Papers by Sergio de la Fuente
![Research paper thumbnail of Dynamics of mitochondrial [Ca2+] measured with the low-Ca2+-affinity dye rhod-5N](https://attachments.academia-assets.com/48632292/thumbnails/1.jpg)
Available methods to measure mitochondrial [Ca 2+ ] ([Ca 2+ ] M ) include both targeted proteins ... more Available methods to measure mitochondrial [Ca 2+ ] ([Ca 2+ ] M ) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca 2+ ] M values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca 2+ -affinity dye rhod-5N provides [Ca 2+ ] M values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca 2+ -affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5 mM. Addition of Ca 2+ buffers containing between 4.5 and 10 M [Ca 2+ ] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca 2+ ] M up to the 100 M-1 mM range, which were dependent on mitochondrial membrane potential. Ca 2+ release from mitochondria was largely dependent on [Na + ]. We have then used rhod-5N loaded cells to investigate the [Ca 2+ ] M response to agonist stimulation at the single-cell and subcellular level.The [Ca 2+ ] M peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25 M. In the presence of the Ca 2+ uniporter stimulator kaempferol, the [Ca 2+ ] M peaks induced by histamine were also highly variable, and the mean [Ca 2+ ] M peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca 2+ ] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca 2+ ] M peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca 2+ ] M increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.
Biophysical Journal, 2016
Diagnosticos Enfermeros En Atencion Primaria Aplicacion Practica Jornadas De Enfermeria Comunitaria En Castilla Y Leon Ponferrada 17 Y 18 De Noviembre De 2000 2000 Isbn 84 7719 918 3 Pags 225 226, 2001
Información del artículo Publicaciones sobre diagnósticos de enfermería: ¿interesan?, ¿cuánto?, ¿... more Información del artículo Publicaciones sobre diagnósticos de enfermería: ¿interesan?, ¿cuánto?, ¿evolucionan?
Acta Obstetricia Et Gynecologica Scandinavica, Aug 1, 2001
To examine fetal outcome in hydrops fetalis in relation to gestational age at diagnosis and follo... more To examine fetal outcome in hydrops fetalis in relation to gestational age at diagnosis and following investigation and treatment. All cases of hydrops fetalis presenting to the Fetal Medicine Unit during the last seven years, between 1993 and 1999, were identified from the Fetal Medicine Database. During this time 87 of 13,980 patients who attended the Fetal Medicine Unit had hydrops fetalis. The cases were examined for gestational age at presentation according to etiology and fetal survival following investigation and treatment. The fetal survival rates for non-immune cases of hydrops before and after 24 weeks were compared. The cause of hydrops was determined antenatally in 71 of the 87 (82%) cases. Of the 51 cases presenting before 24 weeks' gestation, 23 (45%) were due to chromosomal abnormality. After 24 weeks, fetal tachyarrhythmias and hydrothorax were the most common causes and accounted for 14 (38%) of the 36 cases. Thirty-four cases (39%) of hydrops received intrauterine treatment. The survival rates excluding chromosomal abnormalities in the non-immune cases before and after 24 weeks' gestation were 31% and 48%, respectively, and were not significantly different. The survival rate in cases of hydrops fetalis may be improved with appropriate prenatal investigation and therapy. The etiology of hydrops is different before and after 24 weeks, and even when cases of chromosomal abnormality are excluded the survival rate is similar before and after 24 weeks.
The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians, Jan 26, 2016
To evaluate prenatal predictors of postnatal survival in fetuses with agenesis of ductus venosus ... more To evaluate prenatal predictors of postnatal survival in fetuses with agenesis of ductus venosus (ADV). This retrospective study reviewed our experience and the literature between 1991 and 2015. Prenatal findings were evaluated and perinatal morbidity and mortality was documented. A total of 259 cases were included in the present analysis from our centers and 49 published studies (15 patients from our retrospective cohort review and 244 from literature review). The intrahepatic and extrahepatic shunts were present in 32.0% (73/226) and 67.7% (153/226), respectively. Cardiomegaly (n=64/259, 24.7%), hydrops (n=31/259, 12.0%) and amniotic fluid abnormalities (n=22/259, 8.5%) were among the most frequent initial ultrasound findings. 147 fetuses (56.8%) had ADV without structural anomalies while 112 (43.2%) had associated anomalies (cardiac anomalies (n=66), extra-cardiac anomalies (n=19), and both cardiac and extra-cardiac anomalies (n=27). The mean gestational age at ultrasound diagnos...
Revista chilena de obstetricia y ginecología
ABSTRACT
PLoS ONE, 2014
The effect of the intake of antioxidant polyphenols such as resveratrol and others on survival an... more The effect of the intake of antioxidant polyphenols such as resveratrol and others on survival and different parameters of life quality has been a matter of debate in the last years. We have studied here the effects of the polyphenols resveratrol and kaempferol added to the diet in a murine model undergoing long-term hypercaloric diet. Using 50 mice for each condition, we have monitored weight, survival, biochemical parameters such as blood glucose, insulin, cholesterol, triglycerides and aspartate aminotransferase, neuromuscular coordination measured with the rotarod test and morphological aspect of stained sections of liver and heart histological samples. Our data show that mice fed since they are 3-months-old with hypercaloric diet supplemented with any of these polyphenols reduced their weight by about 5-7% with respect to the controls fed only with hypercaloric diet. We also observed that mice fed with any of the polyphenols had reduced levels of glucose, insulin and cholesterol, and better marks in the rotarod test, but only after 1 year of treatment, that is, during senescence. No effect was observed in the rest of the parameters studied. Furthermore, although treatment with hypercaloric diets induced large changes in the pattern of gene expression in liver, we found no significant changes in gene expression induced by the presence of any of the polyphenols. Thus, our data indicate that addition of resveratrol or kaempferol to mice food produces an initial decrease in weight in mice subjected to hypercaloric diet, but beneficial effects in other parameters such as blood glucose, insulin and cholesterol, and neuromuscular coordination, only appear after prolonged treatments.
Obstetrics & Gynecology, 2005
To report our experience with the management of twin pregnancies discordant for lower urinary tra... more To report our experience with the management of twin pregnancies discordant for lower urinary tract obstruction. Cases of twin pregnancies discordant for lower urinary tract obstruction were identified from our fetal medicine database. Information on ultrasonographic findings, antenatal course, pregnancy complications, and perinatal outcome was obtained by reviewing medical records or contacting the referring obstetricians. Five twin pregnancies discordant for lower urinary tract obstruction were diagnosed between 11 and 15 weeks of gestation. There were 3 dichorionic and 2 monochorionic pregnancies (1 diamniotic and 1 monoamniotic). The dichorionic pregnancies were managed conservatively, resulting in a pregnancy loss of both twins in 1 case, a single fetal death at 29 weeks in 1 case, and an early neonatal death due to lung hypoplasia of the affected twin in 1 case. On the other hand, both monochorionic twin pregnancies were managed with serial vesicocenteses. In both cases, the prenatal course was complicated, 1 by premature rupture of the membranes and the other by cord entanglement, requiring delivery at 29 and 31 weeks, respectively. Among the 4 continuing pregnancies with complete perinatal outcome, none of the affected twins survived, and the structurally normal twins were delivered between 29 and 36 weeks and discharged from the hospital in good condition. Twin pregnancies discordant for lower urinary tract obstruction are at high risk of perinatal death and premature delivery. Prenatal intervention seems not to be associated with an improved perinatal outcome of the affected twin, but it may be beneficial in selected cases to attain viability of the unaffected twin.
Neurobiology of Disease, 2013
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![Research paper thumbnail of Dynamics of mitochondrial [Ca2+] measured with the low-Ca2+-affinity dye rhod-5N](https://attachments.academia-assets.com/48632317/thumbnails/1.jpg)
Cell Calcium, 2012
Available methods to measure mitochondrial [Ca 2+ ] ([Ca 2+ ] M ) include both targeted proteins ... more Available methods to measure mitochondrial [Ca 2+ ] ([Ca 2+ ] M ) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca 2+ ] M values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca 2+ -affinity dye rhod-5N provides [Ca 2+ ] M values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca 2+ -affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5 mM. Addition of Ca 2+ buffers containing between 4.5 and 10 M [Ca 2+ ] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca 2+ ] M up to the 100 M-1 mM range, which were dependent on mitochondrial membrane potential. Ca 2+ release from mitochondria was largely dependent on [Na + ]. We have then used rhod-5N loaded cells to investigate the [Ca 2+ ] M response to agonist stimulation at the single-cell and subcellular level.The [Ca 2+ ] M peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25 M. In the presence of the Ca 2+ uniporter stimulator kaempferol, the [Ca 2+ ] M peaks induced by histamine were also highly variable, and the mean [Ca 2+ ] M peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca 2+ ] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca 2+ ] M peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca 2+ ] M increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.
Cell Calcium, 2013
We use here a new very low-Ca 2+ -affinity targeted aequorin to measure the [Ca 2+ ] in the endop... more We use here a new very low-Ca 2+ -affinity targeted aequorin to measure the [Ca 2+ ] in the endoplasmic reticulum ([Ca 2+ ] ER ). The new aequorin chimera has the right Ca 2+ -affinity to make long-lasting measurements of [Ca 2+ ] ER in the millimolar range. Moreover, previous Ca 2+ -depletion of the ER is no longer required. The steady-state [Ca 2+ ] ER obtained is 1-2 mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca 2+ ] ER among different regions of the ER. About half of the ER had a [Ca 2+ ] ER of 1 mM or below, and the rest had [Ca 2+ ] ER values above 1 mM and in some parts even above 2 mM. About 5% of the ER was also found to have high [Ca 2+ ] ER levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca 2+ of the ER was almost linearly dependent on the extracellular [Ca 2+ ] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca 2+ ] microdomains connecting the store operated Ca 2+ channels with the ER Ca 2+ pumps may be important during refilling.
![Research paper thumbnail of Monitoring mitochondrial [Ca2+] dynamics with rhod-2, ratiometric pericam and aequorin](https://attachments.academia-assets.com/48632322/thumbnails/1.jpg)
Cell Calcium, 2010
The dynamics of mitochondrial [Ca 2+ ] ([Ca 2+ ] M ) plays a key role in a variety of cellular pr... more The dynamics of mitochondrial [Ca 2+ ] ([Ca 2+ ] M ) plays a key role in a variety of cellular processes. The most important methods available to monitor [Ca 2+ ] M are fluorescent dyes such as rhod-2 and specifically targeted proteins such as aequorin and pericam. However, significant discrepancies, both quantitative and qualitative, exist in the literature between the results obtained with different methods. We have made here a systematic comparison of the response of several fluorescent dyes, rhod-2 and rhod-FF, and two Ca 2+ -sensitive proteins, aequorin and pericam. Our results show that measurements obtained with aequorin and pericam are consistent in terms of dynamic Ca 2+ changes. Instead, fluorescent dyes failed to follow Ca 2+ changes adequately, especially during repetitive stimulation. In particular, measures obtained with rhod-2 or rhod-FF evidenced the previously reported Ca 2+ -dependent inhibition of mitochondrial Ca 2+ uptake, but data obtained with aequorin or pericam under the same conditions did not. The reason for the loss of response of fluorescent dyes is unclear. Loading with these dyes produced changes in mitochondrial morphology and membrane potential, which were small and reversible at low concentrations (1-2 M), but produced large and prolonged damage at higher concentrations. In addition, cells loaded with low concentrations of rhod-2 suffered large changes in mitochondrial morphology after light excitation. Our results suggest that [Ca 2+ ] M data obtained with these dyes should be taken with care.
Biochemical Journal, 2014
MICU1 (Ca2+ uptake protein 1, mitochondrial) is an important regulator of the MCU (Ca2+ uniporter... more MICU1 (Ca2+ uptake protein 1, mitochondrial) is an important regulator of the MCU (Ca2+ uniporter protein, mitochondrial) that has been shown recently to act as a gatekeeper of the MCU at low [Ca2+]c (cytosolic [Ca2+]). In the present study we have investigated in detail the dynamics of MCU activity after shRNA-knockdown of MICU1 and we have found several new interesting properties. In MICU1-knockdown cells, the rate of mitochondrial Ca2+ uptake was largely increased at a low [Ca2+]c (<2 μM), but it was decreased at a high [Ca2+]c (>4 μM). In the 2-4 μM range a mixed behaviour was observed, where mitochondrial Ca2+ uptake started earlier in the MICU1-silenced cells, but at a lower rate than in the controls. The sensitivity of Ca2+ uptake to Ruthenium Red and Ru360 was similar at both high and low [Ca2+]c, indicating that the same Ca2+ pathway was operating in both cases. The increased Ca2+-uptake rate observed at a [Ca2+]c below 2 μM was transient and became inhibited during Ca2+ entry. Development of this inhibition was slow, requiring 5 min for completion, and was hardly reversible. Therefore MICU1 acts both as a MCU gatekeeper at low [Ca2+]c and as a cofactor necessary to reach the maximum Ca2+-uptake rate at high [Ca2+]c. Moreover, in the absence of MICU1, the MCU becomes sensitive to a slow-developing inhibition that requires prolonged increases in [Ca2+]c in the low micromolar range.
Biochemical Journal, 2012
Mitochondria have a very large capacity to accumulate Ca(2+) during cell stimulation driven by th... more Mitochondria have a very large capacity to accumulate Ca(2+) during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca(2+)](M) (mitochondrial [Ca(2+)]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca(2+)](M) during prolonged stimulation has been previously precluded by the high Ca(2+) affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca(2+)] in the millimolar range for long periods of time without problems derived from aequorin consumption. We show in the present study that addition of Ca(2+) to permeabilized HeLa cells triggers an increase in [Ca(2+)](M) up to an steady state of approximately 2-3 mM in the absence of phosphate and 0.5-1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca(2+)] reaches 0.5-1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca(2+)](M) levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca(2+) uniporter largely reduced the rate of [Ca(2+)](M) increase, but the final steady-state [Ca(2+)](M) reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca(2+)](M) without problems derived from aequorin consumption.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
We have investigated the kinetics of mitochondrial Ca 2+ influx and efflux and their dependence o... more We have investigated the kinetics of mitochondrial Ca 2+ influx and efflux and their dependence on cytosolic [Ca 2+ ] and [Na + ] using low-Ca 2+ -affinity aequorin. The rate of Ca 2+ release from mitochondria increased linearly with mitochondrial [Ca 2+ ] ([Ca 2+ ] M ). Na + -dependent Ca 2+ release was predominant al low [Ca 2+ ] M but saturated at [Ca 2+ ] M around 400 μM, while Na + -independent Ca 2+ release was very slow at [Ca 2+ ] M below 200 μM, and then increased at higher [Ca 2+ ] M , perhaps through the opening of a new pathway. Half-maximal activation of Na + -dependent Ca 2+ release occurred at 5-10 mM [Na + ], within the physiological range of cytosolic [Na + ]. Ca 2+ entry rates were comparable in size to Ca 2+ exit rates at cytosolic [Ca 2+ ] ([Ca 2+ ] c ) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca 2+ ] c . As a consequence, the presence of [Na + ] considerably reduced the rate of [Ca 2+ ] M increase at [Ca 2+ ] c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca 2+ ] c . Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca 2+ ] M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca 2+ buffering, and comparison of our results with data on total mitochondrial Ca 2+ fluxes indicate that the mitochondrial Ca 2+ bound/Ca 2+ free ratio is around 10-to 100-fold for most of the observed [Ca 2+ ] M range and suggest that massive phosphate precipitation can only occur when [Ca 2+ ] M reaches the millimolar range.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
tissues, heart, liver and kidney, the PBR is also known to be highly expressed in blood cells. In... more tissues, heart, liver and kidney, the PBR is also known to be highly expressed in blood cells. In this work, we demonstrate by flow cytometry experiments, that SSR180575, at low concentrations, is able to protect polymorphonuclear leukocytes (PMNs) against TNFαinduced apoptosis in whole blood. Thus, in a new context, SSR180575 again shows potent anti-apoptotic properties. Moreover, TNFαinduced PMN apoptosis appears to be a good surrogate marker for determining SSR180575 blood availability and activity in treated patients.
Acta Obstetricia et Gynecologica Scandinavica, 2001
To examine fetal outcome in hydrops fetalis in relation to gestational age at diagnosis and follo... more To examine fetal outcome in hydrops fetalis in relation to gestational age at diagnosis and following investigation and treatment. All cases of hydrops fetalis presenting to the Fetal Medicine Unit during the last seven years, between 1993 and 1999, were identified from the Fetal Medicine Database. During this time 87 of 13,980 patients who attended the Fetal Medicine Unit had hydrops fetalis. The cases were examined for gestational age at presentation according to etiology and fetal survival following investigation and treatment. The fetal survival rates for non-immune cases of hydrops before and after 24 weeks were compared. The cause of hydrops was determined antenatally in 71 of the 87 (82%) cases. Of the 51 cases presenting before 24 weeks' gestation, 23 (45%) were due to chromosomal abnormality. After 24 weeks, fetal tachyarrhythmias and hydrothorax were the most common causes and accounted for 14 (38%) of the 36 cases. Thirty-four cases (39%) of hydrops received intrauterine treatment. The survival rates excluding chromosomal abnormalities in the non-immune cases before and after 24 weeks' gestation were 31% and 48%, respectively, and were not significantly different. The survival rate in cases of hydrops fetalis may be improved with appropriate prenatal investigation and therapy. The etiology of hydrops is different before and after 24 weeks, and even when cases of chromosomal abnormality are excluded the survival rate is similar before and after 24 weeks.
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Papers by Sergio de la Fuente