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Biomedical subjects

R Franco

Publications and source records attributed to R Franco.

At least 163 records · Page 9Linked to original sources
PubMed1990

Solubilization of A1 adenosine receptor from pig brain: characterization and evidence of the role of the cell membrane on the coexistence of high- and low-affinity states.

The present solubilization strategy recognizes the important role of detergent cocktails in the solubilization and subsequent stability of adenosine A1, receptors from pig brain cortical membranes. The 3-[3-(cholamidopropyl)dimethylammonio]-1-propane-sulfonate-digitonin mixture produced the extraction of up to 52% of the receptor with an enrichment of 1.2-fold with respect to crude membranes. The binding activity of the soluble extract was very stable even in the absence of glycerol. In crude membranes the existence of high- and low-affinity states was detected, but in the soluble extract and in the detergent-treated membranes only the high-affinity state was detected. Association-dissociation curves showed that in crude membranes no interconversion between high- and low-affinity sites is produced by the association of the ligand [3H]R-N6-phenylisopropyladenosine. These results suggest that the high- and low-affinity states are different conformations induced by the structure of the membrane. The modulation of the binding activity by (Gpp(NH)p) 5'-guanylylimidodiphosphate and Mg2+ was studied. In crude membranes Gpp(NH)p shifted the high-affinity state to the low-affinity state, whereas the contrary occurred when Mg2+ was used. The effect of both Mg2+ and Gpp(NH)p was also assayed with the soluble extract and with the detergent-treated membranes. In addition to a decrease of the overall binding capacity, Gpp(NH)p promoted a conversion to all low-affinity states in the detergent-treated membranes or to all very-low-affinity sites in the soluble extract. Mg2+ and Gpp(NH)p counteracted their effects in intact membranes, whereas Mg2+ could not reverse the uncoupling effect of Gpp(NH)p with solubilized or detergent-treated membranes. Thus, it is suggested that Mg2+ acts at sites other than guanine-nucleotide-sensitive sites. If high-affinity states correspond to receptor/G protein complexes and low-affinity states correspond to the uncoupled receptor, we should conclude that Mg2+, as well as the loss of membrane integrity, favours the interaction of A1 receptor molecule with G protein.

Animals↗
PubMed1990

Quantum chemical study of the electronic and conformational characteristics of adenosine and 8-substituted derivatives: functional implications in the mechanism of reaction of adenosine deaminase.

A quantum chemical study of 10 substrates of adenosine deaminase is performed. The conformational preference around the glycosidic bond of several 8-substituted derivatives of adenosine is studied using semiempirical modified neglect of diatomic overlap (MNDO) and Austin model 1 (AM1) methods. All the compounds studied show preference for the anti conformation; the syn - anti energetic differences calculated are small and in excellent agreement with experimental data. A relationship between the ab initio molecular electrostatic potential minimum energy of N3 and the syn - anti energetic difference is found. A highly significant relationship is also found between the ab initio net charge over the purine and pyrimidine rings and the logarithm of the maximum rate of deamination (log Vm) of the nucleosides by adenosine deaminase. In contrast, no significant relationship is found between the anti preference of 8-substituted derivatives of adenosine and their log Vm of deamination.

Adenosine↗
PubMed1990

A method for binding parameters estimation of A1 adenosine receptor subtype: a practical approach.

Working with pig brain striatum in which A1 and A2 adenosine receptor subtypes coexist, we describe an uncomplicated method for unequivocally obtaining the equilibrium parameters (KD and binding capacity) of A1 receptor without interference from ligand binding to A2 receptor. Also, the equilibrium parameter estimation method we propose avoids the experimental determination of nonspecific binding by the inclusion of the corresponding unknown parameter in the function. This not only saves time but also avoids the use of expensive radioligands in saturation experiments. The method is suitable for any system with two different receptor subtypes for the same physiological ligand, and good estimates of the equilibrium parameters corresponding to the subtype displaying the higher affinity for the ligand can be obtained.

Adenosine↗
PubMed1990

Is adenosine deaminase involved in adenosine transport?

The hypothesis that adenosine metabolizing enzymes may have a key role in the transport of adenosine is discussed. The enhancement of adenosine transport by inhibitors of adenosine deaminase (the enzyme which deaminates adenosine to inosine) and the ecto-localization of adenosine deaminase suggest a contribution of the enzyme in taking up nucleosides. Two possible mechanisms are suggested: 1) transport and deamination of adenosine as a coupled process, or 2) uptake of inosine after cleavage of adenosine by ecto-adenosine deaminase. In both cases, the so-called adenosine deaminase binding protein which is a membrane protein could be the real nucleoside transporter. This behaviour of adenosine deaminase as an ectoenzyme anchored to a membrane protein remembers the behaviour of periplasmic binding proteins of bacteria. Thus, adenosine deaminase as well as, for instance, adenosine kinase would be a kind of 'periplasmic proteins' of eukaryotic cells. The function of adenosine deaminase and adenosine kinase would then be to take adenosine and give it to the true transporters.

Adenosine↗
PubMed1990

Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography.

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.

Adenosine Deaminase↗
PubMed1990

Effects of a vasopressin antagonist with combined antipressor and antiantidiuretic activities in rats with left ventricular dysfunction.

These experiments assessed the hemodynamic and aquaretic effects of an arginine vasopressin (AVP) antagonist with dual V1V2-receptor inhibiting properties in rats with congestive heart failure resulting from ischemic cardiomyopathy. The compound d(CH2)5-D-Tyr(Et)VAVP was used in these studies. Rats with limited or extensive myocardial infarcts (i.e., with less than 50% or greater than 66% necrosis of the left ventricular wall, respectively, induced by left coronary ligation) and sham-operated controls received the AVP antagonist (100 micrograms/kg i.v.) 4 weeks later. This agent produced an 18% increase in cardiac output (p less than 0.05) and 13% decrease in systemic vascular resistance in the severely damaged rats, both changes being significantly different from those seen in the normal controls or the rats with limited infarcts. All animals exhibited increases in urinary output of 4-10-fold over baseline. We conclude that the hemodynamic and renal effects of this agent are beneficial in animals with left ventricular dysfunction.

Animals↗
PubMed1990

Expression of multiple alpha 2-adrenergic receptor messenger RNA species in rat tissues.

We used a human platelet alpha 2-adrenergic receptor probe to study the tissue distribution and messenger RNA (mRNA) forms of the rat alpha 2-adrenergic receptor. Under stringent conditions of hybridization and washing, we detected an mRNA species of 3.8 kb. The abundance of this form follows the order spleen, kidney, brain stem and cortex, and skeletal muscle and lung and is consistent with the reported abundance and tissue distribution of the alpha 2 receptor activity. A 3.0 kb mRNA form was also detected in cerebral cortex and brain stem and a 4.1 kb mRNA form was observed in kidney under less stringent hybridization conditions. The tissue distribution of the 3.0 kb form is different from that of alpha 1- and beta-adrenergic receptors and the D2 dopaminergic receptor. The mRNA analysis combined with Southern blot analysis of rat and human genomic DNA indicate that: 1) in addition to a 3.8 kb rat alpha 2-adrenergic receptor transcript, there are other mRNA forms in the rat that do not correspond to previously described adrenergic receptor mRNA species and 2) more than one alpha 2-adrenergic receptor gene in the rat is expressed in a tissue-specific manner.

Animals↗
PubMed1990

Association of adenosine deaminase with erythrocyte and platelet plasma membrane: an immunological study using light and electron microscopy.

Adenosine deaminase has been localized in the plasma membrane of erythrocytes and platelets by means of immunological techniques using light and electron microscopy with cells in suspension. In erythrocytes, adenosine deaminase is associated with the external side of the plasma membrane. In platelets, the enzyme is associated with the external side of the plasma membrane, which is known to extend through the canalicular system of these cells. These results confirm our previous findings, based on biochemical studies, concerning the attachment of the enzyme to cell membranes.

Adenosine Deaminase↗
PubMed1990

Slight differences between adenosine deaminases from different species an immunochemical study.

IgGs against adenosine deaminase from rat brain, rat liver, mouse duodenum and human erythrocyte were purified from rabbit antisera with yields of 82-87%. The inhibition of adenosine deaminase by the antienzyme is studied, and it is demonstrated that rat and mouse antibodies are tight-binding inhibitors. These antibodies inhibit either the rat or the mouse enzymes and do not inhibit the human erythrocytes enzyme. The human antibody does not inhibit either the human or the rat or mouse enzyme. These results indicate that some differences in antigenic behaviour near the active site must be encountered among species. Comparing the sequenced of the two products corresponding to two adenosine deaminase genes recently sequenced (human and murine) a hypothesis concerning the localization of the adenosine deaminase active site is proposed.

Adenosine Deaminase↗
PubMed1990

[Isotopic radioangiography in hepatic cirrhosis. Changes induced by somatostatin].

The value of first step isotopic angiography (FSIA) with 99-TC in the study of altered liver kidney and spleen circulation in patients with liver cirrhosis and alcoholic hepatitis is analyzed. This technique is used to evaluate the effect of a 250 mcg IV bolus of somatostatin on the activity/time curve at the three circulatory levels. The ratio of basal hepatic artery/portal flow in cirrhotics was 59 + 11/41 + 11% and 39 + 6/61 + 4 in alcoholic hepatitis, significantly different from normal controls (p less than 0.001). In cirrhotics the degree of alteration in FSIA correlated with the severity of liver disease. Somatostatin significantly improved the ratio of basel hepatic artery/portal flow and diminished the pressure at the root of suprahepatic veins (18.5 +/- - v.s. 16 +/- mmHg, p greater than 0.001). Neither the slopes nor the time of maximum isotopic activity of renal and splenic activity/time curves were modified. We concluded highlighting the clinical value of FSIA as a non invasive test in the study of chronic liver diseases.

Adult↗
PubMed1990

[Otitis externa maligna. Our experience].

The AA. expose the experience gained through a group of 9 cases of malignant external otitis. They review the previous circumstances, assess the results of medical and/or surgical treatment and the evolution of the processes, emphasizing the importance of prognostic factors. Twenty-two per cent was the rate mortality.

Aged↗
PubMed1989

Use of implicit methods from general sensitivity theory to develop a systematic approach to metabolic control. I. Unbranched pathways.

It is shown that metabolic control theory (MCT), is its present form, is a particular case of general sensitivity theory, which studies the effects of parameter variations on the behavior of dynamic systems. It has been shown that metabolic control theory is obtained from this more general theory for the particular case of steady-state and linear relationships between velocities and enzyme concentrations. In such conditions the relationships between elasticities and flux control coefficients are easily obtained. These relationships are in the form of a matrix product constructed in a priori form. Relationships between combined response coefficients and concentration control coefficients are presented. The use of implicit methodology from general sensitivity theory provides a generalization of MCT, which is applied to unbranched pathways. For this particular case, provided the matrices have been properly constructed, the matrix of global properties (flux and concentration control coefficients) can be obtained by inversion of the matrix of local properties (elasticities). The theorems of MCT (concentration summation, flux summation, flux connectivity, and concentration connectivity) applicable for unbranched pathways are directly obtained by inspection of the matrix product. With these results, the present theoretical basis of MCT is extended with a more structured framework that allows a wider range of application. The results make clearer the relatedness of MCT to the more general approach provided by biochemical systems theory (BST).

Kinetics↗
PubMed1989

Use of implicit methods from general sensitivity theory to develop a systematic approach to metabolic control. II. Complex systems.

In the accompanying paper (Cascante et al., this issue) we have used general sensitivity theory to develop a matrix algebra that, in the case of sequential reactions, directly relates global and local properties of a given system. In complex biochemical systems this direct relationship is not possible due to the existence of linear dependencies among fluxes and among metabolite concentrations (conserved aggregate concentrations in BST or moiety-conserved concentrations in MCT). In this paper our matrix algebra is applied to conserved cycles and branched pathways, and it is shown that with minor modifications it again relates global properties to the local properties of the enzymes in the system. In the case of conserved cycles, elasticities become modified due to the existence of linear dependencies among the concentration variables in the cycle. In branched pathways, new matrix elements involving ratios of fluxes appear. With these modifications, one can show that the so-called theorems of metabolic control theory specific to these types of pathways are special cases of more general relationships. Rules for the construction of matrices relating global and local properties are given that apply to an arbitrary system of cycles and branches. The implicit approach developed in these papers, which is a generalization of that used in MCT, allows one to make more direct comparisons with the general explicit approach originally developed in BST.

Kinetics↗
PubMed1989

Heterogeneity of the gradients performed by the freeze-thaw method.

Gradients produced by the freeze-thaw method were analyzed at various rates of freezing. The shape of the gradient depended on the rate of freezing. At high rates the pattern did not change but at low rates the steepness of the gradient increased with time. Solutes concentrated at the bottom in a fashion which depended on the density of the solvent and on the rate of freezing. It should also be noted that the gradient was not uniform over the entire surface as the concentration of solute increased near the wall of the test tube.

Antibodies↗
PubMed1989

Interdependence between cooperativity and control coefficients.

Influence of the concentration of internal metabolites on the control coefficient (defined as fractional change in flux per fractional change in enzyme activity) and regulatory properties of a given enzyme have been studied theoretically using a cyclic model of three enzymes. This model is useful to investigate the properties of the flux control coefficient for an enzyme following different rate equations. Enzymes can have high or low values of control coefficient irrespective of the type of kinetic equation, but the results obtained show that the sensitivity of these values to substrate variations is strongly dependent on its rate equation. These results help identify which kinetic equation allows the best control of a given metabolic pathway. These results have been applied to the purine nucleotide cycle. It is demonstrated that the best control of the cycle is reached when the irreversible reaction catalyzed by AMP deaminase follows a rate law that corresponds to a rational function of 2:2 degree with respect to AMP concentration.

AMP Deaminase↗
PubMed1989

TEFOOL/2: a program for theoretical drug design on microcomputers.

TEFOOL/2, a program written in BASIC, is presented in this paper. The purpose of TEFOOL/2 is to provide people interested in drug design with an easy-to-handle program where some of the most important techniques in QSAR are included. The program permits the selection of the training series, performs regression calculations and searches for optimum substituents. The latter is achieved by using either a Hansch's strategy or geometrical procedures. The program is interactive and can be implemented on an IBM-PC or compatible microcomputer. Although TEFOOL/2 has been developed for its application in drug design studies, its great flexibility makes it suitable for application to any experimental design or optimization process.

Algorithms↗