytolytic effects of extracellular ATP, (ii). regulation of ectokinase substrate concentration, (iii). termination of purinergic signaling, (iv). involvement in signal transduction, and (v). involvement in cellular adhesion. In this review, the biochemical properties and possible functions of the ecto-ATPases of different protozoa are summarized.

Adenosine Triphosphatases↗

PubMedDate not supplied

Ecto-phosphatase activity on the cell surface of Crithidia deanei.

In the present work we have partially characterized an ecto-phosphatase activity in Crithidia deanei, using viable parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 3.55 +/- 0.47 nmol Pi/h x 10(8) cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this phosphatase activity and the value of the apparent Km for p-NPP was 5.35 +/- 0.89 mM. This phosphatase activity was inhibited by the product of the reaction, the inorganic phosphate. Experiments using classical inhibitors of acid phosphatases, such as ZnCl2 and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and ammonium molybdate, showed a decrease in this phosphatase activity, with different patterns of inhibition.

Animals↗

PubMedDate not supplied

An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence.

This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 +/- 13.9 nmol Pi/h x 10(7) cells). This ecto-enzyme was stimulated by ZnCl2, CaCl2 and MgCl2, was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. D-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity.

Adenosine Triphosphatases↗