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A De Flora

Publications and source records attributed to A De Flora.

At least 55 records · Page 3Linked to original sources
PubMed1995

Self-aggregation of purified and membrane-bound erythrocyte CD38 induces extensive decrease of its ADP-ribosyl cyclase activity.

The transmembrane glycoprotein CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain both the synthesis and the hydrolysis of cyclic ADP-ribose (cADPR). The complete reaction, converting NAD+ to nicotinamide and ADP-ribose, reproduces an NAD+glycohydrolase (NADase) reaction. CD38 purified from human erythrocyte membranes has been recently shown to undergo stable oligomerization induced by either NAD+ or beta-mercaptoethanol. We demonstrate that oligomerization is also triggered by reduced glutathione (GSH) and that the GSH-induced self-aggregation of purified CD38 is accompanied by extensive and comparable decrease of its ADP-ribosyl cyclase and NADase activities. GSH-induced oligomerization of CD38 and strong enzyme inactivation take place also in situ on erythrocyte membranes.

ADP-ribosyl Cyclase↗
PubMed1995

Primary structure of human erythrocyte nicotinamide adenine dinucleotide phosphate (NADP[H])-binding protein FX: identification with the mouse tum- transplantation antigen P35B.

Human erythrocytes contain a nicotinamide adenine dinucleotide phosphate (NADP[H])-binding protein, FX, whose levels are significantly increased in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals bearing the mediterranean variant of G6PD. Elucidation of the still unknown biologic functions of FX was approached by means of amino acid sequencing of its 25 tryptic peptides. Searching in the EMBL data bank allowed identification of extensive homology between these tryptic peptides and all sequence-aligned regions encompassing the complete structure of a putative protein encoded by the P35B gene in the mouse. This gene, which differs from the normal allele by a point mutation, has been previously cloned from a tum- variant of the murine tumor cell line P815, so defined because it is associated with low tumorigenicity compared with the progenitor P815. The reported P35B cDNA contains an open reading frame (ORF) of 813 bp and encodes a putative protein of 271 amino acids (30 kD), whereas FX protein is 320 amino acids in length (35.81 kD, in good agreement with previous studies). However, a single base shift at position 4,752 of the P35B gene suppresses the stop codon after Phe 271 and allows continuation of the ORF for up to 320 amino acids to reach the same length as FX. The remarkably high extent (92%) of homology indicates that erythrocyte FX protein is the human homolog of the P35B gene product.

Amino Acid Sequence↗
PubMed1995

Copper(II) complexes encapsulated in human red blood cells.

Copper(II) complexes were encapsulated in human red blood cells in order to test their possible use as antioxidant drugs by virtue of their labile character. ESR spectroscopy was used to verify whether encapsulation in red blood cells leads to the modification of such complexes. With copper(II) complexes bound to dipeptides or tripeptides, an interaction with hemoglobin was found to be present, the hemoglobin having a strong coordinative site formed by four nitrogen donor atoms. Instead, with copper(II) complexes with TAD or PheANN3, which have the greatest stability. ESR spectra always showed the original species. Only the copper(II) complex with GHL gave rise to a complicated behavior, which contained signals from iron(III) species probably coming from oxidative processes. Encapsulation of all copper(II) complexes in erythrocytes caused a slight oxidative stress, compared to the unloaded and to the native cells. However, no significant differences were observed in the major metabolic properties (GSH, glycolytic rate, hexose monophosphate shunt, Ca(2+)-ATPase) of erythrocytes loaded with different copper(II) complexes, with the exception of methemoglobin levels, which were markedly increased in the case of [Cu(GHL)H-1] compared to [Cu(TAD)]. This latter finding suggests that methemoglobin formation can be affected by the type of complex used for encapsulation, depending on the direct interaction of the copper(II) complex with hemoglobin.

Antioxidants↗
PubMed1994

A novel dimeric fluoropyrimidine molecule behaves as a remote precursor of 5-fluoro-2'-deoxyuridine in human erythrocytes.

A new dimeric fluoropyrimidine molecule (5-fluoro-2'-deoxyuridilyl-(5'-->3')-5-fluoro-2'-deoxy-5'-uridylic acid, Compound 1) was chemically synthesized from two separately deblocked 5-fluoro-2'-deoxyuridine mononucleotide moieties. Other structurally related nucleotides, 5-fluoro-2'-deoxyuridine-5'-diphosphate (FdUDP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine-3',5'-bisphosphate were also synthesized. The structures of all synthesized molecules were verified by mass spectrometric analyses and were consistent with expected molecular mass values. The metabolic patterns of conversion of Compound 1 were investigated both in human erythrocyte lysates and in intact erythrocytes previously loaded with this molecule according to a highly conservative encapsulation procedure. In hemolysates, Compound 1 was transformed to 5-fluoro-2'-deoxyuridine (FUdR) and to 5-fluorouracil (FU) through the intermediate formation of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). In intact red cells, Compound 1 still generated FUdR (and to a lesser extent FU), that was then released outside. The conversion pathway involves a phosphodiesterase-catalysed hydrolysis of Compound 1 into two FdUMP molecules, followed by further dephosphorylation to FUdR and by partial conversion to FU. Unlike hemolysates, Compound 1-loaded intact erythrocytes featured transient formation of FdUDP and FdUTP, both metabolites representing storage compounds for the final and sustained production of FUdR and FU. Therefore, human erythrocytes can behave as bioreactors ensuring the time-controlled production and delivery of the two powerful antitumor drugs FUdR and FU from encapsulated Compound 1. This new molecule and other compounds as well (e.g. FdUDP and FdUTP) can be viewed as useful pre-prodrugs, exploitable for intraerythrocytic bioconversion reactions.

Erythrocytes↗
PubMed1994

Extracellular ATP potentiates nitric oxide synthase expression induced by lipopolysaccharide in RAW 264.7 murine macrophages.

Inducible nitric oxide synthase (iNOS) activity in the murine macrophage cell line RAW 264.7 was increased from two- to four-fold after co-exposure of the cells to low doses of bacterial lipopolysaccharide (LPS) and micromolar ATP, compared to LPS alone. Extracellular ATP and its analogs "per se", i.e. without LPS, were not able to induce iNOS activity. The stimulating effect of UTP too, the concentration range of activity (1-100 mM nucleotides) and the rank of potency (ATP-gamma-S = AMP-PNP > ATP = ADP >> AMP-CPP = UTP) seem to indicate an involvement of P2y-type purinergic receptors. GTP, CTP and adenosine were virtually ineffective. These data suggest that binding of extracellular nucleotides to purinergic receptors may increase nitric oxide production by macrophages. This effect might occur in pathological conditions (i.e. inflammation/infection or trauma) where significant amounts of intracellular ATP can be released due to cellular damage.

Adenine Nucleotides↗
PubMed1994

Self-aggregation of the transmembrane glycoprotein CD38 purified from human erythrocytes.

The 46 kDa human transmembrane glycoprotein CD38 is a multicatalytic enzyme exhibiting ADPribosyl cyclase, cyclic ADPribose hydrolase and NAD(+)-glycohydrolase activities at its extracellular domain. When CD38, purified to homogeneity from human erythrocyte membranes, was incubated with NAD+ or beta-mercaptoethanol, extensive aggregation took place. Addition of both compounds to CD38 led to the formation of still larger aggregates (over 300 nm), which were resistant to TCA precipitation. Extensive and stable CD38 self-aggregation was shown by, i) SDS-PAGE and autoradiography of the [32P]NAD(+)-incubated CD38, ii) SDS-PAGE followed by immunochemical detection of CD38 on the transblots, iii) direct electron microscopy on negatively stained CD38 samples. Self-aggregation of CD38 might be correlated with its putative function as a transducer of activation and proliferation signals in a number of hematopoietic cells.

ADP-ribosyl Cyclase↗
PubMed1994

Synergistic cytotoxicity of carboplatin and divicine on murine erythroleukemic cells.

Carboplatin (CBDCA) and the pyrimidine aglycone divicine displayed cytotoxic effects of murine erythroleukemic cells (MELC), with ID50 values of 158 and 37 microM, respectively. Combination of CBDCA and divicine, at a 2:1 ratio, increased cytotoxicity considerably. Under specific conditions of time schedule of administration, the association of CBDCA and divicine resulted in a clear synergistic activity. Alkaline elution studies on both unirradiated and gamma-irradiated MELC demonstrated opposite patterns of DNA damage with the two molecules. Thus, CBDCA elicited DNA interstrand crosslinks (ISC), while divicine resulted in DNA single strand breaks (SSB). Association of both molecules led in the unirradiated cells to a higher SSB frequency than recorded with divicine alone. Accordingly, intracellular activation of CDBCA by redox cycling of divicine seems not to be involved. Rather, intracellular platinum appears to enhance cytotoxicity of divicine.

Animals↗
PubMed1993

A single protein immunologically identified as CD38 displays NAD+ glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities at the outer surface of human erythrocytes.

The three ectoenzyme activities, NAD+ glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase were purified to homogeneity from solubilized human erythrocyte membranes. The purification procedure involved three sequential chromatography steps on hydroxylapatite, immobilized Cu++ and immobilized anti-CD38 monoclonal antibody resins. The final step yielded a single 46 kDa protein displaying all three enzymatic activities. Since the protein bound specifically to the anti-CD38 resin, it was immunologically identified as CD38, a 46 kDa surface antigen involved in activation and proliferation of lymphocyte populations.

ADP-ribosyl Cyclase↗
PubMed1993

Enhanced formation of reactive species from cis-diammine-(1,1-cyclobutanedicarboxylato)-platinum(II) (carboplatin) in the presence of oxygen free radicals.

Experiments were designed to investigate the influence of oxygen free radicals on the rate of conversion of the anticancer drug cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) to reactive species able to bind to DNA. A system containing the Fe-EDTA chelate and ascorbate was used to generate free radicals. The rate of drug conversion to by-products, during incubation in chloride-free phosphate buffer at 37 degrees, was determined by HPLC analysis and found to be approximately 10 times faster in the presence of the free radical generating system, compared to CBDCA alone. The hydroxyl radical scavenger, mannitol, was able to reduce the rate of CBDCA conversion significantly, while an enhancing effect was observed in the presence of superoxide dismutase. The platinum containing species, which are formed in the presence of free radicals, were demonstrated to react with isolated salmon sperm DNA. The rate of platinum binding to DNA during incubation of CBDCA in the presence of the Fe-EDTA/ascorbate system was markedly enhanced. No effect on platinum binding to DNA during incubation with cis-diamminedichloroplatinum(II) (CDDP) in the same experimental conditions was observed, thus excluding an increased susceptibility of DNA itself to binding of platinum, due to DNA damage induced by free radicals. These findings support the hypothesis that the increased conversion of CBDCA, previously observed in our laboratory, which occurs in the presence of hemoglobin could be mediated by a Fenton-like reaction resulting in oxygen free radical production, thus providing potential clues to improvements in the clinical use of this drug.

Animals↗
PubMed1993

Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins.

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.

Adenine Nucleotides↗
PubMed1993

Production and hydrolysis of cyclic ADP-ribose at the outer surface of human erythrocytes.

Hemoglobin-free membranes from human erythrocytes are able to convert beta-NAD+ to cyclic ADP-ribose, a calcium mobilizer as potent as inositol 1,4,5-trisphosphate. Identification of cyclic ADP-ribose was based on HPLC analyses and its Ca(2+)-mobilizing activity on sea urchin egg microsomes. Erythrocyte membranes also hydrolyze cyclic ADP-ribose to ADP-ribose. By comparing the cyclic ADP-ribose-synthesizing and -hydrolyzing activities on unsealed and right-side-out resealed ghosts, it can be concluded that both are localized at the extracellular side of the membrane. This is confirmed by the demonstration of both enzyme activities on the surface of intact human red cells. Identification of the two enzymes involved in cyclic ADP-ribose metabolism might suggest some physiological role of this nucleotide in red cells.

ADP-ribosyl Cyclase↗
PubMed1993

Adenosine diphosphate ribulose in human erythrocytes: a new metabolite with membrane binding properties.

Incubation of ADPribose with yeast phosphoriboisomerase resulted in the formation of an adenylic nucleotide that was identified with ADPribulose by mass spectrometry. Synthesis of [32P]ADPribulose from [32P]NAD+ by the combined activities of commercial NAD+ glycohydrolase and phosphoriboisomerase allowed us to use it as a labeled internal standard throughout the procedure of purification from trichloroacetic acid extracts of human red blood cells. ADPribulose was purified by means of three sequential reverse phase HPLC separations and its concentration in human erythrocytes was estimated to be 0.11 +/- 0.1 microM. Unsealed erythrocyte ghosts did not transform ADPribulose, which bound to specific membrane proteins with a trichloroacetic and formic acid-resistant binding. The labeled proteins were identified as spectrin, bands 3, 4.1, 4.2 and Glyceraldehyde 3-phosphate dehydrogenase on the basis of their relative mobilities on SDS-PAGE.

Adenosine Diphosphate Ribose↗
PubMed1992

Presence and turnover of adenosine diphosphate ribose in human erythrocytes.

ADP-ribose was detected in human red blood cells (RBC) at 0.45 +/- 0.1 microM concentrations. These levels could be estimated after purification of ADP-ribose by means of three sequential HPLC fractionations of RBC extracts. Extraction was performed by sonication of RBC either in trichloroacetic acid, followed by centrifugation, or in carbonate-bicarbonate buffer, pH 10.0, followed by rapid ultrafiltration. Neither procedure of extraction caused artefactual formation of ADP-ribose. Prolonged incubation of intact RBC in isotonic buffer containing labeled orthophosphate resulted in the slow incorporation of radioactivity into ADP-ribose. Identification of the labeled ADP-ribose was confirmed upon incubation of the purified metabolite with nucleotide pyrophosphatase, yielding radioactive 5'-AMP and ribose 5-phosphate, while its exposure to a nonspecific deaminase resulted in the quantitative formation of labeled inosine diphosphate ribose.

Adenosine Diphosphate↗
PubMed1992

Enhanced intracellular activation of carboplatin in hemoglobin-synthesizing versus undifferentiated erythroleukemic cells.

The formation of DNA interstrand cross-links following exposure of target cells to the antineoplastic drugs Cisplatin and Carboplatin was measured in murine erythroleukemic cells either native or induced to differentiate and to synthesize hemoglobin by treatment with hexamethylene bisacetamide. The uptake of both drugs was identical in the uninduced and the induced cells, thus excluding differences in permeability due to differentiation. Both cell types formed comparable levels of DNA interstrand cross-links when submitted to treatment with Cisplatin. Conversely, significant differences were observed after exposure to Carboplatin. Specifically, the induced, hemoglobin containing cells showed significantly enhanced intracellular activation of the drug and subsequent binding to DNA, compared to the uninduced cells. These data suggest that, in agreement with previous results obtained with erythrocytes, the hemoglobin present in the committed cells enhances the aquation process that mediates the activation of Carboplatin.

Acetamides↗
PubMed1992

Mechanisms of perturbation of erythrocyte calcium homeostasis in favism.

Favism is an acute hemolytic anemia triggered by ingestion of fava beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0-12.9 mumol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 +/- 2.03 mumol Pi/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.

Calcimycin↗