Articles

Dynamics of nitric oxide in the active site of reduced cytochrome c oxidase aa3
- Vos Marten H.
- Lipowski Gérard
- Lambry Jean-Christophe
- Martin Jean-Louis
- Liebl Ursula
Biochemistry, American Chemical Society , 2001, 40 (26), pp.7806 . Nitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase. The dynamics of NO in and near this site are not well understood. We performed flash photolysis studies of NO from heme a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy. The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale [Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184]. A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps. The presence of this recombination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed. Increasing the NO concentration increases the recombination yield on the hundreds of picoseconds time scale. This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (submicromolar) concentrations. Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are discussed. (10.1021/bi010060x)
DOI : 10.1021/bi010060x
Experimental observation of nonlinear circular dichroism in a pump-probe experiment
- Mesnil H.
- Schanne-Klein Marie-Claire
- Hache François
- Alexandre M.
- Lemercier G.
- Andraud Chantal
Chemical Physics Letters, Elsevier , 2001, 338 (4-6), pp.269-276 . We present experimental evidence of nonlinear optical activity in a time-resolved pump-probe experiment carried out in a liquid of chiral molecules. By modulating the polarization of the probe or of the pump, we measure a variation of the circular dichroism (CD) induced by the pump. Application of these techniques to time-resolved spectroscopy of excited molecules is discussed. Cop. 2001 Elsevier Science B.V. (10.1016/S0009-2614(01)00239-1)
DOI : 10.1016/S0009-2614(01)00239-1
Coherent broadband pulse shaping in the mid infrared
- Belabas Nadia
- Likforman Jean-Pierre
- Canioni Lionel
- Bousquet Bruno
- Joffre Manuel
Optics Letters, Optical Society of America - OSA Publishing , 2001, 26 (10), pp.743 . We demonstrate broadband infrared pulse shaping by difference-frequency mixing of two visible phase-locked linearly chirped pulses in GaAs. Control of the temporal profile of the emitted field is achieved through this direct tailoring of the exciting visible intensity. The results are in agreement with a simulation with no adjustable parameter. (C) 2001 Optical Society of America. (10.1364/OL.26.000743)
DOI : 10.1364/OL.26.000743
Alterations of transmembrane currents in frog atrial heart muscle induced by photoexcited gymnochrome a purified from the crinoid, Gymnochrinus richeri
- Sauviat Martin-Pierre
- Benoit Anne-Gaëlle
- Debitus Cécile
- Pouny I.
- Laurent Dominique
Photochemistry and Photobiology, Blackwell Wiley [1962-....] , 2001, 74 (2), pp.115-119 . The effects of gymnochrome A were tested on the electrical activity of the frog atrial heart muscle. Gymnochrome A (1-5 µM) did not alter the resting potential. Gymnochrome A (5 µM) slowed the initial depolarizing phase of the spontaneously beating action potential. Under voltage-clamp conditions gymnochrome A (5 µM) did not affect the electrical constant of the membrane and the kinetic parameters of the peak Na+ current (INa) recorded in the Ringer solution containing tetraethylammonium (2 mM) and Cd2+ (1 mM) but shifted the membrane potential at which the current both activated and reached its maximal value toward more negative membrane potentials. It did not alter the reversal potential for INa, indicating that the selectivity of the Na+ channels had not changed. These observations suggest that gymnochrome A binds to the membrane and shifts the activation of INa on the voltage axis by modifying the free negative fixed charges present at the membrane surface rather than by occupying a specific site on the Na+ channel. Photoexcited gymnochrome A transiently triggered an early outward current which lengthened the time-to-peak of INa and decreased its amplitude. In addition, photoexcited gymnochrome A blocked the background K+ current. This is, to our knowledge, the first time that such effects are reported on the cardiac muscle. These observations suggest that the photoexcitation of gymnochrome produces physico-chemical effects which lead to intracellular changes. Further experiments are required to determine their nature. (10.1562/0031-8655(2001)0740115AOTCIF2.0.CO2)
DOI : 10.1562/0031-8655(2001)0740115AOTCIF2.0.CO2
Magnetic chiroptical effects in surface second harmonic reflection
- Schanne-Klein Marie-Claire
- Hache François
- Brotin Thierry
- Andraud Chantal
- Collet A.
Chemical Physics Letters, Elsevier , 2001, 338 (2-3), pp.159-166 . We perform surface second harmonic reflection (SHR) to study a chiral stilbene. It exhibits a strong circular intensity difference in the second harmonic signal, but no linear intensity difference and no rotation of polarization. This SHR optical activity is characteristic of strong magnetic effects and proves that the molecule under study exhibits a one-electron chirality. (C) 2001 Elsevier Science B.V. All rights reserved. (10.1016/S0009-2614(01)00275-5)
DOI : 10.1016/S0009-2614(01)00275-5
Control of Nitric Oxide Dynamics by Guanylate Cyclase in Its Activated State
- Négrerie Michel
- Bouzhir Latifa
- Martin Jean-Louis
- Liebl Ursula
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2001, 276 (50), pp.46815 . Soluble guanylate cyclase (sGC) is the target of nitric oxide (NO) released by nitric-oxide synthase in endothelial cells, inducing an increase of cGMP synthesis in response. This heterodimeric protein possesses a regulatory subunit carrying a heme where NO binding occurs, while the second subunit harbors the catalytic site. The binding of NO and the subsequent breaking of the bond between the proximal histidine and the heme-Fe2+ are assumed to induce conformational changes, which are the origin of the catalytic activation. At the molecular level, the activation and deactivation mechanisms are unknown, as is the dynamics of NO once in the heme pocket. Using ultrafast time-resolved absorption spectroscopy, we measured the kinetics of NO rebinding to sGC after photodissociation. The main spectral transient in the Soret band does not match the equilibrium difference spectrum of NO-liganded minus unliganded sGC, and the geminate rebinding was found to be monoexponential and ultrafast (t = 7.5 ps), with a relative amplitude close to unity (0.97). These characteristics, so far not observed in other hemoproteins, indicate that NO encounters a high energy barrier for escaping from the heme pocket once the His-Fe2+ bond has been cleaved; this bond does not reform before NO recombination. The deactivation of isolated sGC cannot occur by only simple diffusion of NO from the heme; therefore, several allosteric states may be inferred, including a desensitized one, to induce NO release. Thus, besides the structural change leading to activation, a consequence of the decoupling of the proximal histidine may also be to induce a change of the heme pocket distal geometry, which raises the energy barrier for NO escape, optimizing the efficiency of NO trapping. The nonsingle exponential character of the NO picosecond rebinding coexists only with the presence of the protein structure surrounding the heme, and the single exponential rate observed in sGC is very likely to be due to a closed conformation of the heme pocket. Our results emphasize the physiological importance of NO geminate recombination in hemoproteins like nitric-oxide synthase and sGC and show that the protein structure controls NO dynamics in a manner adapted to their function. This control of ligand dynamics provides a regulation at molecular level in the function of these enzymes. (10.1074/jbc.M102224200)
DOI : 10.1074/jbc.M102224200
Stimulated scattering and its dynamics in semiconductor microcavities at 80 K under nonresonant excitation conditions
- Alexandrou Antigoni
- Bianchi G.
- Peronne Emmanuel
- Hallé B.
- Boeuf F.
- André R.
- Romestain R.
- Dang Le Si
Physical Review B: Condensed Matter and Materials Physics (1998-2015), American Physical Society , 2001, 64 (23), pp.2333181 . We have observed stimulated scattering in a II-VI microcavity at 80 K under nonresonant excitation conditions. This stimulated scattering manifests itself through probe amplification in a pump-probe reflectivity experiment and through a highly nonlinear emission at an energy corresponding to a k? ? 0 state on the lower polariton branch. We were able to follow the onset and decline of stimulated scattering by measuring the temporal evolution of the probe amplification. (10.1103/PhysRevB.64.233318)
DOI : 10.1103/PhysRevB.64.233318
NFkappaB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts.
- Vos M
- Govers R
- Gröne H J
- Kleij L
- Schurink M
- de Weger R
- Goldschmeding R
- Rabelink T J
FASEB Journal, Federation of American Society of Experimental Biology , 2000, pp.815-22 . Monocyte influx secondary to ischemia-reperfusion conditions the renal allograft to rejection by presentation of antigens and production of cytokines. Monocyte influx depends on NFkappaB-dependent transcription of genes encoding adhesion molecules and chemokines. Here we demonstrate that cationic liposomes containing phosphorothioated oligodeoxynucleotides (ODN) with the kappaB binding site serving as competitive binding decoy, can prevent TNF-alpha-induced NFkappaB activity in endothelial cells in vitro. In an allogenic rat kidney transplantation model (BN to LEW), we show that perfusing the renal allograft with this decoy prior to transplantation abolishes nuclear NFkappaB activity in vivo and inhibits VCAM-1 expression in the donor endothelium (P<0.05). At 24 h postreperfusion, periarterial infiltration of monocytes/macrophages was significantly reduced in decoy ODN-treated allografts compared to control allografts (3.7+/-0.7 vs. 9.2+/-1.2 macrophages/vessel; P<0.01). At 72 h, there was a reduction of tubulointerstitial macrophage infiltration in decoy ODN-treated kidneys compared to controls (75.6+/-13.9 vs. 120.0+/-11.2 macrophages/tubulointerstitial area; P<0.05). In conclusion, perfusion of the renal allograft with NFkappaB decoy ODN prior to transplantation decreases the initial inflammatory response in a stringent, nonimmunosuppressed allogenic transplantation model. Therefore, the NFkappaB decoy approach may be useful to explore the role of endothelium and macrophages in graft rejection and may be developed into a graft-specific immunosuppressive strategy allowing reduction of systemic immunosuppression on organ transplantation.
Insensitivity to anti–Müllerian hormone due to a mutation in the human anti–Müllerian hormone receptor
- Imbeaud Sandrine
- Faure Emmanuelle
- Lamarre Isabelle
- Mattei Marie-Geneviève
- Di Clemente Nathalie
- Tizard Richard
- Carré-Eusèbe Danièle
- Carré-Eusèbe C
- Belville Corinne
- Tragethon Lars
- Tonkin Christopher
- Nelson Janice
- Mcauliffe A
- Bidart Jean-Michel
- Lababidi Abdul
- Josso Nathalie
- Cate Richard
- Picard Jean-Yves
Nature Genetics, Nature Publishing Group , 1995, 11 (4), pp.382 - 388 . Anti-Müllerian hormone (AMH) and its receptor are involved in the regression of Müllerian ducts in male fetuses. We have now cloned and mapped the human AMH receptor gene and provide genetic proof that it is required for AMH signalling, by identifying a mutation in the AMH receptor in a patient with persistent Müllerian duct syndrome. The mutation destroys the invariant dinucleotide at the 5' end of the second intron, generating two abnormal mRNAs, one missing the second exon, required for ligand binding, and the other incorporating the first 12 bases of the second intron. The similar phenotypes observed in AMH-deficient and AMH receptor-deficient individuals indicate that the AMH signalling machinery is remarkably simple, consisting of one ligand and one type II receptor. (10.1038/ng1295-382)
DOI : 10.1038/ng1295-382