Search PubMed⌕ Search

Biomedical subjects

G Fassina

Publications and source records attributed to G Fassina.

At least 109 records · Page 6Linked to original sources
PubMed1987

Antagonism between (-)-N6-phenylisopropyladenosine and the calcium channel facilitator Bay K 8644, on guinea-pig isolated atria.

Antagonism between (-)-N6-phenylisopropyladenosine (PIA) and the dihydropyridine calcium channel facilitator Bay K 8644 was investigated in guinea-pig spontaneously beating or electrically driven isolated atria, taken from normal and from reserpine-treated animals. PIA (3-100 nM) produced a dose-dependent decrease in contractile tension and frequency in spontaneously beating atria being more effective in reserpinized preparations. Bay K 8644 (5-200 nM) produced an increase in contractile tension in both normal and reserpinized atria. In electrically driven left atria the positive inotropic effect of Bay K 8644 was similar to that in spontaneously beating preparations. The positive chronotropic effect of Bay K 8644 was slight and variable. PIA produced a rightward parallel shift of the concentration-response curves for the positive inotropic effects of Bay K 8644 in all experimental conditions. In spontaneously beating atria from normal guinea-pigs, the Schild regression plot was linear and its slope near to unity; pA2 of PIA 8.63 +/- 0.05 (IC50 2.35 +/- 0.25 nM). In electrically driven atria the antagonism by PIA of the effects of Bay K 8644 was apparently competitive, and the IC50 of PIA was 18.6 +/- 0.4 nM. PIA antagonized the positive chronotropic effect of Bay K 8644 in spontaneously beating preparations, both from normal and from reserpine-treated animals. Carbachol did not modify the positive inotropic effects of Bay K 8644. These data indicate that PIA may interact with Bay K 8644 at the level of the slow calcium channels, and may decrease the transmembrane calcium flux into the cell.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Correlation between sites of limited proteolysis and segmental mobility in thermolysin.

Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes.

Amino Acid Sequence↗
PubMed1986

Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex.

Incubation of the neutral metalloendopeptidase thermolysin at pH 7.2 in the presence of EDTA and/or low concentrations of calcium ions produces fast enzyme inactivation as a result of autolysis. The 'nicked' protein is a folded species composed of three tightly associated protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by amino acid analysis after acid hydrolysis, end-group determination and partial amino acid sequencing. The results of these analyses indicated that the nicked protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the polypeptide chain loop involved in the binding of Ca(4) in native thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked thermolysin are quite similar to those of the intact protein, as judged by spectroscopic measurements and by the fact that rabbit antibodies against native thermolysin recognize and precipitate the nicked protein in immunodiffusion assays. The nicked protein was much less stable to heat and unfolding agents than intact thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native thermolysin by the four bound calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against autolysis.

Amino Acids↗
PubMed1986

Quantitative high-performance affinity chromatography: evaluation of use for analyzing peptide and protein interactions.

To examine analytical high-performance affinity chromatography as a microscale method for characterizing macromolecular interactions, the chromatographic behavior was evaluated of Arg8-vasopressin on bovine neurophysin II covalently immobilized in its monomer form on several new high-flow and pressure-resisting affinity supports. Zonal elution of both tritiated and unlabeled peptide hormone and an extension of theoretical treatment of analytical affinity chromatography allowed determination of equilibrium dissociation constants of hormone binding to immobilized bovine neurophysin II. Microamounts of hormone, ranging from 0.05 to 15 micrograms, were eluted within 20-30 min, with a quantitative recovery of the amount injected. For zones containing more than 5 micrograms, continuous elution monitoring was possible by ultraviolet absorbance, providing greater speed and accuracy in data analysis. The values obtained for the equilibrium dissociation constants were in good agreement with those previously measured in solution. The above hormone-protein evaluation system has led to identification of several pressure-resistant affinity supports, including silica-, agarose- and glass-based matrices, which are appropriate for use with high-performance liquid chromatographic instrumentation for affinity chromatographic analysis of macromolecular interactions.

Animals↗
PubMed1985

A caffeine analogue (1,3,7-trimethyl-6-thioxo-2-oxopurine) with a negative inotropic and chronotropic effect.

Cardiac effects of thio-xanthine derivatives, S-caffeine and S-theophylline, were studied on isolated guinea-pig atria and on partially purified cardiac cAMP phosphodiesterase enzymes. Theophylline and caffeine were taken as reference compounds. On electrically driven left atria S-caffeine (0.01-1 mmol/l) decreased contractile tension in a concentration dependent manner. On spontaneously beating atria, the same concentrations of S-caffeine showed negative inotropic as well as negative chronotropic effects. On electrically driven left atria, S-theophylline (0.01-1 mmol/l) increased heart contractile tension but, at higher concentrations, a reversal of the stimulating effect was observed. Both S-caffeine and S-theophylline inhibited bovine heart cAMP phosphodiesterase activity to a comparable extent. Their inhibitory potencies were about three and nine times higher than those of theophylline or caffeine but consistently lower than that of IBMX. The results show that the replacement of O with S in the methylxanthine molecule drastically modifies the effects induced by the drugs on cardiac function without changing those on cAMP phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Assay of phenacetin genotoxicity using in vitro and in vivo test systems.

Phenacetin was assayed in a battery of five short-term tests. (1) In a DNA-repair test using various Escherichia coli strains, the drug was not directly genotoxic nor did it induce nonreparable DNA damage in the presence of rat liver S9 fractions, while it was weakly active following activation with hamster liver S9. (2) In the Ames reversion test (strains TA97, TA98, TA100, and TA102 of Salmonella typhimurium, phenacetin reverted only TA100, and only in the presence of hamster liver S9. Mutagenicity was related to the concentration both of the drug and of the above metabolic system. There was no activation with hamster kidney S9, uninduced chicken liver S9, or with a variety of liver S9 preparations from rats treated with enzyme inducers (Aroclor 1254, phenobarbital, or 3-methylcholanthrene) and/or glutathione depletors (diethyl maleate or buthionine sulfoximine). Hamster liver S9 compared favorably to rat and even more to chicken liver S9 fractions also in activating various promutagens [3-amino-1-methyl-SH-pyrido (4,3-b)-indole, 2-aminofluorene, aflatoxin B1, benzo[a]pyrene, and benzo[a]pyrene-trans-7,8-diol] and in decreasing the mutagenicity of direct-acting compounds (4-nitroquinoline N-oxide and sodium dichromate). (3) Phenacetin was borderline positive in a forward mutation test (6-thioguanine resistance) in V79 cells, only in the presence of hamster liver S9, and gave negative results in the presence of rat liver S9 or without any metabolic system. (4) Following in vivo treatment, the alkaline elution assay did not reveal any DNA fragmentation in bone-marrow cells of ip-treated mice or in liver cells of rats treated by gavage. Apparent DNA damage was instead observed in the kidneys of rats receiving the drug by gavage or in the liver following ip administration. However, the effect was prevented (liver) or reduced (kidney) by preliminary perfusion of the organs, which discards (liver) or makes uncertain (kidney) the hypothesis of a true in vivo DNA damage. (5) Phenacetin ip induced in mouse bone-marrow cells a poor yet statistically significant increase in sister chromatid exchanges.

Animals↗
PubMed1984

Prostacyclin and lipolysis in rat fat cells.

In rat fat cells incubated for 15 min at 37 degrees and pH 8.5, glycerol release was highly stimulated both by norepinephrine and by theophylline. Prostacyclin (PGI2) (10(-8)-10(-7)M) did not alter the basal rate of glycerol release but potentiated the lipolytic effect of 2 X 10(-6)M norepinephrine. The rate of norepinephrine-induced glycerol release was increased by PGI2 during 10 min of incubation and then maintained for the next 5 min. Lipolysis induced by concentrations of norepinephrine which produced maximal effects was not altered by PGI2. PGI2 (10(-7)-10(-6)M) also potentiated the effect of 5 X 10(-4)M theophylline on glycerol release, but antagonized the stimulation induced by a maximally effective concentration of the methylxanthine (2 X 10(-3)M). Incubation of the cells with norepinephrine in the presence of 2 X 10(-4) or 5 X 10(-4)M theophylline caused a loss of the potentiating effect of PGI2 on norepinephrine-induced lipolysis. In the presence of 10(-3)M theophylline, the lipolytic action of norepinephrine was inhibited by PGI2. In fat cells incubated with adenosine deaminase (0.5 U/ml), 2.5 X 10(-7)M PGI2 did not alter the response to 5 X 10(-4)M theophylline and inhibited the effect of norepinephrine both in the absence and in the presence of theophylline. The present results show that, under appropriate experimental conditions, PGI2 may act as a lipolytic agent in isolated fat cells and that some kind of interaction exists between stimulation of methylxanthine-sensitive adenosine receptors and stimulation of PGI2 receptors.

Adipose Tissue↗
PubMed1984

Dual effect of (-)-N6-phenylisopropyl adenosine on guinea-pig trachea.

The effect of (-)-N6-phenylisopropyl adenosine (PIA), a metabolically stable P1-receptor agonist, was investigated on guinea-pig isolated trachea. PIA showed two opposite effects: contraction, evident at low concentrations (10(-7) to 2-5 X 10(-6) M), and relaxation at higher doses. Relaxation by PIA was antagonized in an apparently competitive manner by two antagonists of extracellular (P1) adenosine receptors: theophylline (Theo) and 8-phenyltheophylline (PT). Contraction by PIA was not inhibited by methylxanthines and was not mediated by stimulation of cholinergic or histaminergic systems. Inhibitors of arachidonic acid cascade acting at different levels, i.e. indomethacin, nordihydroguaiaretic acid (NDGA) and BW755C, all inhibited the contraction by PIA, while they potentiated the relaxation in a concentration-dependent manner. Mepacrine, an inhibitor of phospholipase A2, inhibited the contraction by PIA, but did not affect the relaxation. These results indicate that the contractile effect induced by PIA is supported by an indirect mechanism involving the release of arachidonic acid derivatives (via P2-purinoceptor?). Thus the balance between the two opposite effects of adenosine on tracheal tone is possibly modulated by the prostaglandin turnover.

Adenosine↗
PubMed1983

Age-related changes in rat adipose tissue in response to fasting: protein, lactate and pyruvate levels.

1. The evolution with age of the metabolic response of adipose tissue to fasting has been investigated in two groups of rats, at different ages, fed and fasted. 2. The protein tissue content increases in response to fasting in young rats but not in adult-old ones. This indicates a lower lipomobilizing response to starvation with increasing age. 3. In contrast to young rats, the adult rat lactate is not increased by fasting while pyruvate is increased. 4. As a result of lactate and pyruvate variations, NAD/NADH is also changed: after fasting it decreases in young rats, while it increases in older rats. 5. Absolute NAD level is decreased by fasting both in young and older rats. 6. These data provide experimental support for the decreased sensitivity of fat pads from older animals to stimuli affecting lipolysis.

Adipose Tissue↗
PubMed1983

Loss of hepatic monooxygenase activities, glutathione, and 'green pigment' formation after the administration of vinyl-cyclooctane to mice.

Vinylcyclooctane, when administered to mice at 500 mg/kg, produced reduction of microsomal cytochrome P-450, heme, aminopyrine-N-demethylase and ethoxycoumarin-O-deethylase activities with respect to control values; furthermore the hepatic reduced glutathione level was depleted suggesting that glutathione is involved in the vinylcyclooctane metabolism. The reduction of cytochrome P-450 and monooxygenase activities was accompanied by the formation of abnormal 'green pigments'.

Animals↗
PubMed1983

Positive inotropic effect of a stable analogue of PGI2 and of PGI2 on isolated guinea pig atria. Mechanism of action.

Fluoroprostacyclin (10,10-difluoro-13,14-dehydroprostacyclin) a stable analogue of PGI2, induced an increase of contractile tension and a positive chronotropic effect on spontaneously beating guinea-pig atria. The increase in contractile tension was proportional to the dose (10(-8) to 2.5 X 10(-7) M) and never exceeded 40% of absolute initial controls. The chronotropic positive effect was low (12 +/- 4%) and not dose-dependent. The positive inotropic effect was also evident on electrically driven atria and was greater at the slower rates of stimulation. Atria from guinea pigs pretreated with reserpine were still sensitive to fluoroprostacyclin, although to a lower degree. The use of appropriate agonists and antagonists excluded the possibility that beta and alpha receptors are involved in the positive inotropic effect of fluoroprostacyclin. PGI2 showed the same behavior as fluoro-PGI2. Prostacyclins were able to reverse the decrease of contractility induced by verapamil, nifedipine, tetrodotoxin and xylocaine. But the reversal by PGI2 was no longer evident when the atrial contractility was completely abolished by the presence of these drugs in high concentrations. The inotropic effect of prostacyclins was more evident when Ca2+ was reduced in the medium. These results demonstrate that the cardiostimulant effect of prostacyclins is not simply adrenergic in origin. A direct effect on ion movements, mainly dependent on Ca2+ transmembranes fluxes, may be largely responsible for their positive inotropic effect.

Adrenergic alpha-Agonists↗
PubMed1983

New derivatives of methyl-xanthines: effect of thiocaffeine thiotheophylline and 8-phenyltheophylline on lipolysis and on phosphodiesterase activities.

The effects of some old and new methylxanthines and 6-thioxanthines, i.e. theophylline (TH), caffeine (CAFF), thiotheophyl line (S-TH), thiocaffeine (S-CAFF), 8-phenyltheophylline (8-PT), 3-isobutyl-1-methyl-xanthine (IBMX) were compared at the level of adipose tissue on spontaneous and norepinephrine-induced lipolysis as well as on fat cell phosphodiesterases. These agents stimulated lipolysis. 8-PT was the most potent; thiocaffeine and thiotheophylline the least potent; IBMX and theophylline had intermediate potencies. The order of potency of the same drugs in potentiating norepinephrine-stimulated lipolysis was: IBMX greater than 8-PT greater than S-CAFF greater than greater than S-TH greater than CAFF greater than TH. The rank order of potency to inhibit cAMP phosphodiesterases was: IBMX greater than S-TH and S-CAFF greater than TH much greater than 8-PT (uneffective). Thus (a) thiocaffeine and thiotheophylline were more potent than the parent compound theophylline in inhibiting cAMP-PDE, although their per se stimulating effect on lipolysis was much lower. This indicates that in the thioxanthines the stimulus per se on lipolysis can be dissociated from their effectiveness as inhibitors of PDE. (b) In contrast, 8-PT (a blocker of adenosine receptors) has a potent lipolytic action per se and a potentiating effect on norepinephrine-induced lipolysis, even if deprived of effect on PDE. This indicates that the potentiating power of methylxanthines on norepinephrine-stimulated lipolysis is not strictly dependent on their anti-PDE activities. These results suggest that in adipose tissue (more similar than adipocytes to the in vivo conditions) the anti-adenosine potency of methylxanthines is the prominent factor in stimulating basal lipolysis. Both anti-adenosine and anti-PDE activities are involved in modulating hormone-induced lipolysis.

Adipose Tissue↗