Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p < 0·05) increased in NP, HP, GDM and HIP groups when compared with the CG, while the addition of 10 µM of the same agonist caused significant (p < 0·05) elevations in HP, GDM and HIP groups when compared with CG. Furthermore, ADA activity was significantly (p < 0·05) enhanced in NP, HP, GDM and HIP groups when compared with CG. In this study, the increased platelet aggregation and ADA activity in pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of NTPDase (NTPDase1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5) activity in human lymphocytes.

Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis (P<0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis (P<0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate (P<0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P1,P5-di(adenosine-5')pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 microg/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K(m) values of 77.6+/-10.2 and 106.8+/-21.0 microM, and V(max) values of 68.9+/-8.1 and 99.4+/-8.5 (mean+/-S.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72+/-0.24% (mean+/-S.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis.

HIV infection is associated with increased NTPDase activity that correlates with CD39-positive lymphocytes.

Infection with the human immunodeficiency virus (HIV) results in alterations in immune cells such as an increase or decrease of cytokine secretion and immunodeficiency. HIV causes a state of chronic cellular activation that can induce apoptosis in lymphocyte T-helpers, making the patient susceptive to opportunistic infections. The biochemical mechanisms involved in this immune response to HIV have been researched. Here, we have shown for the first time that ATP and ADP hydrolysis are essential for the immune response to HIV. Our results clearly indicate an increase of NTPDase-1 (EC 3.6.1.5) activity in lymphocytes of HIV-positive patients, confirmed by an enhanced CD39 expression on its surface. These results suggest that NTPDase-1 may be important to keep an adequate balance between the generation and consumption of ATP and to preserve cellular integrity and immune response to the HIV infection.

NTPDase and 5'-nucleotidase activities in platelets of human pregnants with a normal or high risk for thrombosis.

The nucleotide degrading enzymes, ectonucleotidases, present on the platelet surface of human pregnant with a normal (without complications) or high risk for thrombosis (hypertension and gestational diabetes) were studied. NTPDase (E.C. 3.6.1.5, CD39) and 5'-nucleotidase (E.C. 3.1.3.5, CD73) activities of four patient groups, non-pregnant (NP, n = 18), pregnant without complications (P, n = 25), pregnant with hypertension (HP, n = 15) and pregnant with gestational diabetes mellitus (GDP, n = 10), were analyzed. Increased NTPDase activities were observed in the groups P (37.0%, S.D. = 2.03 and 34.0%, S.D. = 3.19), HP (40.0%, S.D. = 3.32 and 56.0%, S.D. = 3.25) and GDP (23.0%, S.D. = 2.30 and 42.0%, S.D. = 2.26) in comparison to the control group NP (p < 0.01, S.D. = 1.92 and S.D. = 2.48) when ATP and ADP were used as substrate, respectively. AMP was used as substrate to determine the 5'-nucleotidase activities, which showed to be elevated in the groups P (45.0%, S.D. = 1.73), HP (54.0%, S.D. = 2.64) and GDP (68.0%, S.D. = 1.69) when compared to the control group NP (p < 0.01, S.D. = 1.26). However, no statistically significant differences were observed between the groups P, HP and GDP. As a consequence, the enhanced ATP, ADP and AMP hydrolysis was ascribed to the pregnancy itself, independent of a normal or high risk for thrombosis. The enhanced NTPDase and 5'-nucleotidase activities in platelets suggest that these enzymes are involved in the thromboregulation process in the pregnancy.

Effects per se of organic solvents in the cerebral acetylcholinesterase of rats.

Acetylcholinesterase (AChE) was studied in different rat brain regions (cerebellum, hypothalamus, striatum, hippocampus and cortex) in the presence of different organic solvents normally used in the in vitro assay. The organic solvents used were acetone (C3H6O), acetonitrile (C2H3N), ethyl alcohol (C2H6O), isopropyl alcohol (C3H8O), methyl alcohol (CH4O), tert-butyl alcohol (C4H10O) and dimethyl sulfoxide (DMSO, C2H6OS) ranging from 0.6 to 10%. Ethyl and methyl alcohol presented no effect on AChE activity at any of the concentrations and brain structures tested. In the hippocampus, isopropyl alcohol did not demonstrate a significant inhibitory effect, even at high concentrations. Tert-butyl alcohol presented an interesting result, increased AChE activity (P < .05) in the hypothalamus (1.8%), cortex (1.8 and 2.5) and striatum (1.2, 1.8 and 2.5%) and decreased activity at a concentration of 10% in the cortex (P < .05) and striatum (P < .01). Acetone and acetonitrile presented similar results, both significantly inhibiting AChE in all structures (5%, P < .05 and 10%, P < .01). DMSO exhibited a highly inhibitory effect at practically all concentrations tested (P < .01). In conclusion, for testing new compounds on AChE activity in vitro, methyl and ethyl alcohol may be the best organic solvent choice.