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E Chacon

Publications and source records attributed to E Chacon.

15 recordsLinked to original sources
PubMed2005

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods.

There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.

Animals↗
PubMed2000

Aggregation models at high packing fraction

Dense phases of micellar aggregates have strong molecular correlation at two different levels: that of the molecules forming a micelle and that between micelles, leading to a possible phase transition from a micellar fluid to a micellar crystal. The global phase diagram may also include lamellar and other dense phases, which do not have a micellar structure. We present here a generic approach to deal with these systems through a two-level density-functional description, to first describe an isolated micellar aggregate and then the dense micellar system, obtaining the free energy in a self-consistent way from the molecular interactions. Nonmicellar dense phases are included with the same density-functional approach applied at the first level. The results are shown to be very accurate for a one-dimensional model with exact solution, and the method is then applied to a three-dimensional amphiphile model that had been successfully used to describe the properties of diluted amphiphile solutions.

Journal Article↗
PubMed1999

High throughput liquid chromatography-mass spectrometry assessment of the metabolic activity of commercially available hepatocytes from 96-well plates.

We have assessed the metabolic activity of freshly isolated and commercially preserved rat, monkey, and human primary hepatocytes in a 96 well plate format utilizing eight beta-adrenolytic drugs as model compounds. Sample introduction from 96 well plates, HPLC solvent delivery, mass spectrometric (MS) detection, and/or UV detection were fully integrated and operated unattended. After drugs were incubated with hepatocytes for three or six hr, LC-MS analyses were carried out to determine the amount of drug which was not metabolized. Two LC-MS methods were used which had a sample throughput of 4 samples/hr and 12 samples/hr. Under optimal conditions, this hepatic assay could screen 300 samples/week or 1200 samples/month. Although freshly isolated hepatocytes were more active, commercially available rat, monkey, and human primary isolated hepatocytes metabolized drug substrates in similar relative rank orders. This drug-hepatocyte assay provides useful information for prioritizing pharmaceutical leads in relative rank orders or in a high/low manner according to their resistance toward liver metabolism.

Animals↗
PubMed1998

Mitochondrial Ca2+ transients in cardiac myocytes during the excitation-contraction cycle: effects of pacing and hormonal stimulation.

Using laser scanning confocal microscopy, our objective was to measure mitochondrial, nuclear, and cytosolic free ionized Ca2+ in adult rabbit cardiac myocytes loaded with Ca2+-indicating fluorophores. When myocytes were loaded with Fluo 3 at 37 degrees C, the fluorophore was loaded extensively into the cytosol and nucleus, but poorly into mitochondria, and Fluo 3 fluorescence transients after field stimulation were confined to the cytosol and nucleus. In contrast, after loading at 4 degrees C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 microM) increased the magnitude of Ca2+ transients and their subsequent rate of decay, an effect more marked in the cytosol and nucleus than in mitochondria. As pacing frequency was increased from 0.5 to 2 Hz, diastolic mitochondrial Ca2+ rose markedly in the absence but not in the presence of isoproterenol. Resting Ca2+ estimated by Indo 1 ratio imaging using UV/visible laser scanning confocal microscopy was about 200 nM in all compartments. During field stimulation, Ca2+ transiently increased to 671, 522, and 487 nM in cytosol, interfibrillar mitochondria, and perinuclear mitochondria, respectively. Isoproterenol increased these respective peak values to 1280, 750, and 573 nM. These results were consistent with those obtained in Fluo 3 experiments. We conclude that rapid mitochondrial Ca2+ transients occur during excitation-contraction coupling in adult rabbit cardiac myocytes, which may be important in matching mitochondrial metabolism to myocardial ATP demand during changes in cardiac output.

Aniline Compounds↗
PubMed1996

Mitochondrial free calcium transients during excitation-contraction coupling in rabbit cardiac myocytes.

Mitochondrial free Ca2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co-loaded with Fluo-3 to measure free Ca2+ and tetramethylrhodamine methylester to identify mitochondria, we measured cytosolic and mitochondrial Ca2+ transients during the contractile cycle. In resting cells, cytosolic and mitochondrial Fluo-3 signals were similar. During electrical pacing, transients of Fluo-3 fluorescence occurred in both the cytosolic and mitochondrial compartments. Both the mitochondrial and the cytosolic transients were potentiated by isoproterenol. These experiments show directly that mitochondrial free Ca2+ rises and falls during excitation-contraction coupling in cardiac myocytes and that changes of mitochondrial Ca2+ are kinetically competent to regulate mitochondrial metabolism on a beat-to-beat basis.

Aniline Compounds↗
PubMed1996

The pH paradox in ischemia-reperfusion injury to cardiac myocytes.

During myocardial ischemia, a large reduction of tissue pH develops, and tissue pH returns to normal after reperfusion. In recent studies, we evaluated the role of pH in ischemia/reperfusion injury to cultured cardiac myocytes and perfused papillary muscles. Acidosis (pH < or = 7.0) protected profoundly against cell death during ischemia. However, the return from acidotic to normal pH after reperfusion caused myocytes to lose viability. This worsening of injury is a 'pH paradox' and was mediated by changes of intracellular pH (pH(i)), since manipulations that caused pH(i), to increase more rapidly after reperfusion accelerated cell killing, whereas manipulations that delayed the increase of pH(i) prevented loss of myocyte viability. Specifically, inhibition of the Na+/H+ exchanger with dimethylamiloride or HOE694 delayed the return of physiologic pH(i) after reperfusion and prevented reperfusion-induced cell killing to both cultured myocytes and perfused papillary muscle. Dimethylamiloride and HOE694 did not reduce intracellular free Ca2+ during reperfusion. By contrast, reperfusion with dichlorobenzamil, an inhibitor of Na+/Ca2+ exchange, decreased free Ca2+ but did not reduce cell killing. Thus, the pH paradox is not Ca(2+)-dependent. Our working hypothesis is that ischemia activates hydrolytic enzymes, such as phospholipases and proteases, whose activity is inhibited at acidotic pH. Upon reperfusion, the return to normal pH releases this inhibition and hydrolytic injury ensues. Increasing pH(i) may also induce a pH-dependent mitochondrial permeability transition and activate the myofibrillar ATPase, effects that increase ATP demand and compromise ATP supply. In conclusion, acidotic pH is generally protective in ischemia, whereas a return to physiologic pH precipitates lethal reperfusion injury to myocytes.

Animals↗
PubMed1994

Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study.

Exploiting the optical sectioning capabilities of laser scanning confocal microscopy and using parameter-specific fluorescent probes, we determined the distribution of pH, free Ca2+, electrical potential, and volume inside cultured adult rabbit cardiac myocytes during ATP depletion and reductive stress with cyanide and 2-deoxyglucose ("chemical hypoxia"). During normoxic incubations, myocytes exhibited a cytosolic pH of 7.1 and a mitochondrial pH of 8.0 (delta pH = 0.9 units). Sarcolemmal membrane potential (delta psi) was -80 mV, and mitochondrial delta psi was as high as -100 mV, yielding a mitochondrial protonmotive force (delta p) of -155 mV (delta P = delta psi - 60 delta pH). After 30 min of chemical hypoxia, mitochondrial delta pH decreased to 0.5 pH units, but mitochondrial delta psi remained essentially unchanged. By 40 min, delta pH was collapsed, and mitochondrial and cytosolic free Ca2+ began to increase. Mitochondrial and sarcolemmal delta psi remained high. as Ca2+ rose, myocytes shortened, hypercontracted, and blebbed with a 30% decrease of cell volume. After hypercontraction, extensive mitochondrial Ca2+ loading occurred. After another few minutes, mitochondrial depolarized completely and released their load of Ca2+. After many more minutes, the sarcolemmal permeability barrier broke down, and viability was lost. These studies demonstrate a sequence of subcellular ionic and electrical changes that may underlie the progression to irreversible hypoxic injury.

Animals↗
PubMed1993

Intracellular pH and Ca2+ homeostasis in the pH paradox of reperfusion injury to neonatal rat cardiac myocytes.

Ischemia is characterized by anoxia and a large decrease of tissue pH. After a critical period of ischemia, reperfusion precipitates irreversible injury. Previous work showed that reperfusion injury to cultured neonatal myocytes was precipitated by a rapid return to physiological pH, a "pH paradox" (Bond, J., B. Herman, and J. Lemasters. Biochem. Biophys. Res. Commun. 179: 798-803, 1991). The aim of this study was to measure intracellular pH (pHi) and cytosolic free Ca2+ during the pH paradox of reperfusion injury to cultured neonatal rat cardiac myocytes. pHi and free Ca2+ were measured by ratio imaging of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and fura 2 fluorescence. To simulate ATP depletion and acidosis of ischemia, myocytes were incubated with 20 mM 2-deoxyglucose plus 2.5 mM NaCN at pH 6.2. During simulated ischemia, pHi dropped to < 6.5 and subsequently remained constant. During this time, some blebbing but little hypercontraction occurred. After 3 or 4 h of simulated ischemia, inhibitors were removed and cells were incubated at pH 7.4 to simulate reperfusion. pHi began to increase, blebbing accelerated, and myocytes hypercontracted. As pHi increased, viability was lost. The same occurred if pH was increased but metabolic inhibitors were not removed. Monensin, a Na(+)-H+ ionophore, accelerated the increase of pH after reperfusion and hastened cell killing. Hypercontraction, blebbing, and loss of viability did not occur when inhibitors were removed at pH 6.2 or in the presence of dimethylamiloride, an inhibitor of Na(+)-H+ exchange. Protection was associated with maintenance of an acidotic pHi. Free Ca2+ progressively increased during simulated ischemia. After simulated reperfusion, free Ca2+ increased further.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1992

A digitized-fluorescence-imaging study of mitochondrial Ca2+ increase by doxorubicin in cardiac myocytes.

The objective of the present study was to investigate the role of mitochondrial Ca2+ in doxorubicin-induced cell injury. The effect of doxorubicin on cultured cells was investigated by digitized fluorescence imaging. The Ca2+ sensitive fluorescent dye fura-2 was used to estimate cytosolic, mitochondrial and total cellular Ca2+. Rhodamine 123 was used to estimate the mitochondrial membrane potential, and cellular ATP was determined by h.p.l.c. The data showed that doxorubicin induced greater-than-2-fold increases in mitochondrial Ca2+ before changes in cytosolic Ca2+ could be detected. An increase in mitochondrial Ca2+ paralleled the observed dissipation in mitochondrial membrane potential. Cellular ATP levels appeared to decrease as a result of mitochondrial dysfunction, which in turn produced greater-than-2-fold increases in cytosolic Ca2+. The data suggest that doxorubicin-induced alterations in mitochondrial Ca2+ homoeostasis are associated with a dissipation in energy conservation, which may result in cell injury.

Adenosine Triphosphate↗
PubMed1992

Radiation-induced syntheses in cometary simulated models.

The behavior of an aqueous-dominant multicomponent cometary model is examined at high doses of ionizing radiation. The system is composed of a water mixture of HCN (0.2 mol dm-3), CH3CN (0.04 mol dm-3), C2H5CN (0.02 mol dm-3), CH3OH (0.12 mol dm-3) and HCO2H (0.01 mol dm-3. It was exposed to gamma rays at doses up to 18.5 MGy. The chemical kinetic database used in the computer treatment of experimental data consists of 79 reactions. A complex mixture of products has been synthesized: gases, amino acids, carboxylic acids and polymeric material. The results suggest that the pristine material in cometary nuclei may have been chemically altered by the action of cosmic rays and embedded radionuclides.

Amino Acids↗
PubMed1991

Mitochondrial regulation of superoxide by Ca2+: an alternate mechanism for the cardiotoxicity of doxorubicin.

Mitochondrial Ca2+ accumulation and the formation of reactive oxygen species are processes dependent on the electron transport system. The production of superoxide by respiring rat heart mitochondria was decreased by either chelating extramitochondrial Ca2+ with EGTA or by blocking mitochondrial Ca2+ uptake with ruthenium red. Mitochondrial experiments with doxorubicin showed an enhanced stimulation of reactive oxygen species, which was also inhibited by EGTA or ruthenium red. Myocardial cell cultures treated with doxorubicin showed an enhanced formation of intracellular reactive oxygen species, which preceded cell damage. Ruthenium red not only attenuated the enhanced formation of intracellular reactive oxygen species, but also increased cell viability. The relationship between mitochondrial Ca2+ transport and the formation of superoxide suggests that a disruption in mitochondrial Ca2+ homeostasis by doxorubicin may be involved in the release of reactive oxygen species and its cardiotoxicity.

Animals↗
PubMedDate not supplied

Inhibition of Na+/H+ exchange preserves viability, restores mechanical function, and prevents the pH paradox in reperfusion injury to rat neonatal myocytes.

Rat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors--a 'pH paradox'. In this study, we evaluated the effect of two Na+/H+ exchange inhibitors (dimethylamiloride and HOE694) and a Na+/Ca2+ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca2+ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30-60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca2+ began to increase uniformly throughout the cell. Free Ca2+ reached levels estimated to exceed 2 microM by 4 h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca2+ in dichlorobenzamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 microM dimethylamiloride or 20 microM HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca2+ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca2+ transients. Our results indicate that Na+/H+ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.

Amiloride↗