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PubMedDate not supplied

Immunogold labeling and cerium cytochemistry of the enzyme ecto-5'-nucleotidase in promastigote forms of Leishmania species.

We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane.

5'-Nucleotidase↗

PubMed1983

Ecto-5'-nucleotidase activities in lymphocytes and B lymphoblastoid cell lines from patients with congenital agammaglobulinemia.

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in Epstein-Barr virus (EBV)-induced B lymphoblastoid cell lines (LCL) derived from bone marrow cells and peripheral lymphocytes of four patients with congenital agammaglobulinemia (CAG). In control subjects there was no appreciable difference in the range of ecto-5'-NT activity between immunoglobulin (Ig)-producing B LCL and their peripheral lymphocytes. Though peripheral lymphocytes of the patients showed lower ecto-5'-NT activities than those of control subjects, ecto-5'-NT activities in Ig-producing B LCL from peripheral lymphocytes of the patients fell into the same range as those in B LCL from control subjects. Ecto-5'-NT activities in non-Ig producing precursor B cell lines derived from bone marrow cells of the patients also registered within the range of the enzyme activity of Ig-producing B LCL from control subjects. B LCL from mothers of the patients, who were presumed to be heterozygotes, also had the same range of ecto-5'-NT activity as the control subjects. These results indicate that ecto-5'-NT does not appear to be involved in the pathogenesis of congenital agammaglobulinemia.

Agammaglobulinemia↗

PubMed1994

The in vitro effect of 13 metals on the activity of erythrocyte pyrimidine 5'-nucleotidase (P5N).

The in vitro effect of 13 metal ions on the activity of erythrocyte pyrimidine 5'-nucleotidase (P5N) was investigated. Particular metals belonging to the boron group have recently been used as materials in a semi-conductor manufacturing. Of these metal ions, Ga exhibited no inhibition of erythrocyte P5N, while in extensively inhibited the erythrocyte P5N activity at concentrations of 10(-4) to 10(-3) M. In addition, the activity of erythrocyte P5N was inhibited by 90-96% when the metal ions such as Cu, Ag, Cd, Hg or Pb were added to the incubation mixture at concentrations of 10(-4) to 10(-3) M. On the other hand, there was no difference between sulfate and nitrate in the in vitro effect of the metal ions on the activity of erythrocyte P5N.

5'-Nucleotidase↗

PubMed2002

Evaluation of activity of erythrocyte pyrimidine 5'-nucleotidase (P5N) in lead exposed workers: with focus on the effect on hemoglobin.

Anemia that accompanies lead poisoning is in part the result of various inhibitory effects of lead on heme biosynthesis. Lead also increases the rate of red blood cell destruction due to the profoundly depressed activities of erythrocyte pyrimidine 5'-nucleotidase (P5N) activities. We studied parameters of the two metabolic pathways in the workers exposed to lead to evaluate P5N in the lead exposed workers and which pathway has an effect on hemoglobin (Hb) level. 29 male workers in the secondary lead smelting as high exposure group, 46 male workers in the manufacturer of inorganic pigment as low exposure group and 56 clerical male workers from another plant as non-exposed group were studied. Activity of P5N, lead concentration in whole blood (PbB), zinc protoporphyrin (ZPP), Hb, and ferritin were determined. In the present study, P5N activity of nucleotide metabolic pathway correlated with Hb after controlling indices of iron deficiency anemia (ferritin) occurring concurrently and heme biosynthetic pathway (ZPP) in the high exposure group while heme biosynthetic pathway did not correlate with Hb after controlling other two variables in exposure groups. These findings suggest that P5N rather than heme biosynthetic pathway has a major effect on Hb level even in workers without manifest hemolytic anemia.

5'-Nucleotidase↗

PubMed1993

Increased activity of 5' nucleotidase in serum of patients with sickle cell anaemia.

Fifty-nine patients with homozygous sickle cell anaemia, 17 heterozygous individuals and 22 controls were investigated in respect to serum (S) 5'nucleotidase (5'NT, EC 3.1.3.5). The patients showed a significantly higher mean value of S-5'NT compared to the controls. However, this rise was heterogeneous as it occurred only among a subgroup of patients. The heterozygous individuals were not different from either the patients or the controls generating a situation which puts the heterozygous individuals in an intermediate position between the patients and the controls. S-5'NT showed significant correlation with S-bilirubin, S-aspartate aminotransferase, S-alanine aminotransferase and especially S-gammaglutamyl transferase. However, it was not correlated with S-alkaline phosphatase, which is another marker for hepatobiliary disease. These results suggest that the liver involvement in a subgroup of patients with sickle cell anaemia is a mixture of hepatocyte damage and the biliary tree involvement.

5'-Nucleotidase↗

PubMed2004

IFN-alpha induced adenosine production on the endothelium: a mechanism mediated by CD73 (ecto-5'-nucleotidase) up-regulation.

CD73 (ecto-5'-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-alpha produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-alpha treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1beta, TNF-alpha, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-alpha. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-alpha is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.

5'-Nucleotidase↗

PubMed1985

A new 5'-nucleotidase inhibitor, nucleoticidin. II. Physico-chemical properties and structure elucidation.

A novel 5'-nucleotidase inhibitor, named nucleoticidin, was isolated from a fermentation broth of Pseudomonas sp. The molecular weight was estimated by gel filtration to be over 1,000,000. Nucleoticidin is composed of D-glucose and D-mannose at a molar ratio of 1.7 to 1.0. Combined analyses using chemical and physico-chemical methods, such as gas liquid chromatography and mass fragmentography, revealed that nucleoticidin has a structural unit with mannosyl residues at the terminal of a (1----4) linked D-glucosyl main chain with beta-configuration.

Anti-Bacterial Agents↗

PubMedDate not supplied

Serum 5-nucleotidase in some rachitic and jaundiced Egyptian children.

5-Nucleotidase (5-Nase) was determined in the serum of 67 Egyptian Children: 15 cases as a control group, 10 cases with infantile rickets, 8 cases with the so-called physiological jaundice of the newborn, 4 cases with rhesus incompatibility, and 30 cases with infective hepatits. In rachitic cases and in cases with icterus neonatorum, 5-Nase was not increased, while in cases with infective hepatitis it showed a statistically significant elevation when compared with the control group.

Child↗

PubMed1999

In vitro effects of selected flavonoids on the 5'-nucleotidase activity.

A series of structurally related flavonoids and related compounds were evaluated whether they have inhibitory properties on the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5, 5'-NT) activity. Some of the flavonoids tested inhibit the enzyme such as quercetin, morin, apigenin, chrysin, myricetin, luteolin, diosmetin, (+/-)naringenin and diosmin. Rutin, naringin, hyperosid, (+/-)catechin, caffeic acid and rosmarinic acid had no inhibitory effect on the 5'-NT activity. Myricetin and quercetin were the most potent inhibitors for 5'-NT with IC50 values of 1.1 and 1.4 microM, respectively. Kinetic analysis showed a mixed type of inhibitor for both myricetin (Ki = 1.5 microM at pH 7.45), and quercetin (Ki = 0.6 microM at pH 7.45). The K(m) value for 5'-adenosine monophosphate (5-AMP) was determined with 77 microM at pH 7.45. The differential inhibitory potencies of flavonoids seem to be structurally related (hydroxylation pattern). The results demonstrate that some flavonoids are strong inhibitors of 5'-NT activity which can be correlated to their pharmacological effects.

5'-Nucleotidase↗

PubMedDate not supplied

Expression of CD 73 (ecto-5'-nucleotidase) in 165 glioblastomas by immunohistochemistry and electronmicroscopic histochemistry.

BACKGROUND: CD 73 (5'-nucleotidase) is an ectoenzyme, which is expressed on normal and neoplastic glial plasma membranes. The enzyme binds to intracellular filamentous actin and the extracellular matrix proteins laminin and fibronectin. CD 73 is a signalling pathway metabolite in the immune response of lymphocytes. The ectoenzyme catalyzes the conversion of purine and pyrimidine ribo- and deoxyribo-nucleoside monophosphates (AMP, GMP, IMP) and leads to elevation of the corresponding nucleosides (adenosine) in the extracellular space and might therefore modulate neuronal signalling and vascular perfusion. CD 73 has also been called a cellular motility factor. There is an increasing amount of evidence for the modulatory role of PKC-mediated CD 73 activity in ischemia, regeneration and repair, glioma cell proliferation and a possible invasion promoting feature of the ectoenzyme. The aim of the present study was to investigate the expression patterns of CD 73 together with the labelling of PKC and EGFR. The latter is known as a marker for primary glioblastomas. PATIENTS AND METHODS: We investigated the expression of CD 73 in 165 glioblastoma specimens together with the expression patterns of PKC and EGFR by immunocytochemistry on cryosections with a 4-step grading evaluation by two independent observers. CD 73 was further investigated morphologically by electron-microscopic histochemistry in cell cultures of glioblastoma specimens. RESULTS: With these methods it was possible to demonstrate a dense labelling pattern of glioblastoma specimens with anti-CD 73. 95.7% of the glioblastomas were identified with staining products, 63% with labelling grades 2 and 3. The dense staining of the endoplasmatic reticulum, vesicles, caveolar structures and glial membranes was demonstrated by electron-microscopic histochemistry. Some free enzymatic activity was located bound to the ECM components. We observed a significant coexpressions of CD 73 with PKC (p = 0.001) and CD 73 with EGFR (p = 0.022), which is a prospective marker for a high rate of early recurrency. CONCLUSIONS: The CD 73 activity was densely distributed on the membranes of glioblastoma cells in vivo and in cell cultures. The electron-microscopic histochemical studies could demonstrate enzymatic activity at the cell membranes and in vesicular structures and caveolae. Free staining deposits located on ECM components may result in a migration- and infiltration-promoting activity. The CD 73 expression could be correlated with the expression grades of PKC and EGFR. The latter has been identified as a prognostic factor which is expressed mainly on primary glioblastomas. PKC is a known tumour metabolite in several proliferation promoting pathways of EGF receptor signalling.

5'-Nucleotidase↗

PubMed1999

[Pyrimidine-5'-nucleotidase deficiency--congenital hemolytic anemia with basophilic stippling of erythrocytes].

The disease is caused by a deficiency of the enzyme pyrimidine-5'-nucleotidase in the erythrocytes. About 50 cases have been published. We have diagnosed the disease in a pair of siblings from Numedal, Norway. Although this is a rare disorder it has a world-wide distribution. It is inherited as an autosomal recessive disease, and with genetic heterogeneity. In studies of iron balance we have found increased excretion of iron and hemoglobin in the urine, and free hemoglobin in plasma, indicating extravascular hemolysis. Kidney biopsy shows iron depositions in epithelial cells in proximal kidney tubules, localized to lysosomes. An evaluation by ordinary clinical examinations and laboratory tests do not indicate a progression of the disease over a ten-year period.

5'-Nucleotidase↗

PubMed2000

5' nucleotidase and adenosine during retinal vasculogenesis and oxygen-induced retinopathy.

PURPOSE: 5' nucleotidase (5'N) is a major source of the vasogenic substance adenosine in most tissues. The distribution and relative levels of 5'N and adenosine were examined in neonatal dog inner retina during normal vasculogenesis and oxygen-induced retinopathy (OIR). METHODS: Animals ranging in age from 1 to 22 days of age were used in this study. Adenosine immunolocalization was performed on frozen sections with an antibody against adenosine conjugated to laevulinic acid using a streptavidin peroxidase technique. Triplicate room air control animals at different postnatal ages and triplicate oxygen-treated animals at different time points during or after hyperoxic insult were analyzed. Adenosine immunoreactivity (AI) and 5'N enzyme histochemical reaction product were quantified using microdensitometry. Adjacent sections were incubated for von Willebrand factor to label blood vessels. RESULTS: During normal vasculogenesis, AI was most prominent within the inner retina. The peak of immunoreactivity was located at the border of vascularized retina throughout the period of primary retinal vasculogenesis (1-15 days of age). At 22 days when vasculogenesis was complete, AI decreased within the inner retina. The highest 5'N activity was localized to inner Muller cell processes in inner retina and decreased after vasculogenesis was complete. In animals killed after 4 days of oxygen breathing, the vasoobliterative stage of OIR, AI and 5'N activity were reduced throughout the retina. During the vasoproliferative stage, AI was markedly elevated at the edge of reforming vasculature as well as throughout the more posterior inner retina where 5'N activity also was elevated. AI was also in intravitreal neovascularization. CONCLUSIONS: Peak adenosine levels in the inner retina correlated temporally with active vasculogenesis. Adenosine and 5'N levels were reduced in hyperoxia and then returned to above normal levels during the vasoproliferative stage of OIR.

5'-Nucleotidase↗

PubMed2000

Human erythrocyte pyrimidine 5-nucleotidase, PN-I, is identical to p36, a protein associated to lupus inclusion formation in response to alpha-interferon.

Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an alpha-interferon-induced protein. PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library. PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme. The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36. Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I. Molecular properties of the two proteins were essentially the same. We conclude that p36 and PN-I are identical proteins. (Blood. 2000;96:1596-1598)

5'-Nucleotidase↗

PubMed2000

Can 5' nucleotidase estimation be a predictor of liver metastases?

The diagnosis of hepatic metastases is important and has prognostic significance in clinical medicine. A variety of biochemical tests have been used to diagnose liver metastases before surgery and during follow up. However, recently, with the introduction of high resolution imaging modalities like CT Scan/Ultrasonography, the value of these biochemical tests declined. Ultrasonography/CT Scan can pick up liver metastases upto 0.5 cm diameter. Many a times bigger lesions and obviously smaller lesions can be missed by these screening methods. Various biochemical markers were proposed to pick up liver metastases. In this study, the estimation of 5'nucleotidase is found to be the most sensitive predictor of liver metastases when compared to conventional markers and imaging modalities.

5'-Nucleotidase↗

PubMed2001

[Distribution of 5'-nucleotidase activity in greater omentum of rats].

The aim of the present study is to demonstrate the cellular basis of 5'-nucleotidase (5'-Nase) activity in the greater omentum of rats. Enzyme histochemistry for 5'-Nase showed that lymphatic vessels in the omentum as well as lymphocytes in the milky spots were positively stained. Electron microscopic observation revealed-5'-Nase activity at the luminal surface of the lymphatic endothelial cells, pinocytotic vesicles in the endothelial cells and the surface of fibroblasts located at the intercellular space of adipose cells. Fibroblasts extended long cytoplasmic processes toward adipose cells and inflammatory cells. These findings suggest that lymphatic endothelial cells as well as fibroblasts in the omentum may play an important role in regulation of metabolism and immune mechanisms in the greater omentum by supplying adenosin.

5'-Nucleotidase↗

PubMed2000

[Assessment of erythrocyte pyrimidine 5'-nucleotidase test for screening of occupational lead poisoning].

OBJECTIVE: To determine the diagnostic value of red cell P5'-N for detecting occupational lead poisoning. METHODS: Red cell pyrimidine 5;-nucleotidase, blood lead and urine lead levels, free erythrocyte protoporphyrin (FEP), zinc protoporphyrin (ZPP) and urine delta-aminolevulinic acid levels were determined in 157 lead exposed workers and 30 non-exposed healthy people. According to the golden standard; i.e. blood lead >/= 1.93 micromol/L and >/= 2.90 micromol/L, the diagnostic value of red cell P5'N and other indicators for detecting blood lead >/= 1.93 micromol/L and >/= 2.90 micromol/L was compared by receiver operative characteristic curve (ROC) software. After determining the optimal cut-points of the above indicators for detecting the blood lead >/= 1.93 micromol/L and >/= 2.90 micromol/L, the sensitivity of the parallel tests for detecting the blood lead >/= 1.93 micromol/L and >/= 2.90 micromol/L was calculated and compared by EPINFOR software. RESULTS: For detecting blood lead level >/= 1.93 micromol/L, the areas under ROC curve (AUC(ROC)) of P5'N was significantly larger than that of urinary lead and urinary delta-ALA (P < 0.05). The sensitivity of parallel tests with ZPP and P5'N was significantly larger than that of parallel tests with Upb and Udelta-ALA. For detecting blood lead level >/= 2.90 micromol/L, the areas under ROC curve (AUC(ROC))of P5'N was significantly larger than that of urinary lead, FEP and urinary delta-ALA (P < 0.05). The sensitivity of parallel tests with P5'N and ZPP was significantly larger than that of parallel tests with Upb and Udelta-ALA, and that of parallel tests with ZPP and FEP (P < 0.05). CONCLUSION: For detecting blood lead >/= 1.93 micromol/L and >/= 2.90 micromol/L, red cell P5'N is more accurate. It is worth being applied in detecting occupational lead poisoning. For detecting blood lead >/= 1.93 micromol/L and >/= 2.90 micromol/L, the parallel tests with P5'N and ZPP are more accurate.

5'-Nucleotidase↗

PubMed2002

Expression of adenosine deaminase and 5'-nucleotidase in artificially induced deciduoma in rat and hamster.

Enzymes adenosine deaminase (ADA) and 5-nucleotidase (5-'NT) are known to play active role in tissue/cell proliferation and differentiation. To validate this the two enzymes were studied in artificially induced deciduoma of rat and hamster. The deciduoma was induced by traumatizing one of the uterine horns of progesterone primed animals. Non traumatized horn served as control. The animals were later maintained on progesterone, given alone (Gr.I) or conjointly with estrogen (Gr.II). The weight of each uterine horn was recorded to determine the formation of deciduoma. There was no marked difference between the weights of traumatized and control horn on day 2 post-traumatization (PT), but a progressive rise was noticed after this day in both species. The ADA activity however differed, day and species wise. While in the rats of Gr.I it was low in the traumatized horn on all the days, in the hamsters it was remarkably high from day 2 to 6 PT. In the rats of Gr.II also the activity though was low in the traumatized horn, but on day 2 and 4 only; on day 6 and 7 PT it increased markedly. In hamster, on the contrary, again the enzyme activity was remarkably high on all the three days. The 5'-NT activity, however, did not show any marked difference between the two horns under Gr.I and II in both species. It was rather high in the control horn of each group. The results suggest: (I) the progesterone alone though produces a significant rise in the uterine weight of traumatized horn in both species, the ADA activity increases only in hamster, (2) under the conjoint treatment also the enzyme activity remains high in hamster; and (3) the activity of enzyme 5'-NT does not alter during the deciduoma formation in both the species.

5'-Nucleotidase↗

PubMed1975

[Comparison of topographical locations of reticular cells with 5'-nucleotidase activity in spleen white pulp and lymph nodes in man and rat].

The cells connections occuring in thymus-dependent areas of the splenic white pulp of rat between 5'nucleotidasic reticular cells and small lymphocytes allowed to research this same associated cells in the rat lymph node and the human splenic white pulp and lymph node. The locations of the thymus- dependent areas seem different in man and rat.

Animals↗