Smith Peter J. S.

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Smith
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Peter J. S.
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  • Preprint
    Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase
    ( 2011-06) Alavian, Kambiz N. ; Li, Hongmei ; Collis, Leon P. ; Bonanni, Laura ; Zeng, Lu ; Sacchetti, Silvio ; Lazrove, Emma ; Nabili, Panah ; Flaherty, Benjamin ; Graham, Morven ; Chen, Yingbei ; Messerli, Shanta M. ; Mariggio, Maria A. ; Rahner, Christoph ; McNay, Ewan ; Shore, Gordon ; Smith, Peter J. S. ; Hardwick, J. Marie ; Jonas, Elizabeth A.
    Anti-apoptotic BCL-2 family proteins such as Bcl-xL protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-xL enhances the efficiency of energy metabolism. Our evidence suggests that Bcl-xL interacts directly with the beta subunit of the F1FO ATP synthase, decreasing an ion leak within the F1FO ATPase complex and thereby increasing net transport of H+ by F1FO during F1FO ATPase activity. By patch clamping submitochondrial vesicles enriched in F1FO ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-xL increases the membrane leak conductance. In addition, recombinant Bcl-xL protein directly increases ATPase activity of purified synthase complexes, while inhibition of endogenous Bcl-xL decreases F1FO enzymatic activity. Our findings suggest that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-xL expressing neurons.
  • Preprint
    Electrokinetic measurements of membrane capacitance and conductance for pancreatic β-cells
    ( 2005-10-31) Pethig, Ronald ; Jakubek, L. M. ; Sanger, R. H. ; Heart, E. ; Corson, Erica D. ; Smith, Peter J. S.
    Membrane capacitance and membrane conductance values are reported for insulin secreting cells (primary β-cells and INS-1 insulinoma cells) determined using the methods of dielectrophoresis and electrorotation. The membrane capacitance value of 12.57 (± 1.46) mF/m2 obtained for β-cells, and the values 9.96 (± 1.89) mF/m2 to 10.65 (± 2.1) mF/m2 obtained for INS-1 cells, fall within the range expected for mammalian cells. The electrorotation results for the INS-1 cells lead to a value of 36 (± 22) S/m2 for the membrane conductance associated with ion channels, if values in the range 2nS to 3 nS are assumed for the membrane surface conductance. This membrane conductance value falls within the range reported for INS cells obtained using the whole-cell patch-clamp technique. However, the total ‘effective’ membrane conductance value of 601 (± 182) S/m2 obtained for the INS-1 cells by dielectrophoresis is significantly larger (by a factor of around three-fold) than the values obtained by electrorotation. This could result from an increased membrane surface conductance, or increased passive conduction of ions through membrane pores, induced by the larger electric field stresses experienced by cells in the dielectrophoresis experiments.
  • Preprint
    Ion trapping with fast-response ion-selective microelectrodes enhances detection of extracellular ion channel gradients
    ( 2008-11) Messerli, Mark A. ; Collis, Leon P. ; Smith, Peter J. S.
    Previously, 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.
  • Preprint
    Construction, Theory, and Practical Considerations for using Self-referencing of Ca2+-Selective Microelectrodes for Monitoring Extracellular Ca2+ Gradients
    ( 2010-10) Messerli, Mark A. ; Smith, Peter J. S.
    Ca2+ signaling in the extra- and intracellular domains is linked to Ca2+ transport across the plasma membrane. Non-invasive monitoring of these resulting extracellular Ca2+ gradients with self-referencing of Ca2+-selective microelectrodes is used for studying Ca2+ signaling across Kingdoms. The quantitated Ca2+ flux enables comparison with changes to intracellular [Ca2+] measured with other methods and determination of Ca2+ transport stoichiometry. Here we review the construction of Ca2+-selective microelectrodes, their physical characteristics and their use in self-referencing mode to calculate Ca2+ flux. We also discuss potential complications when using them to measure Ca2+ gradients near the boundary layers of single cells and tissues.
  • Preprint
    Windows to cell function and dysfunction : signatures written in the boundary layers
    ( 2010-01-26) Smith, Peter J. S. ; Collis, Leon P. ; Messerli, Mark A.
    The 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 whose profiles both 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 with 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.
  • Preprint
    Dielectrophoretic tweezer for isolating and manipulating target cells
    ( 2010-06-03) Menachery, Anoop ; Graham, David M. ; Messerli, Shanta M. ; Pethig, Ronald ; Smith, Peter J. S.
    The ability to isolate and accurately position single cells in three dimensions is becoming increasingly important in many areas of biological research. We describe the design, theoretical modeling and testing of a novel dielectrophoretic (DEP) tweezer for picking out and relocating single target cells.. The device is constructed using facilities available in most electrophysiology laboratories, without the requirement of sophisticated and expensive microfabrication technology, and offers improved practical features over previously reported DEP tweezer designs. The DEP tweezer has been tested using transfected HEI 193 human schwannoma cells, with visual identification of the target cells being aided by labeling the incorporated gene product with a green fluorescent protein.
  • Preprint
    Dielectrophoretic assembly of insulinoma cells and fluorescent nanosensors into three-dimensional pseudo-islet constructs
    ( 2007-11-14) Pethig, Ronald ; Menachery, Anoop ; Heart, E. ; Sanger, R. H. ; Smith, Peter J. S.
    Dielectrophoretic forces, generated by radio-frequency voltages applied to micromachined, transparent, indium tin oxide electrodes, have been used to condense suspensions of insulinoma cells (BETA-TC-6 and INS-1) into a 10x10 array of threedimensional cell constructs. Some of these constructs, measuring approximately 150 μm in diameter and 120 μm in height, and containing around 1000 cells, were of the same size and cell density as a typical islet of Langerhans. With the dielectrophoretic force maintained, these engineered cell constructs were able to withstand mechanical shock and fluid flow forces. Reproducibility of the process required knowledge of cellular dielectric properties, in terms of membrane capacitance and membrane conductance, which were obtained by electrorotation measurements. The ability to incorporate fluorescent nanosensors, as probes of cellular oxygen and pH levels, into these ‘pseudo-islets’ was also demonstrated. The footprint of the 10x10 array of cell constructs was compatible with that of a 1536 microtitre plate, and thus amenable to optical interrogation using automated plate reading equipment.