RESUMEN
BACKGROUND: Seed germination is a crucial process in the plant life cycle when a dramatic variation of type and sugar content occurs just as the seed is hydrated. The production of hexose 6 phosphate is a key node in different pathways that are required for a successful germination. Hexokinase (HXK) is the only plant enzyme that phosphorylates glucose (Glc), so it is key to fueling several metabolic pathways depending on their substrate specificity, metabolite regulatory responses and subcellular localization. In maize, the HXK family is composed of nine genes, but only six of them (ZmHXK4-9) putatively encode catalytically active enzymes. Here, we cloned and functionally characterized putative catalytic enzymes to analyze their metabolic contribution during germination process. RESULTS: From the six HXKs analyzed here, only ZmHXK9 has minimal hexose phosphorylating activity even though enzymatic function of all isoforms (ZmHXK4-9) was confirmed using a yeast complementation approach. The kinetic parameters of recombinant proteins showed that ZmHXK4-7 have high catalytic efficiency for Glc, fructose (Fru) and mannose (Man), ZmHXK7 has a lower Km for ATP, and together with ZmHXK8 they have lower sensitivity to inhibition by ADP, G6P and N-acetylglucosamine than ZmHXK4-6 and ZmHXK9. Additionally, we demonstrated that ZmHXK4-6 and ZmHXK9 are located in the mitochondria and their location relies on the first 30 amino acids of the N-terminal domain. Otherwise, ZmHXK7-8 are constitutively located in the cytosol. HXK activity was detected in cytosolic and mitochondrial fractions and high Glc and Fru phosphorylating activities were found in imbibed embryos. CONCLUSIONS: Considering the biochemical characteristics, location and the expression of ZmHXK4 at onset of germination, we suggest that it is the main contributor to mitochondrial activity at early germination times, at 24 h other ZmHXKs also contribute to the total activity. While in the cytosol, ZmHXK7 could be responsible for the activity at the onset of germination, although later, ZmHXK8 also contributes to the total HXK activity. Our observations suggest that the HXKs may be redundant proteins with specific roles depending on carbon and ATP availability, metabolic needs, or sensor requirements. Further investigation is necessary to understand their specific or redundant physiological roles.
Asunto(s)
Citosol/fisiología , Germinación/fisiología , Hexoquinasa/metabolismo , Semillas/fisiología , Zea mays/enzimología , Zea mays/fisiología , Citosol/enzimología , Citosol/metabolismo , Germinación/genética , Hexoquinasa/genética , Mitocondrias/enzimología , Mitocondrias/metabolismo , Semillas/enzimología , Semillas/metabolismo , Zea mays/metabolismoRESUMEN
Bacteria are able to synchronize the population behavior in order to regulate gene expression through a cell-to-cell communication mechanism called quorum sensing. This phenomenon involves the production, detection and the response to extracellular signaling molecules named autoinducers, which directly or indirectly regulate gene expression in a cell density-dependent manner. Quorum sensing may control a wide range of biological processes in bacteria, such as bioluminescence, virulence factor production, biofilm formation and antibiotic resistance. The autoinducers are recognized by specific receptors that can either be membrane-bound histidine kinase receptors, which work by activating cognate cytoplasmic response regulators, or cytoplasmic receptors acting as transcription factors. In this review, we focused on the cytosolic quorum sensing regulators whose three-dimensional structures helped elucidate their mechanisms of action. Structural studies of quorum sensing receptors may enable the rational design of inhibitor molecules. Ultimately, this approach may represent an effective alternative to treat infections where classical antimicrobial therapy fails to overcome the microorganism virulence.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Citosol/fisiología , Percepción de Quorum/fisiología , Transducción de Señal/fisiologíaRESUMEN
Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter⻹), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected by in silico analysis and further identified by tandem mass spectrometry de novo sequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways.
Asunto(s)
Antibacterianos/farmacología , Citosol/fisiología , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Magaininas/farmacología , Proteoma/genética , Aminoácidos/metabolismo , Pared Celular/metabolismo , Pared Celular/ultraestructura , Simulación por Computador , Electroforesis en Gel de Poliacrilamida , Metabolismo Energético/genética , Fermentación , Pruebas de Sensibilidad Microbiana , Nitrógeno/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
In fear conditioning, aversive stimuli are readily associated with contextual features. A brief reexposure to the training context causes fear memory reconsolidation, whereas a prolonged reexposure induces memory extinction. The regulation of hippocampal gene expression plays a key role in contextual memory consolidation and reconsolidation. However, the mechanisms that determine whether memory will reconsolidate or extinguish are not known. Here, we demonstrate opposing roles for two evolutionarily related transcription factors in the mouse hippocampus. We found that nuclear factor-κB (NF-κB) is required for fear memory reconsolidation. Conversely, calcineurin phosphatase inhibited NF-κB and induced nuclear factor of activated T-cells (NFAT) nuclear translocation in the transition between reconsolidation and extinction. Accordingly, the hippocampal inhibition of both calcineurin and NFAT independently impaired memory extinction, whereas inhibition of NF-κB enhanced memory extinction. These findings represent the first insight into the molecular mechanisms that determine memory reprocessing after retrieval, supporting a transcriptional switch that directs memory toward reconsolidation or extinction. The precise molecular characterization of postretrieval processes has potential importance to the development of therapeutic strategies for fear memory disorders.
Asunto(s)
Extinción Psicológica/fisiología , Memoria/fisiología , Recuerdo Mental/fisiología , Factores de Transcripción/fisiología , Animales , Western Blotting , Calcineurina/genética , Calcineurina/fisiología , Inhibidores de la Calcineurina , Núcleo Celular/metabolismo , Núcleo Celular/fisiología , Condicionamiento Operante/fisiología , Citosol/metabolismo , Citosol/fisiología , Interpretación Estadística de Datos , Ensayo de Cambio de Movilidad Electroforética , Extinción Psicológica/efectos de los fármacos , Miedo/fisiología , Hipocampo/fisiología , Masculino , Memoria/efectos de los fármacos , Recuerdo Mental/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , FN-kappa B/fisiología , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/fisiología , Translocación GenéticaRESUMEN
Clathrin-coated vesicle endocytosis is thought to be crucial for the maintenance of synaptic transmission and for the cell plasticity at the nervous system. In this study, we demonstrated that acute intrastriatal administration of quinolinic acid (QUIN), an agonist of the N-methyl-D: -aspartate receptor, induces a decrease of the coat protein AP-2 expression and affects their interaction with membranes. By western blot analysis we observed that at 24 h after QUIN intrastriatal injection, alpha1 subunit of AP-2 and alpha2, at lesser extent, were reduced in the striatal membranes. The decrease of both subunits expression was extended to 48 h after treatment, although the soluble proteins were mostly affected. Other areas of the brain were not affected by the treatment, except the cerebellum, where a significant increase of soluble AP-2 (both subunits) was observed at 48 h after injection. Another coat protein, as the phosphoprotein AP-180, was not affected by the injection of QUIN. We also confirmed that QUIN injection causes increasing loss of striatal neurons after the administration of the toxin. We concluded that QUIN may affect the endocytotic machinery of the striatum, by inducing changes in the AP-2 behaviour. Consequently, the internalization of NMDAR and/or AMPAR may be affected, by QUIN, contributing to the excitotoxic effect of the drug.
Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Membrana Celular/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Neurotoxinas/farmacología , Ácido Quinolínico/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Animales , Western Blotting , Recuento de Células , Muerte Celular/efectos de los fármacos , Membrana Celular/fisiología , Cerebelo/efectos de los fármacos , Cerebelo/fisiología , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Citosol/efectos de los fármacos , Citosol/fisiología , Masculino , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Calcium release from intracellular stores plays a key role in the regulation of a variety of cellular activities. In various cell types this release occurs through inositol-triphosphate (IP3) receptors which are Ca2+ channels whose open probability is modulated by the cytosolic Ca2+ concentration itself. Thus, the combination of Ca2+ release and Ca2+ diffusion evokes a variety of Ca2+ signals depending on the number and relative location of the channels that participate of them. In fact, a hierarchy of Ca2+ signals has been observed in Xenopus laevis oocytes, ranging from very localized events (puffs and blips) to waves that propagate throughout the cell. In this cell type channels are organized in clusters. The behavior of individual channels within a cluster cannot be resolved with current optical techniques. Therefore, a combination of experiments and mathematical modeling is unavoidable to understand these signals. However, the numerical simulation of a detailed mathematical model of the problem is very hard given the large range of spatial and temporal scales that must be covered. In this paper we present an alternative model in which the cluster region is modeled using a relatively fine grid but where several approximations are made to compute the cytosolic Ca2+ concentration ([Ca;{2+}]) distribution. The inner-cluster [Ca;{2+}] distribution is used to determine the openings and closings of the channels of the cluster. The spatiotemporal [Ca;{2+}] distribution outside the cluster is determined using a coarser grid in which each (active) cluster is represented by a point source whose current is proportional to the number of open channels determined before. A full reaction-diffusion system is solved on this coarser grid.
Asunto(s)
Canales de Calcio/fisiología , Calcio/fisiología , Modelos Biológicos , Xenopus laevis/fisiología , Animales , Señalización del Calcio , Citosol/fisiología , Femenino , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Oocitos/fisiologíaRESUMEN
The intrinsic apoptotic pathway is characterized by the release of several mitochondrial intermembrane proteins into the cytosol of dying cells. It is unclear whether the release of these proteins follows a common or specific pathway. In the present report we show that survivin and, to a lesser extent, the survivin splice variant survivin DeltaEx3 regulate the specific liberation of second mitochondria-derived activator of caspase/direct IAP binding protein with low pI (Smac/DIABLO), an inhibitor of apoptosis proteins binding protein, during apoptosis induced by etoposide, a DNA damaging agent. This antineoplastic drug induced posttranscriptional upregulation of survivin and survivin DeltaEx3. In turn, mitochondrial survivin associated with Smac/DIABLO, delaying its release. In addition, cytosolic survivin also stabilized the cytosolic levels of released Smac/DIABLO. These results provide an explanation for the observed differences in the release of mitochondrial intermembrane proteins in various apoptotic models and present a new mechanism for the anti-apoptotic effects of survivin in cancer cells.
Asunto(s)
Apoptosis/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/fisiología , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Caspasas/metabolismo , Citocromos c/metabolismo , Citosol/fisiología , Activación Enzimática , Etopósido/farmacología , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis , Mitocondrias/efectos de los fármacos , Estaurosporina/farmacología , SurvivinRESUMEN
In treatment of hemorrhagic shock, small-volume infusion of 7.5% NaCl gives immediate hemodynamic improvement, but in vitro experiments suggest it depresses the hemostatic system. Since previous reports showed that hyperosmotic glycine solutions preserved the platelet function better than hyperosmotic NaCl solutions, we investigated whether glycine changes the intracellular calcium ([Ca]i) signal. Platelets were incubated in hyperosmotic solutions containing sodium glycine or glycine base and stimulated with 0.1 IU/ml thrombin. [Ca]i increases were compared with an isosmotic control. Platelets incubated in zero calcium/EGTA were used to study separately the effect of glycine on calcium mobilization from intracellular stores and extracellular calcium entry. When NaCl was replaced by sodium glycine, the [Ca]i increase produced by thrombin was enhanced, because the calcium entry increased without changes in the mobilization of stored calcium. The addition of 50 mmol/l glycine base to the HEPES-buffered media increases the thrombin-induced entry of calcium or manganese. This study demonstrates that hyperosmotic glycine solutions increase the entry of calcium. This effect contrasts with the impairment of the thrombin-induced calcium signals by NaCl. The addition of low amounts of glycine in resuscitation solutions would be useful to reduce dysfunctional inflammatory responses without the risk of bleeding; however, concentrated solutions could cause toxic effects.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Glicinérgicos/farmacología , Glicina/farmacología , Activación Plaquetaria/efectos de los fármacos , Citosol/fisiología , Humanos , Soluciones Hipertónicas/farmacología , Manganeso/metabolismo , Trombina/fisiologíaRESUMEN
Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca(2+) enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca(2+)-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca(2+)-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.
Asunto(s)
Factor Inductor de la Apoptosis/fisiología , Apoptosis/fisiología , Fosfolipasas A/fisiología , Linfocitos T Citotóxicos/fisiología , Animales , Calcio/metabolismo , Línea Celular , Sistema Libre de Células , Citosol/fisiología , Citotoxicidad Inmunológica , Proteína Ligando Fas/fisiología , Fosfolipasas A2 Grupo II , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Proteínas Citotóxicas Formadoras de Poros/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Linfocitos T Citotóxicos/enzimología , Temperatura , Receptores Señuelo del Factor de Necrosis Tumoral/deficiencia , Receptores Señuelo del Factor de Necrosis Tumoral/genéticaRESUMEN
We focused our attention on Ca(2+) release from the endoplasmic reticulum through a cluster of inositol(1,4,5)-trisphosphate (IP(3)) receptor channels. The random opening and closing of these receptors introduce stochastic effects that have been observed experimentally. Here, we present a stochastic version of Othmer-Tang model (OTM) for IP(3) receptor clusters. We address the average behavior of the channels in response to IP(3) stimuli. In our stochastic simulation we found that the fraction of open channels versus [IP(3)] follows a Hill curve, whose associate Hill coefficient increases when intracellular Ca(2+) level increase. This finding suggests that feedback from cytosolic Ca(2+) plays a key role in the channel response to IP(3). We also study several aspects of the stochastic properties of Ca(2+) release and we compare with experimental observations.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Canales de Calcio/fisiología , Simulación por Computador , Citosol/fisiología , Retículo Endoplásmico/metabolismo , Retroalimentación Fisiológica , Receptores de Inositol 1,4,5-Trifosfato , Modelos Biológicos , Procesos EstocásticosRESUMEN
The pancreatic acinar unit is a classical example of a polarized tissue. Even in isolation, these cells retain their polarity, and this has made them particularly useful for Ca2+ signaling studies. In 1990, we discovered that this cell has the capability of producing both local cytosolic and global Ca2+ signals. The mechanisms underlying this signal generation have now been established. Furthermore, it has become clear that the local signals are sufficient for the control of both fluid and enzyme secretion, whereas prolonged global signals are dangerous and give rise to acute pancreatitis, a disease where the pancreas digests itself.
Asunto(s)
Señalización del Calcio/fisiología , Citosol/fisiología , Retículo Endoplásmico/fisiología , Inositol 1,4,5-Trifosfato/fisiología , Páncreas Exocrino/citología , Calcio/metabolismo , Comunicación Celular/fisiología , Compartimento Celular/fisiología , Membrana Celular/fisiología , Polaridad Celular/fisiología , Humanos , Potenciales de la Membrana/fisiología , Páncreas Exocrino/fisiopatología , Pancreatitis/fisiopatologíaRESUMEN
In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.
Asunto(s)
Canales de Calcio/fisiología , Señalización del Calcio/fisiología , Retículo Endoplásmico/fisiología , Neuronas/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Citosol/metabolismo , Citosol/fisiología , Electrofisiología , Retículo Endoplásmico/metabolismo , Inositol 1,4,5-Trifosfato/fisiología , Potenciales de la Membrana/fisiología , Neuronas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
The transient increase in the expression of transcription factors encoded by immediate-early genes has been considered to play a critical role in the coordination of early gene expression during the hypertrophic growth of cardiac myocytes. Here, we investigated the regulation of c-Jun and its upstream activators JNKs in the myocardium of rats subjected to acute pressure overload induced by transverse aortic constriction. Western blotting and immunohistochemistry analysis demonstrated that both JNK1 and JNK2 were transiently activated by pressure overload, but only JNK1 was activated at the nuclei of cardiac myocytes. JNK1 activation was paralleled by phosphorylation of c-Jun at serine-63 in the myocardial nuclear fraction and by an increase in c-Jun expression in cardiac myocytes. A consistent increase in DNA binding of activator protein-1 (AP-1) complex was observed after 10 and 30 min of pressure overload and Supershift assays confirmed that c-Jun was a major component of activated AP-1 complex. Moreover, experiments performed with the specific JNK inhibitor SP-600125 abolished c-Jun phosphorylation and markedly attenuated its expression as well as the expression of the fetal gene beta-myosin heavy chain. Overall, these findings demonstrate a molecular basis for load-induced activation of c-Jun in cardiac myocytes and its connection with the regulation of fetal gene, characteristic of the acute response to pressure overload.
Asunto(s)
Corazón/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Antracenos/farmacología , Secuencia de Bases , Núcleo Celular/fisiología , Citosol/fisiología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Corazón/efectos de los fármacos , Corazón/fisiopatología , Masculino , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Modelos Animales , Cadenas Pesadas de Miosina/efectos de los fármacos , Cadenas Pesadas de Miosina/genética , Oligodesoxirribonucleótidos , Fosforilación , Presión , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Ratas Wistar , Función Ventricular Izquierda/fisiologíaRESUMEN
In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca2+-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+]I-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.
Asunto(s)
Animales , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canales de Calcio/metabolismo , Neuronas/fisiología , Neuronas/metabolismo , Retículo Endoplásmico/fisiología , Citosol/fisiología , Citosol/metabolismo , /fisiologíaRESUMEN
In the present study, we examined the effect of the intraperitoneal administration of vitamin E (100 mg/kg weight/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of rat liver microsomes . We also analyzed the effect of hepatic cytosolic proteins on this process. The results indicate that the ascorbate induced light emission was 76% lower in microsomes (1 mg protein) obtained from vitamin E treated animals when compared with controls. In the presence of cytosolic protein (1 mg) the chemiluminescence of control microsomes diminished 55.8 and 59.5% when cytosol from controls and treated animals was used, respectively. The chemiluminescence of vitamin E microsomes diminished 25.03 and 22.08% when both types of cytosol were added to the medium. Dialyzed or treated at 70 degrees C cytosol was also able to inhibit the lipid peroxidation of either control or vitamin E rat liver microsomes. By means of gas chromatography we analyzed the fatty acid composition of native and peroxidated microsomes from both animal groups. The peroxidation affected principally arachidonic acid and its diminution was more evident in the control microsomes than in the microsomes from the vitamin E treated group. By HPLC we analyzed the vitamin E content in all subcellular fractions employed. In microsomes from the vitamin E-group, the content of vitamin was 11 times higher than in the control ones (0.678 +/- 0.1038 vs. 0.062 +/- 0.0045 microg alpha-tocopherol/mg protein, respectively), while levels in the cytosol from the vitamin E-group were only 2 times higher than in the control cytosol (0.057 +/- 0.0051 vs. 0.025 +/- 0.0015 microg alpha-tocopherol/mg protein, respectively).
Asunto(s)
Ácido Ascórbico/farmacología , Citosol/fisiología , Hierro/farmacología , Peroxidación de Lípido/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Proteínas/fisiología , Vitamina E/administración & dosificación , Animales , Inyecciones Intraperitoneales , Masculino , Microsomas Hepáticos/química , Microsomas Hepáticos/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Trypanosoma cruzi trypomastigotes survive inside macrophages by promoting fusion between the parasitophorous vacuole and mature host lysosomes upon internalization. Since trypomastigotes can evade the lytic pathway, the earliest steps of endocytosis, such as early endosome fusion, may be affected. To test this hypothesis, we used an in vitro early endosome fusion assay. Our results show that trypomastigote-infected macrophage cytosols cannot promote fusion between early endosomes, compared to mock-infected cytosols (heat-killed trypomastigotes were used in the parasite-macrophage interaction assay). GTP gamma S addition potentiates the fusogenic activity driven by trypomastigote-infected macrophage cytosol-mediated assays, unlike the biphasic fusogenic effect obtained with GTP gamma S treatment of macrophage cytosol controls. Calcium-stimulated early endosome fusogenic processes are not affected in the assays mediated by infected macrophage cytosol. We conclude that GTP-regulated factors, and not calcium-regulated elements, are involved in the inhibition of the early endosome fusogenic process by the trypomastigote-infected macrophage cytosol. This primary impediment to the progress of a normal endocytosis may be a relevant step required for the lysosomal recruitment-fusion of the host lysosomes upon trypomastigote infection and further survival of the parasite within its host.
Asunto(s)
Citosol/fisiología , Endosomas/fisiología , Macrófagos/parasitología , Fusión de Membrana/fisiología , Trypanosoma cruzi/fisiología , Animales , Calcio/fisiología , Citosol/parasitología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/fisiología , Macrófagos/ultraestructura , RatonesRESUMEN
The effect of dexamethasone on the oxidative desaturation of [1-14C]palmitic to palmitoleic acid on rat liver microsomes, was studied. After 12 h of dexamethasone injection (1 mg/rat) a significant increase in delta 9-desaturase activity, was observed. This effect was also produced by a factor present in a 110,000 X g supernatant soluble fraction obtained after washing crude microsomes from dexamethasone-treated rats with a low ionic strength solution. The dexamethasone-induced factor was present not only in the liver cytosolic fraction of treated animals but also in the cytosol of isolated HTC cells previously incubated with the hormone. Dexamethasone would act via a newly synthesized modulatory factor. The effect depends on an unchanged protein structure, since its biological activity is impaired by trypsin digestion.
Asunto(s)
Dexametasona/farmacología , Ácido Graso Desaturasas/metabolismo , Hígado/fisiología , Microsomas Hepáticos/enzimología , Proteínas/fisiología , Animales , Citosol/fisiología , Femenino , Cinética , Microsomas Hepáticos/efectos de los fármacos , Biosíntesis de Proteínas , Proteínas/aislamiento & purificación , Ratas , Ratas Endogámicas , Estearoil-CoA DesaturasaRESUMEN
Efecto del hipotiroidismo y la malnutrición sobre el transporte núcelocitoplasmático de ARN "in vitro". La tiroidectomía neonatal produce en la rata de 10 días de edad modificaciones en la actividad específica del ARN "de marcación rápida", tanto a nivel nuclear como microsomal. La radiactividad del ARN ribosomal, cuya síntesis nuclear no es afectada por el hipotiroidismo, se ve disminuida a nivel microsomal, indicando retraso en el transporte a través de la membrana nucelar. La salida del ARN "de marcación rápida" desde el núcleo no se modifica bajo estas mismas condiciones. La mala nutrición produce cambios similares a los observados en el hipotiroidismo, excepto que la malnutrición, además, disminuye la liberación nuclear de mARN. Alteraciones a nivel de los factores citosólicos parecen ser la causa de la menor liberación nuclear del ARN ribosomal de cerebro de rata hipotiroidea, mientras que cambios en mecanismos intranucleares aún no conocidos serían los responsables de las modificaciones en el transporte de ARN ribosomal de cerebro de rata malnutrida
Asunto(s)
Ratas , Animales , Cerebro/metabolismo , Citosol/fisiología , Hipotiroidismo/fisiopatología , Desnutrición Proteico-Calórica/fisiopatología , ARN/metabolismo , Fraccionamiento Celular , Hormonas Tiroideas/deficiencia , Membrana Nuclear , Proteínas/deficienciaRESUMEN
Efecto del hipotiroidismo y la malnutrición sobre el transporte núcelocitoplasmático de ARN "in vitro". La tiroidectomía neonatal produce en la rata de 10 días de edad modificaciones en la actividad específica del ARN "de marcación rápida", tanto a nivel nuclear como microsomal. La radiactividad del ARN ribosomal, cuya síntesis nuclear no es afectada por el hipotiroidismo, se ve disminuida a nivel microsomal, indicando retraso en el transporte a través de la membrana nucelar. La salida del ARN "de marcación rápida" desde el núcleo no se modifica bajo estas mismas condiciones. La mala nutrición produce cambios similares a los observados en el hipotiroidismo, excepto que la malnutrición, además, disminuye la liberación nuclear de mARN. Alteraciones a nivel de los factores citosólicos parecen ser la causa de la menor liberación nuclear del ARN ribosomal de cerebro de rata hipotiroidea, mientras que cambios en mecanismos intranucleares aún no conocidos serían los responsables de las modificaciones en el transporte de ARN ribosomal de cerebro de rata malnutrida (AU)
Asunto(s)
Ratas , Animales , Cerebro/metabolismo , Citosol/fisiología , Desnutrición Proteico-Calórica/fisiopatología , Hipotiroidismo/fisiopatología , ARN/metabolismo , Fraccionamiento Celular , Hormonas Tiroideas/deficiencia , Membrana Nuclear , Proteínas/deficienciaRESUMEN
The rapid restoration of liver protein mass observed in protein-depleted mice when they are fed with an adequate diet is quantitatively explained by a large decrease in the average rate of breakdown of total liver proteins. This study was performed in order to know whether this inhibition of breakdown affects in the same way all the protein constituents of the tissue, or only affects a group of these proteins belonging to a particular subcellular fraction. Subcellular fractions were obtained by differential centrifugation. The relative rates of breakdown of their proteins were estimated by the conservation of radioactivity in these proteins previously labelled by the administration of NaH14CO3 to mice. The results obtained indicated: 1) a general decrease in the rate of breakdown of proteins of subcellular fractions from re-fed livers compared with livers of protein-depleted mice; 2) a decrease of breakdown of proteins from cytosol in re-fed mice which is higher as lower is the molecular weight of the proteins subunits.