RESUMEN
In 1980, Roger Tsien published a paper, in this journal [Tsien, R. Y. (1980) Biochemistry, 19 (11), 2396], titled "New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures". These new buffers included 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or BAPTA, which is still widely used today. And so, the world was set alight with new ways in which to visualize Ca2+. The ability to watch fluctuations in intracellular Ca2+ revolutionized the life sciences, although the fluorescent indicators used today, particularly in neurobiology, no longer rely exclusively on BAPTA but on genetically encoded fluorescent Ca2+ indicators. In this Perspective, we reflect on the origins of Ca2+ imaging with a special focus on the contributions made by Roger Tsien, from the early concept of selective Ca2+ binding described in Biochemistry to optical Ca2+ indicators based on chemically synthesized fluorophores to genetically encoded fluorescent Ca2+ indicators.
Asunto(s)
Calcio/metabolismo , Colorantes Fluorescentes/química , Microscopía Intravital/métodos , Imagen Óptica/métodos , Calcio/química , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Historia del Siglo XX , Microscopía Intravital/historia , Imagen Óptica/historiaRESUMEN
Myosin-IC (Myo1c) has been proposed to function in delivery of glucose transporter type 4 (GLUT4)-containing vesicles to the plasma membrane in response to insulin stimulation. Current evidence suggests that, upon insulin stimulation, Myo1c is phosphorylated at Ser701, leading to binding of the signaling protein 14-3-3ß. Biochemical and functional details of the Myo1c-14-3-3ß interaction have yet to be described. Using recombinantly expressed proteins and mass spectrometry-based analyses to monitor Myo1c phosphorylation, along with pulldown, fluorescence binding, and additional biochemical assays, we show here that 14-3-3ß is a dimer and, consistent with previous work, that it binds to Myo1c in the presence of calcium. This interaction was associated with dissociation of calmodulin (CaM) from the IQ motif in Myo1c. Surprisingly, we found that 14-3-3ß binds to Myo1c independent of Ser701 phosphorylation in vitro Additionally, in contrast to previous reports, we did not observe Myo1c Ser701 phosphorylation by Ca2+/CaM-dependent protein kinase II (CaMKII), although CaMKII phosphorylated four other Myo1c sites. The presence of 14-3-3ß had little effect on the actin-activated ATPase or motile activities of Myo1c. Given these results, it is unlikely that 14-3-3ß acts as a cargo adaptor for Myo1c-powered transport; rather, we propose that 14-3-3ß binds Myo1c in the presence of calcium and stabilizes the calmodulin-dissociated, nonmotile myosin.
Asunto(s)
Proteínas 14-3-3/metabolismo , Miosina Tipo I/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Secuencias de Aminoácidos , Calcio/química , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Dimerización , Ácido Egtácico/química , Humanos , Espectrometría de Masas , Miosina Tipo I/química , Miosina Tipo I/genética , Fosforilación , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , UltracentrifugaciónRESUMEN
Light has been instrumental in the study of living cells since its use helped in their discovery in the late 17th century. Further, combining chemical technology with light microscopy was an essential part of the Nobel Prize for Physiology in 1906. Such landmark scientific findings involved passive observation of cells. However, over the past 50 years, a "second use" of light has emerged in cell physiology, namely one of rational control. The seminal method for this emerged in late 1970s with the invention of caged compounds. This was the point when "caged compounds" were defined as optical probes in which the active functionality of a physiological signaling molecule was blocked with a photochemical protecting group. Caged compounds are analogous to prodrugs; in both, the activity of the effector is latent. However, caged compounds, unlike prodrugs, use a trigger that confers the power of full temporal and spatial manipulation of the effects of release of its latent biological cargo. Light is distinct because it is bio-orthogonal, passes through living tissue (even into the cell interior), and initiates rapid release of the "caged" biomolecule. Further, because light can be directed to broad areas or focused to small points, caged compounds offer an array of timing scenarios for physiologists to dissect virtually any type of cellular process.The collaborative interaction between chemists and physiologists plays a fundamental role in the development of caged compounds. First, the physiologists must define the problem to be addressed; then, with the help of chemists, decide if a caged compound would be useful. For this, structure-activity relationships of the potential optical probe and receptor must be determined. If rational targets seem feasible, synthetic organic chemistry is used to make the caged compound. The crucial property of prephotolysis bio-inertness relies on physiological or biochemical assays. Second, detailed optical characterization of the caged compound requires the skill of photochemists because the rate and efficiency of uncaging are also crucial properties for a useful caged compound. Often, these studies reveal limitations in the caged compound which has been developed; thus, chemists and physiologists use their abilities for iterative development of even more powerful optical probes. A similar dynamic will be familiar to scientists in the pharmaceutical industry. Therefore, caged compound development provides an excellent training framework for (young) chemists both intellectually and professionally. In this Account, I draw on my long experience in the field of making useful caged compounds for cell physiology by showing how each probe I have developed has been defined by an important physiological problem. Fundamental to this process has been my initial training by the pioneers in aromatic photochemistry, Derek Bryce-Smith and Andrew Gilbert. I discuss making a range of "caged calcium" probes, ones which went on to be the most widely used of all caged compounds. Then, I describe the development of caged neurotransmitters for two-photon uncaging microscopy. Finally, I survey recent work on making new photochemical protecting groups for wavelength orthogonal, two-color, and ultraefficient two-photon uncaging.
Asunto(s)
Quelantes/química , Neuronas/fisiología , Acetatos/química , Potenciales de Acción , Animales , Calcio/química , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Etilenodiaminas/química , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Neuronas/citología , Neuronas/efectos de los fármacos , Neurotransmisores/química , Neurotransmisores/metabolismo , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Ultrafast Ca2+ imaging using low-affinity fluorescent indicators allows the precise measurement of the kinetics of fast Ca2+ currents mediated by voltage-gated Ca2+ channels. Thus far, only a few indicators provided fluorescence transients with sufficient signal-to-noise ratio necessary to achieve this measurement, with Oregon Green BAPTA-5N exhibiting the best performance. Here we evaluated the performance of the low-affinity Ca2+ indicator Cal-520FF to record fast Ca2+ signals and to measure the kinetics of Ca2+ currents. Compared to Oregon Green BAPTA-5N and to Fluo4FF, Cal-520FF offers a superior signal-to-noise-ratio providing the optimal characteristics for this important type of biophysical measurement. This ability is the result of a relatively high fluorescence at zero Ca2+, necessary to detect enough photons at short exposure windows, and a high dynamic range leading to large fluorescence transients associated with short Ca2+ influx periods. We conclude that Cal-520FF is at present the optimal commercial low-affinity Ca2+ indicator for ultrafast Ca2+ imaging applications.
Asunto(s)
Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Colorantes Fluorescentes/química , Imagen Óptica , Calcio/química , Ácido Egtácico/químicaRESUMEN
Ascorbic acid (AA) has antioxidant properties. However, in the presence of Fe2+/Fe3+ ions and H2O2, it may behave as a pro-oxidant by accelerating and enhancing the formation of hydroxyl radicals (â¢OH). Therefore, in this study we evaluated the effect of AA at concentrations of 1 to 200 µmol/L on â¢OH-induced light emission (at a pH of 7.4 and temperature of 37 °C) from 92.6 µmol/L Fe2+-185.2 µmol/L EGTA (ethylene glycol-bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid)-2.6 mmol/L H2O2, and 92.6 µmol/L Fe3+-185.2 µmol/L EGTA-2.6 mmol/L H2O2 systems. Dehydroascorbic acid (DHAA) at the same range of concentrations served as the reference compound. Light emission was measured with multitube luminometer (AutoLumat Plus LB 953) for 120 s after automatic injection of H2O2. AA at concentrations of 1 to 50 µmol/L and of 1 to 75 µmol/L completely inhibited light emission from Fe2+-EGTA-H2O2 and Fe3+-EGTA-H2O2, respectively. Concentrations of 100 and 200 µmol/L did not affect chemiluminescence of Fe3+-EGTA-H2O2 but tended to increase light emission from Fe2+-EGTA-H2O2. DHAA at concentrations of 1 to 100 µmol/L had no effect on chemiluminescence of both systems. These results indicate that AA at physiological concentrations exhibits strong antioxidant activity in the presence of chelated iron and H2O2.
Asunto(s)
Antioxidantes/química , Antioxidantes/farmacología , Ácido Ascórbico/química , Ácido Ascórbico/farmacología , Ácido Egtácico/química , Compuestos Férricos/química , Compuestos Ferrosos/química , Peróxido de Hidrógeno/química , Radical Hidroxilo/efectos adversos , Radical Hidroxilo/antagonistas & inhibidores , Radical Hidroxilo/química , Luminiscencia , Mediciones LuminiscentesRESUMEN
The healthy prostate contains the highest concentration of mobile zinc in the body. As this level decreases dramatically during the initial development of prostate cancer, inâ vivo detection of prostate zinc content may be applied for diagnosis of prostate cancer. Using 19 F ion chemical exchange saturation transfer magnetic resonance imaging (iCEST MRI) and TF-BAPTA as a fluorinated Zn-binding probe with micromolar sensitivity, we show that iCEST MRI is able to differentiate between normal and malignant prostate cells with a 10-fold difference in contrast following glucose-stimulated zinc secretion inâ vitro. The iCEST signal decreased in normal prostate cells upon downregulation of the ZIP1 zinc transporter. Inâ vivo, using an orthotopic prostate cancer mouse model and a transgenic adenocarcinoma of the mouse prostate (TRAMP) model, a gradual decrease of >300 % in iCEST contrast following the transition of normal prostate epithelial cells to cancer cells was detected.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Imagen por Resonancia Magnética/métodos , Neoplasias de la Próstata/patología , Zinc/química , Animales , Línea Celular Tumoral , Medios de Contraste/química , Modelos Animales de Enfermedad , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Flúor/química , Humanos , Masculino , Ratones , Ratones Transgénicos , Neoplasias de la Próstata/metabolismoRESUMEN
Sample preparation has become an important part of bone proteomics and paleoproteomics and remains one of the major challenges to maximizing the number of proteins characterized from bone extractions. Most paleoproteomic studies have relied on in-solution digestion with the inclusion of filter-aided sample preparation (FASP) as effective methods to detect the proteome. However, neither of these are optimal because few proteins have been detected utilizing only in-solution digestion and the molecular weight cutoff of FASP may miss remaining fragments of proteins in fossil bone. The recently developed single-pot, solid-phase-enhanced sample preparation (SP3) overcomes these issues by not relying on molecular weight while still controlling where the proteins are digested. Here, historical human bones were extracted with either 500 mM tetrasodium EDTA or 400 mM ammonium phosphate dibasic, 200 mM ammonium bicarbonate, 4 M guanidine HCl and digested with the SP3 method. Across all samples, 78 ± 7 (400-200-4) and 79 ± 17 (EDTA) protein accessions were identified, including previously difficult to detect proteins such as osteopontin. SP3 also effectively removed 90% or more of the coextracting humic substances (based on reduced absorbance) from extracted proteins. The utility of SP3 for maximizing the number of protein detections in historical bones is promising for future paleoproteomic studies.
Asunto(s)
Colágeno Tipo I/aislamiento & purificación , Fémur/química , Fósiles , Osteopontina/aislamiento & purificación , Paleontología/métodos , Proteoma/aislamiento & purificación , Extracción en Fase Sólida/métodos , Bicarbonatos/química , Ácido Egtácico/química , Peroné/química , Guanidina/química , Historia Antigua , Humanos , Sustancias Húmicas/análisis , Sustancias Húmicas/historia , Fosfatos/química , Tibia/químicaRESUMEN
In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd=561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to -900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.
Asunto(s)
Calcio/metabolismo , Fluorometría/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuroimagen/métodos , Potenciales de Acción/fisiología , Animales , Calcio/análisis , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Dendritas/metabolismo , Dendritas/fisiología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Ácido Egtácico/metabolismo , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Indicadores y Reactivos/química , Indicadores y Reactivos/metabolismo , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Reproducibilidad de los ResultadosRESUMEN
Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra-weak photon emission (UPE). We investigated the UPE from the Fe2+-EGTA (ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid)-H2O2 system with a multitube luminometer (Peltier-cooled photon counter, spectral range 380 to 630 nm). The UPE of 92.6 µmol/L Fe2+-185.2 µmol/L EGTA-2.6 mmol/L H2O2 reached 4319 ± 755 relative light units during 2 min measurement and was about seven times higher (p < 0.001) than the UPE of incomplete systems (Fe2+-H2O2, EGTA-H2O2) and medium alone. Substitution of Fe2+ with Cr2+, Co2+, Mn2+ or Cu2+ as well as of EGTA with EDTA (ethylenediaminetetraacetic acid) or citrate completely abolished UPE. Experiments with ROS scavengers revealed the dependence of UPE on hydroxyl radicals suggesting occurrence of oxidative attack and cleavage of the ether bond in EGTA backbone structure and formation of triplet excited carbonyl groups with subsequent light emission. Plant phenolics (ferulic, chlorogenic and caffec acids) at concentration 87 µmol/L and ascorbate at 0.46 mmol/L inhibited UPE by 90 ± 4%, 90 ± 5%, 97 ± 2% and 92 ± 1%, respectively. Quenching of UPE from Fe2+-EGTA-H2O2 system can be used for evaluation of antioxidant activity of phytochemicals.
Asunto(s)
Antioxidantes/farmacología , Fenoles/farmacología , Plantas/química , Antioxidantes/química , Ácido Egtácico/química , Peróxido de Hidrógeno/química , Hierro/química , Luz , Luminiscencia , Estrés Oxidativo/efectos de los fármacos , Fenoles/química , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND/AIMS: We investigated how diosgenin, a steroidal sapogenin, has anti-tumor necrosis factor-α (TNF-α) effects in human aortic endothelial cells (HAECs). METHODS: Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Intracellular Ca2+ was measured using Fluo-4 AM. Immunofluorescence staining was performed for a disintegrin and metalloprotease 10 (ADAM10). RESULTS: Diosgenin (1 â¼ 100 nM) induced ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) expression. Upon treatment with diosgenin, extracellular Ca2+ entered into the cells via L-type calcium channels, whereas diosgenin-induced ectodomain shedding of TNFR1 was almost completely inhibited by BAPTA-AM (intracellular Ca2+ chelator), verapamil (L-type calcium channel antagonist) and the absence of extracellular Ca2+. Diosgenin caused translocation of ADAM10 to the cell surface, which was mediated by extracellular Ca2+ influx. Depletion of ADAM10 prevented diosgenin-induced ectodomain shedding of TNFR1 and abolished the inhibitory effect of diosgenin on TNF-α-induced ICAM-1 expression. Diosgenin did not induce extracellular Ca2+ influx and ectodomain shedding of TNFR1 in cells depleted of 1,25D3-membrane associated rapid response steroid-binding receptor (1,25D3-MARRS receptor/ERp57). CONCLUSION: Diosgenin elicits L-type calcium channel-mediated extracellular Ca2+ influx, and thereby induces ADAM10-mediated ectodomain shedding of TNFR1. This effect of diosgenin was exerted through 1,25D3-MARRS receptor/ERp57.
Asunto(s)
Proteína ADAM10/metabolismo , Transporte Biológico/efectos de los fármacos , Diosgenina/farmacología , Proteína Disulfuro Isomerasas/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteína ADAM10/antagonistas & inhibidores , Proteína ADAM10/genética , Calcio/química , Calcio/metabolismo , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Ácido Egtácico/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Microscopía Confocal , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Verapamilo/farmacologíaRESUMEN
Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca(2+) chelator BAPTA or an inhibitor of endoplasmic reticulum Ca(2+)-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca(2+) entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca(2+) channel-mediated Ca(2+) influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Membrana Celular/enzimología , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Fosfopiruvato Hidratasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Biotinilación , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Quelantes/química , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Exosomas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/química , Inflamación , Células MCF-7 , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteína ORAI1 , Molécula de Interacción Estromal 1 , Ácido Tricloroacético/químicaRESUMEN
We have designed a nitroaromatic photochemical protecting group that absorbs visible light in the violet-blue range. The chromophore is a dinitro derivative of bisstyrylthiophene (or BIST) that absorbs light very effectively (ε440 = 66,000 M(-1) cm(-1) and two-photon cross section of 350 GM at 775 nm). We developed a "caged calcium" molecule by conjugation of BIST to a Ca(2+) chelator that upon laser flash photolysis rapidly releases Ca(2+) in <0.2 ms. Using the patch-clamp method the optical probe, loaded with Ca(2+), was delivered into acutely isolated mouse cardiac myocytes, where either one- and two-photon uncaging of Ca(2+) induced highly local or cell-wide physiological Ca(2+) signaling events.
Asunto(s)
Compuestos de Calcio/química , Tiofenos/química , Compuestos de Calcio/síntesis química , Quelantes/química , Ácido Egtácico/química , Luz , Procesos Fotoquímicos , Tiofenos/síntesis químicaRESUMEN
Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 µM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.
Asunto(s)
Calorimetría/métodos , Ligandos , Proteínas/química , Cloruro de Calcio/química , Simulación por Computador , Ácido Edético/análogos & derivados , Ácido Edético/química , Ácido Egtácico/química , Inhibidores Enzimáticos , Unión Proteica , Proteínas/metabolismo , TermodinámicaRESUMEN
We present a simplified reaction network in a single well-mixed volume that captures the general features of CaMKII dynamics observed during both synaptic input and spine depolarization. Our model can also account for the greater-than-control CaMKII activation observed with added EGTA during depolarization. Calcium input currents are modeled after experimental observations, and existing models of calmodulin and CaMKII autophosphorylation are used. After calibration against CaMKII activation data in the absence of chelators, CaMKII activation dynamics due to synaptic input via n-methyl-d-aspartate receptors are qualitatively accounted for in the presence of the chelators EGTA and BAPTA without additional adjustments to the model. To account for CaMKII activation dynamics during spine depolarization with added EGTA or BAPTA, the model invokes the modulation of CaV2.3 (R-type) voltage-dependent calcium channel (VDCC) currents observed in the presence of EGTA or BAPTA. To our knowledge, this is a novel explanation for the increased CaMKII activation seen in dendritic spines with added EGTA, and suggests that differential modulation of VDCCs by EGTA and BAPTA offers an alternative or complementary explanation for other experimental results in which addition of EGTA or BAPTA produces different effects. Our results also show that a simplified reaction network in a single, well-mixed compartment is sufficient to account for the general features of observed CaMKII dynamics.
Asunto(s)
Quelantes del Calcio/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Dendritas/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Animales , Canales de Calcio Tipo R/química , Canales de Calcio Tipo R/metabolismo , Quelantes del Calcio/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Dendritas/efectos de los fármacos , Ácido Egtácico/química , Modelos BiológicosRESUMEN
We have shown that Mg/EGTA (5 mM Mg(2+) and 1.5 mM EGTA) could effectively promote the adhesion of integrin αLß2 to its ligand ICAM-1 but could not promote that of the αMß2 to denatured BSA. In order to determine the structural differences between αL and αM that specifically contribute to Mg/EGTA sensitivity, a series of αL/αM chimeras were constructed. Our results showed that αLß2 with αM calf-1 domain completely lost the response to Mg/EGTA activation. In the reverse experiment, αMß2 would require the presence of both the αL calf-1 and calf-2 domain to initiate the Mg/EGTA sensitivity.
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Adhesión Celular/fisiología , Ácido Egtácico/química , Ácido Egtácico/metabolismo , Antígeno-1 Asociado a Función de Linfocito/química , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Magnesio/química , Magnesio/metabolismo , Sitios de Unión , Células HEK293 , Humanos , Unión Proteica , Relación Estructura-ActividadRESUMEN
The mechanism underlying the inflammatory role of TRPA1 in lung epithelial cells (LECs) remains unclear. Here, we show that cigarette smoke extract (CSE) sequentially induced several events in LECs. The Ca(2+) influx was prevented by decreasing extracellular reactive oxygen species (ROS) with the scavenger N-acetyl-cysteine, removing extracellular Ca(2+) with the chelator EGTA, or treating with the TRPA1 antagonist HC030031. NADPH oxidase activation was abolished by its inhibitor apocynin, EGTA, or HC030031. The increased intracellular ROS was halted by apocynin, N-acetyl-cysteine, or HC030031. The activation of the MAPKs/NF-κB signaling was suppressed by EGTA, N-acetyl-cysteine, or HC030031. IL-8 induction was inhibited by HC030031 or TRPA1 siRNA. Additionally, chronic cigarette smoke (CS) exposure in wild-type mice induced TRPA1 expression in LECs and lung tissues. In CS-exposure trpa1 (-/-) mice, the increased BALF level of ROS was similar to that of CS-exposure wild-type mice; yet lung inflammation was lessened. Thus, in LECs, CSE may initially increase extracellular ROS, which activate TRPA1 leading to an increase in Ca(2+) influx. The increased intracellular Ca(2+) contributes to activation of NADPH oxidase, resulting in increased intracellular ROS, which activate the MAPKs/NF-κB signaling leading to IL-8 induction. This mechanism may possibly be at work in mice chronically exposed to CS.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Pulmón/patología , Proteínas del Tejido Nervioso/metabolismo , Humo/efectos adversos , Canales de Potencial de Receptor Transitorio/metabolismo , Acetanilidas/química , Acetofenonas/química , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar , Canales de Calcio/genética , Quelantes/química , Quimiocina CXCL2/metabolismo , Ácido Egtácico/química , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación , Interleucina-8/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NADPH Oxidasas/metabolismo , Proteínas del Tejido Nervioso/genética , Estrés Oxidativo , Purinas/química , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
We proposed a new indicator for evaluation of functional activity of Ca(2+) in human blood serum based on lysis of sheep erythrocytes with 10% human blood serum in the presence of 0.55 mM ethylene glycol tetraacetic acid at 37°C for 10 min. After incubation, the degree of sheep erythrocytes lysis inhibition is estimated: inhibition of complement hemolytic activity <30% is considered as high functional Са(2+) activity, inhibition by 31-70% corresponds to normal activity, and >71% indicates low activity. The comparative studies of complement activating function of heterophilic antibodies, complement system reactivity in the presence of 0.29 M NaCl, and functional activity of ionized Ca(2+) make possible estimation of the individual's immune status.
Asunto(s)
Calcio/farmacología , Animales , Calcio/química , Activación de Complemento/efectos de los fármacos , Ácido Edético/química , Ácido Egtácico/química , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , OvinosRESUMEN
The effect of cations and anions on the activity of Aspergillus flavus var. oryzae and Bacillus subtilis α-amylases showed that the tested enzymes are sensitive to most of cations and resistant to anions. The most significant inhibitory effects on the activity of A. flavus var. oryzae α-amylase have been demonstrated by Al3+ and Fe3+ ions, while on the activity of B. subtilis α-amylase - Hg2+, Cu2+ and Fe3+ ions. Inactivation of A. flavus var. oryzae and B. subtilis α-amylases in the presence of EGTA is indicated on the presence within their structure of metal ions. An important role in the enzymatic catalysis of both enzymes play carboxyl groups as evidenced by their inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. Inhibition of B. subtilis α-amylase by p-chloromercuribenzoate, N-ethylmaleimide and sodium sulfite is indicated on the probable involvement of the sulfhydryl groups in the functioning of the enzyme. Unlike most studied glycosidases the tested enzymes do not contain histidine imidazole group in the active center.
Asunto(s)
Aspergillus flavus/química , Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas Fúngicas/química , alfa-Amilasas/química , Aluminio/química , Aspergillus flavus/enzimología , Bacillus subtilis/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/aislamiento & purificación , Biocatálisis , Carbodiimidas/química , Dominio Catalítico , Cationes , Cobre/química , Ácido Egtácico/química , Pruebas de Enzimas , Etilmaleimida/química , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/aislamiento & purificación , Hierro/química , Cinética , Mercurio/química , Sulfitos/química , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/aislamiento & purificación , Ácido p-Cloromercuribenzoico/químicaRESUMEN
Desmin forms the intermediate filament system of muscle cells where it plays important role in maintaining mechanical integrity and elasticity. Although the importance of intermediate-filament elasticity in cellular mechanics is being increasingly recognized, the molecular basis of desmin's elasticity is not fully understood. We explored desmin elasticity by molecular combing with forces calculated to be as large as 4nN. Average filament contour length increased 1.55-fold axial on average. Molecular combing together with EGTA-treatment caused the fragmentation of the filament into short, 60 to 120-nm-long and 4-nm-wide structures. The fragments display a surface periodicity of 38nm, suggesting that they are composed of laterally attached desmin dimers. The axis of the fragments may deviate significantly from that of the overstretched filament, indicating that they have a large orientational freedom in spite of being axially interconnected. The emergence of protofibril fragments thus suggests that the interconnecting head or tail domains of coiled-coil desmin dimers are load-bearing elements during axial stretch.