The expression of NTPDase1 and -2 of Leishmania infantum chagasi in bacterial and mammalian cells: Comparative expression, refolding and nucleotidase characterization.

Visceral Leishmaniasis (VL) represents an important global health problem in several warm countries around the world. The main targets in this study are the two nucleoside triphosphate diphosphohydrolases (NTPDases) from Leishmania infantum chagasi that are the main etiologic agent of VL in the New World. These enzymes, called LicNTPDase1 and -2, are homologous to members 5 and 6 of the mammalian E-NTPDase/CD39 superfamily of enzymes. These enzymes hydrolyze nucleotides and accordingly can participate in the purine salvage pathways and in the modulation of purinergic signaling through the extracellular nucleotide-dependent host immune responses. They can therefore affect adhesion and infection of host cells and the parasite virulence. To further characterize these enzymes, in this work, we expressed LicNTPDase1 and -2 in the classical bacterial system Escherichia coli and mammalian cell system COS-7 cells. Our data demonstrate that changes in refolding after expression in bacteria can increase the activity of recombinant (r) rLicNTPDase2 up to 20 times but has no significant effect on rLicNTPDase1. Meanwhile, the expression in COS-7 led to a significant increase in activity for rLicNTPDase1.

Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases.

BACKGROUND AND PURPOSE: ARL 67156, 6-N,N-Diethyl-D-beta-gamma-dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto-ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156. EXPERIMENTAL APPROACH: The effect of ARL 67156 on recombinant NTPDase1, 2, 3 & 8 (mouse and human), NPP1, NPP3 and ecto-5'-nucleotidase (human) have been evaluated. The inhibition of the activity of NTPDases (using the following substrates: ATP, ADP, UTP), NPPs (pnp-TMP, Ap(3)A) and ecto-5'-nucleotidase (AMP) was measured by colorimetric or HPLC assays. KEY RESULTS: ARL 67156 was a weak competitive inhibitor of human NTPDase1, NTPDase3 and NPP1 with K(i) of 11+/-3, 18+/-4 and 12+/-3 microM, respectively. At concentrations used in the literature (50-100 microM), ARL 67156 partially but significantly inhibited the mouse and human forms of these enzymes. NTPDase2, NTPDase8, NPP3 and ecto-5'-nucleotidase activities were less affected. Importantly, ARL 67156 was not hydrolysed by either human NTPDase1, 2, 3, 8, NPP1 or NPP3. CONCLUSIONS AND IMPLICATIONS: In cell environments where NTPDase1, NTPDase3, NPP1 or mouse NTPDase8 are present, ARL 67156 would prolong the effect of endogenously released ATP on P2 receptors. However, it does not block any ectonucleotidases efficiently when high concentrations of substrates are present, such as in biochemical, pharmacological or P2X(7) assays. In addition, ARL 67156 is not an effective inhibitor of NTPDase2, human NTPDase8, NPP3 and ecto-5'-nucleotidase.

Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8.

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with K (m) values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X(1-7) and P2Y(2,4,11) receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y(1,12,13) receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y(6) receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-8.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5'-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Increased plasma glutathione peroxidase activity in patients with acute myocardial infarction.

BACKGROUND: Aim of the study was to determine the concentration of selenium (Se) and the activity of glutathione peroxidase (GSH-Px) in patients with acute myocardial infarction (AMI) and to observe the behavior of these parameters during thrombolysis therapy. MATERIAL AND METHODS: The study comprised two groups of AMI patients and a control group. The first group consisted of 49 patients from whom blood samples were taken after admission to the intensive care unit and subsequently after 3, 7, 14 and 30 days of hospitalization. In the second group of patients (n = 18) blood was taken for measuring only the GSH-Px activity in plasma. In this group blood samples were collected after admission to the hospital, 6, 12, 24, 48 hours, 3, 7, 14 and 30 days later. Control group comprised of 58 healthy subjects. Se levels in whole blood and plasma were measured spectrofluorometrically with 2,3-diaminonaphthalene as a complexing reagent. GSH-Px activity in red cells and plasma was measured spectrofluorometrically with t-butyl hydroperoxide as substrate. RESULTS: In the first group of patients Se concentrations in whole blood and plasma as well as GSH-Px activities in red cells and plasma did not differ significantly from healthy subjects. Both Se levels and GSH-Px activities were stable during the entire period of the study. In the second group of patients, however, plasma GSH-Px activity increased after admission and reached the highest value after 48 hours. This activity was significantly higher compared to healthy subjects (p < 0.004) and to the mean initial activity of this group (p < 0.02). In the later period the activity decreased to the values of healthy subjects. CONCLUSION: We suggest that the increased activity of GSH-Px in plasma of AMI patients is the response of the organism to the increased levels of reactive oxygen species produced during reperfusion and thrombolysis.

[PLED pattern and its clinical significance in stroke patients]. / Zmiany w eeg typu PLED i ich kliniczne znaczenie u chorych z udarem mózgu.

The pathophysiological connection between periodic lateralized epileptiform discharges (PLED) and epileptic seizures is still not clear. In the study clinical data and EEG findings were analysed in 22 patients aged 43-90 years with a history of stroke in whom EEG disclosed PLED. Eleven patients were studied in the acute phase of stroke and 11 were studied years after stroke when the diagnosis was established of poststroke epilepsy. In 2 patients in acute stroke group single epileptic seizures occurred and 5 had partial status epilepticus. In the group with poststroke epilepsy 4 had single seizures and 4 had epileptic status with partial epilepsy seizures. Thus, in 15 out of 22 patients PLEDs were noted after epileptic seizures. In all cases PLED appearance was connected with consciousness disturbances, lasting 1 to 17 days. In 6 cases PLED pattern was interrupted by seizure activity over one hemisphere, in 3 of them partial epileptic seizures were associated with it. In acute phase of stroke neuroimaging demonstrated the presence of fresh ischaemic foci, but in cases of poststroke epilepsy no such fresh foci were observed. These results suggest that PLED frequently can be associated with epilepsy, and in some patients it can be a bioelectrical manifestation of partial status epileptic.

[Electroencephalographic studies of family members of patients with juvenile myoclonic epilepsy]. / Badania elektroencefalograficzne u czlonków rodzin pacjentów z mlodziencza padaczka miokloniczna.

Juvenile myoclonic epilepsy (JME) is a common idiopathic generalized epilepsy (IGE); it has a clinical and probably a strong genetic relation to the other IGE forms. Generalized spike/polyspike-wave discharges (SW/PSW) are typical of all IGEs. The aim of our study was to determine the incidence of epilepsy and SW/PSW in EEG of family members of 12 JME patients. 35 first degree relatives aged over 15 years were examined. 40 min EEG with 5 min HV were recorded. IGE was diagnosed in 3 (8.6%) persons: JME in 2 and childhood absence epilepsy (CAE) in 1 person. Six more relatives (17.1%) had typical SW/PSW traits in EEG. Thus the IGE features were found in 9 (25.7%) individuals--members of 7 out of 12 families (58%). EEG of 7 other relatives (20%) revealed non-specific episodic diffuse or focal abnormalities. The above results reveal higher incidence of different kinds of ICEs and typical EEG traits in families of JME patients. This findings confirm familial susceptibility to IGE and may be helpful in genetical counselling.