REFROIDISSEMENT MOLECULAIRE INDUIT PAR CHAMP ELECTRIQUE ; MISE EN EVIDENCE PAR SPECTROSCOPIE NON LINEAIRE CARS

National audienceNous avons montré qu'un refroidissement moléculaire pouvait être induit par un champ électrique statique appliqué à des molécules non polaires d'huile de paraffine. Cet effet a été mis en évidence grâce à une mesure de spectroscopie CARS (Coherent Anti-Stokes Raman Scattering). De manière complémentaire, l'exposition de molécules à un champ électrique permet de contrôler leur orientation et ainsi améliorer ou diminuer l'amplitude de leur signature vibrationnelle

Activation Process of [NiFe] Hydrogenase Elucidated by High-Resolution X-Ray Analyses: Conversion of the Ready to the Unready State

SummaryHydrogenases catalyze oxidoreduction of molecular hydrogen and have potential applications for utilizing dihydrogen as an energy source. [NiFe] hydrogenase has two different oxidized states, Ni-A (unready, exhibits a lag phase in reductive activation) and Ni-B (ready). We have succeeded in converting Ni-B to Ni-A with the use of Na2S and O2 and determining the high-resolution crystal structures of both states. Ni-B possesses a monatomic nonprotein bridging ligand at the Ni-Fe active site, whereas Ni-A has a diatomic species. The terminal atom of the bridging species of Ni-A occupies a similar position as C of the exogenous CO in the CO complex (inhibited state). The common features of the enzyme structures at the unready (Ni-A) and inhibited (CO complex) states are proposed. These findings provide useful information on the design of new systems of biomimetic dihydrogen production and fuel cell devices

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound d-Fructose Dehydrogenase with Structural Bioelectrochemistry

電極を基質認識できる酵素の反応メカニズムを解明 --次世代バイオセンシングにつながる基盤技術--. 京都大学プレスリリース. 2023-10-16.Flavin adenine dinucleotide-dependent d-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of direct electron transfer (DET)-type bioelectrocatalysis, was investigated from the perspective of structural biology, bioelectrochemistry, and protein engineering. DET-type reactions offer several benefits in biomimetics (e.g., biofuel cells, bioreactors, and biosensors) owing to their mediator-less configuration. FDH provides an intense DET-type catalytic signal; therefore, extensive research has been conducted on the fundamental principles and applications of biosensors. Structural analysis using cryo-electron microscopy and single-particle analysis has revealed the entire FDH structures with resolutions of 2.5 and 2.7 Å for the reduced and oxidized forms, respectively. The electron transfer (ET) pathway during the catalytic oxidation of d-fructose was investigated by using both thermodynamic and kinetic approaches. Structural analysis has shown the localization of the electrostatic surface charges around heme 2c in subunit II, and experiments using functionalized electrodes with a controlled surface charge support the notion that heme 2c is the electrode-active site. Furthermore, two aromatic amino acid residues (Trp427 and Phe489) were located in a possible long-range ET pathway between heme 2c and the electrode. Two variants (W427A and F489A) were obtained by site-directed mutagenesis, and their effects on DET-type activity were elucidated. The results have shown that Trp427 plays an essential role in accelerating long-range ET and triples the standard rate constant of heterogeneous ET according to bioelectrochemical analysis

Identification of intracellular squalene in living algae, Aurantiochytrium mangrovei with hyper-spectral coherent anti-Stokes Raman microscopy using a sub-nanosecond supercontinuum laser source

We applied hyper-spectral coherent anti-Stokes Raman scattering imaging to intracellular lipid identification in living microalgae, Aurantiochytrium mangrovei 18W-13a. Two different lipids, squalene and triacylglycerol, were found inside living cells with clear vibrational contrast. Based on the endogenous lipid band as a result of the cis C[DOUBLE BOND]C stretch vibrational mode, squalene and triacylglycerol were clearly distinguished in different intracellular areas. In particular, squalene was detected solely in vacuoles as lipid particles, which was also supported by electron microscopy

Backward multiplex coherent anti-Stokes Raman (CARS) spectroscopic imaging with electron-multiplying CCD (EM-CCD) camera

A multiplex CARS imaging system, equipped with an EM-CCD camera, was developed to improve the sensitivity of backward CARS imaging in biological analysis using an inverted microscope. The signal-to-noise ratio was improved by a factor of ca. 3 compared to a conventional CCD mode through the use of EM gain. When imaging epithelial cells in the backward CARS configuration, intracellular organelles such as lipid droplets and nuclei were spectroscopically identified with an exposure time of only 100 ms/pixel.</p

Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family

AbstractBy DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase Cα (PKCα) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCα activation in PCs

Contrast-tuneable microscopy for single-shot real-time imaging

A novel real image in-line laser holography enables a tuneable image contrast, edge sharpness, and visualization of sub-wavelength structures, using a simple pair of filters and large-diameter lenses that can incorporate higher-order scattered light. Demonstrated also are the accuracy in object sizing and the ease of imaging along the focal depth, based on a single-shot imaging via holographic principle. In addition, the use of broad, collimated laser beam for irradiation has led to a wider field of view, making it particularly useful for an extensive monitoring of, and sweeping search for, cells and microbial colonies and for the real-time imaging of cancer-cell dynamics