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R Franco

Publications and source records attributed to R Franco.

At least 19 recordsLinked to original sources
PubMed1992

A model for adenosine transport and metabolism.

1. A model is presented for adenosine transport and metabolism in different steady states. The model considers steady-state equations for metabolic enzymes based on information from the literature on their kinetic behaviour. 2. Assuming that extracellular adenosine and inosine are translocated by three transporters, we have devised rate equations for these nucleoside transporters which are valid when both nucleosides are present. Since the Na(+)-independent transporter can either incorporate nucleosides into the cell or release them, various conditions have been simulated in which inosine was either incorporated or released. 3. Control analyses are reported which show that the fluxes towards intracellular adenine nucleosides are controlled by ecto-5'-nucleotidase in some circumstances and by the nucleoside transporters in others. The nucleoside transporter is responsible for five fluxes (two Na+ dependent adenosine transport mechanisms, a Na(+)-dependent inosine transport, a Na(+)-independent adenosine transport and a Na(+)-independent inosine influx or efflux) but the control is not always positive for all these fluxes. The control patterns of these five fluxes indicate that, in the presence of extracellular adenosine and inosine, the intracellular metabolism of adenine derivatives would be highly dependent on the extracellular and intracellular concentrations of both nucleosides, on the ectoenzymes (5'-nucleotidase and adenosine deaminase) and on the transporter. 4. Predictions of the model were examined. The results indicate that a change in one independent variable (extracellular AMP concentration) makes the system evolve towards a new steady state which is far from the initial one and has a different control pattern. In contrast, simulation of inhibition of the carriers produces only slight modification of the fluxes since the concentrations of the metabolites change to counteract the effect. Thus, for instance, a 50% inhibition of the three carriers does not affect the flux towards intracellular adenine nucleotides. Finally, our model has confirmed that the evolution of the concentration of extracellular adenosine, when an increase in extracellular AMP is produced, agrees with the behaviour expected for a neurohormone.

Adenosine↗
PubMed1992

The nickel site in active Desulfovibrio baculatus [NiFeSe] hydrogenase is diamagnetic. Multifield saturation magnetization measurement of the spin state of Ni(II).

The magnetic properties of the nickel(II) site in active Desulfovibrio baculatus (DSM 1743) [NiFeSe] hydrogenase have been measured using the multifield saturation magnetization technique. The periplasmic [NiFeSe] hydrogenase was isolated from bacteria grown in excess selenium in the presence of 57Fe. Saturation magnetization data were collected at three fixed fields (1.375, 2.75, 5.5 tesla) over the temperature range from 2 to 100 K. Mössbauer and EPR spectroscopies were used to characterize the magnetic state of the two [4Fe-4S] clusters of the enzyme and to quantitate the small amounts of iron impurities present in the sample. The nickel(II) site was found to be diamagnetic (low spin, S = 0). In combination with recent results from extended x-ray absorption fine structure studies, this magnetic state indicates that the nickel(II) site of active D. baculatus [NiFeSe] hydrogenase is five-coordinate.

Desulfovibrio↗
PubMed1992

Optimal association-saturation procedure for estimating association and dissociation rate parameters in receptor studies. Application to solubilized A1 adenosine receptors.

A method of obtaining estimates of the maximum binding and association and dissociation rate constants for a receptor-ligand interaction is described. This new procedure, the association-saturation method, is based on an exact mathematical equation which defines the model without simplifications. The method proposed is readily applicable to any system consisting of one ligand and one receptor. With only four determinations, each involving one ligand concentration and an association time, it is possible to determine the number of specific binding sites, the dissociation and association rate constants and the equilibrium dissociation constant with accuracy. No dissociation curve is required, and only one point of the association curve for each ligand concentration is necessary. In addition to the higher confidence in the estimates of the parameter values obtained, this method leads to important savings in radiolabelled compounds and experimental time. The results were compared with those obtained with standard association-dissociation curves and saturation isotherms. The optimum number of replicates of each experimental point to obtain reliable estimates of different parameters is discussed on the basis of simulation studies. The performance of the procedure is analysed by means of association-saturation experiments with the agonist [adenine-2,8-3H,ethyl-2-3H]N6-phenylisopropyladenosine and solubilized A1 adenosine receptors from pig brain cortex.

Animals↗
PubMed1992

Modulation of adenosine agonist [3H]N6-(R)-phenylisopropyladenosine binding to pig brain cortical membranes by changes of membrane fluidity and of medium physicochemical characteristics.

The binding of [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) to pig brain cortical membranes was investigated as a function of membrane fluidity and of the physicochemical characteristics of the medium. The two affinity states of the A1 adenosine receptor behave differently in the experiments performed. Increases (up to 6.7 poises) or reduction (up to 1.3 poises) in membrane microviscosity did not lead to significant variation of equilibrium parameter values except for a slight modification of the low-affinity Kd value at 1.3 poises. Addition of cholesterol to raise the microviscosity up to 9.0 poises led to a marked decrease of binding to the high-affinity state without modifying the Kd. Furthermore, the Kd value for the low-affinity state rose markedly. Addition of sucrose, which modifies the conductance, viscosity and density of the aqueous medium, did not lead to significant changes when used at a concentration of 0.25 M, either in isolation or in radioligand binding assay medium. The presence of 0.32 M sucrose in the isolation medium led to a 30% fall in the total binding without affecting the distribution and Kd values for either affinity state very much. However, the presence of greater than or equal to 0.32 M sucrose in the binding assay medium produced the disappearance of the low-affinity states and the appearance of high-affinity states; only one affinity state was found to have a somewhat increased Kd value. On the other hand, increases of the medium conductance did not lead to the disappearance of the low-affinity state although some decrease of the binding was observed at high Tris concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Studies with biotinylated RBC: (1) use of flow cytometry to determine posttransfusion survival and (2) isolation using streptavidin conjugated magnetic beads.

Methods are reported for the quantitation and isolation of biotinylated red blood cells (B- RBC). The first method is for determination of posttransfusional survival of rabbit RBC by flow cytometry. The survival of B-RBC was measured using both fresh and paraformaldehyde-fixed cells with similar results. The posttransfusion survival of rabbit RBC measured in this way was normal. There was no indication of increased cell destruction due to antibodies directed against B-RBC and no evidence for loss of biotin from circulating cells. The second methodology is for the isolation of B-RBC from blood with streptavidin-coated magnetic beads. At least eighty percent of positive cells were recovered with very few false positives. Both methods may be helpful in the study of resealed erythrocytes.

Animals↗
PubMed1992

Sickle cells modified by an osmotic pulse in the presence of inositol hexaphosphate have decreased intracellular hemoglobin concentration and decreased in vitro sickling without prolonged in vivo survival.

An osmotic pulse was used to modify red blood cells (RBC) from two patients with sickle cell disease, resulting in an increased volume and decreased hemoglobin content. This treatment yielded cells which were divided into two populations, one in which RBC had markedly decreased hemoglobin concentration and another in which the cells appeared to be unmodified. Morphological sickling at low oxygen partial pressure was markedly decreased. However, there was no evidence for increased RBC lifespan when the 51Cr-labeled, modified cells were reinfused.

Anemia, Sickle Cell↗
PubMed1992

The distribution of A1 adenosine receptor and 5'-nucleotidase in pig brain cortex subcellular fractions.

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [3H]R-PIA binding, the activity of adenosine metabolizing enzymes (5'-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A1 adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A1 adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding parameters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5'-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.

5'-Nucleotidase↗
PubMed1992

Theoretical study about the variability of the genome of foot-and-mouth disease viruses.

1. The possibilities of change in amino acids of a protein are discussed in terms of a point mutation. 2. Whereas Met and Trp are forced to change due to a point mutation, other amino acids (Ala, Arg, Gly, Leu, Pro and Val) have a probability of 1/3 to survive in the sequence. 3. On basis of these considerations, the genome from 5 strains (CSP, C3Ind, O1K, A10 and A12) of the foot-and-mouth disease virus was studied. 4. A hypothetical genealogic tree for these strains is suggested, where CSP and C3Ind are close and also A10 and A12. O1K is closer to A10 and A12 than to CSP or C3Ind.

Aphthovirus↗
PubMed1992

Interaction of trans-acting factors with the proximal promoter of the mouse alpha-fetoprotein gene.

1. The alpha-Fetoprotein (AFP) gene is expressed during fetal life, but not in adult cells. Also, the AFP gene is expressed in most hepatomas. 2. Using gel retardation (band-shift) assays under very stringent conditions we have compared the binding of trans-acting factors to the proximal enhancer (-202, +34) region of the AFP gene. 3. We have detected the presence of two retarded bands in experiments performed with adult rat hepatocytes and the Fa32 cell line (which does not produce AFP) but only one band is observed with the HepG2 cell line (which produces AFP) and fetal liver. 4. We relate the two retarded bands to a glucocorticoid response element and, tentatively, to the C/EBP trans-acting fractor.

Animals↗
PubMed1992

The adenosine receptors present on the plasma membrane of chromaffin cells are of the A2b subtype.

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.

2-Chloroadenosine↗
PubMed1992

Characterization of adenosine receptors in brush-border membranes from pig kidney.

1. The adenosine receptors from pig kidney proximal tubules have been studied in membrane vesicle preparations derived from either luminal (brush-border membranes-BBM-) or basolateral (BL) sides. There was a substantial amount of A2-like NECA binding in both preparations, but the A1 subtype of adenosine receptors was not found in either BBM or BL membranes. The use of [3H]-CGS21680 which is a more specific ligand for A2a receptors revealed true adenosine receptors in the BBM. 2. The kinetic parameters for [3H]-CGS21680 binding to pig renal BBM were: Bmax = 1.48 pmol mg-1 protein and Kd = 150 nM. In the presence of Gpp(NH)p the affinity decreased (Kd = 220 nM), whereas the addition of Mg2+ induced a marked increase in affinity (Kd = 83 nM). These equilibrium constants are higher than those found for the A2a adenosine receptors present in pig brain striatal membranes (Kd = 12 nM), and are close to those found in rat renal BBM (Kd = 90 nM). 3. The order of potency of agonist and antagonists was not consistent with the presence of either A1 or A2 receptors, but it was very similar to the agonist order of potency for the A3 receptor subtype. Furthermore, the blockade of the [3H]-CGS21680 binding by both cholera and pertussis toxin further supports the view that the subtypes present in BBM are neither A1 nor A2. 4. Overall the results suggest the presence in BBM of an A3 receptor, or of a new subtype of adenosine receptor, which is linked to G proteins sensitive to both cholera and pertussis toxins.

Adenosine↗
PubMed1991

N-ethylmaleimide affects agonist binding to A1 adenosine receptors differently in the presence than in the absence of ligand.

The effect of sulphydryl reagents (N-ethylmaleimide-NEM-4- hydroxymercuriobenzoate-HMB- and 5-5'-dithio-bis-2-nitrobenzoate-DTNB-) on agonist and antagonist binding to A1 adenosine receptors from pig brain was studied. The action of the mercurial agent HMB was found to be strong and seemed to be nonspecific. The effects of either NEM or DTNB were milder and more specific. The characterization of the agonist binding in membranes pretreated with moderate concentrations of DTNB and NEM led to reduced affinities for both high- and low-affinity sites without marked modifications of maximal binding or of proportion of affinity states. These results for NEM are surprising since the compound is usually used to mimick the effects of Gpp(NH)p, i.e. to shift high-affinity states to low-affinity states. It was found that this Gpp(NH)p-like effect of NEM is only possible when the compound is included in the assay medium. Similarly, Gpp(NH)p produces the uncoupling of the receptor molecule from G protein if included in the assay medium. Thus, membranes pretreated with Gpp(NH)p exhibited both affinity states and with similar equilibrium binding parameter values to those of the crude membranes.

Adenine↗
PubMed1991

The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state.

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.

Animals↗
PubMed1991

Biochemical systems theory: increasing predictive power by using second-order derivatives measurements.

Models based on the power-law formalism provide a useful tool for analyzing metabolic systems. Within this methodology, the S-system variant furnishes the best strategy. In this paper we explore an extension of this formalism by considering second-order derivative terms of the Taylor series which the power-law is based upon. Results show that the S-system equations which include second-order Taylor coefficients give better accuracy in predicting the response of the system to a perturbation. Hence, models based on this new approach could provide a useful tool for quantitative purposes if one is able to measure the required derivatives experimentally. In particular we show the utility of this approach when it comes to discriminating between two mechanisms that are equivalent in the S-system a representation based on first-order coefficients. However, the loss of analytical tractability is a serious disadvantage for using this approach as a general tool for studying metabolic systems.

Kinetics↗
PubMed1991

Fitting integrated enzyme rate equations to progress curves with the use of a weighting matrix.

A method is presented for fitting the pairs of values product formed-time taken from progress curves to the integrated rate equation. The procedure is applied to the estimation of the kinetic parameters of the adenosine deaminase system. Simulation studies demonstrate the capabilities of this strategy. A copy of the FORTRAN77 program used can be obtained from the authors by request.

Adenosine Deaminase↗
PubMed1991

Control analysis of transition times. Extension of analysis and matrix method.

The present theoretical basis of Control Analysis is extended with the definition of Transition Time Response Coefficients. Some new relationships between local and global coefficients defined in Control Analysis are presented. These relationships are in the form of matrix products constructed in a priori form. The use of these straightforward relationships is shown in an exemplary application corresponding to an experimental system consisting of the glycolytic degradation from glucose to glyceraldehyde-3-phosphate.

Animals↗
PubMed1991

Effect of phospholipases and proteases on the [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) binding to A1 adenosine receptors from pig cerebral cortex.

The effect of phospholipases and proteases on the membrane-bound and solubilized A1 adenosine receptor has been studied. Phospholipids modulate the [3H]N6-(R)-phenylisopropyladenosine binding to A1 adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that 1) there is co-solubilization of receptor and phospholipids; 2) the phospholipid requirements are different for the coupled and the uncoupled receptor; 3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and 4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the Kd value of the high-affinity state by modifying both k1 and k-1. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A1 adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered.

Animals↗