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Biomedical subjects

M C García

Publications and source records attributed to M C García.

At least 37 records · Page 2Linked to original sources
PubMed2001

Mitochondria regulate inactivation of L-type Ca2+ channels in rat heart.

1. L-type Ca2+ channels play an important role in vital cell functions such as muscle contraction and hormone secretion. Both a voltage-dependent and a Ca2+-dependent process inactivate these channels. Here we present evidence that inhibition of the mitochondrial Ca2+ import mechanism in rat (Sprague-Dawley) ventricular myocytes by ruthenium red (RR), by Ru360 or by carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreases the magnitude of electrically evoked transient elevations of cytosolic Ca2+ concentration ([Ca2+]c). These agents were most effective at stimulus rates greater than 1 Hz. 2. RR and CCCP also caused a significant delay in the recovery from inactivation of L-type Ca2+ currents (I(Ca)). This suggests that sequestration of cytosolic Ca2+, probably near the mouth of L-type Ca2+ channels, into mitochondria during cardiac contractile cycles, helps to remove the Ca2+-dependent inactivation of L-type Ca2+ channels. 3. We conclude that impairment of mitochondrial Ca2+ transport has no impact on either L-type Ca2+ currents or SR Ca2+ release at low stimulation frequencies (e.g. 0.1 Hz); however, it causes a depression of cytosolic Ca2+ transients attributable to an impaired recovery of L-type Ca2+ currents from inactivation at high stimulation frequencies (e.g. 3 Hz). The impairment of mitochondrial Ca2+ uptake and subsequent effects on Ca2+ transients at high frequencies at room temperature could be physiologically relevant since the normal heart rate of rat is around 5 Hz at body temperature. The role of mitochondria in clearing Ca2+ in the micro-domain near L-type Ca2+ channels could be impaired during high frequencies of heart beats such as in ventricular tachycardia, explaining, at least in part, the reduction of muscle contractility.

Animals↗
PubMed2001

Long-term depolarization regulates the alpha1s, subunit of skeletal muscle Ca2+ channels and the amplitude of L-type Ca2+ currents.

The effects of long-term depolarization on the level of alpha1s and on L-type Ca2+ currents of skeletal muscle were investigated. Long-term depolarization (14 h) caused a 50% decrease of alpha1s, revealed with the Western blot technique. This decline was prevented by preincubation with the Ca2+ channel blocker nifedipine. Electrophysiological experiments using the voltage-clamp technique were performed to measure the actions of long-term depolarization on Ca2+ currents and charge movement. A progressive decline in the amplitude of the Ca2+ currents by depolarizations lasting 0.5-14 h was observed. Similar to Western blot results, the fall in current amplitude was prevented by nifedipine, and it depended on external Ca2+. The nonlinear charge mobilized by step pulses was also significantly reduced (50%) by long-term depolarization. It is suggested that alpha1s subunit is down-regulated by long-term depolarization by a very stringent mechanism and that, in this process, Ca2+ ions permeating through L-type channels play a key role. A new role for the L-type Ca2+ current in skeletal muscle fibers in which the channels are self-regulated is proposed.

Aniline Compounds↗
PubMed2001

Effect of cyclic 3',5'-adenosine monophosphate, glucocorticoids, and insulin on leptin messenger RNA levels and leptin secretion in cultured human trophoblast.

Leptin is a polypeptide hormone originally thought to be produced exclusively by adipocytes. However, both leptin mRNA and leptin protein were identified in human placental trophoblast cells, suggesting a potential role in human pregnancy. In the present report, we examined the regulation of leptin mRNA levels and secretion by cAMP, glucocorticoids, and insulin in term human placental tissue. Placentae were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin concentrations were measured by ELISA in the cultured media of trophoblast maintained in monolayer culture for 24, 48, and 72 h. Likewise leptin mRNA levels in these cultured human trophoblast cells were determined by reverse transcription-polymerase chain reaction. Treatment with forskolin and (Bu)(2) cAMP led to a time- and dose-dependent increase in leptin release, significant after 48 and 72 h. Moreover, incubation with forskolin for 48 h also clearly increased leptin mRNA concentration. Leptin secretion and mRNA levels were also assessed after treatment with insulin or dexamethasone. We found a time- and dose-dependent increase in leptin release, significant after 48 and 72 h. Leptin mRNA levels were also increased after these treatments. All this supports a stimulatory role of cAMP pathway, insulin and dexamethasone in the leptin mRNA levels, and leptin release in trophoblast cells in vitro.

Bucladesine↗
PubMed2000

Characterization of commercial soybean products by conventional and perfusion reversed-phase high-performance liquid chromatography and multivariate analysis.

Conventional and perfusion reversed-phase high-performance liquid chromatography are used to characterize commercial soybean products for human consumption. For this purpose, previously optimized methods of conventional and perfusion chromatography applied to the separation of soybean proteins are employed. Sixty different samples corresponding to 26 different trademarks of soybean products [soybean protein isolate, soybean flour, textured soybean, soybean milks (liquid and powdered), and soybean infant formulas] are analyzed. Characterization of soybean products is carried out on the basis of their protein profiles obtained by both chromatographic methods. Data obtained are processed using multivariate methods such as principal components and discriminant analysis. Perfusion chromatography enables a further and faster characterization of commercial soybean products than conventional chromatography, of great value in the quality control of this kind of product.

Chromatography, High Pressure Liquid↗
PubMed2000

Determination by perfusion reversed-phase high-performance liquid chromatography of the soybean protein content of commercial soybean products prepared directly from whole soybeans.

The use of soybean flour as external standard for the determination of soybean proteins in soybean products directly prepared from whole soybeans is investigated. For that purpose a perfusion reversed-phase high-performance liquid chromatography method consisting of a linear binary gradient acetonitrile-water (both with 0.1% trifluoroacetic acid) in 3 min at a flow-rate of 3 ml/min, and a temperature of 60 degrees C is used. Samples dissolved in water are directly injected in the chromatographic system. The method is validated by evaluating detection limits, precision, and accuracy and applied to the quantitation of soybean proteins in soybean products directly prepared from whole soybeans.

Calibration↗
PubMed2000

Perfusion chromatography: an emergent technique for the analysis of food proteins.

Perfusion chromatography is a technique arised to overcome the problem associated with mass transfer in the separation of large molecules such as proteins by high-performance liquid chromatography (HPLC). Perfusion media are constituted by two set of pores: throughpores (6000-8000 A) and diffusive pores (800-1500 A) which enable better access of macromolecules to the inner of the particle by the combination of convective and diffusive flow. As a consequence, times required for a chromatographic separation are reduced. Perfusion media are available in different chromatographic modes: reversed-phase, ion-exchange, hydrophobic interaction, and affinity. From the theoretical models developed to explain the dynamic of retention of solutes in perfusive supports, it was derived that efficiency of a separation was independent of the flow-rate and only depended slightly on the particle diameter. Furthermore, loading capacity was also independent of the superficial velocity. All these advantages have promoted the use of this chromatographic technique for the separation of biomolecules both in analytical and preparative chromatography. Characteristics of perfusion chromatography make this technique very interesting for the analysis of food proteins. Perfusion chromatography enables the assessment of protein composition of a foodstuff at sufficient speed and low cost to be suitable in routine analysis.

Chromatography, Liquid↗
PubMed2000

Leptin regulation of prepro-orexin and orexin receptor mRNA levels in the hypothalamus.

The aim of this study was to determine the effects of leptin treatment on prepro-orexin and orexin receptor expression in the rat hypothalamus. Adult male rats, food-deprived for 48 and 72 h, were treated one time with vehicle or leptin (10 microg, icv). Prepro-orexin mRNA content was measured by semiquantitative RT-PCR, Northern blot, and in situ hybridization; orexin receptor 1 and 2 mRNA content was quantified by Northern blot and/or semiquantitative RT-PCR. Our results indicate that leptin inhibits a fasting-induced increase in prepro-orexin mRNA and orexin receptor 1 mRNA levels in the rat hypothalamus, while orexin receptor 2 mRNA levels were unchanged in all situations evaluated. These data provide direct evidence for an additional mechanism of adaptation of the hypothalamus to food deprivation and for a new effect of leptin in the regulation of food intake.

Animals↗
PubMed1999

Dihydropyridine-sensitive ion currents and charge movement in vesicles derived from frog skeletal muscle plasma membranes.

1. Whole-cell voltage clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes to characterize the electrophysiological properties of dihydropyridine (DHP) receptors. This preparation allows control of the composition of the internal medium and the recording of currents, without the influence of the sarcoplasmic reticulum (SR). 2. In solutions containing Ba2+, Bay K 8644-sensitive, L-type inward currents were recorded. Peak Ba2+ currents (IBa) averaged 3.0 microA microF-1 and inactivated in a voltage-dependent manner. Half-maximal steady-state inactivation occurred at -40 mV. No major facilitation of tail currents was observed. 3. The time course of activation of L-type Ca2+ channels was voltage dependent and 10 times faster than that in muscle fibres; the current density values were also much lower. 4. Lowering [Mg2+]i from 2 to 0.1 mM shifted the time to peak of IBa versus voltage relation by -13 mV. 5. In solutions that contained mostly impermeant ions, non-linear capacitive currents were recorded. Charge movement with properties resembling charge 1 was observed in polarized vesicles. The charge movement depended on voltage with Boltzmann parameters: Qmax (maximum charge), 45.6 nC microF-1; V (potential at which Q = 0.5 Qmax), -58.4 mV; and k (slope factor), 22. 3 mV. There was no indication of the presence of Qgamma (the 'hump' component of charge movement). 6. In depolarized vesicles, non-linear currents were observed during hyperpolarizing pulses. The currents produced an excessive charge during 'on' transients only. Charge during 'off' transients was linear from -180 to +60 mV. There was no evidence of the presence of charge 2.

Algorithms↗
PubMed1999

A long-term blockade of L-type calcium currents upregulates the number of Ca2+ channels in skeletal muscle.

The effects of a long-term blockade of L-type Ca2+ channels on membrane currents and on the number of dihydropyridine binding sites were investigated in skeletal muscle fibers. Ca2+ currents (ICa) and intramembrane charge movement were monitored using a voltage-clamp technique. The peak amplitude of ICa increased by more than 40% in fibers that were previously incubated for 24 hr in solutions containing the organic Ca2+ channel blocker nifedipine or in Ca2+-free conditions. A similar incubation period with Cd2+, an inorganic blocker, produced a moderate increase of 20% in peak ICa. The maximum mobilized charge (Qmax) increased by 50% in fibers preincubated in Ca2+-free solutions or in the presence of Cd2+. Microsomal preparations from frog skeletal muscle were isolated by differential centrifugation. Preincubation with Cd2+ prior to the isolation of the microsomal fraction doubled the number of 3H-PN200-110 binding sites and produced a similar increase in the values of the dissociation constant. The increase in the number of binding sites is consistent with the increase in the peak amplitude of ICa as well as with the increase in Qmax.

Action Potentials↗
PubMed1999

Characterization and quantitation of soybean proteins in commercial soybean products by capillary electrophoresis.

Capillary electrophoresis was applied for the first time to determine soybean proteins in commercial soybean products. The most suitable conditions for the analysis of these products in less than 7 min were 0.05 M phosphate buffer (pH 8) with 1 M urea; detection wavelength, 254 nm; applied voltage, 20 kV; and temperature, 30 degrees C. Quantitation of soybean proteins was achieved using referenced conditions by means of the method of standard additions, using as standard a soybean protein isolate. This method was validated and applied to the quantitation of soybean proteins in commercial products derived from soybean protein isolate and soybean seeds.

Buffers↗
PubMed1999

In vitro synthesis of type I collagen: quantification of carboxyterminal propeptide of procollagen type I versus tritiated proline incorporation.

Radioimmunoassay of the carboxyterminal propeptide of human type I procollagen has been recently introduced to measure in vitro synthesis of type I collagen by osteoblasts and fibroblasts. However, it has not been assessed whether the equivalent results are obtained with this new assay and with tritiated proline incorporation to collagen protein. To this purpose, both methods were used to quantify synthesis of type I collagen in normal human osteoblast cultures to which fetal calf serum and human serum were added in order to stimulate protein synthesis. A positive correlation in the results obtained by both methods was obtained (r = 0.95, P = 0.0001). Given the technical advantages of detection of levels to carboxyterminal propeptide of type I procollagen, we consider that this is the technique of choice for the quantification of in vitro synthesis of type I collagen by normal human osteoblasts.

Aged↗
PubMed1998

Ultrarapid detection of bovine whey proteins in powdered soybean milk by perfusion reversed-phase high-performance liquid chromatography.

A perfusion reversed-phase high-performance liquid chromatographic method has been developed to simultaneously separate soybean and bovine whey proteins (alpha-lactalbumin and beta-lactoglobulins (A + B)) in a very short analysis time (approximately 5 min). The method consisted of a linear binary gradient water-acetonitrile-0.10% trifluoroacetic acid at a flow-rate of 3 ml/min, with the column thermostated at 60 degrees C, and ultraviolet detection at 254 nm. This method enables the rapid detection of adulterations of powdered soybean milks by addition of bovine whey proteins. When bovine whey proteins were too low to be detected by direct injection of the sample, a previous acidic precipitation step was required in order to concentrate these proteins. Quantitatve analysis of bovine whey proteins was also successfully performed. In fact, it was possible to detect about 1% and 1.3% of alpha-lactalbumin and beta-lactoglobulins, respectively, in a commercial powdered soybean milk in which these proteins were included in its formulation. Results were compared with those obtained by conventional reversed-phase high-performance liquid chromatography.

Animals↗
PubMed1998

Involvement of GABA and glutamate receptors in the blood pressure responses to intrathecally injected sodium nitroprusside in anesthetized rats.

In pentobarbital-anesthetized rats the intrathecal (i.t.) injection of the nitric oxide (NO) donor, sodium nitroprusside (125, 250 and 500 nmol), induced a dose-dependent hypotensive response followed by a dose-dependent pressor effect. The pressor response to sodium nitroprusside (250 nmol) was reduced to 30% of the control value by the selective antagonist for AMPA/kainate receptors, 6.7-dinitroquinoxaline-2,3-dione (50 nmol, i.t.), whereas it was not modified by the selective NMDA receptor antagonist, 2-amino-5-phosphono-valeric acid (30 nmol, i.t.). The hypotensive effect of sodium nitroprusside was antagonized by the GABA(A) receptor antagonists, bicuculline (4.4 nmol, i.t.) and picrotoxin (4.4 nmol, i.t.), and also by the GABA(B) receptor antagonist, 2-hydroxy saclofen (113 nmol, i.t.). The blood pressure responses to sodium nitroprusside were not modified by blockade of muscarinic receptors with methyl atropine (164 nmol, i.t.), or of nicotinic receptors with hexamethonium (211 nmol, i.t.), of alpha1-adrenoceptors with prazosin (3.1 nmol, i.t.), of alpha2-adrenoceptors with yohimbine (2.8 micromol/kg, i.v.), of 5-HT receptors with methysergide (5.1 micromol/kg, i.v.), or of glycine receptors with strychnine (65 nmol, i.t.). It is concluded that NO generated from sodium nitroprusside in the spinal cord exerts inhibitory and excitatory effects on blood pressure probably through the release of GABA and glutamate, respectively. The inhibitory action on blood pressure involves the stimulation of spinal GABA(A) and GABA(B) receptors whereas the excitatory response to glutamate appears to be mediated through the activation of spinal AMPA/kainate receptors.

Animals↗
PubMed1998

Opposite roles of D1 and D5 dopamine receptors in locomotion revealed by selective antisense oligonucleotides.

Contralateral rotations induced by the D1-like agonist SKF 38393 in unilaterally 6-hydroxydopamine-lesioned rats were completely prevented by the administration of the D1-like antagonist SCH 23390. A similar result was obtained after intracerebroventricular administration of an antisense oligodeoxynucleotide for the D1 receptor (D1R-as). Contrariwise, administration of a D5R-as potentiated the effects of SKF 38393, showing a 60% increase in the rotational scores. Both effects were reversible upon cessation of D1R-as or D5R-as treatment and were also specific since rotational scores in rats treated with vehicle or with a randomly designed oligodeoxynucleotide were not modified. These results suggest that whereas D1 receptors play a facilitatory role in locomotion, D5 receptors exert an inhibitory effect.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1998

Chronic levodopa is not toxic for remaining dopamine neurons, but instead promotes their recovery, in rats with moderate nigrostriatal lesions.

Orally administered levodopa remains the most effective symptomatic treatment for Parkinson's disease (PD). The introduction of levodopa therapy is often delayed, however, because of the fear that it might be toxic for the remaining dopaminergic neurons and, thus, accelerate the deterioration of patients. However, in vivo evidence of levodopa toxicity is scarce. We have evaluated the effects of a 6-month oral levodopa treatment on several dopaminergic markers, in rats with moderate or severe 6-hydroxydopamine-induced lesions of mesencephalic dopamine neurons and sham-lesioned animals. Counts of tyrosine hydroxylase (TH)-immunoreactive neurons in the substantia nigra and ventral tegmental area showed no significant difference between levodopa-treated and vehicle-treated rats. In addition, for rats of the sham-lesioned and severely lesioned groups, immunoradiolabeling for TH, the dopamine transporter (DAT), and the vesicular monoamine transporter (VMAT2) at the striatal level was not significantly different between rats treated with levodopa or vehicle. It was unexpected that quantification of immunoautoradiograms showed a partial recovery of all three dopaminergic markers (TH, DAT, and VMAT2) in the denervated territories of the striatum of moderately lesioned rats receiving levodopa. Furthermore, the density of TH-positive fibers observed in moderately lesioned rats was higher in those treated chronically with levodopa than in those receiving vehicle. Last, that chronic levodopa administration reversed the up-regulation of D2 dopamine receptors seen in severely lesioned rats provided evidence that levodopa reached a biologically active concentration at the basal ganglia. Our results demonstrate that a pharmacologically effective 6-month oral levodopa treatment is not toxic for remaining dopamine neurons in a rat model of PD but instead promotes the recovery of striatal innervation in rats with partial lesions.

Amphetamine↗
PubMed1998

A perfusion reversed-phase chromatographic method for ultrarapid determination of soybean proteins in soybean infant formulas and soybean milks: method development and validation.

Perfusion reversed-phase high-performance liquid chromatography (perfusion RP-HPLC) is applied for the first time to the determination of soybean proteins in commercial samples prepared from soybean protein isolate. A linear binary gradient of acetonitrile-water-0.1% trifluoroacetic acid at a flow rate of 3 mL/min and a temperature of 60 degrees C is optimized to analyze soybean proteins in approximately 3 min. Direct injection of samples into the chromatographic system is possible by dissolving them in water. Quantitation of soybean proteins is achieved by the proposed method using a soybean protein isolate as an external standard. The method is validated and applied to the quantitation of soybean proteins in infant formulas and powdered and liquid soybean milks.

Calibration↗
PubMed1998

Effects of oral phosphorus supplementation on mineral metabolism of renal transplant recipients.

BACKGROUND: Persistent hyperparathyroidism (HPT) is frequently observed in kidney transplant recipients. Hypophosphataemia is a common biochemical consequence of HPT. Theoretically, oral phosphorus administration may induce negative effects on the control of HPT, though this point has never been demonstrated in kidney-transplant recipients. This study was designed to evaluate the effects of oral phosphorus supplementation on the mineral metabolism of successful kidney transplant recipients. METHODS: Thirty-two kidney transplant recipients with serum creatinine < 2 mg/dl and serum phosphate levels <3.5 mg/dl were included in the study. After a washout period in which oral phosphorus supplementation was discontinued, the following parameters were determined (F0 period): serum calcium, phosphate, alkaline phosphatase, uric acid, bicarbonate, PTH, 1,25-dihydroxyvitamin D3 (1,25 (OH)(2)D) and 25-hydroxyvitamin D3 (25OHD). Creatinine clearance, calcium, and phosphate excretion were determined from a 24-h urine sample. The same determinations were repeated (F1 period) after all patients received 1.5 g of oral phosphorus for 15 days. For data analysis, patients were divided into two subgroups (optimal and suboptimal) according to allograft function (Ccr>or < 70 ml/min/1.73 m2). RESULTS: In the F0 period, only nine of 32 patients had PTH levels within the normal range (<65 pg/ml). The mean concentrations of PTH, 1,25(OH)(2)D and 25OHD were 132+/-97pg/ml, 40.5+16pg/ml and 12.5+/-8.2 ng/ml respectively. Phosphorus supplementation led to significant reductions in serum calcium and 1,25(OH)(2)D concentrations, as well as in urinary calcium excretion in the whole group. On the contrary, serum phosphate, PTH, and urinary phosphate excretion increased significantly. The percentage increase in PTH concentrations after phosphorus supplementation were similar in patients with optimal and suboptimal allograft function (33 vs 36%). The reduction of 1,25 (OH)(2)D concentrations after phosphorus supplementation was observed mainly in the subgroup with optimal allograft function (21% reduction with respect to baseline values), while the mean 1,25(OH)(2)D concentrations in patients with suboptimal allograft function scarcely changed (1.4% increase). Changes in 1,25(OH)(2)D concentrations after phosphorus supplementation, expressed as a percentage of the initial concentrations, correlated positively with the percentage changes in PTH concentrations for the whole group, as well as for each subgroup. The best determinants for the percentage and the absolute increase in PTH concentration after phosphorus supplementation was the final serum phosphate concentration (F=4.84, r=0.37, P=0.035) and the increase in serum phosphate (F=7.69, r=0.45, P= 0.009) respectively. CONCLUSIONS: Oral phosphorus supplementation led to a significant increase in the PTH concentration of kidney transplant recipients. The mean 1,25(OH)(2)D concentration decreased mainly in recipients with optimal allograft function. The counterbalance effect of PTH on 1,25(OH)(2)D production may account for the relative preservation of 1,25(OH)2D levels in recipients with suboptimal allograft function.

Administration, Oral↗