Ca2+ influx via the Na+/Ca2+ exchanger is enhanced in malignant hyperthermia skeletal muscle.

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca(2+) dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca(2+) depending on cellular activity. Resting intracellular calcium ([Ca(2+)]r) and sodium ([Na(+)]r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na(+)]e elevates [Ca(2+)]r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca(2+) or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca(2+)]r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca(2+)]r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca(2+)]r in MHS muscle fibers and decreases the amplitude of [Ca(2+)]r rise triggered by halothane, but had no effect on [Ca(2+)]r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca(2+) transient elicited by high [K(+)]e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca(2+)]r and the Ca(2+) transient area induced by high [K(+)]e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca(2+) transients associated with K(+)-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.

High-yield production of manganese peroxidase, lignin peroxidase, and versatile peroxidase in Phanerochaete chrysosporium.

The white-rot fungus Phanerochaete chrysosporium secretes extracellular oxidative enzymes during secondary metabolism, but lacks versatile peroxidase, an enzyme important in ligninolysis and diverse biotechnology processes. In this study, we report the genetic modification of a P. chrysosporium strain capable of co-expressing two endogenous genes constitutively, manganese peroxidase (mnp1) and lignin peroxidase (lipH8), and the codon-optimized vpl2 gene from Pleurotus eryngii. For this purpose, we employed a highly efficient transformation method based on the use of shock waves developed by our group. The expression of recombinant genes was verified by PCR, Southern blot, quantitative real-time PCR (qRT-PCR), and assays of enzymatic activity. The production yield of ligninolytic enzymes was up to four times higher in comparison to previously published reports. These results may represent significant progress toward the stable production of ligninolytic enzymes and the development of an effective fungal strain with promising biotechnological applications.

A novel and highly efficient method for genetic transformation of fungi employing shock waves.

Genetic transformation of filamentous fungi is an essential tool in many areas such as biotechnology, medicine, phytopathology and genetics. However, available protocols to transform fungi are inefficient, laborious and have low reproducibility. We report the use of underwater shock waves as a novel method to transform filamentous fungi. An experimental piezoelectric shock wave generator was designed to expose fungal conidia to heterologous DNA. The device was successfully tested in Aspergillus niger, Fusarium oxysporum, Trichoderma reesei and Phanerochaete chrysosporium. The transformation frequency per number of conidia was between two and four orders of magnitude higher in comparison to previously published methods. For example, the frequency of transformation in A. niger was improved up to 5400-fold as compared with Agrobacterium protocols. Transformation was verified by expression of the green fluorescent protein, PCR and Southern blot. Our method offers new possibilities for fast, easy and efficient genetic manipulation of diverse fungal species.

Comparative characterization of nine novel GH51, GH54 and GH62 α-l-arabinofuranosidases from Penicillium subrubescens.

α-l-Arabinofuranosidases (ABFs) are important enzymes in plant biomass degradation with a wide range of applications. The ascomycete fungus Penicillium subrubescens has more α-l-arabinofuranosidase-encoding genes in its genome compared to other Penicillia. We characterized nine ABFs from glycoside hydrolase (GH) families GH51, GH54 and GH62 from this fungus and demonstrated that they have highly diverse specificity and activity levels, indicating that the expansion was accompanied by diversification of the enzymes. Comparison of the substrate preference of the enzymes to the expression of the corresponding genes when the fungus was grown on either of two plant biomass substrates did not show a clear correlation, suggesting a more complex regulatory system governing l-arabinose release from plant biomass by P. subrubescens.

The Role of the Na+/Ca2+ Exchanger in Aberrant Intracellular Ca2+ in Cardiomyocytes of Chagas-Infected Rodents.

Chagas disease is produced by the parasite Trypanosoma cruzi (T. cruzi), which is the leading cause of death and morbidity in Latin America. We have shown that in patients with Chagas cardiomyopathy, there is a chronic elevation of diastolic Ca2+ concentration ([Ca2+]d), associated with deterioration to further address this issue, we explored the role Na+/Ca2+ exchanger (NCX). Experiments were carried out in noninfected C57BL/6 mice and infected with blood-derived trypomastigotes of the T. cruzi Y strain. Anesthetized mice were sacrificed and the cardiomyocytes were enzymatically dissociated. Diastolic [Ca2+] ([Ca2+]d) was measured using Ca2+ selective microelectrodes in cardiomyocytes from control mice (CONT) and cardiomyocytes from T. cruzi infected mice in the early acute phase (EAP) at 20 dpi, in the acute phase (AP) at 40 dpi, and in the chronic phase (CP) at 120 dpi. [Ca2+]d was 1.5-times higher in EAP, 2.6-times in AP, and 3.4-times in CP compared to CONT. Exploring the reverse mode activity of NCX, we replaced extracellular Na+ in equivalent amounts with N-methyl-D-glucamine. Reduction of [Na+]e to 65 mM caused an increase in [Ca2+]d of 1.7 times in cardiomyocytes from CONT mice, 2 times in EAP infected mice, 2.4 times in AP infected mice and 2.8 in CP infected mice. The Na+ free solution caused a further elevation of [Ca2+]d of 2.5 times in cardiomyocytes from CONT, 2.8 times in EAP infected mice, 3.1 times in AP infected mice, and 3.3 times in CP infected mice. Extracellular Ca2+ withdrawal reduced [Ca2+]d in both CONT and cardiomyocytes from Chagas-infected mice and prevented the increase in [Ca2+]d induced by Na+ depletion. Preincubation with 10µM KB-R7943 or in 1µM YM-244769 reduced [Ca2+]d in cardiomyocytes from infected mice, but not control mice. Furthermore, both NCX blockers prevented the increase in [Ca2+]d associated with exposure to a solution without Na+. These results suggest that Ca2+ entry through the reverse NCX mode plays a significant role in the observed [Ca2+]d dyshomeostasis in Chagas infected cardiomyocytes. Additionally, NCX inhibitors may be a viable therapeutic approach for treating patients with Chagas cardiomyopathy.

Recombinant production and characterization of six novel GH27 and GH36 α-galactosidases from Penicillium subrubescens and their synergism with a commercial mannanase during the hydrolysis of lignocellulosic biomass.

α-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular α-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All α-galactosidases exhibited high affinity to pNPαGal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining α-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other α-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal α-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses.

Myceliophthora thermophila Xyr1 is predominantly involved in xylan degradation and xylose catabolism.

BACKGROUND: Myceliophthora thermophila is a thermophilic ascomycete fungus that is used as a producer of enzyme cocktails used in plant biomass saccharification. Further development of this species as an industrial enzyme factory requires a detailed understanding of its regulatory systems driving the production of plant biomass-degrading enzymes. In this study, we analyzed the function of MtXlr1, an ortholog of the (hemi-)cellulolytic regulator XlnR first identified in another industrially relevant fungus, Aspergillus niger. RESULTS: The Mtxlr1 gene was deleted and the resulting strain was compared to the wild type using growth profiling and transcriptomics. The deletion strain was unable to grow on xylan and d-xylose, but showed only a small growth reduction on l-arabinose, and grew similar to the wild type on Avicel and cellulose. These results were supported by the transcriptome analyses which revealed reduction of genes encoding xylan-degrading enzymes, enzymes of the pentose catabolic pathway and putative pentose transporters. In contrast, no or minimal effects were observed for the expression of cellulolytic genes. CONCLUSIONS: Myceliophthora thermophila MtXlr1 controls the expression of xylanolytic genes and genes involved in pentose transport and catabolism, but has no significant effects on the production of cellulases. It therefore resembles more the role of its ortholog in Neurospora crassa, rather than the broader role described for this regulator in A. niger and Trichoderma reesei. By revealing the range of genes controlled by MtXlr1, our results provide the basic knowledge for targeted strain improvement by overproducing or constitutively activating this regulator, to further improve the biotechnological value of M. thermophila.

The presence of trace components significantly broadens the molecular response of Aspergillus niger to guar gum.

Guar gum consists mainly of galactomannan and constitutes the endosperm of guar seeds that acts as a reserve polysaccharide for germination. Due to its molecular structure and physical properties, this biopolymer has been considered as one of the most important and widely used gums in industry. However, for many of these applications this (hemi-)cellulosic structure needs to be modified or (partially) depolymerized in order to customize and improve its physicochemical properties. In this study, transcriptome, exoproteome and enzyme activity analyses were employed to decipher the complete enzymatic arsenal for guar gum depolymerization by Aspergillus niger. This multi-omic analysis revealed a set of 46 genes encoding carbohydrate-active enzymes (CAZymes) responding to the presence of guar gum, including CAZymes not only with preferred activity towards galactomannan, but also towards (arabino-)xylan, cellulose, starch and pectin, likely due to trace components in guar gum. This demonstrates that the purity of substrates has a strong effect on the resulting enzyme mixture produced by A. niger and probably by other fungi as well, which has significant implications for the commercial production of fungal enzyme cocktails.

Enhanced Delignification of Lignocellulosic Biomass by Recombinant Fungus Phanerochaete chrysosporium Overexpressing Laccases and Peroxidases.

Ligninolytic enzyme production and lignin degradation are typically the rate-limiting steps in the biofuel industry. To improve the efficiency of simultaneous bio-delignification and enzyme production, Phanerochaete chrysosporium was transformed by shock wave-induced acoustic cavitation to co-overexpress 3 peroxidases and 1 laccase and test it on the degradation of sugarcane bagasse and wheat bran. Lignin depolymerization was enhanced by up to 25% in the presence of recombinant fungi in comparison with the wild-type strain. Sugar release on lignocellulose was 2- to 6-fold higher by recombinant fungi as compared with the control. Wheat bran ostensibly stimulated the production of ligninolytic enzymes. The highest peroxidase activity from the recombinant strains was 2.6-fold higher, whereas the increase in laccase activity was 4-fold higher in comparison to the control. The improvement of lignin degradation was directly proportional to the highest peroxidase and laccase activity. Because various phenolic compounds released during lignocellulose degradation have proven to be toxic to cells and to inhibit enzyme activity, a significant reduction (over 40%) of the total phenolic content in the samples treated with recombinant strains was observed. To our knowledge, this is the first report that engineering P. chrysosporium enhances biodegradation of lignocellulosic biomass.

Altered Ca2+ homeostasis in human uremic skeletal muscle: possible involvement of cADPR in elevation of intracellular resting [Ca2+].

BACKGROUND: Patients with chronic renal failure may develop muscle weakness and fatigability due to disorders of skeletal muscle function, collectively known as the uremic myopathy. Cyclic adenosine diphosphate-ribose (cADPR), an endogenous metabolite of beta-NAD+, activates Ca2+ release from intracellular stores in vertebrate and invertebrate cells. The current study investigated the possible role of cADPR in uremic myopathy. METHODS: We have examined the effect of cADPR on myoplasmic resting Ca2+ concentration ([Ca2+]i) in skeletal muscle obtained from control subjects and uremic patients (UP). [Ca2+]i was measured using double-barreled Ca2+-selective microelectrodes in muscle fibers, prior to and after microinjections of cADPR. RESULTS: Resting [Ca2+]i was elevated in UP fibers compared with fibers obtained from control subjects. Removal of extracellular Ca2+, or incubation of cells with nifedipine, did not modify [Ca2+]i in UP or control fibers. Microinjection of cADPR produced an elevation of [Ca2+]i in both groups of cells. This elevation was not mediated by Ca2+ influx, or inhibited by heparin or ryanodine. [cADPR]i was determined to be higher in muscle fibers from UP compared to those from the control subjects. Incubation of cells with 8-bromo-cADPR, a cADPR antagonist, partially reduced [Ca2+]i in UP muscle fibers and blocked the cADPR-elicited elevation in [Ca2+]i in both groups of muscle cells. CONCLUSION: Skeletal muscles of the UP exhibit chronic elevation of [Ca2+]i that can be partially reduced by application of 8-bromo-cADPR. cADPR was able to mobilize Ca2+ from intracellular stores, by a mechanism that is independent of ryanodine or inositol trisphosphate receptors. It can be postulated that an alteration in the cADPR-signaling pathway may exist in skeletal muscle of the patients suffering from uremic myopathy.

Efecto de la reestructuración cognitiva sobre las distorsiones cognitivas de los adolescentes frente al duelo por fallecimiento de sus padres / Cognitive restructuring effects on cognitive distortions in adolescents in grief over parent's death

El objetivo del estudio fue evaluar los efectos de la técnica reestructuración cognitiva sobre las distorsiones cognitivas en los adolescentes frente al duelo por el fallecimiento de sus padres. Se realizó una investigación de tipo descriptiva, con diseño cuasi­experimental, de corte transversal, dirigida a una muestra intencional conformada por 10 estudiantes de bachillerato de instituciones públicas y privadas quienes perdieron a alguno de sus padres. Se diseñó un instrumento de preguntas cerradas con una confiabilidad de 0,94. El instrumento se aplicó a los adolescentes antes y después del abordaje con la técnica de reestructuración cognitiva para así explorar la valoración cognitiva elaborada en su respectivo proceso de duelo. Entre los resultados obtenidos se aprecia que 35% evidencian distorsiones cognitivas antes del estudio (razonamiento emocional, sobregeneralización, pensamiento dicotómico y personalización) mientras que después del abordaje sólo en 25% persistieron estas distorsiones. En cuanto a la dimensión estímulos, el 63% se afectaban por las conversaciones sobre sus padres fallecidos y sólo 25% lo hacían después del abordaje. Tristeza e ira fue detectada en 63% de los adolescentes previo abordaje y luego sólo en 48% de ellos. El análisis de los datos reveló la efectividad de la técnica en estudio para modificar las distorsiones cognitivas en este grupo de adolescentes(AU)

The goal of this study is to evaluate the effects of cognitive restructuring on cognitive distortions in adolescents in grief over their parent's death. A descriptive, cross-sectional, with quasi-experimental design was performed on 10 high school students of public and private institutions who lost one or both parents. An instrument of closed questions was designed with a reliability of 0.94. The results show that 35% of adolescents had cognitive distortions before the study (emotional reasoning, overgeneralization, dichotomous thinking and personalization); after therapy 25% persisted with these distortions. Regarding the dimension stimuli, 63% were affected by conversations about their deceased parents; after therapy, only 25% were still affected. Sadness and anger was detected in 63% of adolescents; after the application of the technique, these emotional expressions presented only in 48% of participants. The analysis of the data revealed the effectiveness of this technique to modify cognitive distortions in this group of adolescents(AU)

Recombinant expression of four oxidoreductases in Phanerochaete chrysosporium improves degradation of phenolic and non-phenolic substrates.

Phanerochaete chrysosporium belongs to a group of lignin-degrading fungi that secretes various oxidoreductive enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP). Previously, we demonstrated that the heterologous expression of a versatile peroxidase (VP) in P. chrysosporium recombinant strains is possible. However, the production of laccases (Lac) in this fungus has not been completely demonstrated and remains controversial. In order to investigate if the co-expression of Lac and VP in P. chrysosporium would improve the degradation of phenolic and non-phenolic substrates, we tested the constitutive co-expression of the lacIIIb gene from Trametes versicolor and the vpl2 gene from Pleurotus eryngii, and also the endogenous genes mnp1 and lipH8 by shock wave mediated transformation. The co-overexpression of peroxidases and laccases was improved up to five-fold as compared with wild type species. Transformant strains showed a broad spectrum in phenolic/non-phenolic biotransformation and a high percentage in synthetic dye decolorization in comparison with the parental strain. Our results show that the four enzymes can be constitutively expressed in a single transformant of P. chrysosporium in minimal medium. These data offer new possibilities for an easy and efficient co-expression of laccases and peroxidases in suitable basidiomycete species.

Tandem shock waves to enhance genetic transformation of Aspergillus niger.

Filamentous fungi are used in several industries and in academia to produce antibiotics, metabolites, proteins and pharmaceutical compounds. The development of valuable strains usually requires the insertion of recombinant deoxyribonucleic acid; however, the protocols to transfer DNA to fungal cells are highly inefficient. Recently, underwater shock waves were successfully used to genetically transform filamentous fungi. The purpose of this research was to demonstrate that the efficiency of transformation can be improved significantly by enhancing acoustic cavitation using tandem (dual-pulse) shock waves. Results revealed that tandem pressure pulses, generated at a delay of 300 µs, increased the transformation efficiency of Aspergillus niger up to 84% in comparison with conventional (single-pulse) shock waves. This methodology may also be useful to obtain new strains required in basic research and biotechnology.

Targeting of envelope domain III protein of DENV type 2 to DEC-205 receptor elicits neutralizing antibodies in mice.

Dengue virus (DENV) is the causal agent of severe disease and, in some cases, mortality in humans, but no licensed vaccines against dengue are available. An effective vaccine against dengue requires long-term humoral and cellular immune responses. Several viral proteins have been the subjects of intense research, especially the envelope (E) protein, aimed at developing a vaccine. Domain III of the envelope protein (EDIII) has been identified as a potential candidate because it is involved in binding to host cell receptors and contains epitopes that elicit virus neutralizing antibodies. However, this domain is not sufficiently antigenic when is expressed and administered as antigen to elicit a strong immune response. One alternative to enhance immunogenicity is to target the antigen to dendritic cells to induce T-cells for broad antibody responses. In this work, a single chain antibody fragment (scFv) raised against the DEC-205 receptor fused with the EDIII was successfully expressed in Nicotiana benthamiana. The recombinant protein was expressed and purified from the plant and evaluated in BALB/c mice to test its immunogenicity and ability to induce neutralizing antibodies against DENV. The mice immunized with the recombinant protein produced specific and strong humoral immune responses to DENV. Only two immunizations were required to generate a memory response to DENV without the presence of adjuvants. Also, recognition of the recombinant protein with sera from DENV-infected patients was observed. These findings suggest that this strategy has potential for development of an effective vaccine against DENV.

[Dysfunction of diastolic [Ca²âº] in cardiomyocytes isolated from chagasic patients]. / Disfunción de la [Ca(2+)] diastólica en cardiomiocitos aislados de pacientes chagásicos.

INTRODUCTION AND OBJECTIVES: Chagas is an endemic disease in Latin America, caused by the parasite Trypanosoma cruzi, which usually affects the functioning of the heart. We have studied the regulation of intracellular calcium in cardiomyocytes isolated from chagasic patients with different degrees of heart dysfunction. METHODS: Calcium selective microelectrodes were used to simultaneously measure diastolic calcium concentration ([Ca²âº](d)) and resting membrane potential in endomyocardial biopsies obtained from chagasic patients and controls. RESULTS: The [Ca²âº](d) increased by 123%, 295%, and 738% in chagasic patients in functional class I, II, and III, respectively, in relation to controls. Membrane potential showed a partial depolarization of 6% in functional class I, 10% in functional class II, and 22% in functional class III, compared to control values. Alteration in the [Ca²âº](d) was partially reverted by 1-[6-[[(17ß)-3-metoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), a ß-phospholipase C antagonist, and by 2-aminoethoxydiphenyl-borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker. Phenylephrine, an agent that induces a rapid transient increase in 1,4,5-trisphosphate intracellular content, produced a rise in [Ca²âº](d), higher in chagasic cardiomyocytes than in controls, and its effect was fully inhibited by 2-APB. CONCLUSIONS: In cardiomyocytes from chagasic patients there is a dysfunction of the regulation of the [Ca²âº](d), which correlates with the cardiac abnormalities observed in the different stages of the disease. This disturbance in the regulation of intracellular calcium appears to be associated with alterations in the regulation of intracellular messenger inositol 1,4,5-trisphosphate.

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i.

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca(2+) concentration ([Ca(2+)](i)) using double-barreled, Ca(2+)-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca(2+)](i) was approximately twofold in MHS compared with MHN quiescent myoballs (232 +/- 35 vs. 112 +/- 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca(2+)](i) in both groups; however, the concentration required to cause a rise in [Ca(2+)](i) elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca(2+) buffer BAPTA reduced [Ca(2+)](i), raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca(2+)](i), and reduced the amount of Ca(2+) release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca(2+)](i) levels in these cells.

Disfunción de la [Ca 2+] diastólica en cardiomiocitos aislados de pacientes chagásicos / Dysfunction of diastolic [Ca2+] in cardiomyocytes isolated from chagasic patients

Introducción y objetivos. La enfermedad de Chagas es un mal endémico en Latinoamérica, causado por el parásito Trypanosoma cruzi, que generalmente afecta al funcionamiento del corazón. Estudiamos la regulación del calcio intracelular en cardiomiocitos de pacientes chagásicos con diversos grados de deterioro funcional. Métodos. Se utilizaron microelectrodos selectivos para el calcio para determinar simultáneamente la concentración diastólica de calcio ([Ca2+]d) y el potencial de membrana en biopsias endomiocárdicas de pacientes chagásicos y controles. Resultados. La [Ca2+]d aumentó el 123, el 295 y el 738% en los pacientes chagásicos de los grupos funcionales I, II y III, respectivamente, respecto a los controles. El potencial de membrana mostró una parcial despolarización de un 6% en el grupo funcional I, el 10% en el II y el 22% en el III respecto a los controles. La [Ca2+]d se revirtió parcialmente con 1-[6-[[(17ß)-3-metoxiestra-1,3,5(10)-trieno-17-il]amino]hexil]-1H-pirrole-2,5-diona (U-73122), antagonista de la betafosfolipasa C, y 2-aminoetoxidifenil borato (2-APB), inhibidor de los receptores de inositol 1,4,5-trifosfato. La fenilefrina, fármaco que induce una rápida elevación del contenido intracelular del inositol 1,4,5-trifosfato, incrementó la [Ca2+]d en cardiomiocitos controles y chagásicos, que fue mayor en los cardiomicitos chagásicos y se inhibió por 2-APB. Conclusiones. En cardiomiocitos de pacientes chagásicos hay una disfunción de la regulación de la [Ca2+]d correlacionada con las alteraciones cardiacas observadas en las diferentes fases de la enfermedad. Esta perturbación en dicha regulación se asocia, aparentemente, a una alteración en la regulación del mediador intracelular inositol 1,4,5-trifosfato (AU)

Introduction and objectives: Chagas is an endemic disease in Latin America, caused by the parasite Trypanosoma cruzi, which usually affects the functioning of the heart. We have studied the regulation of intracellular calcium in cardiomyocytes isolated from chagasic patients with different degrees of heart dysfunction. Methods: Calcium selective microelectrodes were used to simultaneously measure diastolic calcium concentration ([Ca2+]d) and resting membrane potential in endomyocardial biopsies obtained from chagasic patients and controls. Results: The [Ca2+]d increased by 123%, 295%, and 738% in chagasic patients in functional class I, II, and III, respectively, in relation to controls. Membrane potential showed a partial depolarization of 6% in functional class I, 10% in functional class II, and 22% in functional class III, compared to control values. Alteration in the [Ca2+]d was partially reverted by 1-[6-[[(17ß)-3-metoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), a b-phospholipase C antagonist, and by 2-aminoethoxydiphenyl- borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker. Phenylephrine, an agent that induces a rapid transient increase in 1,4,5-trisphosphate intracellular content, produced a rise in [Ca2+]d, higher in chagasic cardiomyocytes than in controls, and its effect was fully inhibited by 2-APB. Conclusions: In cardiomyocytes from chagasic patients there is a dysfunction of the regulation of the [Ca2+]d, which correlates with the cardiac abnormalities observed in the different stages of the disease. This disturbance in the regulation of intracellular calcium appears to be associated with alterations in the regulation of intracellular messenger inositol 1,4,5-trisphosphate (AU)

Liberación anormal de CA²+ inducida por el inositol trifosfato en fibras estriadas susceptibles a hipertermia maligna / Abnormal liberation of CA²+ induced by the triphosphate inositol in striated fibers susceptible to malignant lypetermia

La hipertermia maligna es un síndrome farmocogenético asociado con la alteración de la regulación del calcio mioplásmico. Una modificación en el metabolismo del inositol 1,4,5 trifosfato (InsP3), hasido asociada con la fisiopatología de la hipertermia maligna. El objetivo del trabajo fue estudiar el efecto del InsP3 sobre la concentración intracelular de calcio ([CA²+]i) en fibras musculares obtenidas de sujetos no susceptibles y susceptibles a hipertermia maligna. La [CA²]i fue cuantificada mediante el uso de microelectrodos sensibles a CA²+. En susceptibles a hipertermia maligna la [CA²+]i fue más elevada que en las fibras musculares no suceptibles hipertemia maligna. La microinyección de InsP3 0,5 y 1 µM indujo una elevación significativa de la [CA²+]i, en ambos grupos musculares. Sin embargo, este incremento fue mayor en las fibras susceptibles a hipertermia maligna que en las no susceptibles a hipertermia maligna. La incubación de los músculos en soluciones con bajo contenido en Ca²+ o en nifedipina (10 µM) no modificó la elevación de [Ca²+]i mediana por el InsP3. El tratamiento con dantrolene (50 µM) redujo la [Ca²+]i, y bloqueó la elevación de la [Ca²+]i, inducida por el InsP3 en ambos grupos. Estos resultados sugieren (i) el posible papel del InsP3 como mediador químico en la liberación de Ca²+ desde los depósitos intracelulares; (ii) que el InsP3 podrá jugar un papel importante en la fisiopatología de la hipertermia maligna y (iii) que el efecto profiláctico y terapeútico del dantrolene podría estar relacionado con su efecto inhibitorio sobre la liberación de calcio intracelular mediada por el InsP3

Cambios en la [Ca²+]I: inducidos por el dantrolene en fibras esqueléticas susceptibles al síndrome de hipertermia maligna / Changes in the [Ca²+]I: armature the dantrolene in skeletal fibers from patients susceptible to syndrome of hyperthermia malignant

La hipertermia maligna es una miopatía de carácter hereditario, que puede ser desencadenada cuando individuos susceptibles son expuestos a relajantes musculares del tipo despolarizante y/o agentes anestésicos halogenados. Dantrolene representa el único fármaco capaz de prevenir o revertir el síndrome de hipertermia maligna. Nosotros hemos estudiado el efecto del dantrolene sobre la concentración intracelular de calcio libre ([Ca²+]i) en biosias musculares obtenidas de pacientes susceptibles a hipertermia maligna, antes y después de la administración oral de dantrolene (1,2, y 2,5 mh/kg de peso). La [Ca²+]i fue determinada mediante el uso de microelectrodos de doble barrera selectivos al ion calcio. La administración oral del dantrolene 1 mg/kg redujo [Ca²+]i de 413 ñ 10nM (n=15) a 325 ñ 9 nM (n=11), 2 mg/kg de 405 ñ 10 nM (n=11) a 203 ñ 11 nM (n=11) y 2,5 mg/kg de 395 ñ 15 nM (n=11) a 109 ñ 2 nM (n=14). El efecto del dantrolene sobre la [Ca²+]i no fue mediado por cambios en el potencial de membrana. Estos resultados demuestran la efectividad del dantrolene por vía oral en reducir la [Ca²+]i en fibras musculares esqueléticas obtenidas de pacientes susceptibles al síndrome de hipertermia maligna y explican el efecto beneficioso de este relajante muscular en el tratamiento prequirúrgico de estos pacientes