Articles

Mechanistic and structural basis for inhibition of thymidylate synthase ThyX
- Basta Tamara
- Boum Yap
- Briffotaux Julien
- Becker Hubert F.
- Lamarre-Jouenne Isabelle
- Lambry Jean-Christophe
- Skouloubris Stéphane
- Liebl Ursula
- Graille Marc
- van Tilbeurgh Herman
- Myllykallio Hannu
Open Biology, Royal Society , 2012, 2 (10), pp.120120 . Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 angstrom. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e. g. in Mycobacterium tuberculosis and Helicobacter pylori, our studies have also potential to pave the way towards the development of new anti-microbial compounds. (10.1098/rsob.120120)
DOI : 10.1098/rsob.120120
Observing the Confinement Potential of Bacterial Pore-Forming Toxin Receptors Inside Rafts with Nonblinking Eu3+-Doped Oxide Nanoparticles
- Türkcan Silvan
- Masson Jean-Baptiste
- Casanova Didier
- Mialon Geneviève
- Gacoin Thierry
- Boilot Jean-Pierre
- Popoff Michel R.
Biophysical Journal, Biophysical Society , 2012, 102 (10), pp.2299-2308 . We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and -toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 0.05 m2. Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 0.01 m2/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 0.03 pN/m at 37C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family. (10.1016/j.bpj.2012.03.072)
DOI : 10.1016/j.bpj.2012.03.072
Adaptive Optics for Biomedical Microscopy
- Booth Martin J.
- Débarre Delphine
- Jesacher Alexander
Optics and photonics news, Optical Society of America - OSA Publishing , 2012, 23 (1), pp.22-29 . Over the last decade, researchers have applied adaptive optics--a technology that was originally conceived for telescopes--to high-resolution microscopy in order to overcome the problems caused by specimen-induced aberrations. © 2012 Optical Society of America (10.1364/OPN.23.1.000022)
DOI : 10.1364/OPN.23.1.000022
Photoinduced dynamics of oxyluciferin analogues: Unusual enol "super"photoacidity and evidence for keto-enol isomerization
- Solntsev K.M.
- Laptenok Sergey P.
- Naumov P.
Journal of the American Chemical Society, American Chemical Society , 2012, 134 (40), pp.16452-16455 . The first systematic pico-nanosecond time-resolved spectroscopic study of the firefly emitter oxyluciferin and two of its chemically modified analogues revealed that in the excited state the enol group is more acidic than the phenol group. The 6'-dehydroxylated derivative, in which only the 4-enolic hydroxyl proton is acidic, has an experimentally determined pK a* of 0.9 in dimethyl sulfoxide and an estimated pK a* of -0.3 in water. Moreover, this compound provided direct evidence that in a nonpolar, basic environment the keto form in the excited state can tautomerize into the enol, which subsequently undergoes excited-state proton transfer (ESPT) to produce enolate ion. This observation presents the first experimental evidence of excited-state keto-enol tautomerization of a firefly fluorophore, and it could be important in resolving the enol-keto conundrum related to the color-tuning mechanism of firefly bioluminescence. The 6'-dehydroxylated form of oxyluciferin adds a very rare case of a stable enol to the family of "super"photoacids. Cop. 2012 American Chemical Society. (10.1021/ja3045212)
DOI : 10.1021/ja3045212
Picosecond to second dynamics reveals a structural transition in clostridium botulinum no-sensor triggered by the activator BAY-41-2272
- Yoo Byung-Kuk
- Lamarre Isabelle
- Rappaport Fabrice
- Nioche Pierre
- Raman C.S.
- Martin Jean-Louis
- Negrerie Michel
ACS Chemical Biology, American Chemical Society , 2012, 7 (12), pp.2046-2054 . Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor that synthesizes cGMP upon NO activation. In synergy with the artificial allosteric effector BAY 41-2272 (a lead compound for drug design in cardiovascular treatment), sGC can also be activated by carbon monoxide (CO), but the structural basis for this synergistic effect are unknown. We recorded in the unusually broad time range from 1 ps to 1 s the dynamics of the interaction of CO binding to full length sGC, to the isolated sGC heme domain ß1(200) and to the homologous bacterial NO-sensor from Clostridium botulinum. By identifying all phases of CO binding in this full time range and characterizing how these phases are modified by BAY 41-2272, we show that this activator induces the same structural changes in both proteins. This result demonstrates that the BAY 41-2272 binding site resides in the ß1(200) sGC heme domain and is the same in sGC and in the NO-sensor from Clostridium botulinum. Cop. 2012 American Chemical Society. (10.1021/cb3003539)
DOI : 10.1021/cb3003539
Genomic binding of Pol III transcription machinery and relationship with TFIIS transcription factor distribution in mouse embryonic stem cells
- Carrière Lucie
- Graziani Sébastien
- Alibert Olivier
- Ghavi-Helm Yad
- Boussouar Fayçal
- Humbertclaude Hélène
- Jounier Sylvie
- Aude Jean-Christophe
- Keime Celine
- Murvai Janos
- Foglio Mario
- Gut Marta
- Gut Ivo
- Lathrop Mark
- Soutourina Julie
- Gérard Matthieu
- Werner Michel
Nucleic Acids Research, Oxford University Press , 2012, 40 (1), pp.270-283 . (10.1093/nar/gkr737)
DOI : 10.1093/nar/gkr737
Imaging and 3D morphological analysis of collagen fibrils
- Altendorf Hellen
- Decencière Etienne
- Jeulin Dominique
- de Sa Peixoto Paolo
- Deniset-Besseau Ariane
- Angelini E.
- Mosser G.
- Schanne-Klein Marie-Claire
Journal of Microscopy, Wiley , 2012, 247 (2), pp.161-175 . The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants. (10.1111/j.1365-2818.2012.03629.x)
DOI : 10.1111/j.1365-2818.2012.03629.x
Diffraction from a subwavelength elliptic aperture: Analytic approximate aperture fields
- Masson Jean-Baptiste
- Gallot Guilhem
Journal of the Optical Society of America. A Optics, Image Science, and Vision, Optical Society of America , 2012, 29 (9), pp.2005-2014 . An analytical approximate solution of the electromagnetic field on a subwavelength elliptical hole in a thin perfectly conducting screen is presented. Illumination is a linear polarized, normally incident plane wave. A polynomial development method is used and allows one to obtain an easy-to-use analytical solution of the fields, which can be used to build analytical expressions of aperture fields for apertures in anisotropic structures. © 2012 Optical Society of America. (10.1364/josaa.29.002005)
DOI : 10.1364/josaa.29.002005
A Bayesian Inference Scheme to Extract Diffusivity and Potential Fields from Confined Single-Molecule Trajectories
- Tuerkcan Silvan
- Alexandrou Antigoni
- Masson Jean-Baptiste
Biophysical Journal, Biophysical Society , 2012, 102 (10), pp.2288-2298 . Currently used techniques for the analysis of single-molecule trajectories only exploit a small part of the available information stored in the data. Here, we apply a Bayesian inference scheme to trajectories of confined receptors that are targeted by pore-forming toxins to extract the two-dimensional confining potential that restricts the motion of the receptor. The receptor motion is modeled by the overdamped Langevin equation of motion. The method uses most of the information stored in the trajectory and converges quickly onto inferred values, while providing the uncertainty on the determined values. The inference is performed on the polynomial development of the potential and on the diffusivities that have been discretized on a mesh. Numerical simulations are used to test the scheme and quantify the convergence toward the input values for forces, potential, and diffusivity. Furthermore, we show that the technique outperforms the classical mean-square-displacement technique when forces act on confined molecules because the typical mean-square-displacement analysis does not account for them. We also show that the inferred potential better represents input potentials than the potential extracted from the position distribution based on Boltzmann statistics that assumes statistical equilibrium. (10.1016/j.bpj.2012.01.063)
DOI : 10.1016/j.bpj.2012.01.063
Asynchronous optical sampling with arbitrary detuning between laser repetition rates
- Antonucci Laura
- Solinas Xavier
- Bonvalet Adeline
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2012, 20 (16), pp.17928-17937 . A method of asynchronous optical sampling based on free-running lasers with no requirement on the repetition rates is presented. The method is based on the a posteriori determination of the delay between each pair of pulses. A resolution better than 400 fs over 13 ns total delay scan is demonstrated. In addition to the advantages of conventional asynchronous sampling techniques, this method allows a straightforward implementation on already-existing laser systems using a fiber-based setup and an appropriate acquisition procedure. (C) 2012 Optical Society of America (10.1364/OE.20.017928)
DOI : 10.1364/OE.20.017928
Nitric oxide binding to the cardiolipin complex of ferric cytochrome
- Silkstone G.
- Kapetanaki Sofia M.
- Husu I.
- Vos Marten H.
- Wilson M.T.
Biochemistry, American Chemical Society , 2012, 51 (34), pp.6760-6766 . Cardiolipin, a phospholipid specific to the mitochondrion, interacts with the small electron transfer heme protein cytochrome c through both electrostatic and hydrophobic interactions. Once in a complex with cardiolipin, cytochrome c has been shown to undergo a conformational change that leads to the rupture of the bond between the heme iron and the intrinsic sulfur ligand of a methionine residue and to enhance the peroxidatic properties of the protein considered important to its apoptotic activity. Here we report that the ferric cytochrome c/cardiolipin complex binds nitric oxide tightly through a multistep process in which the first step is the relatively slow displacement (5 s-1) from heme coordination of an intrinsic ligand that replaces methionine in the complex. Nanosecond photolysis of the nitrosyl adduct demonstrated that a fraction of the nitric oxide escapes from the heme pocket and subsequently recombines to the heme in second-order processes (k = 1.8 × 106 and 5.5 × 105 M-1 s-1) that, under these conditions, were much faster than recombination of the intrinsic ligand with which they compete. Ultrafast (femtosecond) laser photolysis showed that the geminate recombination of nitric oxide to the heme occurred with time constants (? = 22 and 72 ps) and that ~23% of the photolyzed nitric oxide escaped into the bulk phase. This high value for the escape fraction relative to other heme proteins indicates the open nature of the heme pocket in this complex. These results are summarized in a scheme and are discussed in terms of the possible modulation of the apoptotic activity of cytochrome c by nitric oxide. Cop. 2012 American Chemical Society. (10.1021/bi300596)
DOI : 10.1021/bi300596
Ultrafast Ligand Dynamics in the Heme-Based GAF Sensor Domains of the Histidine Kinases DosS and DosT from Mycobacterium tuberculosis.
- Vos Marten H.
- Bouzhir-Sima Latifa
- Lambry Jean-Christophe
- Luo Hao
- Eaton-Rye Julian J.
- Ioanoviciu Alexandra
- Ortiz de Montellano Paul
- Liebl Ursula
Biochemistry, American Chemical Society , 2012, 51 (1), pp.159-166 . The transcriptional regulator DosR from M. tuberculosis plays a crucial role in the virulence to dormancy transition of the pathogen. DosR can be activated by DosT and DosS, two histidine kinases with heme-containing sensor GAF domains, capable of diatomic ligand binding. To investigate the initial processes occurring upon ligand dissociation, we performed ultrafast time-resolved absorption spectroscopy of the isolated sensor domains ligated with O2, NO, and CO. The results reveal a relatively closed heme pocket for both proteins. For DosT the yield of O2 escape from the heme pocket on the picoseconds time scale upon photodissociation was found to be very low (1.5%), similar to other heme-based oxygen sensor proteins, implying that this sensor acts as an effective O2 trap. Remarkably, this yield is an order of magnitude higher in DosS (18%). For CO, by contrast, the fraction of CO rebinding within the heme pocket is higher in DosS. Experiments with mutant DosT sensor domains and molecular dynamics simulations indicate an important role in ligand discrimination of the distal tyrosine, present in both proteins, which forms a hydrogen bond with heme-bound O2. We conclude that despite their similarity, DosT and DosS display ligand-specific different primary dynamics during the initial phases of intraprotein signaling. The distal tyrosine, present in both proteins, plays an important role in these processes. (10.1021/bi201467c)
DOI : 10.1021/bi201467c
Optical activity of metallic helices in the terahertz domain: A theoretical investigation
- Hache François
- Gallot Guilhem
Journal of the Optical Society of America B, Optical Society of America , 2012, 29 (10), pp.2675-2684 . Optical activity in the terahertz spectral domain has recently seen a growing interest, but fine understanding of these phenomena is not yet developed. In this article, we study analytically the response of a metallic helix in the terahertz regime and present a full nonlocal calculation of its chiroptical response. Because we do not use multipolar expansion, this calculation is very general and applies to the case where the helix size is comparable to the wavelength of the light. We calculate the circular birefringence and dichroism in three configurations: propagation along or perpendicular to the helix axis and response of an isotropic distribution of such helices. We obtain analytical expressions and can examine the consequence of the breakdown of the multipolar expansion and the wavelength-dependence of the chiroptical response, as well as give orders of magnitude that compare favorably with experimental data. This calculation is also comforted by a finite element calculation. Cop. 2012 Optical Society of America. (10.1364/JOSAB.29.002675)
DOI : 10.1364/JOSAB.29.002675
Amplification and temporal filtering during gradient sensing by nerve growth cones probed with a microfluidic assay
- Morel Mathieu
- Shynkar Vasyl
- Galas Jean-Christophe
- Dupin Isabelle
- Bouzigues Cédric
- Studer Vincent
- Dahan Maxime
Biophysical Journal, Biophysical Society , 2012, 103 (8), pp.1648-1656 . Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during ?-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA A chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA A receptors. Cop. 2012 Biophysical Society. (10.1016/j.bpj.2012.08.040)
DOI : 10.1016/j.bpj.2012.08.040
Glotaran: A Java-based graphical user interface for the R package TIMP
- Snellenburg Joris J.
- Laptenok Sergey
- Seger Ralf
- Mullen Katharine M.
- van Stokkum Ivo H.M.
Journal of Statistical Software, University of California, Los Angeles , 2012, 49 (3) . In this work the software application called Glotaran is introduced as a Java-based graphical user interface to the R package TIMP, a problem solving environment for fitting superposition models to multi-dimensional data. TIMP uses a command-line user interface for the interaction with data, the specification of models and viewing of analysis results. Instead, Glotaran provides a graphical user interface which features interactive and dynamic data inspection, easier -- assisted by the user interface -- model specification and interactive viewing of results. The interactivity component is especially helpful when working with large, multi-dimensional datasets as often result from time-resolved spectroscopy measurements, allowing the user to easily pre-select and manipulate data before analysis and to quickly zoom in to regions of interest in the analysis results. Glotaran has been developed on top of the NetBeans rich client platform and communicates with R through the Java-to-R interface Rserve. The background and the functionality of the application are described here. In addition, the design, development and implementation process of Glotaran is documented in a generic way.
Transcriptional Activation and Cell Cycle Block Are the Keys for 5-Fluorouracil Induced Up-Regulation of Human Thymidylate Synthase Expression
- Ligabue Alessio
- Marverti G.
- Liebl Ursula
- Myllykallio Hannu
PLoS ONE, Public Library of Science , 2012, 7 (10), pp.e47318 . Background: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. Methodology/Principal Findings: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G 1 phase and hTS is localized in the nuclei during S and G 2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. Conclusions/Significance: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies. Cop. 2012 Ligabue et al. (10.1371/journal.pone.0047318)
DOI : 10.1371/journal.pone.0047318
Multicolor two-photon tissue imaging by wavelength mixing
- Mahou Pierre
- Zimmerley Maxwell
- Loulier Karine
- Matho Katherine S.
- Labroille Guillaume
- Morin Xavier
- Supatto Willy
- Livet Jean
- Débarre Delphine
- Beaurepaire Emmanuel
Nature Methods, Nature Publishing Group , 2012, 9 (8), pp.815-818 . We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos. (10.1038/nmeth.2098)
DOI : 10.1038/nmeth.2098
Picosecond time scale modification of forward scattered light induced by absorption inside particles
- Kervella Myriam
- d'Abzac François-Xavier
- Hache François
- Hespel Laurent
- Dartigalongue Thibault
Optics Letters, Optical Society of America - OSA Publishing , 2011, 20 (1), pp.32-41 . The aim of this work is to evaluate the influence of absorption processes on the Time Of Flight (TOF) of the light scattered out of a thick medium in the forward direction. We use a Monte-Carlo simulation with temporal phase function and Debye modes. The main result of our study is that absorption inside the particle induces a decrease of the TOF on a picosecond time scale, measurable with a femtosecond laser apparatus. This decrease, which exhibits a neat sensitivity to the absorption coefficient of particles, could provide an efficient way to measure this absorption. © 2011 OSA (10.1364/OE.20.000032)
DOI : 10.1364/OE.20.000032
Simple characterisation of a deformable mirror inside a high numerical aperture microscope using phase diversity.
- Débarre D.
- Vieille T.
- Beaurepaire Emmanuel
Journal of Microscopy, Wiley , 2011, 244 (2), pp.136-43 . We present a simple and versatile scheme for characterising amplitude and phase modulation by an active element, such as a deformable mirror, in the pupil plane of a high NA microscope. By placing a mirror in the vicinity of the focal plane of the objective and recording images of the reflected focal spot on a camera, we show that reliable measurements of the influence function of the mirror actuators in the pupil plane of the objective can be obtained using an iterative electric field retrieval algorithm. Compared to direct wavefront sensors, the proposed method allows characterisation for a variety of objectives with different NA and pupil sizes without modification of the setup, requires minimal space inside the microscope, and can be used with pulsed sources such as used for multiphoton microscopy. In order to validate our method, we compare our data to the results obtained with a Shack-Hartmann wavefront sensor, and show that comparable precision is achieved. (10.1111/j.1365-2818.2011.03518.x)
DOI : 10.1111/j.1365-2818.2011.03518.x
Saccharomyces cerevisiae chitin biosynthesis activation by N-acetylchitooses depends on size and structure of chito-oligosaccharides.
- Becker Hubert F.
- Piffeteau Annie
- Thellend Annie
BMC Research Notes, BioMed Central , 2011, 4 (1), pp.454 . ABSTRACT: BACKGROUND: To explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on in vitro chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a Saccharomyces cerevisiae membrane fraction. FINDINGS: N-acetylchito-tetra, -penta and -octaoses resulted in 11 to 25 % [14C]-GlcNAc incorporation into [14C]-chitin, corresponding to an increase in the initial velocity. The activation appeared specific to N-acetylchitooses as it was not observed with oligosaccharides in other series, such as beta-(1,4), beta-(1,3) or alpha-(1,6) glucooligosaccharides. CONCLUSIONS: The effect induced by the N-acetylchitooses was a saturable phenomenon and did not interfere with free GlcNAc and trypsin which are two known activators of yeast chitin synthase activity in vitro. The magnitude of the activation was dependent on both oligosaccharide concentration and oligosaccharide size. (10.1186/1756-0500-4-454)
DOI : 10.1186/1756-0500-4-454
Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis
- Hamdane Djemel
- Argentini Manuela
- Cornu David
- Myllykallio Hannu
- Skouloubris Stéphane
- Hui-Bon-Hoa Gaston
- Golinelli-Pimpaneau Béatrice
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2011, 286 (42), pp.36268-36280 . The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1- carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-l-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m5U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton. (10.1074/jbc.M111.256966)
DOI : 10.1074/jbc.M111.256966
Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos.
- Mahou Pierre
- Olivier Nicolas
- Labroille Guillaume
- Duloquin Louise
- Sintes Jean-Marc
- Peyriéras Nadine
- Legouis Renaud
- Débarre Delphine
- Beaurepaire Emmanuel
Biomedical optics express, Optical Society of America - OSA Publishing , 2011, 2 (10), pp.2837-49 . Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.e. the real part of the third-order nonlinear susceptibility χ((3)). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ((3))(-3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ((3))(-2ω(1) + ω(2);ω(1), -ω(2), ω(1)). We show that FWM images provide χ((3)) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue. (10.1364/BOE.2.002837)
DOI : 10.1364/BOE.2.002837
Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy.
- Goulam Houssen Yannick
- Gusachenko Ivan
- Schanne-Klein Marie-Claire
- Allain Jean-Marc
Journal of Biomechanics, Elsevier , 2011, 44 (11), pp.2047-52 . We continuously monitored the microstructure of a rat-tail tendon during stretch/relaxation cycles. To that purpose, we implemented a new biomechanical device that combined SHG imaging and mechanical testing modalities. This multi-scale experimental device enabled simultaneous visualization of the collagen crimp morphology at the micrometer scale and measurement of macroscopic strain-stress response. We gradually increased the ultimate strain of the cycles and showed that preconditioning mostly occurs in the first stretching. This is accompanied by an increase of the crimp period in the SHG image. Our results indicate that preconditioning is due to a sliding of microstructures at the scale of a few fibrils and smaller, that changes the resting length of the fascicle. This sliding can reverse on long time scales. These results provide a proof of concept that continuous SHG imaging performed simultaneously with mechanical assay allows analysis of the relationship between macroscopic response and microscopic structure of tissues. (10.1016/j.jbiomech.2011.05.009)
DOI : 10.1016/j.jbiomech.2011.05.009
Impact of pulse polarization on coherent vibrational ladder climbing signals
- Nuernberger Patrick
- Vieille Thibault
- Ventalon Cathie
- Joffre Manuel
Journal of Physical Chemistry B, American Chemical Society , 2011, 115 (18), pp.5554 . We report a theoretical study that elaborates the influence of the polarization state of both the pump and the probe pulse in ultrafast coherent vibrational ladder climbing experiments in the mid-infrared. Whereas a subensemble in a randomly oriented sample of molecules is excited by the pump pulse in this multiphoton process, further inhomogeneities such as the spatial profile of the laser beams, the longitudinal attenuation in the sample, and the probe beam polarization have to be taken into account. Analytical expressions for a density function describing the number of molecules that are exposed to an effective pump intensity are introduced, and the variation of the population distribution and the actual transient absorption signal in dependence on the polarization-state combinations for pump and probe pulse are discussed in detail. In simulations on the model system carboxy-hemoglobin, it is demonstrated that the polarization states play important roles both for exciting a certain population distribution and for actually observing it. In particular, it will be discussed under which conditions experimental data indicates a population inversion. Cop. 2011 American Chemical Society. (10.1021/jp1113762)
DOI : 10.1021/jp1113762
Biological applications of rare-earth based nanoparticles
- Bouzigues Cédric
- Gacoin T.
- Alexandrou Antigoni
ACS Nano, American Chemical Society , 2011, 5 (11), pp.8488 . Biomedicine and cell and molecular biology require powerful imaging techniques of the single molecule scale to the whole organism, either for fundamental science or diagnosis. These applications are however often limited by the optical properties of the available probes. Moreover, in cell biology, the measurement of the cell response with spatial and temporal resolution is a central instrumental problem. This has been one of the main motivations for the development of new probes and imaging techniques either for biomolecule labeling or detection of an intracellular signaling species. The weak photostability of genetically encoded probes or organic dyes has motivated the interest for different types of nanoparticles for imaging such as quantum dots, nanodiamonds, dye-doped silica particles, or metallic nanoparticles. One of the most active fields of research in the past decade has thus been the development of rare-earth based nanoparticles, whose optical properties and low cytotoxicity are promising for biological applications. Attractive properties of rare-earth based nanoparticles include high photostability, absence of blinking, extremely narrow emission lines, large Stokes shifts, long lifetimes that can be exploited for retarded detection schemes, and facile functionalization strategies. The use of specific ions in their compositions can be moreover exploited for oxidant detection or for implementing potent contrast agents for magnetic resonance imaging. In this review, we present these different applications of rare-earth nanoparticles for biomolecule detection and imaging in vitro, in living cells or in small animals. We highlight how chemical composition tuning and surface functionalization lead to specific properties, which can be used for different imaging modalities. We discuss their performances for imaging in comparison with other probes and to what extent they could constitute a central tool in the future of molecular and cell biology. Cop. 2011 American Chemical Society. (10.1021/nn202378b)
DOI : 10.1021/nn202378b