53,550 research outputs found

    Structure of the full-length TRPV2 channel by cryo-EM.

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    Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels

    Comparison of Oral, Intranasal and Aerosol Administration of Amiodarone in Rats as a Model of Pulmonary Phospholipidosis.

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    ‘Foamy’ alveolar macrophages (FAM) observed in nonclinical toxicology studies during inhaled drug development may indicate drug-induced phospholipidosis, but can also derive from adaptive non-adverse mechanisms. Orally administered amiodarone is currently used as a model of pulmonary phospholipidosis and it was hypothesized that aerosol administration would produce phospholipidosis-induced FAM that could be characterized and used in comparative inhalation toxicology. Han-Wistar rats were given amiodarone via (1) intranasal administration (6.25 mg/kg) on two days, (2) aerosol administration (3 mg/kg) on two days, (3) aerosol administration (10 mg/kg) followed by three days of 30 mg/kg or (4) oral administration (100 mg/kg) for 7 days. Alveolar macrophages in bronchoalveolar lavage were evaluated by di_erential cell counting and high content fluorescence imaging. Histopathology and mass-spectrometry imaging (MSI) were performed on lung slices. The higher dose aerosolised amiodarone caused transient pulmonary inflammation (p < 0.05), but only oral amiodarone resulted in FAM (p < 0.001). MSI of the lungs of orally treated rats revealed a homogenous distribution of amiodarone and a putative phospholipidosis marker, di-22:6 bis-monoacylglycerol, throughout lung tissue whereas aerosol administration resulted in localization of both compounds around the airway lumen. Thus, unlike oral administration, aerosolised amiodarone failed to produce the expected FAM responses.Peer reviewedFinal Published versio

    Rapid isolation and purification of mitochondria for transplantation using tissue dissociation and differential filtration

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    Researchers have identified several methods for treating acute myocardial infarction (AMI) patients affected by ischemia and reperfusion injury. Some of these therapies include thrombolysis, balloon angioplasty, and coronary arterial bypass graft (CAGB). This lab has previously demonstrated that transplantation of mitochondria into the ischemic zone of a rabbit heart during reperfusion significantly improved recovery as compared to current techniques. In order for this therapy to be translated into the clinic a rapid isolation method for producing highly pure and functional mitochondria will be required. Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 minutes to complete. Herein, a method for rapid isolation of mitochondria from mammalian tissue biopsies is described. In this protocol, manual homogenization is replaced with the tissue dissociator's standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters which eliminates repetitive centrifugation steps. Mitochondrial isolation time is less than 30 minutes compared to 60-100 minutes using alternative methods. This isolation protocol yields approximately 2 x 10^10 viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample

    A unified approach to linking experimental, statistical and computational analysis of spike train data

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    A fundamental issue in neuroscience is how to identify the multiple biophysical mechanisms through which neurons generate observed patterns of spiking activity. In previous work, we proposed a method for linking observed patterns of spiking activity to specific biophysical mechanisms based on a state space modeling framework and a sequential Monte Carlo, or particle filter, estimation algorithm. We have shown, in simulation, that this approach is able to identify a space of simple biophysical models that were consistent with observed spiking data (and included the model that generated the data), but have yet to demonstrate the application of the method to identify realistic currents from real spike train data. Here, we apply the particle filter to spiking data recorded from rat layer V cortical neurons, and correctly identify the dynamics of an slow, intrinsic current. The underlying intrinsic current is successfully identified in four distinct neurons, even though the cells exhibit two distinct classes of spiking activity: regular spiking and bursting. This approach – linking statistical, computational, and experimental neuroscience – provides an effective technique to constrain detailed biophysical models to specific mechanisms consistent with observed spike train data.Published versio

    Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry.

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    The structure of rat liver vault ribonucleoprotein particles was examined using several different staining techniques in conjunction with EM and digestion with hydrolytic enzymes. Quantitative scanning transmission EM demonstrates that each vault particle has a total mass of 12.9 +/- 1 MD and contains two centers of mass, suggesting that each vault particle is a dimer. Freeze-etch reveals that each vault opens into delicate flower-like structures, in which eight rectangular petals are joined to a central ring, each by a thin hook. Vaults examined by negative stain and conventional transmission EM (CTEM) also reveal the flower-like structure. Trypsin treatment of vaults resulted exclusively in cleavage of the major vault protein (p104) and concurrently alters their structure as revealed by negative stain/CTEM, consistent with a localization of p104 to the flower petals. We propose a structural model that predicts the stoichiometry of vault proteins and RNA, defines vault dimer-monomer interactions, and describes two possible modes for unfolding of vaults into flowers. These highly dynamic structural variations are likely to play a role in vault function

    Structural insights on TRPV5 gating by endogenous modulators.

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    TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-Ï€ interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery

    His+ reversions Caused in Salmonella typhimurium by different types of ionizing radiation

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    The yield of his+ reversions in the Ames Salmonella tester strain TA2638 has been determined for 60Co γ rays, 140 kV X rays, 5.4 keV characteristic X rays, 2.2 MeV protons, 3.1 MeV α particles, and 18 MeV/U Fe ions. Inactivation studies were performed with the same radiations. For both mutation and inactivation, the maximum effectiveness per unit absorbed dose was obtained for the characteristic X rays, which have a dose averaged linear energy transfer (LET) of roughly 10 keV/μm. The ratio of the effectiveness of this radiation to γ rays was 2 for inactivation and about 1.4 for the his+ reversion. For both end points the effectiveness decreases substantially at high LET, i.e., for the α particles and the Fe ions. The composition of the bottom and the top agar was the one recommended by Maron and Ames [Mutat. Res. 113, 173-215 (1983)] for application in chemical mutagenicity tests. The experiments with the less penetrating radiations differed from the usual protocol by utilization of a technique of plating the bacteria on the surface of the top agar. As in an earlier study [Roos et al., Radiat. Res. 104, 102-108 (1985)] greatly enhanced yields of mutations, relative to the spontaneous reversion rate, were obtained in these experiments by performing the irradiations 6 h after plating, which differs from the conventional procedure to irradiate the bacteria shortly after plating

    Data Assimilation: A Mathematical Introduction

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    These notes provide a systematic mathematical treatment of the subject of data assimilation
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