Publications

2006

• Photoinduced electron transfer from a novel nanotrigger addressed to the NADPH site within the endothelial NO-synthase to the flavin moieties of the protein
  
  • Beaumont Edward
  • Robin Anne-Claire
  • Berka Vladimir
  • Tsai Ah-Lim
  • Blanchard-Desce Mireille
  • Lambry Jean-Christophe
  • Slama-Schwok Anny
  Nitric Oxide: Biology and Chemistry, Elsevier, 2006, 14 (4), pp.14-15. We developed a new selective molecular tool to trigger enzymatic activity in a synchronous manner and monitor the sequence of the kinetic events by ultra-fast transient spectroscopy. Our approach is based on a synthetic nanotrigger addressing a selected site within proteins, namely the conserved NADPH binding site common to many enzymes involved in bioreductive processes [1]. The nanotrigger combines a "docking" subunit responsible for the recognition of NADPH sites within proteins and a "chromophoric" subunit responsive to light excitation and able to transfer electrons to the flavin moieties of proteins. We present the first spectroscopic data on such a nanotrigger [1] in the presence of the reductase domain of the endothelial nitric oxide synthase eNOSred. (10.1016/j.niox.2006.04.050)
  DOI : 10.1016/j.niox.2006.04.050
• Nouvelle approche des fibroses par microscopie multiphotonique avec génération de second harmonique [New approach of fibrosis by multiphoton microscopy with second harmonic generation]
  
  • Hernest Monica
  • Pena Ana-Maria
  • Strupler Mathias
  • Beaurepaire Emmanuel
  • Martin Jean-Louis
  • Schanne-Klein Marie-Claire
  • Tharaux Pierre-Louis
  Médecine/Sciences, EDP Sciences, 2006, 22 (10), pp.820-821. La fibrose est une réponse adaptative pathologique qui détruit non spécifiquement les tissus. Il s'agit d'un processus universel de réparation des tissus qui survient en réaction à de nombreux types d'agressions telles les contraintes mécaniques, les brûlures, les radiations ionisantes, l'ischémie, l'inflammation. Ces agressions concourent de manière intriquée à la physiopathologie des maladies infectieuses, tumorales ou auto-immunes, de l'hypertension artérielle et des maladies cardio-vasculaires. Le terme de fibrose décrit précisément l'accumulation nouvelle de protéines de la matrice extra-cellulaire selon un arrangement spatial fibrillaire caractéristique. Il s'agit essentiellement de molécules de collagènes de type I et III (voire de type II, V ou XI) synthétisées sous forme de triples hélices elles-mêmes assemblées en fibrilles par les cellules fibroblastiques. L'apparition des collagènes fibrillaires marque un changement qualitatif et quantitatif de composition des collagènes des tissus. Réciproquement, ces changements de la matrice extracellulaire influencent le phénotype des cellules qui y résident. Ainsi dans le rein normal, le collagène de type I n'existe que dans l'adventice artériel. Son apparition au sein des autres structures de cet organe marque une fibrose tubulo-interstitielle qui constitue le meilleur marqueur pronostic défavorable d'une évolution vers l'insuffisance rénale terminale, et ce quelle que soit la maladie causale. Ainsi, comme lors des fibroses compliquant les hépatopathies et les pneumopathies chroniques, les séquelles de brûlures ou d'abrasions cutanées-muqueuses ou encore le remodelage cardiaque et vasculaire, le réarrangement de la géométrie de la matrice extracellulaire altère l'organisation fonctionnelle du tissu considéré. De ce fait, ce processus de réparation a des effets fonctionnels délétères qui constituent un enjeu médical majeur. Les fibrilles de collagène ont des capacités d'auto-assemblage qui sont aussi catalysées et stabilisées ou au contraire empêchées par les enzymes de la matrice extracellulaire. Le développement de la fibrose ou sa régression dépend donc ainsi du bilan des équilibres biologiques de ces mécanismes. Il est donc crucial de caractériser les changements extracellulaires et cellulaires qui font du restutio ad integrum de l'architecture et de la fonction tissulaire un défi biomédical. (10.1051/medsci/20062210820)
  DOI : 10.1051/medsci/20062210820
• Endothelial nitric oxide synthase reduces nitrite anions to NO under anoxia
  
  • Gautier Clément
  • van Faassen E.
  • Mikula I.
  • Martasek P.
  • Slama-Schwok Anny
  Biochemical and Biophysical Research Communications, Elsevier, 2006, 341 (3), pp.816-821. In this work, we demonstrate that endothelial nitric oxide synthase is capable of anoxic reduction of nitrite anions to nitric oxide at physiological pH by absorption and EPR spectroscopy and electrochemical measurements. The nitrite reduction is achieved at the oxygenase domain of the protein and proceeds even in the absence of the tetrahydrobiopterin cofactor. The nitrite pathway increases by sixfold the NO production with respect to the regular arginine pathway under hypoxia, which is largely blocked. Therefore, basal levels of NO release could be sustained by anoxic nitrite reduction. The reaction suggests a new pathway for fast NO delivery under hypoxia, precisely when the vasodilating properties of nitric oxide are most needed. (c) 2006 Elsevier Inc. All rights reserved. (10.1016/j.bbrc.2006.01.031)
  DOI : 10.1016/j.bbrc.2006.01.031
• Microscopies multiharmoniques pour l'imagerie structurale de tissus intacts [Second- and third-harmonic generation microscopies for the structural imaging of intact tissues]
  
  • Débarre Delphine
  • Pena Ana-Maria
  • Supatto Willy
  • Boulesteix Thierry
  • Strupler Mathias
  • Martin Jean-Louis
  • Sauviat Martin-Pierre
  • Schanne-Klein Marie-Claire
  • Beaurepaire Emmanuel
  Médecine/Sciences, EDP Sciences, 2006, 22 (10), pp.845. Depuis son introduction en 1990, la microscopie de fluorescence excitée à deux photons (Fluo-2P) s'est peu à peu imposée comme une méthode incontournable d'imagerie de tissus intacts à l'échelle sub-cellulaire. En effet, la caractéristique la plus remarquable de la microscopie multiphotonique est de maintenir une résolution tridimensionnelle micrométrique lors de l'observation en profondeur d'un milieu optiquement diffusant. Combinée aux technologies de protéines-fusion (type GFP), cette approche est aujourd'hui utilisée dans de nombreux domaines, notamment en neurophysiologie. Un autre attrait de ce type d'imagerie réside dans l'utilisation possible d'autres phénomènes optiques non linéaires (c'est-à-dire impliquant l'interaction simultanée de plusieurs photons avec une molécule observée) comme source de contraste. Ainsi, les microscopies par génération de second harmonique (GSH) et par génération de troisième harmonique (GTH) permettent également d'observer des milieux complexes et fournissent des informations complémentaires par rapport à l'imagerie de fluorescence. Certaines structures cellulaires ou tissulaires fournissent, en effet, ce type de réponse optique sans nécessiter de marquage exogène. La microscopie GSH permet, par exemple, de détecter le collagène fibrillaire et la microscopie GTH permet d'observer sans marquage le développement embryonnaire de petits organismes. One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy. (10.1051/medsci/20062210845)
  DOI : 10.1051/medsci/20062210845
• Ionic contrast terahertz time resolved imaging of frog auricular heart muscle electrical activity
  
  • Masson Jean-Baptiste
  • Sauviat Martin-Pierre
  • Gallot Guilhem
  Applied Physics Letters, American Institute of Physics, 2006, 89 (15), pp.153904. The authors demonstrate the direct, noninvasive and time resolved imaging of functional frog auricular fibers by ionic contrast terahertz (ICT) near field microscopy. This technique provides quantitative, time-dependent measurement of ionic flow during auricular muscle electrical activity, and opens the way of direct noninvasive imaging of cardiac activity under stimulation. ICT microscopy technique was associated with full three-dimensional simulation enabling to measure precisely the fiber sizes. This technique coupled to waveguide technology should provide the grounds to development of advanced in vivo ion flux measurement in mammalian hearts, allowing the prediction of heart attack from change in K+ fluxes. Cop. 2006 American Institute of Physics. (10.1063/1.2360931)
  DOI : 10.1063/1.2360931
• Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples
  
  • Lipowski Gérard
  • Liebl Ursula
  • Guigliarelli Bruno
  • Nitschke Wolfgang
  • Schoepp-Cothenet Barbara
  FEBS Letters, Wiley, 2006, 580 (25), pp.5988. The EPR spectral parameters of aa3 oxidase and cyt c552 from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c552. The orientation of the g-tensors of hemes a and c552 were determined on partially ordered membranes, enriched cyt c552 and a c552:aa3 subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c552 with respect to the parent membrane. Taken together with previous data on the interaction surface between aa3 oxidase and cyt c552, these results allow us to arrive at a single conformation for the c552:aa3 electron transfer complex. Cop. 2006 Federation of European Biochemical Societies. (10.1016/j.febslet.2006.09.038)
  DOI : 10.1016/j.febslet.2006.09.038
• Emission properties and applications of nanostructured luminescent oxide nanoparticles
  
  • Giaume D.
  • Buissette V.
  • Lahlil K.
  • Gacoin T.
  • Boilot J.-P.
  • Casanova Didier
  • Beaurepaire Emmanuel
  • Sauviat Martin-Pierre
  • Alexandrou Antigoni
  Progress in Solid State Chemistry, Elsevier, 2006, 33 (2-4), pp.99. Rare earth doped oxide materials are well known for their numerous applications in light emitting devices. An interesting issue is to study the emission properties of nanoparticles, with the aim to understand the influence of small size and surface effects on the emission processes. These particles could furthermore be used in new applications such as the elaboration of transparent emitting devices or new biological labels. The work presented here concerns highly luminescent rare earth doped yttrium vanadates (YVO4:Eu) and lanthanum phosphate LaPO4:Ce,Tb*xH2O nanoparticles. Simple aqueous colloidal syntheses are used for the elaboration of concentrated colloids based on the progressive decomposition of polymeric precursors at moderate temperature (60-90 °C). Both types of particles exhibit strong emission (quantum yields of 25% and 45% for vanadates and phosphates, respectively), but significantly lower than that for the equivalent bulk materials. The alteration of the emission processes is discussed in terms of surface quenching effects. Improvements are obtained through the elaboration of core/shell nanostructures. Surface derivatization has been achieved through the controlled growth of an organically modified silica shell using a functionalized silane precursor. Two examples are given concerning the applications of those particles. The first one is the elaboration of transparent and highly luminescent thin films, obtained by the dispersion of the functionalized particles in a sol-gel silica matrix. The other one is the use of guanidine functionalized particles as biological labels for the single particle detection of sodium channels in cardiac cells. (10.1016/j.progsolidstchem.2005.11.041)
  DOI : 10.1016/j.progsolidstchem.2005.11.041
• Dynamics of NO rebinding to the heme domain of NO synthase-like proteins from bacterial pathogens
  
  • Gautier Clément
  • Mikula Ivan
  • Nioche P.
  • Martasek P.
  • Raman C.
  • Slama-Schwok Anny
  Nitric Oxide, 2006, à paraître.
• Chirped Molecular Vibration During Photo-Isomerization in Stilbene Derivative in Solution
  
  • Kobayashi T.
  • Colonna Anne
  • Yabushita A.
  • Iwakura I.
  • Tokunaga E.
  , 2006, pp.poster.
• La transglutaminase tissulaire promeut l'organisation fibrillaire du collagène et la progression de la fibrose rénale hypertensive dépendante de l'angiotensine II. (sélectionné pour le prix poster)
  
  • Hernest Monica
  • Pena Ana-Maria
  • Beaurepaire Emmanuel
  • Chatziantoniou C.
  • Martin Jean-Louis
  • Schanne-Klein Marie-Claire
  , 2006.
• Optical in situ size determination of single lanthanide-ion doped oxide nanoparticles
  
  • Casanova Didier
  • Giaume Domitile
  • Beaurepaire Emmanuel
  • Gacoin Thierry
  • Boilot Jean-Pierre
  • Alexandrou Antigoni
  Applied Physics Letters, American Institute of Physics, 2006, 89 (25), pp.253103. We show that the size of a lanthanide-ion doped nanoparticle can be accurately determined from its luminosity. The optically determined size distribution is in very good agreement with the distribution obtained from transmission electron microscopy. These data confirm that single nanoparticles are visualized in microscopy experiments. Nanoparticles as small as 13 nm are detectable with integration times of 500 ms. (c) 2006 American Institute of Physics. (10.1063/1.2405871)
  DOI : 10.1063/1.2405871
• True near field versus contrast near field imaging.
  
  • Masson Jean-Baptiste
  • Gallot Guilhem
  Optics Express, Optical Society of America - OSA Publishing, 2006, 14 (24), pp.11566-11574. We demonstrate that in near field imaging, interaction between light and sample can be divided into two main areas: the true near field and the contrast near field domain. We performed extensive numerical simulations in order to identify the limits of these areas, and to investigate contrast near field imaging in which much easier propagation calculation can be achieved. Finally, we show an application with terahertz axonal imaging. © 2006 Optical Society of America (10.1364/OE.14.011566)
  DOI : 10.1364/OE.14.011566
• Ultrafast Conformational Changes in Carboxy-Myoglobin Studied by Time-Resolved Circular Dichroism
  
  • Dartigalongue Thibault
  • Hache François
  , 2006.
• A Hydrophobic Distal Pocket Affects NO-heme Geminate Recombination Dynamics in Dehaloperoxidase and H64V Myoglobin
  
  • Franzen Stefan
  • Jasaitis Audrius
  • Belyea Jennifer
  • Brewer Scott
  • Casey Romain
  • Macfarlane a W Iv
  • Stanley R.
  • Vos Marten H.
  • Martin Jean-Louis
  Journal of Physical Chemistry B, American Chemical Society, 2006, 110, pp.14483-14493.
• Time-resolved circular dichroism in carbonmonoxy-myoglobin: The central role of the proximal histidine
  
  • Dartigalongue Thibault
  • Hache François
  Chirality, Wiley, 2006, 18 (4), pp.273. A calculation of the circular dichroism (CD) spectra of carbonmonoxy-and deoxy-myoglobin is carried out in relation to a time-resolved CD experiment. This calculation allows us to assign a dominant role to the proximal histidine in the definition of the electronic normal modes and to interpret the transient CD structure observed in a strain of the proximal histidine. This strain builds up in 10 ps and relaxes in 50 ps as the protein evolves towards its deoxy form. Cop. 2006 Wiley-Liss, Inc. (10.1002/chir.20254)
  DOI : 10.1002/chir.20254
• Dynamics of NO rebinding to the heme domain of NO synthase-like proteins from bacterial pathogens
  
  • Gautier Christian
  • Martasek P.
  • Mikula I.
  • Nioche P.
  • Raman C.S.
  • Slama-Schwok Anny
  Nitric Oxide, 2006, 15 (4), pp.312. Some Gram-positive bacterial pathogens harbor a gene that encodes a protein (HNS, Heme domain of NO Synthase-like proteins) with striking sequence identity to the oxygenase domain of mammalian NO synthases (NOS). However, they lack the N-terminal and the Zn-cysteine motif participating to the stability of an active dimer in the mammalian isoforms. The unique properties of HNS make it an excellent model system for probing how the heme environment tunes NO dynamics and for comparing it to the endothelial NO synthase heme domain (eNOSHD) using ultrafast transient spectroscopy. NO rebinding in HNS from Staphylococcus aureus (SA-HNS) is faster than that measured for either Bacillus anthracis (BA-HNS) or for eNOSHD in both oxidized and reduced forms in the presence of arginine. To test whether these distinct rates arise from different energy barriers for NO recombination, we measured rebinding kinetics at several temperatures. Our data are consistent with different barriers for NO recombination in SA-HNS and BA-HNS and the presence of a second NO-binding site. The hypothesis that an additional NO-binding cavity is present in BA-HNS is also consistent with the effect of the NO concentration on its rebinding. The lack of the effect of NO concentration on the geminate rebinding in SA-HNS could be due to an isolated second site. We confirm the existence of a second NO site in the oxygenase domain of the reduced eNOS as previously hypothesized [A. Slama-Schwok, M. Négrerie, V. Berka, J.C. Lambry, A.L. Tsai, M.H. Vos, J.L. Martin, Nitric oxide (NO) traffic in endothelial NO synthase. Evidence for a new NO binding site dependent on tetrahydrobiopterin? J. Biol. Chem. 277 (2002) 7581-7586]. This site requires the presence of arginine and BH4; and we propose that NO dynamic and escape from eNOS is regulated by the active site H-bonding network connecting between the heme, the substrate, and cofactor. Cop. 2006 Elsevier Inc. All rights reserved. (10.1016/j.niox.2006.03.010)
  DOI : 10.1016/j.niox.2006.03.010
• Interferometric Fourier transform coherent anti-stokes Raman scattering
  
  • Cui M.
  • Joffre Manuel
  • Skodack J.
  • Ogilvie Jennifer P.
  Optics Express, Optical Society of America - OSA Publishing, 2006, 14 (18), pp.8448-8458. We present an interferometric time-domain Fourier transform implementation of coherent anti-Stokes Raman scattering (CARS). Based on a single femtosecond laser source, the method provides a straight-forward scheme for obtaining high resolution CARS spectra. We give a theoretical description of the method, and demonstrate good agreement between simulation and experimental CARS spectra. We also discuss the method's relation to other CARS approaches for microscopy and microspectroscopy applications. Cop. 2006 Optical Society of America. (10.1364/OE.14.008448)
  DOI : 10.1364/OE.14.008448
• Generation and complete characterization of intense mid-infrared ultrashort pulses
  
  • Ventalon Cathie
  • Fraser James M.
  • Likforman Jean-Pierre
  • Villeneuve D. M.
  • Corkum Paul B.
  • Joffre Manuel
  Journal of the Optical Society of America B, Optical Society of America, 2006, 23, pp.332.
• SHG microscopy. Quantitative scoring of kidney collagen fibrosis. (prix du meilleur poster)
  
  • Strupler Mathias
  • Hernest Monica
  • Pena Ana-Maria
  • Martin Jean-Louis
  • Beaurepaire Emmanuel
  • Tharaux Pierre-Louis
  • Schanne-Klein Marie-Claire
  , 2006.
• Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy.
  
  • Débarre Delphine
  • Supatto Willy
  • Pena Ana-Maria
  • Fabre Aurelie J
  • Tordjmann Thierry
  • Combettes Laurent
  • Schanne-Klein Marie-Claire
  • Beaurepaire Emmanuel
  Nature Methods, Nature Publishing Group, 2006, 3 (1), pp.47-53. Lipid bodies have an important role in energy storage and lipid regulation. Here we show that lipid bodies are a major source of contrast in third-harmonic generation (THG) microscopy of cells and tissues. In hepatocytes, micrometer-sized lipid bodies produce a THG signal 1-2 orders of magnitude larger than other structures, which allows one to image them with high specificity. THG microscopy with approximately 1,200 nm excitation can be used to follow the distribution of lipid bodies in a variety of unstained samples including insect embryos, plant seeds and intact mammalian tissue (liver, lung). We found that epi-THG imaging is possible in weakly absorbing tissues because bulk scattering redirects a substantial fraction of the forward-generated harmonic light toward the objective. Finally, we show that the combination of THG microscopy with two-photon and second-harmonic imaging provides a new tool for exploring the interactions between lipid bodies, extracellular matrix and fluorescent compounds (vitamin A, NADH and others) in tissues. (10.1038/nmeth813)
  DOI : 10.1038/nmeth813
• Sensitivity of cardiac background inward rectifying K+ outward current (IK1) to the alkaloids lepadiformines A, B, and C
  
  • Sauviat Martin-Pierre
  • Vercauteren J.
  • Grimaud N.
  • Jugé M.
  • Nabil M.
  • Petit J.-Y.
  • Biard J.-F.
  Journal of Natural Products, American Chemical Society, 2006, 69 (4), pp.558. Three marine alkaloids, purified from Clavelina moluccensis, were structurally identified as lepadiformines A, B, and C and studied on frog atrial myocytes IK1, using the patch-clamp technique. Lepadiformine A (0.4 to 3.3 μM) blocked IK1 dose-dependently with an apparent dissociation constant (KD) equal to 1.42 μM and a stoichiometry of 0.77. The block is voltage-dependent, suggesting that lepadiformine A occupies a receptor site located at about two-thirds of the membrane depth. The shortening of the aliphatic chain at position C13 of lepadiformine B decreased the potency of the molecule to block IK1 but not the affinity (KD = 1.56 μM) and stoichiometry (0.72). Additional deletion of the oxygenated side chain at C2 in lepadiformine C markedly decreased the inhibitory effect of the molecule. In conclusion, lepadiformine modulates IK1 response in cardiac muscle. The oxygenated side chain in C2 is implicated in the affinity of lepadiformine, which behaved as an amine, for a receptor located near or inside the IK1 pore, and the aliphatic chain length at position C13 is involved in the degree of IK1 blockage. (10.1021/np050215s)
  DOI : 10.1021/np050215s
• Role of heme iron coordination and protein structure in the dynamics and geminate rebinding of nitric oxide to the H93G myoglobin mutant: Implications for nitric oxide sensors
  
  • Négrerie Michel
  • Kruglik Sergei G.
  • Lambry Jean-Christophe
  • Vos Marten H.
  • Martin Jean-Louis
  • Franzen S.
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2006, 281, pp.10389. The influence of the heme iron coordination on nitric oxide binding dynamics was investigated for the myoglobin mutant H93G (H93G-Mb) by picosecond absorption and resonance Raman time-resolved spectroscopies. In the H93G-Mb, the glycine replacing the proximal histidine does not interact with the heme iron so that exogenous substituents like imidazole may coordinate to the iron at the proximal position. Nitrosylation of H93G-Mb leads to either 6- or 5-coordinate species depending on the imidazole concentration. At high concentrations, (imidazole)-(NO)-6-coordinate heme is formed, and the photoinduced rebinding kinetics reveal two exponential picosecond phases (~10 and ~100 ps) similar to those of wild type myoglobin. At low concentrations, imidazole is displaced by the trans effect leading to a (NO)-5-coordinate heme, becoming 4-coordinate immediately after photolysis as revealed from the transient Raman spectrum. In this case, NO rebinding kinetics remain bi-exponential with no change in time constant of the fast component whose amplitude increases with respect to the 6-coordinate species. Bi-exponential NO geminate rebinding in 5-coordinate H93G-Mb is in contrast with the single-exponential process reported for nitrosylated soluble guanylate cyclase (Negrerie, M., Bouzhir, L., Martin, J. L., and Liebl, U. (2001) J. Biol. Chem. 276, 46815-46821). Thus, our data show that the iron coordination state or the heme iron out-of-plane motion are not at the origin of the bi-exponential kinetics, which depends upon the protein structure, and that the 4-coordinate state favors the fast phase of NO geminate rebinding. Consequently, the heme coordination state together with the energy barriers provided by the protein structure control the dynamics and affinity for NO-binding enzymes. Cop. 2006 by The American Society for Biochemistry and Molecular Biology, Inc. (10.1074/jbc.M513375200)
  DOI : 10.1074/jbc.M513375200