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Biomedical subjects

R Franco

Publications and source records attributed to R Franco.

At least 109 records · Page 6Linked to original sources
PubMed1995

Nicotine gum, 2 and 4 mg, for nicotine dependence. A double-blind placebo-controlled trial within a behavior modification support program.

The effectiveness of nicotine gum in combination with a behavior modification program was studied. The nicotine dependence of participating smokers (N = 322) was assessed. One hundred sixty-eight smokers were labeled as high nicotine dependent and 154 as moderate to low dependent. In a randomized double-blind procedure, the high-dependent smokers were given gum containing 4 mg of nicotine (87) or 2 mg of nicotine (81) and the smokers with medium or low dependence were given gum containing 2 mg (76) or a placebo gum (78). The smokers were also randomized to familiarizing themselves with the medication a week before quit day (112) or to regular use, that is starting gum use on the quit day (122). In the high-dependent group, sustained and chemically verified nonsmoking rates at 6 weeks, 1 year, and 2 years were, respectively, 60%, 39%, and 34% in the subjects given the 4-mg dose compared with 41%, 16%, and 16% for those using the 2-mg dose. In the group with medium or low dependence, the success rates at the same time periods were 70%, 49%, and 39% for the subjects given the 2-mg dose and 38%, 22%, and 17% for those given placebo gum. The differences in success rates were significant at least at the p < 0.02% level for all comparisons. Familiarizing with the gum as compared with regular use gave fewer reports of side effects, 15% vs 34%, p < 0.001. A trend toward better success rates at 6 weeks, although not statistically significant, was observed for the familiarization group, 61% vs 52%. The study shows that high nicotine-dependent smokers need higher doses of nicotine replacement, in this case the 4-mg dose rather than the 2-mg dose, whereas 2 mg is superior to placebo among less dependent smokers. These results compare favorably with those reported from the more recent nicotine patch therapy.

Adult↗
PubMed1994

Solubilization and molecular characterization of the nitrobenzylthioinosine binding sites from pig kidney brush-border membranes.

The nitrobenzylthioinosine binding sites from luminal membranes of proximal tubule of pig kidney were solubilized by treatment of the brush-border membrane vesicles with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) in 2% solution. The high yield solubilization of a stable form of the transporter took place in the presence of adenosine in the medium of incubation with the detergent and the additional presence of glycerol as stabilizer. The solubilization of the NBTI-sensitive nucleoside transporter from pig kidney brush-border membranes did not change the nitrobenzylthioinosine (NBTI) binding characteristics; the only major change was a 3-fold decrease in the affinity. The carrier molecule was cross-linked to [3H]NBTI and by electrophoretic characterization under reducing conditions it displayed a molecular mass of 65 kDa. Treatment of the samples at low temperature prior to electrophoresis gave rise to the appearance of further bands corresponding to dimeric and tetrameric forms which interacted non-covalently. The removal of the N-linked oligosaccharides by treatment with endoglycosidase F shifted the molecular mass to 57 kDa. The chromatographic behaviour of the solubilized transporter was similar to that of human erythrocytes and differed from that found in pig erythrocytes. Since the molecular mass of the monomer before and after treatment with endoglycosidase F is the same for pig erythrocytes and pig kidney luminal membranes, the different chromatographic behaviour might result from tissue differences due to transcriptional variations or to posttranscriptional modifications of the transporter molecule.

Affinity Labels↗
PubMed1994

Mammalian ferrochelatase, a new addition to the metalloenzyme family.

A [2Fe-2S] cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy. In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of [2Fe-2S]+ cluster. Mössbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in 57Fe-enriched medium, demonstrated unambiguously that the cluster is a [2Fe-2S] cluster. No change in the cluster oxidation state was observed during catalysis. The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the [2Fe-2S] center in regulation of mammalian ferrochelatases.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase.

Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney.

Adenine Nucleotides↗
PubMed1994

A1 adenosine receptors can occur manifesting two kinetic components of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX) binding.

The results described in this paper show, for the first time, that A1 adenosine receptors can have two kinetic components for the binding of the antagonist [3H]DPCPX. At low ionic strength (< or = 42 mmol/l), dissociation of [3H]DPCPX bound to A1 receptors fitted better to a two kinetic components model than to a one kinetic component model. The kinetic constants were consistent with comparable Kd values for the two components of the antagonist binding, and therefore these two components cannot be distinguished by saturation isotherm analysis.

Animals↗
PubMed1994

Characterization of the GTP/GDP binding site in the murine CD3-zeta polypeptide chain.

Using a newly developed in situ affinity-labeling method of nucleotide-binding proteins (NTPoxi technique) we discovered that the human T-cell receptor-associated CD3-zeta protein might bind GTP/GDP. To further characterize GTP/GDP binding to CD3-zeta, murine T-cell lines expressing zeta zeta homodimers or CD3-zeta/Fc epsilon R1 gamma heterodimers were used. Specific GTPoxi labeling of CD3-zeta was found in all murine T cells in which a complete CD3-zeta polypeptide chain was expressed, including cells in which CD3-zeta was disulfide bridged to the Fc epsilon R1 gamma chain. In murine T cells the kinetics of labeling of CD3-zeta was similar to that of small G-proteins. Upon activation of murine T cells a slight but significant increase in GTPoxi labeling of CD3-zeta was detected. Whether all 3 so-called 'Reth motifs' (zeta A, zeta B and/or zeta C) were necessary for the binding of GTP/GDP was addressed by using cells expressing truncated CD3-zeta molecules. Whereas truncated CD3-zeta, in which zeta A and part of zeta B were deleted, was still able to bind GTP, upon deletion of all 3 Reth motifs cross-linking by the GTPoxi method became impossible. Regardless of whether this implies a direct or indirect binding of GTP/GDP to CD3-zeta, these nucleotides and their hydrolysis must play an important role in T-cell activation through the TCR/CD3 complex.

Amino Acid Sequence↗
PubMed1994

Regulation of nitrobenzylthioninosine-sensitive adenosine uptake by cultured kidney cells.

The effect of nitrobenzylthioinosine (NBTI) on [3H]adenosine uptake and the characterization of the [3H]NBTI binding in cell (primary cultures and LLC-PK1 cell line) plasma membrane and brush-border membrane (BBM) vesicles from pig renal cortices and LLC-PK1 cells was analyzed. [3H]adenosine uptake was strongly inhibited by NBTI in nonconfluent cells, whereas it was totally insensitive to the reagent in BBM. The concentration dependence of [3H]adenosine uptake in BBM was linear, suggesting simple diffusion. In both cell membranes and BBM high-affinity [3H]NBTI binding was observed. [3H]NBTI binding as well as NBTI-sensitive [3H]adenosine uptake was strongly reduced when cells grew to confluence. Both reduction effects were reproduced by treatment of nonconfluent cells with chlorophenyl adenosine 3',5'-cyclic monophosphate (cAMP), which indicates that the transporter is regulated by a cAMP-dependent protein kinase. To confirm this hypothesis, the binding of [3H]NBTI was analyzed in pig kidney BBM obtained in the presence of orthovanadate and alkaline phosphatase. With respect to control membranes, BBM obtained in the presence of orthovanadate showed a lower maximum number of binding sites (Bmax), whereas those obtained in the presence of alkaline phosphatase showed a slight increase in Bmax for [3H]NBTI binding. Taken together, these results suggest that the reduction in both [3H]NBTI-binding capacity and NBTI-sensitive [3H]adenosine uptake takes place by a mechanism that involves phosphorylation of the transporter molecule or of a protein that interacts with it.

Adenosine↗
PubMed1994

Pressor mechanisms in adriamycin-induced nephropathy with hypertension in rats.

We explored the role of angiotensin II and vasopressin in the maintenance of blood pressure during the nephrotic syndrome of adriamycin-induced nephropathy in rats. All 91 rats treated with adriamycin developed chronic renal failure with nephrotic syndrome, which was more pronounced in the normotensive rats than the 35% who became hypertensive. Angiotensin II blockade with DuP 753 produced a significantly greater hypotensive response in both the adriamycin-hypertensive (-16 +/- 3 mm Hg) and adriamycin-normotensive (-14 +/- 5 mm Hg) groups than the saline-treated controls (-5 +/- 1 mm Hg, P < .05). Vasopressin blockade with either a V1V2 inhibitor or a selective V1 inhibitor produced a hypotensive response in adriamycin-hypertensive rats only (by -16 +/- 4 and -17 +/- 2 mm Hg, respectively, P < .01), although the nonselective vasopressin inhibitor produced similar fluid loss and body weight reduction in all three groups. The data suggest that in adriamycin-induced nephropathy with nephrotic syndrome, angiotensin II contributes to blood pressure maintenance in both hypertensive and normotensive animals, whereas the pressor action of vasopressin contributes to elevated blood pressure in hypertensive animals only.

Angiotensin II↗
PubMed1993

Voltammetric studies of the catalytic electron-transfer process between the Desulfovibrio gigas hydrogenase and small proteins isolated from the same genus.

The kinetics of electron transfer between the Desulfovibrio gigas hydrogenase and several electron-transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, square-wave voltammetry and chronoamperometry. The cytochrome c3 from Desulfovibrio vulgaris (Hildenborough), Desulfovibrio desulfuricans (Norway 4), Desulfovibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron-transfer proteins such as ferredoxin I, ferredoxin II and rubredoxin from D. gigas were tentatively used as redox carriers. Only ferredoxin II was effective in mediating electron exchange between hydrogenase and the working electrode. The second-order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mechanism [Moreno, C., Costa, C., Moura, I., Le Gall, J., Liu, M. Y., Payne, W. J., van Dijk, C. & Moura, J. J. G. (1993) Eur. J. Biochem. 212, 79-86] and the results obtained by cyclic voltammetry were compared with those obtained by chronoamperometry. Values for k of 10(5)-10(6) M-1 s-1 (cytochrome c3 as electron carrier) and 10(4) M-1 s-1 (ferredoxin II as the electron carrier) were determined. The rate-constant values are discussed in terms of the existence of an electrostatic interaction between the electrode surface and the redox carrier and between the redox carrier and a positively charged part of the enzyme.

Catalysis↗
PubMed1993

Characterization of D. desulfuricans (ATCC 27774) [NiFe] hydrogenase EPR and redox properties of the native and the dihydrogen reacted states.

Redox intermediates of D. desulfuricans ATCC 27774 [NiFe] hydrogenase were generated under dihydrogen. Detailed redox titrations, coupled to EPR measurements, give access to the mid-point redox potentials of the iron-sulfur centers and of the Nickel-B signal that represents the ready form of the enzyme. The interaction between the dihydrogen molecule and the nickel centre was probed by the observation of an isotopic effect on the EPR signals detected in turnover conditions, by comparison of the H2O/H2 and D2O/D2-reacted samples.

Binding Sites↗
PubMed1993

Coupling of GTP-binding to the T cell receptor (TCR) zeta-chain with TCR-mediated signal transduction.

The zeta-subunit of the TCR binds GTP and is a well characterized substrate for a TCR-activated tyrosine kinase. To examine the possible coupling of GTP-binding to zeta with TCR-mediated signal transduction, a mutant (termed J32-3.2) of the T cell line Jurkat (J32) was used. Anti-TCR/CD3 stimulation of the TCR/CD3+ J32-3.2 cells resulted in a weak stimulation of both the phosphatidyl inositol and tyrosine kinase signal transduction pathways, as measured by changes in the level of free intracellular calcium, tyrosine phosphorylation of TCR-zeta, CD3-epsilon and ZAP-70, p56lck, or p59fyn tyrosine kinase activity and IL-2 gene activation. The impaired responsiveness of J32-3.2 cells to anti-TCR/CD3 mAb correlated with a low basal level of GTP-binding to zeta. Furthermore, in J32-3.2 cells TCR activation by antibody ligation caused a weaker increase in GTP-binding to the zeta-chain, as compared with that of wild-type J32 cells, which indicates for the first time that GTP-binding to zeta can be modulated by extracellular signals and suggest that the role of GTP-binding to zeta is to couple the TCR to intracellular signal transduction mechanisms.

Amino Acid Sequence↗
PubMed1993

The T cell receptor associated CD3-epsilon protein is phosphorylated upon T cell activation in the two tyrosine residues of a conserved signal transduction motif.

Signal transduction through the T cell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3-zeta chain. Since both CD3-epsilon and the zeta chain contain a tyrosine-based signaling motif, we examine phosphorylation of CD3-epsilon in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3-epsilon in vivo. Induction of CD3-epsilon phosphorylation followed similar kinetics to that of the zeta chain phosphorylation. In contrast to zeta, CD3-epsilon phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide-specific Western blotting established that CD3-epsilon phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C-terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3-epsilon by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.

CD3 Complex↗
PubMed1993

Adenine nucleotides and adenosine metabolism in pig kidney proximal tubule membranes.

Exogenous adenosine triphosphate (ATP) added to brush-border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto-nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto-adenosine deaminase and ecto-adenosine monophosphate (AMP) nucleotidase was shown. The ecto-adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush-border membrane vesicles. Using orthovanadate, levamisole, and alpha, beta-methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto-AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto-AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine.

Adenine Nucleotides↗
PubMed1993

Surface expression of adenosine deaminase in mitogen-stimulated lymphocytes.

Adenosine deaminase (ADA) expression on the surface of mitogen-stimulated lymphocytes was studied by flow cytometry. The gate for lymphocytes was located by cell size (forward scatter), cytoplasmic complexity (side scatter) and by expression of the markers CD2, CD4, CD8 and CD19. After mitogenic proliferation two populations appeared, one corresponding to non-stimulated cells, and the other consisting of larger cells which showed relatively high expression of adenosine deaminase on their surface. The increase was similar to that observed for CD71 expression, and paralleled the increase in 3H-thymidine incorporation. There was a correlation between ADA and CD71 expression (r = 0.92 for phytohaemagglutinin (PHA) and 0.97 for pokeweed mitogen (PWM)). These results suggest a role for ecto-adenosine deaminase in lymphocyte proliferation and/or triggering.

5'-Nucleotidase↗
PubMed1993

Role of histidine residues in agonist and antagonist binding sites of A1 adenosine receptor.

The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N6-[adenine-2,8-3H, ethyl-2-3H]phenylisopropyladenosine ([3H]R-PIA) and antagonist 8-cyclopentyl-1,3-[3H]-dipropylxanthine ([3H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [3H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [3H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A1 adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (KD) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

[Bladder fibroma. Report of a case].

Analysis of one case of benign vesical primitive connective tumour, with pathoanatomical description of vesical fibroma. The interest of this case rests on its rarity and uncertain origin, still subject to controversy. The paper presents the tumour's clinical history, pathological anatomy, therapeutical approach and prognosis.

Female↗
PubMed1993

Thermodynamic analysis of agonist and antagonist binding to membrane-bound and solubilized A1 adenosine receptors.

The thermodynamic properties of the agonist [Adenine-2,8-3H, ethyl-2(3)-H]-N6-phenylisopropyladenosine ([3H]R-PIA) and the antagonist 8-Cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX) binding to membrane-bound and 3-3[-(choloamidopropyl)-dimethylammoniol-1-propanesulfonate/ digitonin- solubilized A1 adenosine receptors from pig brain cortex were evaluated. Rate constants for [3H]R-PIA and [3H]DPCPX association (k+1) and dissociation (k-1) processes to this receptor subtype were measured from association-dissociation experiments at six different temperatures. The values for equilibrium association constant (KA = 1/KD) were derived from rate constant values (k+1/k-1). The antagonist binding to membrane-bound receptors, the agonist binding to fast kinetic component membrane-bound receptors and the agonist binding to soluble receptors showed a linear temperature-dependence of the standard free-energy change. The first two processes are enthalpy- and entropy-driven, and the third process is enthalpy-driven with entropy working against it. On the other hand, a curvilinear temperature-dependence appears in the agonist binding to slow kinetic component membrane-bound receptors and in the antagonist binding to soluble receptors, but analyzing the semireactions (association-dissociation) involved in each case reveals that the thermodynamic behavior is very different. The thermodynamic similarities and differences are discussed in terms of receptor--G protein interaction.

Animals↗