The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
Detlev Riesner - One of the best experts on this subject based on the ideXlab platform.
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Heat stability of prion rods and recombinant prion protein in water, Lipid and Lipid–water mixtures
Journal of General Virology, 2001Co-Authors: Thomas R. Appel, Michael Andreas Wolff, Friedrich Von Rheinbaben, Michael Heinzel, Detlev RiesnerAbstract:Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27–30, and the corresponding recombinant protein, rPrP(90–231), were autoclaved in water, bovine Lipid or Lipid–water mixtures for 20 min at temperatures from 100 to 170 °C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of Lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or Lipid–water as well as for rPrP(90–231) in Lipid–water. The presence of Lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in Pure Lipid gave different results – not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in Pure Lipid exceeded that in Lipid–water mixtures. Degradation factors larger than 104 were reached at 170 °C in the presence of Lipids and at 150 °C in the absence of Lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 °C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.
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Heat stability of prion rods and recombinant prion protein in water, Lipid and Lipid-water mixtures.
The Journal of general virology, 2001Co-Authors: Thomas R. Appel, Friedrich Von Rheinbaben, Michael Heinzel, Michael Wolff, Detlev RiesnerAbstract:Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP(90-231), were autoclaved in water, bovine Lipid or Lipid-water mixtures for 20 min at temperatures from 100 to 170 degrees C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of Lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or Lipid-water as well as for rPrP(90-231) in Lipid-water. The presence of Lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in Pure Lipid gave different results - not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in Pure Lipid exceeded that in Lipid-water mixtures. Degradation factors larger than 10(4) were reached at 170 degrees C in the presence of Lipids and at 150 degrees C in the absence of Lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 degrees C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.
David Tareste - One of the best experts on this subject based on the ideXlab platform.
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Energetics and dynamics of SNAREpin folding across Lipid bilayers
Nature Structural & Molecular Biology, 2007Co-Authors: Feng Li, Frédéric Pincet, Eric Perez, Thomas J Melia, James E Rothman, David TaresteAbstract:Membrane fusion occurs when SNAREpins fold up between Lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy–versus–distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (∼2–4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k _B T ) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of Pure Lipid bilayers (40–50 k _B T ).
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energetics and dynamics of snarepin folding across Lipid bilayers
Nature Structural & Molecular Biology, 2007Co-Authors: Frédéric Pincet, Eric Perez, Thomas J Melia, James E Rothman, William S Eng, David TaresteAbstract:Membrane fusion occurs when SNAREpins fold up between Lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of Pure Lipid bilayers (40-50 k(B)T).
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Energetics and dynamics of SNAREpin folding across Lipid bilayers
Nature Structural & Molecular Biology, 2007Co-Authors: Frédéric Pincet, Eric Perez, Thomas J Melia, James E Rothman, William S Eng, David TaresteAbstract:Membrane fusion occurs when SNAREpins fold up between Lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy–versus–distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (∼2–4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k _B T ) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of Pure Lipid bilayers (40–50 k _B T ).
J. C. Ruiz-suárez - One of the best experts on this subject based on the ideXlab platform.
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Physical Aspects of the Cut-Off Effect of N-Alcohols in Pure Lipid Membranes
Biophysical Journal, 2014Co-Authors: Francisco J. Sierra-valdez, J. C. Ruiz-suárezAbstract:Nowadays, the molecular nature of general anesthetic target sites remains unknown; some theories profess that the action occurs in proteins, others in Lipids. In particular, for nearly a century it has been known that alcohols can act as general anesthetics. However, as the chain length of an alcohol increases, so does its potency as an anesthetic, only up to a certain chain length beyond which the anesthetic activity disappears (the so-called "cut-off effect"). In the attempt to explain such phenomenon, and based on the proposed anesthetic theories, different explanations have emerged without conclusive arguments, nevertheless, the lack of sufficient evidences supporting the cut-off effect of n-alcohols in general anesthesia make this work worth to be pursue. In the present work, using calorimetry and atomic force microscopy (AFM), we show a systematic study of the interaction of n-alcohols (from methanol, C1, to eicosanol, C20) with Lipid membranes, in order to collaborate in the comprehension of a physical mechanism of the cut-off phenomenon. Our results suggest that the lowering of the melting transition temperature (Tm) of Lipid membranes due to short-chain alcohols, is highly related to their ability to disturb Lipid membranes (as has been shown for a wide variety of anesthetics), whilst, the increase of Tm induced by long-chain ones (from C12), is caused by a stiffening of the Lipid membrane. We also correlate such effects with some physical properties of n-alcohols and the Lipid composition. These results concur with other findings to underwrite the idea that anesthesia does not need a specific binding site in a protein and allow us to speculate that anesthesia only depends on the ability of certain atom or molecule to solubilized in Lipids increasing the disorder of the membrane.
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The influence of non polar and polar molecules in mouse motile cells membranes and Pure Lipid bilayers.
PloS one, 2013Co-Authors: Francisco J. Sierra-valdez, Linda S. Forero-quintero, Patricio A. Zapata-morin, Miguel Costas, Arturo Chavez-reyes, J. C. Ruiz-suárezAbstract:We report an experimental study of mouse sperm motility that shows chief aspects characteristic of neurons: the anesthetic (produced by tetracaine) and excitatory (produced by either caffeine or calcium) effects and their antagonic action. While tetracaine inhibits sperm motility and caffeine has an excitatory action, the combination of these two substances balance the effects, producing a motility quite similar to that of control cells. We also study the effects of these agents (anesthetic and excitatory) on the melting points of Pure Lipid liposomes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and dipalmitoyl phosphatidic acid (DPPA). Tetracaine induces a large fluidization of the membrane, shifting the liposomes melting transition temperature to much lower values. The effect of caffeine is null, but its addition to tetracaine-doped liposomes greatly screen the fluidization effect. A high calcium concentration stiffens Pure Lipid membranes and strongly reduces the effect of tetracaine. Molecular Dynamics Simulations are performed to further understand our experimental findings at the molecular level. We find a strong correlation between the effect of antagonic molecules that could explain how the mechanical properties suitable for normal cell functioning are affected and recovered.
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Noble gases in Pure Lipid membranes
The journal of physical chemistry. B, 2013Co-Authors: Francisco J. Sierra-valdez, J. C. Ruiz-suárezAbstract:The mechanism of how a noble gas modifies the excitability of nerve cells and how such excitability can be recovered under hyperbaric pressure remains unclear. Here we present a calorimetric study where the melting point depression of Pure Lipid membranes induced by noble gases and its recovery with a hydrostatic pressure is addressed. A correlation is found between the electric polarizability (α) of these gases and their effect on the melting transition of the membranes. These results concur with other findings to support the idea that general anesthesia only depends on the ability of a certain atom or molecule to increase the general disorder of the membrane.
David A. Middleton - One of the best experts on this subject based on the ideXlab platform.
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Solid-state NMR reveals structural changes in phospholamban accompanying the functional regulation of Ca2+-ATPase.
The Journal of biological chemistry, 2003Co-Authors: Eleri Hughes, David A. MiddletonAbstract:Abstract Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca2+-ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca2+-ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. Novel backbone amide proton-deuterium exchange CP-MAS NMR experiments on the two PLB analogues co-reconstituted with SERCA1 indicated that labeled residues Leu42 and Leu44 were situated well within the membrane interior, whereas Pro21 and Ala24 lie exposed outside the membrane. Internuclear distance measurements on PLB using rotational resonance NMR indicated that the sequences Pro21–Ala24 and Leu42–Leu44 adopt an α-helical structure in Pure Lipid bilayers, which is unchanged in the presence of Ca2+-ATPase. By contrast, rotational echo double resonance (REDOR) NMR experiments revealed that the sequence Ala24–Gln26 switches from an α-helix in Pure Lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca2+-ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins.
Thomas R. Appel - One of the best experts on this subject based on the ideXlab platform.
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Heat stability of prion rods and recombinant prion protein in water, Lipid and Lipid–water mixtures
Journal of General Virology, 2001Co-Authors: Thomas R. Appel, Michael Andreas Wolff, Friedrich Von Rheinbaben, Michael Heinzel, Detlev RiesnerAbstract:Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27–30, and the corresponding recombinant protein, rPrP(90–231), were autoclaved in water, bovine Lipid or Lipid–water mixtures for 20 min at temperatures from 100 to 170 °C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of Lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or Lipid–water as well as for rPrP(90–231) in Lipid–water. The presence of Lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in Pure Lipid gave different results – not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in Pure Lipid exceeded that in Lipid–water mixtures. Degradation factors larger than 104 were reached at 170 °C in the presence of Lipids and at 150 °C in the absence of Lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 °C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.
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Heat stability of prion rods and recombinant prion protein in water, Lipid and Lipid-water mixtures.
The Journal of general virology, 2001Co-Authors: Thomas R. Appel, Friedrich Von Rheinbaben, Michael Heinzel, Michael Wolff, Detlev RiesnerAbstract:Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP(90-231), were autoclaved in water, bovine Lipid or Lipid-water mixtures for 20 min at temperatures from 100 to 170 degrees C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of Lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or Lipid-water as well as for rPrP(90-231) in Lipid-water. The presence of Lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in Pure Lipid gave different results - not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in Pure Lipid exceeded that in Lipid-water mixtures. Degradation factors larger than 10(4) were reached at 170 degrees C in the presence of Lipids and at 150 degrees C in the absence of Lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 degrees C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.
