NT Activity Research Articles

Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5'-nucleotidase (CD73).

The adenosine producing enzyme ecto-5'-nucleotidase (5'-NT) is not normally expressed during thymocyte development until the medullary stage. To determine whether earlier expression would lead to adenosine accumulation and/or be deleterious for thymocyte maturation, thymic purine metabolism, and T cell differentiation were studied in lckNT transgenic mice overexpressing 5'-NT in cortical thymocytes under the control of the lck proximal promoter. In spite of a 100-fold elevation in thymic 5'-NT activity, transgenic adenosine levels were unchanged and T cell immunity was normal. Inosine, the product of adenosine deamination, was elevated more than twofold, however, indicating that adenosine deaminase (ADA) can prevent the accumulation of adenosine, even with a dramatic increase in 5'-NT activity, and demonstrating the availability of 5'-NT substrates in the thymus for the first time. Thymic adenosine concentrations of mice treated with the ADA inhibitor 2'-deoxycoformycin (dCF) were elevated over 30-fold, suggesting that high ADA activity, rather than an absence of 5'-NT, is mainly responsible for low thymic adenosine levels. The adenosine concentrations in dCF-treated mice are sufficient to cause adenosine receptor-mediated thymocyte apoptosis in vitro, suggesting that adenosine accumulation could play a role in ADA-deficient severe combined immunodeficiency.

Antigenic diversity of Akabane virus detected by monoclonal antibodies

The antigenic properties of 21 Japanese field isolates and two Australian strains of Akabane (AKA) virus (Simbu serogroup, bunyavirus) isolated from 1959 to 1990 were compared by enzyme-linked immunosorbent assay (ELISA), plaque-reduction neutralization (PRNT) and hemagglutination inhibition (HI) tests using monoclonal antibodies (Mabs) to the OBE-1 strain of AKA virus. Sixteen Mabs were established by fusing P3X63Ag8U1 mouse myeloma cells and spleen cells from BALB/c mice immunized with the OBE-1 strain. Of the 16 clones, 13 produced immunoglobulin (Ig) which precipitated glycoprotein G1 and three produced Ig which precipitated nucleoprotein (N). Twelve out of 13 Mabs had both NT and HI activities to not only the homologous OBE-1 strain but also the other isolates. By the competitive binding assay, at least five antigenic regions for G1, and two for N were defined. Some of the anti-G1 Mabs which reacted to the same antigenic region had unique reactivity while anti-N Mabs recognizing the same epitope reacted with almost the same degree to all of the isolates. Finally, nine epitopes of the G1 protein in five different antigenic regions have been identified. There was no striking correlation between isolation date and place of the isolates and their reactivity to Mabs. A most interesting result is that three isolates collected in the same place over a three week period had different reactivity patterns detected by ELISA, showing great antigenic variation of the virus. AKA virus may be a single gene pool consisting of different genotypes in the field.

Effect of Various Types of Exercise Training on 5′-Nucleotidase and Adenosine Deaminase Activities in Rat Heart: Influence of a Single Bout of Endurance Exercise

The aim of the present study was to find out whether activities of the enzymes controlling adenosine metabolism, 5′-nucleotidase (5NT) and adenosine deaminase (ADA), in the left ventricle of the rat's heart change after 6 weeks of endurance or sprint training. Additionally, an influence of a single bout of endurance exercise till exhaustion on activities of these enzymes was investigated in sedentary and trained rats. The rats were divided into three groups: (1) sedentary controls (C), (2) endurance-trained (ET), and (3) sprint-trained (ST). It was shown that both types of training increased 5NT, but did not change ADA activity in the rat heart. Acute exercise till exhaustion did not affect 5NT activity in the heart taken from C and ST rats, but decreased its activity in the ET group. The heart ADA activity after exhaustive exercise increased in C and in ET group, but decreased in ST animals. It is concluded that physical training affects cardiac adenosine metabolism and the type of training may exert an influence on purine nucleotides metabolism in the heart during exhaustive exercise.

Cardioprotection with ischemic preconditioning and MLA: role of adenosine-regulating enzymes?

Both ischemic preconditioning and pretreatment with the endotoxin derivative monophosphoryl lipid A (MLA) protect the heart against infarction, yet the cellular mechanisms responsible for the cardioprotection achieved with either intervention are unknown. Using pentobarbital-anesthetized dogs, we tested the hypothesis that increased activity of 5'-nucleotidase (5'-NT), the enzyme that catalyzes the formation of adenosine from AMP, may play a role. Twenty-two dogs underwent 1 h of coronary occlusion and 4 h of reperfusion: eight controls received no intervention, seven animals were preconditioned with four 5-min episodes of brief ischemia, and seven received MLA (35 micrograms/kg iv) 24 h previously. Collateral blood flow was measured by injection of radiolabeled microspheres, infarct size was delineated by tetrazolium staining, and myocardial 5'-NT activities were measured by quantifying the release of adenosine from AMP. Despite comparable values of collateral blood flow in all groups, infarct size was reduced in preconditioned and MLA-treated dogs vs. controls. In addition, 5'-NT activities were increased throughout the heart with preconditioning and MLA treatment. However, single and multivariate regression analyses revealed no correlation between infarct size and 5'-NT activities for either treatment group. In fact, in the preconditioned cohort, animals with the highest enzyme activities developed the largest infarcts. This dissociation between infarct size and 5'-NT suggests that increased activity of 5'-NT is not the mechanism by which preconditioning or MLA treatment protects the canine heart against infarction.

Decreased Number of CD73 (ecto‐5′‐Nucleotidase) Molecules on Lymphocytes from Patients with Primary Immunoglobulin Deficiencies. Correlation between Number of CD73 Molecules and T‐Lymphocyte Function In Vitro

CD73 is a bifunctional glycosyl phosphatidylinositol anchored leucocyte differentiation antigen which has specific ecto-5'-nucleotidase (ecto-5'-NT) activity and is an accessory T-lymphocyte activation molecule. The aim of the present study was to investigate the CD73 expression on blood mononuclear cells (BMC) from a group of patients with primary immunoglobulin deficiency (IGD). This group of patients had both significantly decreased levels of ecto-5'-NT on BMC (P = 0.002) and decreased numbers of CD73 molecules per CD73+ lymphocyte (P = 0.01). Five of the 10 patients had a decreased percentage of CD73+ lymphocytes. Among B-lymphocytes the patients had normal percentages of CD73+ cells but four of the 10 patients had numbers of CD73 molecules per CD73+ B-lymphocyte below the normal range. Among CD4-lymphocytes three out of 10 patients had percentages of CD73+ below the normal range and four out of 10 patients had decreased percentages of CD73+ CD8-lymphocytes. Significant correlations were found between in vitro proliferative responses to mitogens and the number of CD73 molecules per CD73+ lymphocyte (rs = 0.60, P < 0.01) and per CD73+ CD8-lymphocyte (rs = 0.64, P < 0.02). In addition, a positive correlation was found between ability to proliferate and level of ecto-5'-NT on BMC (rs = 0.53, P < 0.05). Furthermore the ability of BMC to synthesize ecto-5'-NT was studied. During 2 days culture ecto-5'-NT activity increased markedly on BMC from both patients and healthy donors. The level of activity on BMC from all patients attained levels higher than on freshly isolated BMC from healthy donors. This shows that the decreased levels of ecto-5'-NT found on freshly isolated BMC from patients with IGD is due to defective regulation of the enzyme activity in vivo.

IgG Subclass Antibodies to Human Cytomegalovirus (CMV) in Normal Human Plasma Samples and Immune Globulins and their Neutralizing Activities

Abstract.An enzyme-linked immunoabsorbent assay (ELISA) was developed for quantitation of IgG subclass antibodies to human cytomegalovirus (CMV) in human serum or plasma samples and in immune globulin (IG) preparations. The assay was based on the parallel titration of known concentrations of purified IgG subclass myeloma proteins and a specific CMV antiserum. The purified IgG subclass myeloma proteins were captured on an ELISA plate pre-coated with anti-human kappa, anti-human lambda or a mixture of anti-human kappa and lambda antibodies and the specific antiserum was titrated against CMV antigen coated on the plate. IgG subclass antibodies, captured or bound to antigen, were quantitated with IGG subclass heavy chain specific monoclonal antibodies. The method was highly reproducible, specific and sensitive. Using this method, 257 human plasma samples and 50 IG preparations were assayed for CMV specific IgG subclass and IgM antibodies. The major IgG subclass antibody to CMV was IgG1 which represented more than 96% of CMV IgG antibodies, followed by IgG3 (mean CMV IgG3 antibody content was 3% of IgG antibodies in IG preparations and 1·8% in plasma samples). A majority of the samples had low levels of IgG2 antibodies and a few samples exhibited low levels of IgG4 antibodies. IG preparations showed very low levels of CMV IgM antibodies whereas plasma samples had 14·2% of CMV antibodies (IgG and IgM) as IgM antibodies. Virus neutralizing (Nt) activity of these samples showed a significant correlation with CMV IgG1 antibodies. Nine samples of plasma and IGs were further evaluated for Nt activity of IgG1 and IgG3 antibodies by separating IgG3 from the rest of the antibodies with protein A agarose. IgG3 antibodies showed much higher Nt activity than IgG1 antibodies suggesting that enrichment of IgG3 antibodies in IG preparations may be useful in preparing CMV specific IG.

Activities of adenosine deaminase, 5'-nucleotidase, guanase, and cytidine deaminase enzymes in cancerous and non-cancerous human breast tissues.

We measured activities of some DNA turnover enzymes in 9 breast tissues with stage II cancer, 6 breast tissues with stage IIIa cancer, and 9 non-cancerous adjacent breast tissues from the same patients with stage II cancer. We found higher Adenosine Deaminase (ADA) and 5'-Nucleotidase (5'NT) and lower Guanase (GUA) activities in the cancerous tissues compared with the non-cancerous ones. No meaningful differences were however found between Cytidine Deaminase (CD) activities. Regarding the correlation analysis, positive correlations were established between ADA and 5'NT activities of the cancerous tissues (r = 0.45 for the tissues with stage II and r = 0.60 for the tissues with stage IIIa cancer). No meaningful correlations were however found between other enzyme activities. Relating to activity ratios, no meaningful differences were found between ADA/5'NT values in the tissues. GUA/CD ratios were however lower and the other ratios higher in the cancerous tissues. Results indicated that ADA and 5'NT activities increased and GUA activity decreased in the cancerous breast tissues but CD activities did not change in the tissues affected. It has been suggested that increased ADA and 5'NT together with decreased GUA activities might be a physiologic attempt of the cancer cells to provide more substrates needed by cancer cells to accelerate the salvage pathway activity. Furthermore, high ADA activity might also play part in the detoxication process of high amounts of toxic adenosine and deoxyadenosine substrates produced from accelerated purine metabolism in the cancerous tissues.

No correlation between CD73 expression and ecto-5‘-nucleotidase activity on blood mononuclear cellsin vitro

Monoclonal antibodies against ecto-5'-nucleotidase (ecto-5'-NT) have been clustered to CD73. The aim of the present study was to elucidate whether specific ecto-5'-NT activity on blood mononuclear cells (BMC) was correlated with CD73 expression measured by flow cytometry. During culture of CD73-negative lymphocytes the percentage of cells with ecto-5'-NT activity increased without there being a comparable increase in CD73 expression. After 2 days' culture, 9% (3-16, median and range given) demonstrated ecto-5'-NT activity, whereas CD73-positive cells comprised only 1.6% (0.0-3.0). These results show the existence of lymphocytes with ecto-5'-NT activity but without expression of CD73. The ecto-5'-NT activity increased significantly (p<0.02) during culture of unseparated BMC, whereas the number of anti-CD73 binding sites did not change. As a consequence. the number of anti-CD73 binding sites per U ecto-5'-NT activity decreased during culture. Exposure to interleukin-4 or prostaglandin E2 changed the enzymatic activity but not the number of anti-CD73 binding sites. Treatment of BMC with phosphatidylinositol-specific phospholipase-C released 57% (51%-75%) of the ecto-5'-NT activity into the supernatants, without a detectable decrease in CD73 expression. The fact that ecto-5'-NT was removed from the supernatant after precipitation with anti-CD73 showed that the released ecto-5'-NT activity was due to enzymatic activity of CD73 molecules. The results of this study indicate that CD73 exists on BMC in isoforms with distinct capacities to bind mouse monoclonal anti-CD73.

Regulation of low Km (ecto-) 5'-nucleotidase gene expression in leukemic cells.

Ecto-5’-NT has been considered a biochemical marker in leukemias and lymphomas (1). The enzyme activity is low in lymphocytes (2) and decreased in B-cells in patients with X-linked agammaglobulinemia (3), decreased or not detectable in lymphocytes from chronic lymphocytic leukemia and infectious mononucleosis (4) and decreased in CD8+ lymphocytes in AIDS (5). In general, low activity has been observed during development and in undifferentiated cells which increases markedly in some cell types at the final stages of differentiation and during senescence and aging. Although there are some data showing correlation of low ecto-5’-NT activity with efficacy of nucleoside analogues of anti-cancer activity, no specific role which would explain the pattern of distribution of this enzyme has been proposed.

Azathioprine-related bone marrow toxicity and low activities of purine enzymes in patients with rheumatoid arthritis

Azathioprine (AZA) metabolism largely parallels the endogenous purine pathways. To date, thiopurine methyltransferase (TPMT) deficiency has been reported as a cause of AZA-related bone marrow toxicity in 1 patient with rheumatoid arthritis (RA). We therefore studied purine enzyme activities in 3 patients with RA who experienced AZA-related bone marrow toxicity. Lymphocyte activity of purine nucleoside phosphorylase and 5'-nucleotidase (5NT) and erythrocyte activity of TPMT, key enzymes in thiopurine catabolism, were measured in 3 RA patients who had experienced AZA-related bone marrow toxicity and in 16 RA patients without signs of toxicity despite at least 6 months of treatment with AZA. Two patients with AZA-related bone marrow toxicity were found to have a TPMT deficiency, 1 partial and 1 total. In the third patient, 5NT activity was found to be well below the lowest level observed in the control subjects. All 3 patients with severe AZA-related bone marrow toxicity had abnormal purine enzyme activities. Deficiency of purine enzymes, including TPMT and 5NT, may be a cause of AZA-related bone marrow toxicity in patients with RA.

Purine enzymes in rheumatoid arthritis: possible association with response to azathioprine. A pilot study.

To study the possible association of purine enzyme activities with response to azathioprine (AZA) treatment in rheumatoid arthritis (RA) and their correlation with parameters of disease activity. Lymphocyte activities of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), adenine phosphoribosyltransferase (APRT), purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5NT), and erythrocyte activities of thiopurine methyltransferase (TPMT) were measured in 14 healthy controls and 36 patients with RA. Eight patients had not previously been treated with AZA. Response to AZA therapy in 28 patients, determined in a prospective trial, was considered good in nine (group 1), insufficient in seven (group 2). In 12 patients AZA was withdrawn because of adverse reactions (group 3). Disease activity parameters were obtained simultaneously with purine enzyme measurements. Purine enzyme levels in the different groups were compared. Levels of 5NT activity were significantly lower in patients with RA than in healthy controls. PNP activity was higher in patients with RA not using prednisone compared with those who did and healthy controls. No clear correlation between purine enzyme levels and disease activity parameters was found. 5NT activities were significantly higher in group one than in group three (p = 0.012; alpha = 0.017), and almost significantly higher than in group two (p = 0.03; alpha = 0.017). The results indicate that purine enzyme activities in patients with RA differ from healthy controls, are associated with the outcome of AZA treatment and seem not to be associated with disease activity. Our findings may offer a clue to predict the response to AZA therapy in RA.

Effects of Polyunsaturated Fatty Acids on Glial Cell Activation. A Study Using Primary Cultures

The aim of this study was to examine the effects of essential fatty acids (gammalinolenic acid [18:3 n-6; GLA] and alpha-linolenic acid [18:3 n-3; Lin]) on the activation of glial cells, using lipopolysaccharides as the activating agent. Primary cultures of mixed glial cells from rat brain were used as the model. The morphological activation of microglia was the most significant response to the exposure. This activation was followed by an increase in 5’-nucleotidase (5’-NT) activity. The 5’-NT activity was increased by GLA or Lin alone to 250–350% of the control value and further increased by co-incubation with lipoteichoic acid (a lipopolysaccharide) to 500–600% of the control value. The lipopolysaccharide-induced activation of glial cells was also followed by an augmented release of prostaglandin E2. GLA increased the release of prostaglandin E2 in a dose-dependent manner, whereas Lin had no effect on its release. The results show that this model system is useful for studies on factors affecting the activation of glial cells. GLA and Lin did not reverse glial activation induced by lipopolysaccharides under these experimental conditions.

Adenosine deaminase, 5′-nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in gastric juices from patients with gastric cancer, ulcer, and atrophic gastritis

Adenosine deaminase (ADA), 5'-Nucleotidase (5NT), Xanthine oxidase (XO), Cu-Zn Superoxide dismutase (SOD) and Catalase (CAT) activities were determined in gastric juices from patients with gastric cancer, ulcer, gastritis and from healthy subjects. Enzyme activities were given as units per ml gastric juice and units per mg protein in gastric juice. ADA, 5NT and XO activities were found lower and protein concentrations were found higher in the cancer group than controls. There was however no significant difference between Cu-Zn SOD activities of the cancer and control groups. In all groups including control one, we could not find catalase activities in most of the samples. On the other hand, ADA, 5NT activities and protein concentrations in the gastric juice were lower in the gastritis group than control group. In the ulcer group, we found higher Cu-Zn SOD and XO activities and lower 5NT activity and protein concentrations compared with control values. In an attempt to establish statistical correlations between mean enzyme activities, pH and protein concentrations in the gastric juices of the groups, we found noticeable intra and inter-correlations, which indicated possible relations between DNA and free radical metabolizing enzymes.

Acidosis during ischemia promotes adenosine triphosphate resynthesis in postischemic rat heart. In vivo regulation of 5'-nucleotidase.

Capacity for ATP resynthesis during recovery from ischemia or hypoxia is limited to the size of the adenine nucleotide pool, which is determined in part by the activity of cytosolic 5'-nucleotidase (5'-NT): AMP-->adenosine plus inorganic phosphate (Pi). To define in vivo regulation of 5'-NT, we used the tools of 31P nuclear magnetic resonance (NMR), spectroscopy and chemical assay to measure the substrates (AMP), products (Pi, adenosine, and its catabolites), and inhibitors (Pi and H+) of 5'-NT in isolated perfused rat hearts exposed to hypoxia (where pH remains near 7) and no flow, global ischemia (where pH falls to 6.1). We estimated 5'-NT reaction velocity, assessed the relative contributions of Pi and H+ to enzyme inhibition, and defined the consequences of changes in 5'-NT activity on ATP resynthesis after hypoxia and ischemia. We conclude that (a) 5'-NT is activated during hypoxia and early ischemia but is inhibited during prolonged ischemia, (b) H+ (pH < 6.2) is a potent inhibitor of 5'-NT, and (c) differences in AMP accumulation are sufficient to explain the differences in the capacity for net ATP resynthesis in ischemic and hypoxic tissue. These observations have implications for our understanding of heterogeneity of ischemic injury and myocardial protection during ischemia.

Precursor Trapping: A "Neonatal" Mechanism of Myocardial Protection

During induced ischemia for cardiac surgery, 5′-nucleotidase (5NT) catalyzes nucleotide breakdown by dephosphorylating AMP and IMP to diffusible precursors—adenosine and inosine. These precursors become unavailable upon reperfusion washout limiting nucleotide resynthesis, resulting in poor postischemic function. Neonatal hearts, which are more resistant to ischemia than adults, have low 5NT activity, trapping available precursors. Adult rabbit hearts given cardioplegia with a 5NT inhibitor, pentoxifylline, demonstrated improved postischemic contractility, compliance, and myocardial oxygen consumption after 120 min of 34°C ischemia. To determine if this improved function was a result of enhanced nucleotide precursor availability during or following ischemia, total nondiffusible nucleotides, ATP, ADP, AMP, and IMP, and total diffusible nucleotides, adenosine, inosine, hypoxanthine, and xanthine, were measured by HPLC at end ischemia, 1 and 15 min after reperfusion. While all preischemic values were equivalent, pentoxifylline-treated hearts had significantly greater total nondiffusible nucleotides at end ischemia, 1 and 15 min after reperfusion. Additionally, pentoxifylline-treated hearts had significantly greater total diffusible nucleosides at end ischemia and 1 min after reperfusion, but were equal to control at 15 min after reperfusion. Furthermore, coronary sinus effluent had a significantly higher release of total diffusible nucleosides in control vs pentoxifylline-treated hearts. The data indicate that precursor trapping with pentoxifylline prevented nucleotide catabolism to diffusible precursors and enhanced postischemic nucleotide availability. We postulate the increased precursor availability augmented myocardial nucleotide resynthesis and correlated with the improved functional recovery noted. This strategy may have application in adult cardiac surgery.

Relationship of deoxycytidine kinase and cytoplasmic 5'-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine

The agent 2-chlorodeoxyadenosine (2-CdA) has chemotherapeutic activity in hairy cell leukemia (HCL) and in refractory chronic lymphocytic leukemia (CLL). The cytotoxic activity of 2-CdA requires the intracellular accumulation of 2-CdA nucleotides. Deoxycytidine kinase (dCK) and cytoplasmic 5′-nucleotidase (5′-NT) are the principal enzymes that phosphorylate 2-CdA and dephosphorylate 2-CdA 5′-monophosphate, respectively. The net accumulation of 2-CdA nucleotides may therefore depend on both dCK and 5′-NT. The purpose of the present experiments was to determine if there is a relationship between pretreatment levels of dCK and 5′-NT in HCL and in CLL cells, and the clinical outcome of 2- CdA treatment. As measured by a direct immunoassay for dCK in 25 CLL patients, and by a 5′-NT activity assay in 23 patients, mean dCK levels were significantly higher in 2-CdA responders than in nonresponders (P &lt; .01), whereas mean 5′-NT levels were significantly lower in 2-CdA responders than in nonresponders (P &lt; .05). Mean dCK levels were higher in six HCL 2-CdA responders than in one nonresponder, whereas mean 5′- NT levels were lower in the 2-CdA responders than in the nonresponder. These results suggest that both dCK and 5′-NT are determinants of 2-CdA responsiveness.

Relationship of Deoxycytidine Kinase and Cytoplasmic 5’-Nucleotidase to the Chemotherapeutic Efficacy of 2-Chlorodeoxyadenosine

The agent 2-chlorodeoxyadenosine (2-CdA) has chemotherapeutic activity in hairy cell leukemia (HCL) and in refractory chronic lymphocytic leukemia (CLL). The cytotoxic activity of 2-CdA requires the intracellular accumulation of 2-CdA nucleotides. Deoxycytidine kinase (dCK) and cytoplasmic 5'-nucleotidase (5'-NT) are the principal enzymes that phosphorylate 2-CdA and dephosphorylate 2-CdA 5'-monophosphate, respectively. The net accumulation of 2-CdA nucleotides may therefore depend on both dCK and 5'-NT. The purpose of the present experiments was to determine if there is a relationship between pretreatment levels of dCK and 5'-NT in HCL and in CLL cells, and the clinical outcome of 2-CdA treatment. As measured by a direct immunoassay for dCK in 25 CLL patients, and by a 5'-NT activity assay in 23 patients, mean dCK levels were significantly higher in 2-CdA responders than in nonresponders (P < .01), whereas mean 5'-NT levels were significantly lower in 2-CdA responders than in nonresponders (P < .05). Mean dCK levels were higher in six HCL 2-CdA responders than in one nonresponder, whereas mean 5'-NT levels were lower in the 2-CdA responders than in the nonresponder. These results suggest that both dCK and 5'-NT are determinants of 2-CdA responsiveness.

An in vitro method for comparing the efficacy of two preservation solutions in one canine liver using the 5‘-nucleotidase assay

Abstract. The activity and localization of the plasma membrane-bound enzyme 5‘-nucleotidase (5’-NT) in liver tissue are sensitive parameters of ischemic damage. The value of 5‘-NT as a marker of liver graft viability was studied in relation to liver preservation. In six mongrel dogs, the main right and left branches of the portal vein were cannulated and flushed separately in situ with cold University of Wisconsin (UW) solution and Euro-Collins (EC) solution, respectively. After hepatectomy, the right and left liver lobes were split and stored at 5oC in either of the two solutions. 5’-NT activity was demonstrated in cryostat sections of liver tissue using the lead salt method. After 48 h of storage in EC solution, the 5‘-NT score had decreased to 31 ± 16 % (n = 6), whereas in UW solution the 5’-NT score was 76%± 10% (n=6). Significantly (P < 0·05) higher 5‘-NT scores were also found after 24-h and 72-h preservation times in UW versus EC solutions. This result is in keeping with the higher preservation tolerance of liver grafts preserved in UW solution. The 5’-NT assay was studied in relation to graft function in orthotopic liver transplantation experiments in dogs. All dogs with liver grafts preserved in UW solution for 24 h (n= 4) and 48 h (n= 3) survived (> 5 days). Pretransplant 5‘-NT scores ranged from 61% to 100%. The 72-h-preserved livers (n= 5) did not show life-supporting function. Pretransplant 5’-NT scores (33%± 12%, n = 5) were significantly (P < 0·05) decreased. The 5‘-NT score pretransplantation was a more reliable indicator of graft function than peak SGOT values post-transplantation. In conclusion, the 5’-NT assay, in conjunction with the double flush method through the portal vein, provides a simple and rapid “in vitro” method to test solutions for liver preservation.

Responses of limbic and extrapyramidal neurotensin systems to stimulants of abuse. Involvement of dopaminergic mechanisms.

In summary, we have observed that drugs of abuse, which can cause schizophrenia-like paranoia, alter striatal and accumbens NT systems in a similar, dramatic fashion. The NT responses to these drugs, in particular METH, are mediated by activation of DA D1 receptors. We have observed that NMDA-type glutamate receptors are essential for the D1-NT interaction. NMDA receptors are selective, since they do not contribute to the antagonistic effects of DA D2 receptors on NT activity. This observation suggests that NT responses to D1 and D2 regulation are mediated through separate and distinct mechanisms. Finally, we found that the presence of METH dramatically reduces striatal NT release, which most likely leads to NT accumulation in nerve terminals and the observed increase in NT tissue level. The blockade of NT release by a psychotogenic drug, such as METH, is consistent with the hypothesis that NT has antipsychotic activity and a decrease in its release may contribute to some forms of schizophrenia similar to that caused by intense use of the stimulants of abuse.

Relation of 5′-nucleotidase and phosphatase activities with immunophenotype, drug resistance and clinical prognosis in childhood leukemia

Ecto-5′-nucleotidase (ecto-5′NT) catalyzes the extracellular dephosphorylation of nucleotides like IMP. Cytoplasmic 5′NT (cyto-5′NT) and non-specific (e.g. acid- and alkaline) phosphatases (AP) regulate the intracellular degradation of nucleotides. High NT and AP activities might cause a resistance to the thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We studied the relation between these enzymes and immunophenotype, drug resistance and prognosis in 77 children with acute lymphoblastic leukemia (ALL). Enzyme activities were assessed radiochemically; in vitro drug resistance was measured with the MTT assay. AP activities were higher in T-ALL and B-ALL than in precursor B-ALL. Cyto-5′NT activity was very low in all phenotypes and accounted for a significant proportion of total IMPase activity only in the very immature CD10 cμ − precursor B-ALL. CD10 + ALL cases with high ecto-5′NT activities showed a trend ( p = 0.065) for a lower probability of continuous complete remission than those with a low activity. Ecto-5′NT activity was not related to in vitro drug resistance to 6-TG. A weak correlation was found between in vitro 6-TG resistance and cyto-5′NT and AP activities. We conclude that high ecto-5′NT activities do not cause a resistance to 6-thiopurines in childhood ALL. Some patients have high cyto-5′NT and AP activities associated with 6-thiopurine resistance.