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C Hidalgo

Publications and source records attributed to C Hidalgo.

At least 19 recordsLinked to original sources
PubMed1998

Automated determination of phenylcarbamate herbicides in environmental waters by on-line trace enrichment and reversed-phase liquid chromatography-diode array detection.

A fully automated liquid chromatographic method using on-line trace-enrichment, gradient elution and diode array detection is described for the trace-level determination of several phenylcarbamate herbicides, such as carbetamide, propham, desmedipham, phenmedipham, chlorbufam and chlorpropham, in environmental water samples. In this work, two different enrichment pre-columns have been assayed, a 5.8 x 4.6 mm I.D., 10 microns ODS Prelute cartridge and a 10 x 2 mm I.D. cartridge filled with 10 microns PRP-1 polymer, both coupled to a 150 x 4.6 mm I.D. analytical column filled with 5 microns ODS. Using the C18 pre-column, up to 50 ml of water sample could be percolated without peak broadening of any compound. However, a lack of reproducibility was observed in the case of carbetamide, the most polar analyte, after performing recovery experiments by percolating drinking and surface water samples spiked at several levels (0.5 and 4 micrograms l-1). On the other hand, the PRP-1 pre-column allowed the enrichment up to 100 ml of water sample with satisfactory results for every compound, including carbetamide. The procedure was validated by recovery experiments in environmental water samples spiked at 0.2 and 1 microgram l-1 yielding average recoveries between 84-108% with relative standard deviations in the range 2-12%. Detection limits as low as 0.04 microgram l-1 were achieved. It was observed that desmedipham and phenmedipham degraded rapidly in the environmental water samples as showed the degradation studies performed along 24 h in drinking and surface waters spiked at 4 micrograms l-1. Although the standard mixture, prepared in HPLC water, was stable for around one week, in the environmental water matrices more than 95% of each herbicide degraded after 6 h, and new chromatographic peaks corresponding to the degradation products were detected.

Autoanalysis↗
PubMed1998

Changes in luminal pH caused by calcium release in sarcoplasmic reticulum vesicles.

Fast (milliseconds) Ca2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (approximately 0.2 pH units) of the lumen occurred within the same time scale (t(1/2) = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5'-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5-10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.

Adenosine Triphosphate↗
PubMed1998

Sulfhydryl oxidation modifies the calcium dependence of ryanodine-sensitive calcium channels of excitable cells.

The calcium dependence of ryanodine-sensitive single calcium channels was studied after fusing with planar lipid bilayers sarcoendoplasmic reticulum vesicles isolated from excitable tissues. Native channels from mammalian or amphibian skeletal muscle displayed three different calcium dependencies, cardiac (C), mammalian skeletal (MS), and low fractional open times (low Po), as reported for channels from brain cortex. Native channels from cardiac muscle presented only the MS and C dependencies. Channels with the MS or low Po behaviors showed bell-shaped calcium dependencies, but the latter had fractional open times of <0.1 at all [Ca2+]. Channels with C calcium dependence were activated by [Ca2+] < 10 microM and were not inhibited by increasing cis [Ca2+] up to 0.5 mM. After oxidation with 2,2'-dithiodipyridine or thimerosal, channels with low Po or MS dependencies increased their activity. These channels modified their calcium dependencies sequentially, from low Po to MS and C, or from MS to C. Reduction with glutathione of channels with C dependence (native or oxidized) decreased their fractional open times in 0.5 mM cis [Ca2+], from near unity to 0.1-0.3. These results show that all native channels displayed at least two calcium dependencies regardless of their origin, and that these changed after treatment with redox reagents.

Animals↗
PubMed1998

Specific cellular and humoral immune response in Balb/c mice immunised with an expression genomic library of Trypanosoma cruzi.

An expression genomic library of Trypanosoma cruzi (T. cruzi) constructed using pcDNA3 plasmid was used for the immunisation (25 micrograms) of Balb/c mice. Expression of T. cruzi antigens in the muscle of inoculated mice was detected by indirect immunofluorescence 7 days after immunisation. Specific IgG antibodies were significatively increased (P < 0.05) in animals that were reimmunized with 50 micrograms of the genomic library. An antigen specific lymphoproliferative response was detected in one animal of the group inoculated with one dose of the library.

Animals↗
PubMed1998

Cyclic ADP-ribose activates caffeine-sensitive calcium channels from sea urchin egg microsomes.

Adenosine 5'-cyclic diphosphoribose [cyclic ADP-ribose (cADPR)], a metabolite of NAD+ that promotes Ca2+ release from sea urchin egg homogenates and microsomal fractions, has been proposed to act as an endogenous agonist of Ca2+ release in sea urchin eggs. We describe experiments showing that a microsomal fraction isolated from Tetrapigus nyger sea urchin eggs displayed Ca(2+)-selective single channels with conductances of 155.0 +/- 8.0 pS in asymmetric Cs+ solutions and 47.5 +/- 1.1 pS in asymmetric Ca2+ solutions. These channels were sensitive to stimulation by Ca2+, ATP, and caffeine, but not inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The channels were also activated by cADP-ribose in a Ca(2+)-dependent fashion. Calmodulin and Mg2+, but not heparin, modulated channel activity in the presence of cADP-ribose. We propose that these Ca2+ channels constitute the intracellular Ca(2+)-induced Ca2+ release pathway that is activated by cADP-ribose in sea urchin eggs.

Adenosine Diphosphate Ribose↗
PubMed1998

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles.

In cardiac sarcolemmal vesicles, MgATP stimulates Na+/Ca2+ exchange with the following characteristics: 1) increases 10-fold the apparent affinity for cytosolic Ca2+; 2) a Michaelis constant for ATP of approximately 500 microM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 microM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5'-O-(3-thiotriphosphate), without the need for vanadate, but not by beta,gamma-methyleneadenosine 5'-triphosphate; and 6) not affected by unspecific protein alkaline phosphatase but abolished by a phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC effect is counteracted by phosphatidylinositol. In addition, in the absence of ATP, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) was able to stimulate the exchanger activity in vesicles pretreated with PI-PLC. This MgATP stimulation is not related to phosphorylation of the carrier, whereas phosphorylation appeared in the phosphoinositides, mainly PIP2, that coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP and no Ca2+ show a marked synthesis of L-alpha-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 microM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2 increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+ exchanger through a fast phosphorylation chain: a Ca(2+)-independent PIP formation followed by a Ca(2+)-dependent synthesis of PIP2.

Adenosine Triphosphate↗
PubMed1996

Luminal pH regulated calcium release kinetics in sarcoplasmic reticulum vesicles.

Calcium binding to triads isolated from rabbit skeletal muscle followed a single hyperbolic function in the pH range 5.5-8.0. Maximal binding was obtained at pH 8.0; decreasing the pH decreased the binding capacity and, at pH < or = 6.0, increased Kd 2-fold. These results indicate that lowering the pH diminished calcium binding to calsequestrin, since this protein is the primary source of calcium binding sites in triads. Luminal pH had a marked effect on calcium release induced by 2 mM ATP, at pCa 5.0, pH 6.8. At a constant luminal [Ca2+] of 0.1 mM, release rate constants (k) and initial rates of release increased steadily as a function of decreasing luminal pH; at luminal pH 7.5, values of k < 0.4 s-1 were found, whereas at pH 5.5 values of k approximately 10 S-1 were obtained. Increasing luminal [Ca2+] from 0.05 mM to 0.7 mM had no effect on the k values measured at luminal pH 5.5. In contrast, at pH 6.8, increasing luminal [Ca2+] produced a marked increase in k values, that reached maximal values of k approximately 10 S-1 at 0.7 mM luminal [Ca2+]. Control experiments using fluorescent pH indicators showed that luminal pH did not change significantly during calcium release. It is proposed that luminal protons or calcium induces conformational changes in calsequestrin that in turn promote activation of the calcium release channels.

Animals↗
PubMed1996

Calcium dependence of ryanodine-sensitive calcium channels from brain cortex endoplasmic reticulum.

Endoplasmic reticulum vesicles isolated from rat brain cortex and fused with lipid bilayers displayed ryanodine-sensitive calcium channels, with three cytoplasmic calcium dependences. A: Channels (n=5) stimulated by Ca2+ (K0.5=1.2 microM and nHill=1.9) and not inhibited up to 0.5 mM Ca2+. B: Channels (n=14) cooperatively activated (K0.5=6.9 microM and nHill=1.8), and inhibited by Ca2+ (K0.5=152 microM and nHill=1.8). C: Low Po (<0.1) channels (n=22), non-cooperatively activated and inhibited with the same K0.5=26.3 microM Ca2+. These three types of responses to cytoplasmic [Ca2+] may underlie separate calcium release pathways in neurons of rat brain cortex.

Animals↗
PubMed1996

Protons induce calsequestrin conformational changes.

Calsequestrin, a high-capacity, intermediate-affinity, calcium-binding protein present in the lumen of sarcoplasmic reticulum, undergoes extensive calcium-induced conformational changes at neutral pH that cause distinct intrinsic fluorescence changes. The results reported in this work indicate that pH has a marked effect on these calcium-induced intrinsic fluorescence changes, as well as on calorimetric changes produced by the addition of Ca(2+) to calsequestrin. The addition of Ca(2+) at neutral pH produced a marked and cooperative increase in calsequestrin intrinsic fluorescence. In contrast, at pH 6.0 calsequestrin's intrinsic fluorescence was not affected by the addition of Ca(2+), and the same intrinsic fluorescence as that measured in millimolar calcium at neutral pH was obtained. The magnitude and the cooperativity of the calcium-induced intrinsic fluorescence changes decreased as either [H+] or [K+] increased. The evolution of heat production, determined by microcalorimetry, observed upon increasing the molar ratio of Ca(2+) to calsequestrin in 0.15 M KCl, decreased markedly as the pH decreased from pH 8.0 to pH 6.0, indicating that pH modifies the total heat content changes produced by Ca(2+). We propose that protons bind to calsequestrin and induce protein conformational changes that are responsible for the observed proton-induced intrinsic fluorescence and calorimetric changes.

Animals↗
PubMed1996

Ca(2+)- and pH-dependent halothane stimulation of Ca2+ release in sarcoplasmic reticulum from frog muscle.

The effect of halothane on calcium release kinetics was studied in triad-enriched sarcoplasmic reticulum vesicles from frog skeletal muscle. Release from vesicles passively equilibrated with 3 mM 45CaCl2 was measured in the millisecond time range by use of a fast-filtration system. Halothane (400 microM) increased release rate constants at pH 7.1 and 7.4 as a function of extravesicular pCa. In contrast, halothane at pH 6.8 produced the same stimulation of release from pCa 7.0 to 3.0; no release took place in these conditions in the absence of halothane. Halothane shifted the calcium activation curve at pH 7.1, but not at pH 7.4, to the left and increased channel open probability at pH 7.1 in the cis pCa range of 7.0 to 5.0. These results indicate that cytosolic pCa and pH modulate the stimulatory effects of halothane on calcium release. Furthermore, halothane stimulated release in frog skeletal muscle at low pH and resting calcium concentration, indicating that in frog muscle halothane can override the closing of the release channels produced by these conditions, as it does in malignant hyperthermia-susceptible porcine muscle.

Animals↗
PubMed1995

Luminal calcium regulation of calcium release from sarcoplasmic reticulum.

This article discusses how changes in luminal calcium concentration affect calcium release rates from triad-enriched sarcoplasmic reticulum vesicles, as well as single channel opening probability of the ryanodine receptor/calcium release channels incorporated in bilayers. The possible participation of calsequestrin, or of other luminal proteins of sarcoplasmic reticulum in this regulation is addressed. A comparison with the regulation by luminal calcium of calcium release mediated by the inositol 1,4,5-trisphosphate receptor/calcium channel is presented as well.

Animals↗
PubMed1995

Luminal calcium regulates calcium release in triads isolated from frog and rabbit skeletal muscle.

Triads isolated from frog and rabbit skeletal muscle were equilibrated with different external [Ca2+], ranging from 0.025 to 10 mM. Vesicular calcium increased with external [Ca2+] as the sum of a linear plus a saturable component; the latter, which vanished after calsequestrin removal, displayed Bmax values of 182 and 132 nmol of calcium/mg of protein, with Kd values of 1.21 and 1.14 mM in frog and rabbit vesicles, respectively. The effect of luminal [Ca2+] on release kinetics in triads from frog and rabbit skeletal muscle was investigated, triggering release with 2 mM ATP, pCa 5, pH 6.8. In triads from frog, release rate constant (k) values increased sixfold after increasing luminal [Ca2+] from 0.025 to 3 mM. In triads from rabbit, k values increased 20-fold when luminal [Ca2+] increased from 0.05 to 0.7 mM. In both preparations, k values remained relatively constant (10-12 s-1) at higher luminal [Ca2+], with a small decrease at 10 mM. Initial release rates increased with luminal [Ca2+] in both preparations; in triads from rabbit the increase was hyperbolic, and in triads from frogs the increase was sigmoidal. These results indicate that, although triads from frog and rabbit respond differently, in both preparations luminal [Ca2+] has a distinctive effect on release, presumably by regulating sarcoplasmic reticulum calcium channels.

Animals↗
PubMed1994

[Influence of body fat and its distribution on cardiovascular risk factors in healthy subjects].

The aim of this work was to study the association between obesity and body fat distribution with known cardiovascular risk factors. Seven hundred eighty two healthy individuals, 634 men and 148 female age 44 +/- 10 years were studied. Multiple stepwise regression models were performed in which cardiovascular risk factors (total, LDL and HDL cholesterol, triglycerides, fasting and postprandial blood glucose, systolic and diastolic blood pressure) were considered as the dependent variable and age, sex, smoking habits, body mass index (BMI), waist circumference (WC), waist hip ratio (WHR), subscapular/tricipital skinfold ratio (STR) and percentage of total body fat (%BF), derived from the sum of four skinfolds, as the independent variables. Among anthropometric variables, WC was the principal predictor of total cholesterol and basal blood glucose, WHR was the principal predictor of HDL cholesterol (inverse relationship) and triglycerides; BMI was the principal predictor of systolic and diastolic blood pressure; %BF was the principal predictor of post prandial blood glucose. Performing the same analysis in a subgroup of patients with a BMI between 21 and 24, measures of fat distribution continued to be predictors of cardiovascular risk factors. It is concluded that both total body fat and its distribution are related to cardiovascular risk factors and, in some cases, may have an additive effect and should be measured in preventive medical examinations.

Adipose Tissue↗
PubMed1993

pH-sensitive calcium release in triads from frog skeletal muscle. Rapid filtration studies.

Triad vesicles from frog skeletal muscle exhibited calcium-induced calcium release highly sensitive to extravesicular pH; calcium induced release at pH > 7.4 but not at pH 6.8. In contrast, triads isolated from rabbit skeletal muscle exhibited significant calcium-induced calcium release at pH 6.8. At pH 7.4, there was no stimulation of calcium release in triads from frog at pCa 7, maximum stimulation at pCa 5, and complete inhibition at pCa 3. Addition of ATP at pCa 5, pH 6.8, induced calcium release with the same high rate constants in both preparations. In triads from frog, ATP-induced calcium release at pCa 5 had the same kinetics at pH 6.8 and 7.4, whereas ATP-induced calcium release at pCa > 8, pH 6.8, was partial, with a decrease in the amounts released but not in rate constants. In contrast, triads from rabbit displayed the opposite behavior, with a decrease in rate constants but not in the amounts of calcium released at pCa > 8, pH 6.8. In triads from frog ATP-induced calcium release decreased at pCa < 4, to reach total inhibition at pCa 2; addition of magnesium (free > 0.5 mM) completely abolished ATP-induced calcium release at pH 6.8, pCa 7 or 5. The potential physiological relevance of these results is discussed.

Adenosine Triphosphate↗
PubMed1993

Phospholipase C activity in membranes and a soluble fraction isolated from frog skeletal muscle.

Highly purified triads and transverse tubules, as well as soluble fraction isolated from frog skeletal muscle, hydrolyze exogenous phosphatidylinositol 4,5-bisphosphate forming inositol 1,4,5-trisphosphate with maximal rates in the range 0.5-1 nmol/mg per min at pCa 3. Sarcoplasmic reticulum membranes present a minor activity. The hydrolysis rates in triads were 0.072 +/- 0.015 nmol/mg per min at pCa 7, increasing to 0.263 +/- 0.026 nmol/mg per min at pCa 5 with 1.0 mM Mg and 0.1 mM substrate. The phospholipase C activity of isolated transverse tubules at pCa 3 was 0.570 +/- 0.032 nmol/mg per min. Since triads contain 10% transverse tubules, and correcting for the small contribution of sarcoplasmic reticulum, the calculated phospholipase C activity of transverse tubules at pCa 3 is about 10-times higher than the observed values, suggesting loss of activity during isolation. The activation by calcium was also observed in a soluble fraction and was neither replaced nor inhibited by magnesium. No effect of GTP analogs on phospholipase C activity was detected.

Animals↗