NT Activity Research Articles

Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'-nucleotidase and adenosine deaminase.

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryo-decidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast (days 7-13) and the metrial gland (days 11-13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6-11), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of immunomodulators on ecto-5'-nucleotidase activity on blood mononuclear cells in vitro.

In this study the effects of immunomodulators on the ecto-5'-nucleotidase (ecto-5'-NT) activity on blood mononuclear cells (BMC) were examined in vitro. Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) decreased the level of ecto-5'-NT activity on BMC whereas prostaglandin E2 (PGE2) increased the ecto-5'-NT level. All three immunomodulators influenced the ecto-5'-NT activity of isolated monocytes whereas only IL-4 and PGE2 had an effect on the enzyme level on isolated lymphocytes. The effect was dependent upon protein synthesis. The effect was dose dependent: IL-4 was effective at concentrations down to 0.5 U/ml, IFN-gamma down to 40 U/ml and PGE2 at nanomolar concentrations. These data indicate that immunomodulators may also take part in the regulation of ecto-5'-NT activity on BMC in vivo. BMC from 7 patients with different immunodeficiency syndromes showed decreased ecto-5'-NT activity on freshly isolated cells. However, following culture ecto-5'-NT activity was increased above the level found on freshly isolated BMC from healthy persons. On BMC from 3 patients with hypogammaglobulinaemia, the effect of IL-4 on the level of ecto-5'-NT activity was identical to that found on BMC from healthy donors, whereas PGE2 increased ecto-5'-NT activity on BMC from only 1 of the 3 patients investigated. The decreased ecto-5'-NT activity of BMC from patients with immunodeficiency may thus be due to a defective regulation of ecto-5'-NT activity in vivo.

Synovial fluid 5′‐nucleotidase activity. Relationship to other purine catabolic enzymes and to arthropathies associated with calcium crystal deposition

We measured 5'-nucleotidase (5NT) activity in synovial fluid from 159 patients with various diagnoses. The activity of 5NT was compared with activities of nucleotide pyrophosphohydrolase, alkaline and neutral phosphatases, and adenosine deaminase, in the same samples. Higher levels of 5NT activity occurred in synovial fluid from osteoarthritic joints than from joints of patients with gout, pseudogout, or rheumatoid arthritis. The highest levels of 5NT activity were found in synovial fluid from patients with Milwaukee shoulder syndrome and from osteoarthritis patients in whom deposition of calcium-containing crystals was also present.

Expression of 5'-nucleotidase (CD73) related to other differentiation antigens in leukemias of B-cell lineage

Ecto-5'nucleotidase (5'NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of CD19 and HLA-DR. Four stages of B-cell leukemias were defined: stage I (pro-B) as CD10-, cytoplasmic mu- (c mu-), surface Ig- (sIg-); stage II (cALL) as CD10+/c mu-/sIg-; stage III (pre-B) as CD10+ or -/c mu+/sIg-; and stage IV (B) as CD10-/c mu-/sIg+. A linear correlation was found between immunohistochemical and radiochemical determination of 5'NT (r = .86). 5'NT expression was low in T-cell leukemias and stage I, high in stages II and III, and low again in stage IV of B-cell leukemias. 5'NT expression was not related to c mu, CD20, CD21, CD22, CD34, and terminal deoxynucleotidyl transferase (TdT) expression, but was significantly related to CD10 and inversely related to kappa/lambda expression. However, the 5'NT activity in CD10+ leukemias (stages II and III) shows a very wide range. Within the group of CD10+ leukemias no differences were detected between 5'NT+ and 5'NT- cells in their expression of other B-cell antigens. We conclude that the place of 5'NT in leukemias corresponding to early stages of B-cell development has been characterized. 5'NT is expressed in CD10+ stages and decreases before the expression of sIgs. Future studies should make clear whether a high expression of this enzyme in CD10+ stages is a normal maturation phenomenon or a malignant phenomenon.

Expression of 5' -Nucleotidase (CD73) Related to Other Differentiation Antigens in Leukemias of B-Cell Lineage

Abstract Ecto-5′nucleotidase (5′NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of CD19 and HLA-DR. Four stages of B-cell leukemias were defined: stage I (pro-B) as CD10-, cytoplasmic mu- (c mu- ), surface Ig- (sIg-); stage II (cALL) as CD10+/c mu-/sIg-; stage III (pre-B) as CD10+ or -/c mu+/sIg-; and stage IV (B) as CD10-/c mu-/sIg+. A linear correlation was found between immunohistochemical and radiochemical determination of 5′NT (r = .86). 5′NT expression was low in T-cell leukemias and stage I, high in stages II and III, and low again in stage IV of B-cell leukemias. 5′NT expression was not related to c mu, CD20, CD21, CD22, CD34, and terminal deoxynucleotidyl transferase (TdT) expression, but was significantly related to CD10 and inversely related to kappa/lambda expression. However, the 5′NT activity in CD10+ leukemias (stages II and III) shows a very wide range. Within the group of CD10+ leukemias no differences were detected between 5′NT+ and 5′NT- cells in their expression of other B-cell antigens. We conclude that the place of 5′NT in leukemias corresponding to early stages of B-cell development has been characterized. 5′NT is expressed in CD10+ stages and decreases before the expression of sIgs. Future studies should make clear whether a high expression of this enzyme in CD10+ stages is a normal maturation phenomenon or a malignant phenomenon.

Serum 5'nucleotidase activity in rats: a method for automated analysis and criteria for interpretation.

A manual kit for determining serum 5'nucleotidase (5'NT, EC 3.1.3.5) activity was adapted for use with rat samples on a large discrete clinical chemistry analyzer. The precision of the method was good (within-run C.V. = 2.14%; between-run C.V. = 5.5%). A comparison of the new automated method with a manual and semi-automated method gave regression statistics of y = 1.18X -3.66 (Sy. x = 4.54), and y = 0.733X + 1.97 (Sy. x = 1.69), respectively. Temperature conversion factors provided by the kit manufacturer for human samples were determined to be inaccurate for converting results from rat samples. Analysis of components contributing to normal variation in rat serum 5'NT activity showed age and sex to be major factors. Increased serum 5'NT activity was observed in female rats when compared to male rats beginning at about 5 to 6 weeks of age. An analysis of variance of serum 5'NT, alkaline phosphatase, and GGT activities observed over a 9-week period in normal rats suggests several advantages for 5'NT as a predictor of biliary lesions in rats.

Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol‐anchored lymphocyte differentiation antigen ecto‐5′‐nucleotidase (CD73).

A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5'-NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab')2 goat anti-mouse Ig. Both antibodies can be used to purify the three major forms of placental 5'-NT by affinity chromatography. By two-color immunofluorescence, CD73 was found to be expressed on 19 +/- 5% of CD3+, 11 +/- 4% of CD4+, 51 +/- 14% of CD8+, 25 +/- 8% of CD28+, 15 +/- 5% of CD29+, 27 +/- 7% of CD45RA+, and 70 +/- 6% of CD19+ lymphocytes. Within T cells, CD73 expression is restricted to the CD28+ subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+ T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the "light-zone." CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non-keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activation, lymphocyte homing, and/or cell adhesion.

Antibodies to 5'-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate.

Human peripheral blood T cells were stimulated to proliferate when cultured with submitogenic doses of PMA and goat antibodies to 5'-nucleotidase (5'-NT). The degree of proliferation, as measured by [3H]TdR incorporation on day 3, was similar to that achieved by stimulation with PHA. Anti-5'-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6 to 1.0 ng/ml of PMA and 125 micrograms/ml of IgG isolated from a goat anti-5'-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2R expression and IL-2 secretion were also induced by anti-5'-NT antibodies and PMA. Anti-5'-NT-induced proliferation was inhibited greater than 95% by a murine anti-IL-2 receptor mAb and required less than 0.3% monocytes. Similar results have been obtained with a murine mAb specific for 5'-NT. As expected, anti-5'-NT antibodies and PMA did not induce the proliferation of ecto-5'-NT-T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol-specific phospholipase C removed an average of 89% of the 5'-NT activity from the cell surface and also inhibited by 83% the ability of the cells to proliferate in response to anti-5'-NT antibodies and PMA. Thus, the activation signal provided by anti-5'-NT antibodies is apparently transduced, in large part, by a form of the enzyme that is attached to the membrane via glycosyl-phosphatidylinositol linkage. These data suggest that 5'-NT may play a role in lymphocyte activation as has been proposed for other glycosyl-phosphatidylinositol-anchored lymphocyte surface proteins.

Enzymatic imbalance in peripheral blood mononuclear cells isolated from individuals with anti-HIV antibodies

Plasma membrane-bound 5'-nucleotidase (5'-NT), gamma-glutamyltransferase (gamma-GT) and soluble deoxynucleotidyltransferase (TdT) were studied in peripheral blood cells (PBMN) of 35 individuals, 26 male and 9 female, with circulating anti-HIV antibodies. Twenty-six were drug abusers, 2 were drug abusers and homosexuals and 4 were homosexuals. Three did not fall into any risk group. The surface immunologic phenotype of cells stained with the fluorescent monoclonal antibodies Leu 5, Leu 3, Leu 2, Leu 12, Leu M3, Leu M1, anti-CALLA and anti-HLA-DR was delineated by flow cytometry. While the gamma-GT activity did not change, the lymphocyte 5'-NT activity was significantly less than normal in anti-HIV positive individuals and in anti-HIV negative drug abusers. TdT activity was detectable in 14 anti-HIV positive patients (40%), who did not have clinical AIDS. Of 8 patients with AIDS, 3 had a low level of TdT activity but 5 had cells completely devoid of TdT and 5'-NT activity. 5'-nucleotidase activity and the frequency of Leu 2 suppressor antigen bearing cells were the only independent variables that correlated with AIDS incidence.

Functional characterization of ecto-5'-nucleotidase-positive and -negative human T lymphocytes.

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR. However, prior incubation with anti-5'-NT antibodies did inhibit the ability of irradiated T cells to provide help for PWM-stimulated Ig synthesis by as much as 55%. In five separate experiments, ecto-5'-NT-T cells demonstrated an equal or better ability to incorporate [3H]TdR after PHA stimulation or in a MLR, as compared with ecto-5'-NT+ T cells. Similarly, ecto-5'-NT- T cells were not diminished in their ability to provide help for autologous B cells in a PWM-driven system. Clearly, the inability of ecto-5'-NT- T cells from patients with a variety of immunodeficiency diseases to function in these assays cannot be explained solely by their lack of ecto-5'-NT activity. In contrast, ecto-5'-NT-positive and -negative T cells showed markedly different dose-response curves for proliferation in response to PMA. Ecto-5'-NT+ T cells responded to lower doses of PMA (1.0 ng/ml) than did ecto-5'-NT- T cells and showed a two- to eight-fold greater rate of [3H]TdR incorporation at 3 to 10 ng of PMA per ml. Ecto-5'-NT+ T cells may have a protein kinase C that is more accessible or more easily activated or may utilize an alternate pathway of activation when stimulated with low concentrations of PMA.

The generation of alloreactive cytotoxic T lymphocytes requires the expression of ecto-5'nucleotidase activity.

Ecto-5'-nucleotidase (ecto-5'NT) activity is highly expressed by CTL precursor cells. The aim of this work was to investigate whether ecto-5'NT is involved in their functional activation. The inhibition of ecto-5'NT activity by biochemical (alpha,beta-methyleneadenosine 5-diphosphate; AOPCP) or immunologic inhibitors (polyclonal anti-ecto-5'NT serum) suppressed the proliferative and cytotoxic activation of CTL against a lymphoblastic B cell line in primary one-way MLC. The kinetic analysis of suppression showed that AOPCP and anti-5'NT serum had different effects: AOPCP suppressed only the activation of the cytotoxic program whereas anti-5'NT serum suppressed also recognition and binding to the stimulatory/target cell. Inhibition of CTL generation was not a metabolic consequence of the purine salvage blockade. Incubation of AOPCP-treated MLC with hypoxanthine restored proliferation but not cytotoxicity. The ecto-5'NT inhibitors used had no general toxic effect on cell numbers or viability. AOPCP selectively affected CTL generation and displayed no activity against mitogen-induced proliferation, activation of Ts cells, and generation of IL-2-activated killer cells. These data indicate that ecto-5'NT has a critical role in the functional activation of alloreactive CTL.

Induction of adenosine deaminase and 5' nucleotidase activity in cultured human blood monocytes and monocytic leukemia (THP-1) cells by differentiating agents.

The in vitro effect of short-term culture as well as the effect of retinol (ROH), retinoic acid (RA), muramyl dipeptide [( Abu']MDP), lipopolysaccharide (LPS), and gamma interferon (IFN-gamma) on the induction of the purine metabolic enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5'nucleotidase (5NT) in human peripheral blood monocytes (HPBM) was examined. HPBM isolated by centrifugal elutriation were cultured for up to 96 h. Following an initial time lag of 24 h, mean ADA activity from seven separate experiments as measured in nmoles/10(6) cells/h increased from a baseline of 31.3 +/- 9.3 to 57.8 +/- 16.4 (P less than 0.005) at 72 h and to 72 +/- 21.5 (P less than .025) by 96 h. 5NT activity increased from a baseline of 2.2 +/- 0.9 to a maximum of 44 +/- 10.1 by 72 h and then declined to 29 +/- 18 (P less than 0.005) by 96 h, while no significant change in PNP activity was observed. HPBM incubated for 3 d with optimal concentrations of LPS, RA, and IFN-gamma had increases in ADA and 5NT activity ranging from three- to 10-fold compared to HPBM cultured in media alone, whereas no effect was observed with ROH and [Abu']MDP. RA, but not ROH, significantly enhanced ADA activity in a monocytic leukemia cell (THP-1) line. Addition of RA or the tumor promoter, phorbol 12-myristic 13-acetate (PMA), to HPBM or THP-1 cells resulted in significant increases in 5NT activity with opposite effects on ADA activity. These findings suggest that the biological mechanisms associated with differentiation in normal and malignant monocytes seem to be related and that the sequence and degree to which the various differentiation agents induce the enzyme elevations are also related to the mechanisms of activation/differentiation.

The role of 5′nucleotidase in therapy-resistance of childhood leukemia

Purine nucleotides become available for a cell by two routes, namely purine de novo synthesis and the purine salvage pathway. 5′Nucleotidase is a purine pathway enzyme and is present in two forms. Cytoplasmic 5′nucleotidase (cyto 5′NT) catalyzes the intracellular degradation of purine nucleotides into their corresponding nucleosides. The plasmamembrane bound form (ecto 5′NT) has its active site facing the external medium. Its function is the extracellular dephosphorylation of nucleotides, to which cells are generally impermeable, into nucleosides and the transport of these nucleosides through the cell membrane. Lymphoblastic 5′NT activity varies between different children with common-ALL. 5′NT positive cases have a higher relapse rate and thus a poorer prognosis than 5′NT negative cases (10). This can be explained by two hypotheses which are not mutually exclusive: 1. 1. Rescue hypothesis. When purine de novo synthesis is blocked by methotrexate (MTX) and/or 6-mercaptopurine (6-MP), the malignant cell has to rely on the purine salvage pathway. This pathway depends on the ecto-5′NT activity. So, leukemic cells might be resistant to MTX and/or 6-MP because of ecto-5′NT activity. 2. 2. Breakdown hypothesis. Leukemic cells are resistant to 6-MP because of the breakdown of the toxic nucleotide form of 6-MP into the nucleoside form by cyto-5′NT.

Reduction of monocyte 5'nucleotidase activity by gamma-interferon in multiple sclerosis and autoimmune diseases.

Ecto-5'nucleotidase (5'NT) activity in the plasma membrane of peripheral blood monocytes from patients with multiple sclerosis (MS), rheumatoid arthritis (RA), myasthenia gravis (MG), motor neuron disease (MND), and from normal control subjects of similar age was determined by radioisotopic assay. The activity in unstimulated monocytes cultured for 24 and 48 hours was found to be higher than normal in 56% of patients with active relapsing MS, 29% of patients with RA, and 7% of patients in the MG/MND group. While the enzyme activity was reduced after cultivation of the cells with recombinant interferon-gamma (gamma-IFN) in all of the groups studied, the percentage reduction was significantly greater in monocytes from patients with active relapsing MS who were in relapse at the time of sampling (p = 0.03). HLA-DR expression was monitored using immunofluorescence staining with monoclonal antibody and showed that similar numbers of monocytes in all patient and control groups expressed this antigen. Thus, while high monocyte 5'NT activity was found principally in patients with active relapsing MS and in some patients with RA, the monocytes from patients with MS were, in addition, exquisitely sensitive to stimulation with gamma-IFN.

161 ANTI-5′-HUCLEOTIDASE ANTIBODIES CAUSE HUMAN PERIPHERAL BLOOD T CELLS TO PROLIFERATE

Human peripheral blood T cells were stimulated to proliferate when cultured with subraitogenic doses of phorbol myristate acetate (PMA) and goat antibodies to ecto-5′-nucleotidase (ecto-5′-NT). The degree of proliferation, as measured by 3H-thymidine incorporation on day 3, was similar to that achieved by stimulation with phytohemagglutinin (PHA). Anti-5′-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6-1.0 ng PMA/ml and 150 μg/ml of IgG isolated from a goat anti-5′-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2 receptor expression and IL-2 secretion were also induced by anti-5′-NT antibodies and PMA. As expected, anti-5′-NT antibodies and PMA did not induce the proliferation of ecto-5′-NT− T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol (PI)-specific phospholipase C removed 80% of the ecto-5′-NT activity from the cell surface and also inhibited the ability of the cells to proliferate in response to anti-5′-NT antibodies and PMA by 84%. Thus, the activation signal provided by anti-ecto-5′-NT antibodies is apparently transduced by a form of the enzyme which is attached to the membrane via PI-linkage. These data suggest that ecto-5′-NT may play a role in lymphocyte activation as has been proposed for other PI-linked lymphocyte surface proteins including Thy-l, T cell activating protein, TAP, and the rat alloantigen RT-6.

116 SENSITIVITY TO PURINE ANTAGONISTS IN CHILDHOOD LEUKEMIA ASSESSED BY THE AUTOMATED MTT-ASSAY

We showed that in childhood common acute lymphoblastic leukemia (c-ALL), 51 nucleotidase (5′NT) positive cases have a poorer prognosis than 5′NT negative cases. This might be due to the breakdown of the cytotoxic nucleotides of 6-mercaptopurine (6-MP) by high cytoplasmic 5′NT activities. Alternatively, ccto 5′NT can provide purine requirements of the purine salvage pathway and therefore rescue cells from a blockage of purine de novo synthesis by 6-MP and/or methotrexate. To test these hypotheses we need to determine the relation between these enzymes and drug sensitivity. We have adapted the MTT assay, which has only been reported on in studies dealing with established cell lines, to assess the chemosensitivity of cells obtained directly from patients. The assay is based on the reduction of MTT to formazan by living but not by dead cells. Formazan production is quantitated automatically with a microplate spectrophotometer. Incubation of ALL cells with 6-MP (4-125 μg/ml) and 6-thioguanine (1.6-50 μg/ml) for 2-4 days resulted in dose-response curves covering the range from 0% to 100%; cell survival. Comparison of the MTT assay with a dye exclusion assay in 10 patients with ALL demonstrated an identical success rate and comparable dose-response curves for both assays. Because automated quantitation of the chemosensitivity of leukemic cell samples involving about 80 drug concentrations takes only a few minutes with the MTT assay, this assay is a rapid, efficient, and objective method of measuring sensitivity to purine antagonists in ALL patients.

160 FUNCTIONAL CHARACTERIZATION OF ECTO-5′-NUCLEOTIDASE (ECTO-5′-NT) POSITIVE AND NEGATIVE HUMAN LYMPHOCYTES

Human T and B lymphocytes, separated into ecto-5′-NT positive and negative populations with goat antl-5′-NT antibodies and the fluorescence-activated cell sorter, were characterized in functional assays. Ecto-5′-NT− T cells proliferated as well as, or better than, ecto-5′-NT+ T cells after stimulation with phytohemagglutinin or in a mixed lymphocyte reaction. Ecto-5′-NT− T cells also provided more help for pokeweed mitogen (PWM)-stimulated immunoglobulin synthesis than ecto-5′-NT+ T cells. Therefore, the inability of ecto-5′-NT deficient lymphocytes from patients with immunodeficiency diseases to respond in these assays cannot be attributed solely to their reduced ecto-5′-NT activity. In contrast, ecto-5′-NT+ T cells proliferated in response to ten-fold lower doses of phorbol esters than ecto-5′-NT+ T cells, suggesting that ecto-5′-NT+ T cells may utilize unique biochemical pathways of cellular activation or may have a preferential ability to respond to certain stimuli. Ecto-5′-NT positive and negative B cells were tested for the ability to synthesize IgM and IgG after stimulation with PWM and Epstein Barr virus. Although both groups of cells synthesized IgM, the synthesis of IgG was restricted to the ecto-5′-NT+ sub-population. These data provide the first direct evidence that ecto-5′-NT is a marker for the functional maturation of human B cells and demonstrate that ecto-5′-NT is different from other human B cell surface antigens such as IgD, Leu 8, and HB-4 which are lost as B cells mature.

176 PURINE CATABOLIC ENZYMES IN HUMAN SYNOVIAL FLUIDS

Cartilage 5′nucleotidase (5NT) activity has been implicated in calcium pryophosphate dihydrate (CPPD) deposition [Arthritis Rheum 24:492, 1981]. We measured synovial fluid (SF) 5NT and nucleotide pyrophosphohydrolase (NPPH), another cartilage ectoenzyme associated with CPPD deposition, in 173 patients with well-characterized arthropathies. A cytosolic enzyme, adenosine deaminase (ADA), and an ectoenzyme not associated with CPPD deposition, alkaline phosphatase (AP), were measured as controls.SF 5NT and NPPH activities are highest and ADA activities are lowest in joints with noninflammatory SF which contain calcium crystals (Wilcoxon rank-sum, p<0.05). SF activities correspond with those reported in cartilage and suggest a specific association of elevated SF 5NT and NPPH activities with articular calcium crystal deposition.

Further studies on the diagnostic value of γ-glutamyl transpeptidase and 5′-nucleotidase in cattle, sheep and horses

The distribution of 5'-nucleotidase (5'-NT) and gamma-glutamyl transpeptidase (gamma-GT) is similar in the tissues of the sheep, calf and horse, except that there is relatively less gamma-GT in calf liver than in the liver of the other two species. The liver lesion produced by the oral administration of chloroform is similar in the three species and is accompanied by the release of 5'-NT into the plasma of the sheep and calf but not of the horse. Conversely, gamma-GT is released into plasma of the horse but not of the sheep or calf. This difference is not related to the tissue distribution of the two enzymes. The kidney lesion in sheep produced by the intravenous administration of mercuric chloride is accompanied by a reduction in the rate of excretion of an injected dose of inulin and by an increase in the concentration of urea in plasma and in the activity of gamma-GT in plasma and urine. There was no increase in 5'-NT activity in plasma or urine.

Physiological amino acids in the central nervous system of the tarantulas, Aphonopelma chalcodes and Dugesiella echina (Orthognatha: Theraphosidae)

1. 1. An analysis of the physiological (free) amino acids in the central nervous system of the tarantula spiders, Aphonopelma chalcodes and Dugesiella echina (Theraphosidae) was conducted. 2. 2. Nitrogenous waste products (ammonia and urea) together comprised 56% of the total ninhydrin-positive compounds in A. chalcodes and 55% in D. echina. 3. 3. GABA accounted for 6% of the total ninhydrin-positive compounds in the CNS of A. chalcodes, and 7% in D. echina. 4. 4. GABA, aspartate, taurine, glycine and glutamate (which have been shown to function as neurotransmitters and/or comodulators of NT activity) together comprised 25% of the total ninhydrin-positive compounds. 5. 5. The most abundant amino acids were proline, alanine, glutamate and aspartate. Glutamate and aspartate concentrations in the CNS of the more primitive theraphosids are lower than those found in web-building spiders.