1,720,960 research outputs found
Ion trapping with fast-response ion-selective microelectrodes enhances detection of extracellular ion channel gradients
Full text linkPreviously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 µm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10–55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane
Windows to cell function and dysfunction: signatures written in the boundary layers
Full text linkThe medium surrounding cells either in culture or in tissues contains a chemical mix varying with cell state. As solutes move in and out of the cytoplasmic compartment they set up characteristic signatures in the cellular boundary layers. These layers are complex physical and chemical environments the profiles of which reflect cell physiology and provide conduits for intercellular messaging. Here we review some of the most relevant characteristics of the extracellular/intercellular space. Our initial focus is primarily on cultured cells but we extend our consideration to the far more complex environment of tissues, and discuss how chemical signatures in the boundary layer can or may affect cell function. Critical to the entire essay are the methods used, or being developed, to monitor chemical profiles in the boundary layers. We review recent developments in ultramicro electrochemical sensors and tailored optical reporters suitable for the task in hand
Fast response, noninvasive, potentiometric microelectrodes resolve single potassium channel activity in the diffusive boundary layer of a single cell
No full textIon transport across the plasma membrane of cells involves an inevitable chemical modification of the solutions on both sides. Noninvasive, real-time detection of these subtle ionic signatures is most easily achieved in the extracellular diffusive boundary layer with electrochemical detection. In order to perform these measurements near single cells it is critical to use a device that enables high spatial and temporal resolution. While ion-selective microelectrodes (ISMs) provide the high spatial resolution, they give rise to orders of magnitude increase in the time constant and response time of the microsensors when compared to larger sensors. By constructing and using fast response ISMs biological events as brief as 10ms have been resolved. The signal-to-noise ratio is enhanced with self-referencing and signal processing techniques, enabling long-term monitoring of small magnitude, steady ion gradients and rapid ionic transients that reflect the physiological and metabolic activity of single cell
Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity
No full textIn order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O(2) consumption, cytosolic Ca(2+) concentration ([Ca(2+)](i)), and mitochondrial membrane potential (mDeltapsi) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca(2+)](i) and mDeltapsi were monitored with Fluo-4 and TMRE(+), respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca(2+)](i) followed seconds afterwards by an increase in O(2) consumption which reached a maximum level within 1-5 min. A modest increase in mDeltapsi occurred during this time period, and then, shortly before maximal O(2) consumption was reached, the mDeltapsi, as indicated by TMRE(+) fluorescence, dissipated. Maximal O(2) consumption lasted up to 5 min and then declined together with mDeltapsi and ATP levels, while [Ca(2+)](i) further increased. mDeltapsi and [Ca(2+)](i) returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca(2+)](i) increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O(2) consumption, [Ca(2+)](i), and mDeltapsi. The data obtained using this new method are consistent with a model where Ca(2+) influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation
Role for malic enzyme, pyruvate carboxylation, and mitochondrial malate import in glucose-stimulated insulin secretion
No full textPyruvate cycling has been implicated in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. The operation of some pyruvate cycling pathways is proposed to necessitate malate export from the mitochondria and NADP(+)-dependent decarboxylation of malate to pyruvate by cytosolic malic enzyme (ME1). Evidence in favor of and against a role of ME1 in GSIS has been presented by others using small interfering RNA-mediated suppression of ME1. ME1 was also proposed to account for methyl succinate-stimulated insulin secretion (MSSIS), which has been hypothesized to occur via succinate entry into the mitochondria in exchange for malate and subsequent malate conversion to pyruvate. In contrast to rat, mouse beta-cells lack ME1 activity, which was suggested to explain their lack of MSSIS. However, this hypothesis was not tested. In this report, we demonstrate that although adenoviral-mediated overexpression of ME1 greatly augments GSIS in rat insulinoma INS-1 832/13 cells, it does not restore MSSIS, nor does it significantly affect GSIS in mouse islets. The increase in GSIS following ME1 overexpression in INS-1 832/13 cells did not alter the ATP-to-ADP ratio but was accompanied by increases in malate and citrate levels. Increased malate and citrate levels were also observed after INS-1 832/13 cells were treated with the malate-permeable analog dimethyl malate. These data suggest that although ME1 overexpression augments anaplerosis and GSIS in INS-1 832/13 cells, it is not likely involved in MSSIS and GSIS in pancreatic islets
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Modulation of extracellular proton fluxes from retinal horizontal cells of the catfish by depolarization and glutamate
Full text linkSelf-referencing H(+)-selective microelectrodes were used to measure extracellular proton fluxes from cone-driven horizontal cells isolated from the retina of the catfish (Ictalurus punctatus). The neurotransmitter glutamate induced an alkalinization of the area adjacent to the external face of the cell membrane. The effect of glutamate occurred regardless of whether the external solution was buffered with 1 mM HEPES, 3 mM phosphate, or 24 mM bicarbonate. The AMPA/kainate receptor agonist kainate and the NMDA receptor agonist N-methyl-D-aspartate both mimicked the effect of glutamate. The effect of kainate on proton flux was inhibited by the AMPA/kainate receptor blocker CNQX, and the effect of NMDA was abolished by the NMDA receptor antagonist DAP-5. Metabotropic glutamate receptor agonists produced no alteration in proton fluxes from horizontal cells. Depolarization of cells either by increasing extracellular potassium or directly by voltage clamp also produced an alkalinization adjacent to the cell membrane. The effects of depolarization on proton flux were blocked by 10 microM nifedipine, an inhibitor of L-type calcium channels. The plasmalemma Ca(2+/)H(+) ATPase (PMCA) blocker 5(6)-carboxyeosin also significantly reduced proton flux modulation by glutamate. Our results are consistent with the hypothesis that glutamate-induced extracellular alkalinizations arise from activation of the PMCA pump following increased intracellular calcium entry into cells. This process might help to relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons from photoreceptor synaptic terminals. Our findings argue strongly against the hypothesis that protons released by horizontal cells act as the inhibitory feedback neurotransmitter that creates the surround portion of the receptive fields of retinal neuron
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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