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Biomedical subjects

R Franco

Publications and source records attributed to R Franco.

At least 91 records · Page 5Linked to original sources
PubMed1997

Dipropylcyclopentylxanthine triggers apoptosis in Jurkat T cells by a receptor-independent mechanism.

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a xanthine analog used as selective antagonist of adenosine receptors, caused apoptosis in a human leukemia T cell line. Jurkat cells treated with DPCPX underwent apoptosis as demonstrated by flow cytometry, by DNA fragmentation and by accumulation of histones, H2A, H2B, H3 and H4, in the nucleoplasm of cells. Cell cycle and cell sorting analyses indicated an arrest of cells in G(2)/M followed by the appearance of apoptotic cells in G(1) and G(2)/M phases. The mechanism of programmed cell death does not seem to be mediated by signal transduction events at the plasma membrane since it did not involve activation of cell membrane receptors and modification of the intracellular levels of Ca(2+) or cAMP. Apoptosis by incorporation into DNA of a derivative of DPCPX is suggested in basis of the presence of radioactivity label in the DNA obtained from cells preincubated with [(3)H]DPCPX.

Journal Article↗
PubMed1997

Modulation of GH4 cell cycle via A1 adenosine receptors.

Identification and characterization of A1 adenosine receptors (A1Rs) in a tumor cell line derived from rat pituitary (GH4 cells) was performed by ligand binding and immunocytochemistry. Subsequently, the involvement of A1Rs in the regulation of cell proliferation was studied in these cells. The agonist N6-(R)-phenylisopropyladenosine (R-PIA) did not modify the number of cultured cells, but it regulated the kinetics of the cell cycle. By means of experiments of pulse and of pulse and chase with bromodeoxyuridine and further labeling with Hoechst 33258, propidium iodide, and/or fluorescein-conjugated antibodies against bromodeoxyuridine, it was demonstrated that R-PIA, via A1Rs, accelerated progression from G0/G1 to S phase and from S to G2/M phase of the cell cycle, whereas the initiation of a new cycle occurred at the same time in treated and untreated cells. As a consequence, R-PIA did not change the total length of the cycle. This is the first description of cell cycle regulation without modification of cell proliferation. Although pertussis toxin blocked the R-PIA-induced inhibition of cyclic AMP production in these cells, it did not affect the R-PIA action on the cell cycle. In contrast, cholera toxin mimicked the action of R-PIA. Thus, it is likely that regulation of the cell cycle via A1Rs is mediated by heterotrimeric G proteins different from those that mediate inhibition of adenylate cyclase. Due to the fact that cells in G0/G1 phase were less susceptible to secretory signals, adenosine, in an autocrine manner and by regulating the cell cycle kinetics, may contribute to the modulation of the secretory capacity of pituitary cells.

Adenosine↗
PubMed1997

Regulation of L-type calcium channels in GH4 cells via A1 adenosine receptors.

Identification of A1 adenosine receptors (A1Rs) in a tumor cell line derived from rat pituitary (GH4 cells) was performed by ligand binding and immunological experiments. Subsequently, the involvement of A1Rs in the regulation of calcium conductance was studied in these cells. The agonist N6-(R)-(2-phenylisopropyl)adenosine (R-PIA) did not modify the intracellular calcium basal levels, whereas it inhibited the increase produced by 15 mM KCl depolarization. The antagonist 1,3-dipropyl-8-cyclopentylxanthine led to the opening of voltage-dependent cell surface calcium channels in the absence of exogenous KCl. The channels were of the L type because the effect was abolished by calciseptine and by verapamil. These results suggest that endogenous adenosine exerts a tonic inhibitory effect on calcium transport. This was confirmed by the high adenosine concentration found in cell supernatants (up to 1 microM) and by the calcium mobilization produced by exogenously added adenosine deaminase. In depolarizing conditions, the calcium peak in the presence of adenosine deaminase was reduced when cells were preincubated with R-PIA, thus suggesting that A1R activation regulates the intensity of depolarization. These results demonstrate that adenosine is an important regulator of the physiological state of pituitary tumor cells by modulating, in an autocrine manner, the activity of L-type voltage-dependent calcium channels.

Adenosine↗
PubMed1997

Ligand-induced phosphorylation, clustering, and desensitization of A1 adenosine receptors.

Through immunocytochemistry with the use of antibodies against A1 adenosine receptors (A1Rs) and confocal microscopy, we show that stimulation of A1Rs by the agonist (R)-phenylisopropyladenosine [(R)-PIA] caused a rapid (5-15 min) aggregation (clustering) of receptor molecules on the surface of DDT1MF-2 cells. Internalization of the chronically stimulated receptor was slower and occurred concomitantly, with a time-dependent decrease (50%) in the number of cell surface [3H](R)-PIA binding sites. The reduction of binding sites was due partly (30%) to internalization and partly (20%) to the presence of desensitized cell surface receptor molecules that were unable to bind the ligand. Chronic exposure of DDT1MF-2 cells to 50 nM (R)-PIA produced functional desensitization, as deduced from second messenger production assays. Quantification of the content of A1Rs by immunoblotting and flow cytometry in cells pretreated with 50 nM (R)-PIA indicates a time-dependent slow down-regulation of the receptor. Receptor clustering and agonist-induced receptor phosphorylation, which occurred in serine and tyrosine, were simultaneous. The finding that activators of protein kinase A or C were able to induce functional desensitization of A1Rs, phosphorylate A1Rs in serine and threonine, and trigger clustering of the receptor suggests that phosphorylation of A1Rs in serine/threonine is involved in desensitization-related events.

Adenosine↗
PubMed1997

Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells.

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.

Animals↗
PubMed1996

Characterization of representative enzymes from a sulfate reducing bacterium implicated in the corrosion of steel.

This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed.

Corrosion↗
PubMed1996

The cluster-arranged cooperative model: a model that accounts for the kinetics of binding to A1 adenosine receptors.

To explain the equilibrium binding and binding kinetics of ligands to membrane receptors, a number of models have been proposed, none of which is able to adequately describe the experimental findings, in particular the apparent negative cooperativity of ligand binding. In this paper, a new model, the cluster-arranged cooperative model, is presented whose main characteristic is that it explains the existence of negative cooperativity in the binding of ligands to the receptor molecule. The model is based on our findings of agonist binding to A1 adenosine receptors and of ligand-induced clustering of these receptors on the cell surface. The model assumes the existence of two conformational forms of the receptor in an equilibrium which depends on the concentration of the ligand. In this way, negative cooperativity is explained by the transmission of the information between receptor molecules through the structure of the membrane. The model is able to predict the thermodynamic binding and binding kinetics of [3H]-(R)-(phenylisopropyl)adenosine to A1 adenosine receptors in the presence and absence of guanylyl imidodiphosphate. In the presence of the guanine nucleotide analogue, the linear Scatchard plots obtained for [3H]-(R)-(phenylisopropyl)adenosine binding are explained by the disappearance of cooperativity, thus suggesting that G proteins are important for the existence of negative cooperativity in ligand binding. Among other predictions, the model justifies early events in homologous desensitization since high ligand concentrations would lead to the saturation of the receptor in a low-affinity conformation that does not signal. Our model can likely explain the behavior of a number of heptaspanning and tyrosine-kinase receptors exhibiting complex binding kinetics.

Animals↗
PubMed1996

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors.

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A1 adenosine receptor (A1R) is a second ecto-ADA binding protein. Binding of ADA to A1R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A1R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A1R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A1R.

Adenosine Deaminase↗
PubMed1996

Adenosine deaminase interacts with A1 adenosine receptors in pig brain cortical membranes.

Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A1 adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high-affinity site of the receptor, which corresponds to the receptor-G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A1 adenosine receptors to heterotrimeric G proteins.

Adenosine Deaminase↗
PubMed1995

Immunological identification of A1 adenosine receptors in brain cortex.

The A1 adenosine receptor from pig brain cortex has been identified by means of two antipeptide antibodies against two domains of the receptor molecule: PC/10 antiserum was raised against a part of the third intracellular loop, and PC/20 antiserum was raised against a part of the second extracellular loop. PC/10 antibody was able to recognize a 39-kDa band that corresponded to the A1 receptor, as demonstrated by immunoblotting and by immunoprecipitation of the molecule cross-linked to [125I](R)-2-azido-N2-p-hydroxy(phenylisopropyl)adenosine. Besides the 39-kDa band, PC/20 also recognized a 74-kDa form that does not seem to correspond to a receptor-G protein complex. The occurrence of the two bands was detected and analyzed in samples from different species and tissues showing a heterogeneous distribution of both. The 74-kDa form can be converted into the 39-kDa form by treatment with agonists or antagonists of A1 adenosine receptors. These results suggest that A1 adenosine receptor can occur in dimers and that the dimer-monomer conversion might be regulated by adenosine as the physiological ligand. Since the 74-kDa aggregates were not recognized by PC/10, it is likely that part of the third intracellular loop participates in the protein-protein interaction.

Animals↗
PubMed1995

Expression of ecto-adenosine deaminase and CD26 in human T cells triggered by the TCR-CD3 complex. Possible role of adenosine deaminase as costimulatory molecule.

The expression of surface adenosine deaminase (ADA) and CD26 in activated human T cells was studied by flow cytometry. PBLs and CD3+ or CD4+ cells, when subjected to a variety of stimuli (anti-CD3 Abs plus IL-2 or phorbol esters), presented two structurally different cell populations, which differed in size and cellular complexity (populations B1 and B2). In PBLs triggered by an anti-CD3 mAb there was no significant increase of expression of either surface ADA or CD26 in cells of population B1, whose structure is similar to that of nonstimulated cells. In contrast, there was a significant increase in the percentage of expression of ADA and CD26 in the population B2, which corresponds to structurally more complex and larger cells. In the case of activation via TCR-CD3 but in the presence of IL-2 or via phorbol esters, the increase was found in cells from both populations, but B2 cells always showed a higher percentage of expression than B1 cells. The results of increased expression of surface ADA and CD26 were similar in whole T cells or in purer preparations such as CD3+ or CD4+ lymphocytes. Polyclonal Abs against ADA were not able to induce an activation response in T cells even when cross-linked by a secondary Ab. Interestingly, these Abs produced anergy in CD4+ cells subjected to an anti-CD3 stimulus. In contrast, addition of ADA produced an enzyme-independent synergism in the response through the TCR-CD3 complex. In T cells, ADA and CD26 colocalized on the surface of T cells; thus, the effect of exogenous ADA seems to be mediated by CD26 molecules that are not interacting with endogenous ADA (spare CD26 molecules). The presence of spare CD26 molecules on the surface of CD4+ cells was demonstrated by flow cytometry in the presence of exogenous ADA and also by confocal microscopy. The set of results strongly indicates that ADA binding to CD26 produces a costimulatory response in T cell activation events.

Adenosine Deaminase↗
PubMed1995

Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods.

All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mössbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mössbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mössbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are delta = 1.36 +/- 0.03 mm/s and delta EQ = 3.04 +/- 0.06 mm/s, distinct from the larger delta EQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed.

Animals↗
PubMed1995

A model of the pentose phosphate pathway in rat liver cells.

A mathematical model based on kinetic data taken from the literature is presented for the pentose phosphate pathway in fasted rat liver steady-state. Since the oxidative and non oxidative pentose phosphate pathway can act independently, the complete (oxidative+non oxidative) and the non oxidative pentose pathway were stimulated. Sensitivity analyses are reported which show that the fluxes are mainly regulated by D-glucose-6-phosphate dehydrogenase (for the oxidative pathway) and by transketolase (for the non oxidative pathway). The most influent metabolites were the group ATP, ADP, P1 and the group NADPH, NADP+ (for the non oxidative pathway).

Animals↗
PubMed1995

Structure and function of ferrochelatase.

Ferrochelatase is the terminal enzyme of the heme biosynthetic pathway in all cells. It catalyzes the insertion of ferrous iron into protoporphyrin IX, yielding heme. In eukaryotic cells, ferrochelatase is a mitochondrial inner membrane-associated protein with the active site facing the matrix. Decreased values of ferrochelatase activity in all tissues are a characteristic of patients with protoporphyria. Point-mutations in the ferrochelatase gene have been recently found to be associated with certain cases of erythropoietic protoporphyria. During the past four years, there have been considerable advances in different aspects related to structure and function of ferrochelatase. Genomic and cDNA clones for bacteria, yeast, barley, mouse, and human ferrochelatase have been isolated and sequenced. Functional expression of yeast ferrochelatase in yeast strains deficient in this enzyme, and expression in Escherichia coli and in baculovirus-infected insect cells of different ferrochelatase cDNAs have been accomplished. A recently identified (2Fe-2S) cluster appears to be a structural feature shared among mammalian ferrochelatases. Finally, functional studies of ferrochelatase site-directed mutants, in which key amino acids were replaced with residues identified in some cases of protoporphyria, will be summarized in the context of protein structure.

Amino Acid Sequence↗
PubMed1995

Surface adenosine deaminase. A novel B-cell marker in chronic lymphocytic leukemia.

Previous studies found that ADA is present on the surface of mononuclear blood cells from healthy patients. Because the expression of this surface antigen depends upon the cell type, the presence of ADA on the plasma membrane of cells from patients with malignant hematologic diseases was studied by flow cytometry. The highest percentage of expression was found in CLL, whereas the lowest was found in T-cell-derived malignancies. The enzyme expression in immortalized cell lines showed a similar pattern, with the highest expression (95% +/- 5%) in the SKW64 B-derived cell line, the lowest (15% +/- 5%) in Jurkat T-lymphoma derived cells, and the intermediate (32% +/- 8%) in K562 cells derived from a chronic myelogenous leukemia. Double labeling ADA/CD5 and ADA/CD19, as well as the correlation of ADA expression with the expression of other surface markers, indicate that surface ADA might be considered a novel marker for CLL.

Adenosine Deaminase↗
PubMed1995

An ankyrin-related gene (unc-44) is necessary for proper axonal guidance in Caenorhabditis elegans.

Caenorhabditis elegans unc-44 mutations result in aberrant axon guidance and fasciculation with inappropriate partners. The unc-44 gene was cloned by transposon tagging, and verified by genetic and molecular analyses of six transposon-induced alleles and their revertants. Nucleotide sequence analyses demonstrated that unc-44 encodes a series of putative ankyrin-related proteins, including AO49 ankyrin (1815 aa, 198.8 kD), AO66 ankyrin (1867 aa, 204 kD), and AO13 ankyrin (< or = 4700 aa, < or = 517 kD). In addition to the major set of approximately 6 kb alternatively spliced transcripts, minor transcripts were observed at approximately 3, 5, 7, and 14 kb. Evidence is provided that mutations in the approximately 14-kb AO13 ankyrin transcript are responsible for the neuronal defects. These molecular studies provide the first evidence that ankyrin-related molecules are required for axonal guidance.

Amino Acid Sequence↗