Biomedical subjects

J Llopis

Publications and source records attributed to J Llopis.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells.

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.

Amino Acid Sequence↗

PubMed1999

Magnesium-manganese interactions caused by magnesium deficiency in rats.

OBJECTIVE: We investigated the effect of dietary magnesium (Mg) deficiency on the nutritive utilization and tissue distribution of manganese (Mn). METHODS: Wistar rats were fed a Mg-deficient diet (56 mg/kg) for 70 day. Absorbed Mn, Mn balance and Mn content in plasma, whole blood, skeletal muscle, heart, kidney, liver, femoral bone and sternum were determined after 21, 35 and 70 days. RESULTS: The Mg-deficient diet significantly increased Mn apparent absorption and Mn balance from week five until the end of the experimental period. This effect was accompanied by a significant increase in the concentration of Mn in heart at all three time points. Whole blood, skeletal muscle and kidney Mn were significantly increased from day 35, and femur Mn content was increased only at the end of the study (day 70). However, Mn concentration in the sternum decreased significantly from day 35. No changes were found in liver Mn content. CONCLUSION: Mg deficiency increased Mn absorbed, and this favored the deposition of Mn in all tissues studied except the liver and trabecular bone. The lack of response by the liver to increased Mn absorption may have led to the redistribution of this ion to other tissues.

Absorption↗

PubMed1998

Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins.

Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/ V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl- serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Animals↗

PubMed1998

Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression.

Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores and triggers complex waves and oscillations in levels of cytosolic free calcium. To determine which longer-term responses are controlled by oscillations in InsP3 and cytosolic free calcium, it would be useful to deliver exogenous InsP3, under spatial and temporal control, into populations of unpermeabilized cells. Here we report the 15-step synthesis of a membrane-permeant, caged InsP3 derivative from myo-inositol This derivative diffused into intact cells and was hydrolysed to produce a caged, metabolically stable InsP3 derivative. This latter derivative accumulated in the cytosol at concentrations of hundreds of micromolar, without activating the InsP3 receptor. Ultraviolet illumination uncaged an InsP3 analogue nearly as potent as real InsP3, and generated spikes of cytosolic free calcium, and stimulated gene expression via the nuclear factor of activated T cells. The same total amount of InsP3 analogue elicited much more gene expression when released by repetitive flashes at 1-minute intervals than when released at 0.5- or > or = 2-minute intervals, as a single pulse, or as a slow sustained plateau. Thus, oscillations in cytosolic free calcium levels at roughly physiological rates maximize gene expression for a given amount of InsP3.

Animals↗

PubMed1998

Numeric alterations in chromosomes 7 and 8 detected by fluorescent in situ hybridization correlate with high-grade localized prostate cancer.

OBJECTIVE: To compare the ability of flow cytometry (FCM) and fluorescent in situ hybridization (FISH), using a small set of 4 enumeration chromosome probes to detect aneuploidy in prostate tumors, and to correlate it with histological grade and pathological stage. METHODS: Among 28 suitable cases, 21 could be analyzed by FISH and FCM techniques. DNA centromeric probes were used in FISH analysis to enumerate chromosomes 7, 8, 10 and 12. RESULTS: (a) Of the 21 cases studied by FISH, 5 were diploid, 14 aneuploid and 2 were tetraploid. When studied by FCM, these tumors were: 14 diploid, 6 aneuploid, and 1 tetraploid. FISH proved to have a higher ability for detecting DNA aneuploidy than FCM while been equally specific, since all tumors aneuploid by FCM were also found to be aneuploid by FISH. (b) Of the 14 aneuploid tumors, 12 were of high histological grade, while only 2 of the 7 nonaneuploid were of high grade. A statistically significant association was observed between high histological grade and FISH aneuploidy (p = 0.033). (c) All the aneuploid tumors showed chromosome 7 and/or 8 aneusomy. Trisomy 7 and monosomy 8 were the most frequent alterations present in 56 and 42% of the aneuploid tumors, respectively. CONCLUSION: FISH analysis of chromosome 7 and 8 alterations proved to be more sensitive than FCM in the detection of aneuploid prostate tumors. This aneuploidy was significantly associated with a poor pathological prognosis.

Aneuploidy↗

PubMed1997

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Amino Acid Sequence↗

PubMed1997

Changes in bioavailability and tissue distribution of selenium caused by magnesium deficiency in rats.

OBJECTIVE: We investigated the effect of dietary magnesium (Mg) deficiency on the bioavailability and tissue distribution of selenium (Se). METHODS: Wistar rats were fed an Mg-deficient diet for 70 days (D). Selenium content in plasma, whole blood, skeletal muscle, heart, kidney and femoral bone was determined after 7, 35, 49 and 70 days on a Mg-deficient diet. RESULTS: The Mg-deficient diet significantly decreased Se absorption from week 7 until the end of the experimental period; this effect was accompanied by a significant decrease in Se balance. We found a direct linear correlation between Se absorption and retention (balance) (pair-fed controls r = 0.842, p < 0.001; Mg-deficient rats r = 0.959, p < 0.001), and between Se absorbed and plasma Se concentration (controls r = 0.527, p < 0.02; Mg-deficient rats r = 0.704, p < 0.001). In general there were no significant correlations between urinary Se and any other parameter. Tissue analyses showed that after 7 days, Mg deficiency reduced erythrocyte Se and significantly increased plasma and kidney Se. Heart Se was significantly increased only at the end of the study. We found no significant changes in skeletal muscle or bone Se at any time during the experimental period. CONCLUSION: Chronic Mg deficiency decreases Se absorption and retention and erythrocyte concentrations of this mineral, and increases Se in plasma, kidney and heart. Under our experimental conditions, absorption appears to play an important role in the regulation of the tissue distribution of Se.

Animals↗

PubMed1996

Lipid status in a population of Spanish schoolchildren.

We assessed lipid status in a population of 181 schoolchildren and adolescents divided into three age groups: 6-7 years (n = 60), 10-12 years (n = 61) and 13-15 years (n = 60). All subjects were from the Mediterranean coastal area of Vélez-Málaga in southern Spain. Nutrient intakes and biochemical parameters related with lipid status [total cholesterol (TC), HDL-chol, LDL-chol, VLDL-chol and triglycerides (TG)] were measured. Boys and girls aged 6-7 years had the same plasma concentrations of TC and TG; these concentrations were slightly higher than the mean values for age and sex found in earlier studies in Spanish populations. The high TC values were due mainly to increased concentrations of LDL-chol. This age group also had the largest percentage of members (26.6% boys, 36.6% girls) with plasma TC above 200 mg/dl. Of the groups aged 10-12 years, more girls than boys had abnormally high plasma cholesterol (16.1 vs 10%), HDL-chol (6.5 vs 0%) and TG concentrations (6.5 vs 0%). However, more adolescent boys than adolescent girls (aged 13-15 years) had abnormally low plasma concentrations of HDL-chol (10 vs 3.3%).

Adolescent↗

PubMed1996

[Unilateral spontaneous adrenal hematoma: an unusual cause of retroperitoneal hemorrhage].

Presentation of one case of spontaneous retroperitoneal haemorrhage in a 72 year-old male, his first symptom being a left retrothoracic pain of sudden onset. The supplementary studies performed (ultrasound, CAT and MNR) pointed to the adrenal gland as the origin of the haemorrhage. Faced eith the uncertainty of the etiological diagnosis, a left supra-renalectomy was conducted which confirmed the pathological anatomy of the piece and the presence of massive haematoma of the suprarenal gland with no other pathological data. The clinical, diagnostic and therapeutical aspects are discussed.

Adrenal Gland Diseases↗

PubMed1996

Influence of dietary fish on fatty acid composition of the erythrocyte membrane in coronary heart disease patients.

This study examines the effects of dietary fish oil on the fatty acid composition of the erythrocyte membrane in persons with coronary heart disease. Thirty men with myocardial infarction were studied during three consecutive periods, during which they consumed 1) their habitual diet, 2) a diet specially designed to prevent ischaemic cardiopathy, and 3) a diet rich in polyunsaturated fat, in which fish was substituted for meat. At the end of each diet, fatty acid concentrations in the erythrocyte membrane were measured, and a 48 h recall dietary questionnaire was used to determine the types of foods consumed. Despite the increase in the intake of (n-3) polyunsaturated fatty acids (PUFA), the fatty acid composition of the erythrocyte membrane failed to reflect the greater dietary supply of eicosapentaenoic acid, and showed increased concentrations of stearic (C16) and palmitic (C18) acid. The increase in dietary (n-3) polyunsaturated fatty acids markedly increased 22:4 (n-6), 22:5 (n-6), 22:5 (n-3) and 22:6 (n-3) fatty acids.

Adult↗

PubMed1995

Changes in tissue calcium and phosphorus content and plasma concentrations of parathyroid hormone and calcitonin after long-term magnesium deficiency in rats.

OBJECTIVE: The present study was undertaken to investigate the effect of dietary magnesium (Mg) deficiency on plasma levels of parathyroid hormone (PTH) and calcitonin (CT), and the changes in tissue calcium (Ca) and phosphorus (P) content. METHODS: Wistar rats were fed an Mg-deficient diet. Plasma levels of PTH and CT, and Ca and P content in plasma, whole blood, skeletal muscle, heart, kidney, femoral bone and brain, were determined after 7, 35, 42, 49, 56, 63 and 70 days. RESULTS: Mg deficiency significantly increased plasma levels of ionic Ca after 1 week of deficiency, and significantly increased total Ca in plasma and whole blood after 5 weeks. Whole blood and plasma levels of P decreased significantly after 8 and 5 weeks, respectively, in comparison with values obtained in pair-fed control rats. Plasma levels of CT were not modified by hypercalcemia and hypomagnesemia. Plasma PTH decreased significantly during the first 3 weeks, then recovered gradually, reaching control values in week 7 and remaining stable thereafter, despite elevated levels of ionic Ca. The Mg-deficient diet led to calcification in skeletal muscle, heart, kidney and femur, detectable as early as week 5. However, P content increased only in kidney after 1 week, decreased in heart and femur after 5 weeks, and did not change in muscle. The Mg-deficient diet did not significantly modify Ca or P content in brain. CONCLUSION: Mg deficiency led to tissue calcification and decreased P levels in blood, heart, and femur. These changes did not affect CT secretion. PTH appeared to be more closely related with hypomagnesemia than with hypercalcemia.

Animals↗

PubMed1995

Influence of dietary supplementation with fish on plasma fatty acid composition in coronary heart disease patients.

The effects of dietary supplementation with fish on plasma fatty acid levels were studied in 20 coronary heart disease patients who had suffered acute myocardial infarction. The study was divided into three periods: hospital admission, after 8 weeks on a heart-healthy diet designed for patients with ischemic heart disease, and after 4 weeks on an n-3 fatty acid-supplemented diet in which red meat was replaced with lean and fatty fish. At the end of each period, the subjects responded to a 48-hour recall questionnaire, so that we could assess their compliance with the diet, and blood samples were collected for the determination of plasma fatty acids. Stearic fatty acid was significantly decreased after the fish diet. n-7 and n-9 fatty acids showed no significant changes throughout the study. At the end of the 4-week period when the fish diet was consumed, linoleic acid and its long-chain derivative docosapentaenoic acid (22:5 n-6) were significantly increased. The most notable changes in n-3 series fatty acids at the end of the third period were the significant decrease in linolenic acid and the significant increase in its long-chain derivatives eicosapentaenoic (20:5 n-3) and docosahexaneoic acid (22:6 n-3). These changes in plasma fatty acid levels may have beneficial effects on coronary heart disease.

Adult↗

PubMed1995

[Priapism after intracavernous injection of PgE1 in two cases of impotence following total cysto-prostatectomy].

The authors report two cases of priapism of more than six hours, following intracavernous injection of 15 and 10 mcg PgE1 respectively, in six patients with impotence after radical cystoprostatectomy. Bearing in mind the proven safety of intracavernous injection of PgE1 in the treatment and diagnosis of erectile dysfunction, such a proportion of priapism in this group of patients has called our attention. It is probably due to an increased in the PgE1 intracavernous metabolism, due to the ligation of the penile venous drainage during surgery.

Alprostadil↗

PubMed1994

Two separate plasma membrane Ca2+ carriers participate in receptor-mediated Ca2+ influx in rat hepatocytes.

The plasma membrane Ca2+ carrier system involved in receptor-mediated Ca2+ entry was studied. Using the Ca2+ readdition protocol, the rate of cytosolic free Ca2+ concentration ([Ca2+]i) increase in vasopressin-pretreated hepatocytes was significantly higher than in thapsigargin- or 2,5-di(tert-butyl)hydroquinone-pretreated cells. The addition of Mn2+ to unstimulated hepatocytes resulted in a biphasic quench of fura-2 fluorescence. After an initial phase that was fast in rate but of short duration, the rate of fura-2 quench by Mn2+ became much slower and lasted until all the cellular fura-2 was quenched. Pretreatment of the cells with vasopressin only accelerated the rate of the latter phase but not of the initial one. In agonist-stimulated cells, acidification of the extracellular medium or the presence of ruthenium red, econazole or SK&F 96365 decreased the rates of both [Ca2+]i increase and Mn2+ entry upon addition of the respective cation. By contrast, neomycin and N-tosyl-L-phenylalanine chloromethyl ketone markedly decreased the rate of [Ca2+]i increase upon Ca2+ readdition but had no effect on vasopressin-stimulated Mn2+ entry. None of the treatments affected the ability of vasopressin and thapsigargin to mobilize the internal Ca2+ store. It is concluded that in hepatocytes the two pathways of receptor-mediated Ca2+ entry control two distinct yet pharmacologically related cation carriers.

Animals↗

PubMed1994

Receptor-mediated Mn2+ influx in rat hepatocytes: comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt.

In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.

Animals↗

PubMed1994

Iron status in a population of Spanish schoolchildren.

Iron status was assessed in a population of 196 schoolchildren and adolescents divided into three age groups: 6-7 years (n = 60), 10-12 years (n = 63) and 13-15 years (n = 73). All subjects were from the Vélez-Málaga area (province of Málaga) in southern Spain. Nutrient intakes and biochemical parameters related with iron status (number of red cells, hematocrit, hemoglobin, serum iron concentration, total iron-binding capacity and transferrin saturation) were measured. The results showed a lack of correlation between the nutrient intake and biochemical findings. Food intake calculations suggested that adolescent girls between 13 and 15 years of age were at greatest risk for iron deficiency (intakes covered only 71.6% and 84% of the iron and folic acid RDAs, respectively), followed by 10-12 year old girls and 6-7 year old boys. However, groups in which iron deficiency was most frequent according to the biochemical data were 6-7 year old boys (10%), followed by 13-15 year old adolescent boys (2.9%).

Adolescent↗

PubMed1994

The droplet technique: measurement of calcium extrusion from single isolated mammalian cells.

This paper contains a description of the modified droplet technique that is designed to monitor Ca2+ extrusion from single isolated pancreatic acinar cells. A cell loaded with calcium indicator is maintained in a small droplet of solution containing another calcium indicator. Differences in the optical properties of the intracellular and extracellular indicators allows one to monitor simultaneously intracellular and extracellular calcium concentrations. The paper contains a description of the calibration procedure that is used to calculate intracellular and extracellular calcium concentrations. The advantages and disadvantages of different pairs of extracellular and intracellular indicators are discussed.

Calcium↗

PubMed1994

Cyclic AMP stimulates Ca2+ entry in rat hepatocytes by interacting with the plasma membrane carriers involved in receptor-mediated Ca2+ influx.

The regulation of Ca2+ influx in rat hepatocytes by glucagon and cyclic AMP (cAMP) was investigated. Exposing hepatocytes to glucagon resulted in an increase in the initial rate of Ca2+ entry. The concentrations of glucagon producing half-maximal and maximal stimulation of Ca2+ entry were 10(-10) and 10(-8) M, respectively. A similar stimulation of Ca2+ influx was obtained in cells exposed to cAMP analogues or to forskolin. Exposing hepatocytes suspended in nominally Ca(2+)-free medium to glucagon for 3 min produced a 9% decrease in the size of the vasopressin-sensitive Ca2+ pool; in contrast, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2cAMP) slightly augmented the size of this pool. Glucagon and Bt2cAMP synergized the initial vasopressin-stimulated Ca2+ and Mn2+ influx rates, but only moderately increased the initial rate of Ca2+ entry after thapsigargin addition. The glucagon- and Bt2cAMP-stimulated Ca2+ influx was inhibited by the same antagonists of the plasma membrane Ca2+ carriers that mediate Ca2+ entry during stimulation by vasopressin. Thus, cAMP does not stimulate Ca2+ entry through either a capacitative type of mechanism or inositol phosphate turnover. The authors' findings instead suggest that cAMP acts directly, or through protein kinase A on the same Ca2+ carriers that are activated by phospholipase C-linked receptor agonists.

Animals↗