Silvia Sestini

Simple non-radiochemical HPLC-linked method for screening for purine metabolism disorders using dried blood spot.

BACKGROUND Pathologies associated with rare inherited disorders affecting purine metabolic pathways range from renal failure to neurological dysfunction and immunodeficiency. The disorders are usually diagnosed by measuring enzyme activities in hemolysates. A non-radiochemical HPLC-linked method is described for simultaneous determination of the activities of hypoxanthine-guanine phosphoribosyltransferase (HPRT: E.2.4.2.8.), adenine phosphoribosyltransferase (APRT: E.2.4.2.7.), adenosine deaminase (ADA: E.3.5.4.4.) and purine nucleoside phosphorylase (PNP: E.2.4.2.1.) in dried blood spots. METHOD 7-mm-diameter blood spots stored at 4 degrees C or room temperature were transferred to an Eppendorf tube and eluted with 500-microl 0.1 mol/l Tris-HCl buffer, pH 7.4. The eluate was added to substrate solutions and incubated at 37 degrees C. Reaction products were analysed by HPLC. RESULTS AND CONCLUSIONS The enzyme activities tested in spot eluates were similar to those in erythrocyte lysates from the same subjects. None of the enzymatic activities tested were significantly affected by different storage temperatures. The main advantages of the proposed method are small blood volume required, easy sample collection and transfer, and accurate results. The method is therefore suitable for screening inborn errors of purine metabolism even in newborns.

DETERMINING NAD SYNTHESIS IN ERYTHROCYTES

Publisher Summary This chapter describes methods for the study of nicotinamide adenine dinucleotide (NAD) synthesis in human erythrocytes, developed according to two approaches— the determination of intracellular concentrations of pyridine compounds and the rate of conversion of radiolabeled precursors in intact cells, and enzyme activity assays in crude lysates. All determinations are performed by methods involving high-performance liquid chromatography (HPLC). Different methods of obtaining protein-free extracts suitable for HPLC processing are examined. Acid or alkaline extraction provides better results than acetonitrile extraction or boiling, which cause oxidation of NADH or breakdown of NAD. The activities of several enzymes are measured in crude lysates made from fresh or, when suitable, stored erythrocytes: nicotinate phosphoribosyltransferase (NA–PRT), nicotinamide phosphoribosyltransferase (NAm–PRT), nicotinamide-mononucleotide and nicotinate-mononucleotide adenylyltransferase (NMN–AT and NAMN–AT), and NAD synthetase (NAD-s). All assay reactions are started by lysate addition. Hemoglobin content is measured in lysates by the cyanmethemoglobin method and used to quantify enzyme specific activity.

11β-Hydroxysteroid dehydrogenase expression in first trimester human trophoblasts

This study evaluated the levels and the enzymatic characteristics of 11beta-hydroxysteroid dehydrogenase activity (11beta-HSD) of chorionic villi isolated from first trimester human placenta. The results demonstrated a predominant expression of the NAD-dependent dehydrogenase isoform (11beta-HSD2) over the NADP-dependent oxoreductase (11beta-HSD1). Thus, in tissue homogenates exogenous NAD increased the conversion of corticosterone to 11-dehydrocorticosterone of about 14-fold while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in villous tissue while 11beta-HSD1 mRNA levels were undetectable. In addition, immunohistochemical staining localized the 11beta-HSD2 protein to syncytiotrophoblasts and cell columns of the chorionic villi. These results suggest roles for the trophoblast-associated 11beta-HSD2 oxidative activity in modulating the exposure of the embryo to active glucocorticoids in the early gestation and in regulating trophoblasts invasion of the uterine wall.

Progestin regulation of 11β-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells

Abstract This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11β-HSD2 in T-47D cells, while 11β-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11β-HSD catalytic activity was elevated 11-fold, while estrone (E 1 ), estradiol (E 2 ) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11β-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11β-HSD2 gene expression, increasing the steady-state levels of 11β-HSD2 mRNA. Taken together, these results demonstrate that 11β-HSD2 is the 11β-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.

Sheep (Ovis aries) Macrophage Migration Inhibitory Factor: molecular cloning, characterization, tissue distribution and expression in the ewe reproductive tract and in the placenta.

Macrophage Migration Inhibitory Factor (MIF) is a pivotal regulator of innate and acquired immunity affecting the response and behavior of macrophages and lymphocytes. However, a number of studies indicated wider physiological functions for this cytokine to include key-roles in reproductive biology. The present study was designed to clone the coding sequence of sheep MIF, to examine the characteristics of the protein in vitro, and to evaluate its expression in sheep tissues and in the ewe reproductive tract in vivo. Ovine MIF cDNA consisted of 348 nucleotides encoding a 115 amino acids protein with an estimated molecular mass of 12,343 Da and an isoelectric point of 7.68. Sheep MIF shared high amino acid identity with the other mammalian MIF family members and showed parallel functions to human MIF, displaying enzymatic oxoreductase activity and inducing monocyte transmigration. Expression studies detected a MIF transcript in all the sheep tissues examined. Among reproductive tissues, MIF mRNA and protein were detected in the ovary, oviduct, uterus and placenta. These results indicate that sheep MIF shares crucial features with other MIF family members and delineate its potential involvement in several aspects of ovine physiology.

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface‐located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD‐glycohydrolase, nucleotide pyrophosphatase and 5′‐nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5′‐nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U‐[14C]‐adenine‐NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto‐NAD‐hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with 14C‐NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [14C]‐NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with 14C‐adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [14C]‐adenosine yield the same radioactive products as with [14C]‐NAD; the absence of inhibition of [14C]‐adenosine uptake by ADPribose in the presence of α‐β methyleneADP, an inhibitor of 5′ nucleotidase, demonstrates that ADPribose coming from NAD via NAD‐glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent. J. Cell. Biochem. 80:360–366, 2001.

Poly(ADP-ribose) polymerase activity in systemic lupus erythematosus and systemic sclerosis

The aim of this study is to investigate the role of poly(ADP-ribose) polymerase (PARP), involved in DNA repair and in autoimmune pathologic conditions such as systemic lupus erythematosus (SLE) and both limited systemic sclerosis (lSSc) and diffuse systemic sclerosis (dSSc), to assess its possible implication in the pathogenetic processes. The relationship between PARP activity and the intracellular concentration of its substrate nicotinamide adenine dinucleotide (NAD) is also investigated. Peripheral mononuclear cells (PMC) from controls and patients with SLE, lSSc, and dSSc were irradiated with ultraviolet light (UV) and PARP activity was assayed by a radiochemical method. Pyridine nucleotide concentrations were assayed by a high-performance liquid chromatography-linked method. PARP activity was detectable in nonirradiated cells and showed similar values in all groups. The activity significantly increased after UV irradiation in control, SLE, and lSSc cells, but not in dSSc cells. Irradiated PMC from both SLE and dSSc showed lower enzyme activity with respect to irradiated controls. Higher intracellular NAD content was found in all of the pathologic conditions in comparison to values in the control; this difference was statistically significant in dSSc. Our data demonstrate a lower PARP activity in response to UV damage in PMC from patients affected by the above pathologic conditions compared with controls. An inverse relationship between PARP activity and NAD content was also observed.

Enzymatic activities affecting exogenous nicotinamide adenine dinucleotide in human skin fibroblasts

The fate of nicotinamide adenine dinucleotide (NAD), AMP, and ADP‐ribose supplied to intact human skin fibroblasts was monitored, and the concentrations of intra‐ and extracellular pyridine and purine compounds were determined by HPLC analysis. Two enzymatic activities affecting extracellular NAD were detected on the plasma membrane, one hydrolyzing the pyrophosphoric bond and yielding nicotinamide mononucleotide (nucleotide pyrophosphatase) and the other cleaving the glycoside link and releasing nicotinamide (NAD‐glycohydrolase). No AMP or ADP‐ribose was found in the extracellular medium of cells incubated with NAD, the former being completely catabolized to hypoxanthine and the latter degraded to adenine and hypoxanthine.

Significance and Relevance of NAD Synthesis in Human Erythrocyte Life Span

NAD and NADP are known to play a key role in the general metabolism of human erythrocytes, owing to their involvement in all oxidative-reductive reactions occuring both in the energetic catabolism and in the processes preventing the cells from oxidative damage.

NAD metabolism in HPRT-deficient mice

The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT- and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT- mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type.