Nanoporous silica nanoparticles functionalized with a fluorescent turn-on spirorhodamineamide as pH indicators.

We prepared water soluble, biocompatible fluorescent turn-on pH nanosensors and characterized their behavior as a function of changes in pH. The response relies on a halochromic reaction of a spirorhodamineamide derived from the bright and highly chemically and photo-stable rhodamine 6G, encapsulated in core/nanoporous shell silica nanoparticles. The fluorescent sensors displayed a fast response in the pH range of intracellular compartments. The encapsulation conferred solubility in aqueous environments and biocompatibility. We assessed the two main properties of the sensor, namely the useful pH range and the kinetics of the response, and compared them to those of the free probe. We found that such properties are strongly dependent on the functionalization and position in the silica matrix relative to the core/shell structure. Finally, we demonstrated the cellular uptake of the nanosensors, and their localization in lysosomes of living cells, by fluorescence confocal microscopy.

Carboxylated zinc-phthalocyanines--II. Dimerization and singlet molecular oxygen sensitization in hexadecyltrimethylammonium bromide micelles.

The dimerization of the diamide of zinc-tetracarboxyphthalocyanine was studied spectroscopically in hexadecyltrimethylammonium bromide (CTAB) micelles at surfactant concentrations from 0.026 to 0.1 M and dye concentrations between 0.1 and 10 microM. The apparent dimerization constant in CTAB 0.1 M is 8.6 x 10(5) M-1, while the intramicellar dimerization constant is 1.8 x 10(3). The dimer absorption spectrum was also obtained. Singlet molecular oxygen sensitization was studied by steady state photolysis using 1,3-diphenylisobenzofurane as scavenger in 0.1 M CTAB. The usual sensitization mechanism is extended to include dimer reactions. Singlet molecular oxygen sensitization yields for monomer and dimer in the micelles are 0.7 and 0.1, respectively. With the reported values it is possible to calculate the average yield of singlet molecular oxygen production at any surfactant and dye concentrations.

Mechanisms underlying post-rest potentiation in isolated left rat atria.

In the isolated rat left atria the influence of rest intervals on the force developed by the first post rest beat (PRB) was studied. Under control conditions the force developed by the PRB increased (respect to previous steady state at 0.5 Hz) with the increase of the rest interval until 20 sec of pause and decreased with longer intervals. In the presence of caffeine (1 or 4 mM plus high [Ca]0) there was a monotonous fall of the PRB as a function of the rest interval. When extracellular calcium was replaced by Sr the tension developed by PRB vs. rest interval curve rose with a slope lower than the control one and reached the peak at 60 sec. At saturation levels of [Ca]0 the PRB tension development did not vary up to 20 sec pause but the decreasing phase observed after 20 sec of rest interval was still present. At 0.5 mM [Ca]0 the response was similar to control curve. The results in the presence of caffeine and strontium suggest that, in rat atria, the rest potentiation appears to be dependent on the release of calcium from intracellular stores (sarcoplasmic reticulum). This is consistent with the hypothesis proposing that longer resting periods provide a longer interval for the transfer of Ca from uptake to release sites in the sarcoplasmic reticulum.

Mechanisms underlying post-rest potentiation in isolated left rat atria.

In the isolated rat left atria the influence of rest intervals on the force developed by the first post rest beat (PRB) was studied. Under control conditions the force developed by the PRB increased (respect to previous steady state at 0.5 Hz) with the increase of the rest interval until 20 sec of pause and decreased with longer intervals. In the presence of caffeine (1 or 4 mM plus high [Ca]0) there was a monotonous fall of the PRB as a function of the rest interval. When extracellular calcium was replaced by Sr the tension developed by PRB vs. rest interval curve rose with a slope lower than the control one and reached the peak at 60 sec. At saturation levels of [Ca]0 the PRB tension development did not vary up to 20 sec pause but the decreasing phase observed after 20 sec of rest interval was still present. At 0.5 mM [Ca]0 the response was similar to control curve. The results in the presence of caffeine and strontium suggest that, in rat atria, the rest potentiation appears to be dependent on the release of calcium from intracellular stores (sarcoplasmic reticulum). This is consistent with the hypothesis proposing that longer resting periods provide a longer interval for the transfer of Ca from uptake to release sites in the sarcoplasmic reticulum.

[Extraintestinal isolation of Salmonella in chickens: epidemiologic study of 3 salmonellosis outbreaks after ingestion of raw eggs]. / Aislamiento extraintestinal de Salmonella en gallinas: estudio epidemiológico de dos brotes de salmonelosis por consumo de huevo crudo.

A taxonomic analysis of the malolactic microflora present in nine grape samples from different zones of Galicia, was carried out. Nineteen strains were isolated and identified as Lactobacillus plantarum (42%), L. brevis (10.6%), L. casei (5.25%), L. hilgardii (5.25%), Streptococcus cremoris (5.25%), Leuconostoc lactis (5.25%), L. oenos (10.6%), and Pediococcus acidilactici (15.8%).

Papaverine-induced positive inotropism with failure to increase cyclic AMP in rat atria.

Phosphodiesterase inhibition by papaverine is likely to play a minor role in the rat atrial inotropic response because non-significant changes in cyclic AMP were obtained. Isoprenaline however raised the nucleotide levels three-fold and up to seventeen-fold when papaverine was added in a similar preparation. A prominent effect of papaverine was to lengthen relaxation instead of shortening it as did isoprenaline. The results suggest different sites of action, although overlapping effects cannot be excluded.

Effects of papaverine on calcium efflux and contractility in superfused rat left atria.

The effects of papaverine upon force of contraction, maximal rate of contraction, maximal rate of relaxation and 45Ca efflux were studied in isolated superfused rat left atria electrically driven at 1 Hz. Papaverine (3 X 10(-5) mol/l, increased developed tension (from 5.35 +/- 1.17 mN to 7.18 +/- 1.51 mN) by 1.8 +/- 0.41 mN (+33%, p less than 0.01) and maximal rate of contraction (+T) by 34.5 +/- 13% (p less than 0.05). In all experimental conditions tested, papaverine increased the rate of 45Ca efflux. The amount by which papaverine increased 45Ca efflux was 175 +/- 41 nmoles . g wet wt-1 and 304 +/- 76 nmoles . g wet wt-1, in the presence and in the absence of caffeine, respectively. The plot of changes in 45Ca efflux versus changes in developed tension fitted to a straight line (r = +0.56, n = 17, p less than 0.05), with a slope and intercept of 42 nmoles Ca . mN-1 . g wet wt-1 and -0.47 mN respectively, suggesting an association between the changes induced by papaverine in force development and increased 45Ca efflux.

Simultaneous analyses of Ca exchange and mechanical parameters in the rat atria.

In the rat left atrium, electrically driven, a superfusion dropping method was developed. The device essentially consisted of a pair of platinum wire electrodes which held one end of the tissue, while the other end was attached to an isometric tension transducer. Either normal or isotopically labeled solutions were poured on the preparation at preset flow rates by means of a minipump. Effluent was collected fractionally in vials for measurement of radioactivity. Several perfusion flows tested led us to the conclusion that higher rates than 2.26 ml/min did not improve contractile performance. Stability of the preparation was found similar to the classical isolated organ-bath method during a 180 min period of observation. Set-up of the superfused atria reproduced paired determinations of 45Ca efflux, together with the contractile parameters. In addition, the frequency of sampling (intervals of 1 min or less) allowed us to detect changes in the washout pattern of exchangeable 45Ca even during early phases of Ca efflux. Therefore, the method reported herein seems to overcome the serious handicap of the scanty tissue employed.

Antagonism and supersensitivity to phenylephrine-induced chronotropic responses.

Right atria from rats were analyzed for chronotropic responses to phenylephrine in face of various drugs and procedures. Propranolol, 10(-8) M, produced a competitive antagonism against the agonist which concentration-effect curve was closely similar to that obtained from reserpinized animals. Prazosin, but not phentolamine (both 10(-6) M) showed inhibition of the phenylephrine-induced changes in heart rate, as judged by their -log EC50. Either of the alpha-adrenoceptor antagonists exhibited a greater steepness in the curve slope with respect to control. The simultaneous exposure of tissues to phentolamine and propranolol proved to effectively antagonize the chronotropic effect of the agonist. This held true for phentolamine assayed in atria from reserpine-pretreated rats. Previous incubation of tissues with papaverine, 10(-5) M, brought about supersensitivity to phenylephrine which was thoroughly inhibited by either phentolamine or propranolol. These results strongly suggest that beta-adrenoceptor stimulation of heart rate by phenylephrine takes place indirectly via norepinephrine release. There is also alpha 1-adrenoceptor stimulation (blocked by prazosin). Finally, it is hypothesized that supersensitivity develops by papaverine-enhanced Ca2+ influx, since numerous evidences are against a phosphodiesterase inhibition-dependent cAMP accumulation mechanism triggered by papaverine in the presence of phenylephrine.

Antagonism and supersensitivity to phenylephrine-induced chronotropic responses.

Right atria from rats were analyzed for chronotropic responses to phenylephrine in face of various drugs and procedures. Propranolol, 10(-8) M, produced a competitive antagonism against the agonist which concentration-effect curve was closely similar to that obtained from reserpinized animals. Prazosin, but not phentolamine (both 10(-6) M) showed inhibition of the phenylephrine-induced changes in heart rate, as judged by their -log EC50. Either of the alpha-adrenoceptor antagonists exhibited a greater steepness in the curve slope with respect to control. The simultaneous exposure of tissues to phentolamine and propranolol proved to effectively antagonize the chronotropic effect of the agonist. This held true for phentolamine assayed in atria from reserpine-pretreated rats. Previous incubation of tissues with papaverine, 10(-5) M, brought about supersensitivity to phenylephrine which was thoroughly inhibited by either phentolamine or propranolol. These results strongly suggest that beta-adrenoceptor stimulation of heart rate by phenylephrine takes place indirectly via norepinephrine release. There is also alpha 1-adrenoceptor stimulation (blocked by prazosin). Finally, it is hypothesized that supersensitivity develops by papaverine-enhanced Ca2+ influx, since numerous evidences are against a phosphodiesterase inhibition-dependent cAMP accumulation mechanism triggered by papaverine in the presence of phenylephrine.

Papaverine-induced changes on cardiac inotropism with special reference to a D-propranolol antagonism.

Contractile responses were analyzed in the rat paced left atrium. Positive inotropic effects were papaverine dose-dependent with an increase in rate of tension development (dT/dt) and time to peak tension, with prolongation in the total duration of contraction. Reserpinized preparations did not modify papaverine response but several treatments inhibited it, i.e., either doubling or halving [Ca2+]0, as well as the addition of D-propranolol. Incubation of tissues with papaverine (1 hr) changed the usual dose-response curve to isoproterenol into a low, monotonous effect, independent of the agonist dose. Neither high nor low [Ca2+]0 could correct this action. D-propranolol restored the isoproterenol response but significantly blocked it. Under the same conditions, phenylephrine showed similar qualitative effects as above, though no significant differences were found in control vs. the various procedures tested. These results strongly suggest that papaverine provokes an initial calcium release followed by a sustained inhibition. In addition its site of action is the same as that of D-propranolol.

Interactions between sympathomimetic agonists and blocking agents: cardiac effects of phenylephrine and isoproterenol.

In the isolated rat atria the effects of phenylephrine (alpha-agonist) and isoproterenol (beta-agonist) on the contractile force and heart rate were studied either in presence or not of alpha- and/or beta-blockade. Phentolamine 10(-6) M and propranolol 10(-8) M were used as alpha- and beta-antagonists, respectively. The chronotropic dose-response curves were made in both spontaneously beating atria while inotropic effects were derived from left atria driven at 1 Hz, 5 msec and voltage about 20 percent above threshold. Both agonists induced an inotropic effect that could be significantly blocked by phentolamine, propranolol and both antagonists given together. The chronotropic dose-response curve of isoproterenol (pD2 = 10.70 +/- 0.11) was blocked by phentolamine (pD2 = 9.27 +/- 0.08, p less than 0.001) and propranolol (pD2 = 9.21 +/- 0.19, p less than 0.001). Contrarily, pentolamine was unable to shift to the right the chronotropic dose-response curve for phenylephrine (pD2 = 6.48 +/- 0.07 and 6.63 +/- 0.10, respectively). This action of phenylephrine on the heart rate was significantly blocked by propranolol (pD2 = 6.21 +/- 0.13, p less than 0.05) and by propranolol plus phentolamine (pD2 = 6.07 +/- 0.04, p less than 0.001). From the present results it is concluded that in the rat's atrial myocardium there exist alpha- and beta-adrenoceptors mediating the positive inotropic effect of isoproterenol and phenylephrine. Yet, the chronotropic action seems to behave in a different way: while isoproterenol triggered either type of receptors; phenylephrine is only mediated by beta-adrenoceptors.U