Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

AbstractThe last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical

Cell and Tissue Imaging with Molecularly Imprinted Polymers

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Molecular recognition of endocrine disruptors by synthetic and natural 17β-estradiol receptors: a comparative study

International audienceA β-estradiol receptor binding mimic was synthesised using molecular imprinting. Bulk polymers and spherical polymer nanoparticles based on methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, were prepared in acetonitrile. The selectivity was evaluated by radioligand binding assays. The imprinted polymers were very specific to β-estradiol since the control polymers bound virtually none of the radioligand. The bulk polymer was then employed to screen endocrine disrupting chemicals. Structurally related steroids like α-estradiol, estrone and ethynylestradiol showed, respectively, 14.0, 5.0 and 0.7% of relative binding to the β-estradiol polymer, whereas most unrelated chemicals did not bind at all. These results are compared to those obtained with a bioassay using stably transfected yeast cells in culture bearing the human estrogen receptor. The receptor was activated by several estrogen-like chemicals and to a lesser extent by some structurally related chemicals

Evaluation of Performance and Validity Limits of Gas Chromatography electron ionization – Orbitrap Detector for fatty acid methyl esters analyses.

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Guide to the Preparation of Molecularly Imprinted Polymer Nanoparticles for Protein Recognition by Solid-Phase Synthesis

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Iron-sulfur proteins as initiators of radical chemistry

Iron-sulfur proteins are very versatile biological entities for which many new functions are continuously being unravelled. This review focus on their role in the initiation of radical chemistry, with special emphasis on 'radical-SAM' enzymes, since several members of the family catalyse key steps in the biosynthetic pathways of cofactors such as biotin, lipoate, thiamine, heme and the molybdenum cofactor. It will also include other examples to show the chemical logic which is emerging from the presently available data on this family of enzymes. The common step in all the ( quite different) reactions described here is the monoelectronic reductive cleavage of SAM by a reduced [4Fe-4S](1+) cluster, producing methionine and a highly oxidising deoxyadenosyl radical, which can initiate chemically difficult reactions. This set of enzymes, which represent a means to perform oxidation under reductive conditions, are often present in anaerobic organisms. Some other, non-SAM-dependent, radical reactions obeying the same chemical logic are also covered

Core–Shell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum

We describe the application of a fluorescently labeled water-soluble core–shell molecularly imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect trypsin. <i>p</i>-Aminobenzamidine (PAB), a competitive inhibitor of trypsin, was immobilized in the wells of a microtiter plate enabling the capture of trypsin in an oriented position, thus maintaining its native conformation. Fluorescent MIP nanoparticles, which bound selectively to trypsin, were used for quantification. The MIP was prepared by a multistep solid-phase synthesis approach on glass beads functionalized with PAB, orientating all trypsin molecules in the same way. The core–MIP was first synthesized, using a thermoresponsive polymer based on <i>N</i>-isopropylacrylamide, so as to enable its facile liberation from the immobilized template by a simple temperature change. The shell, mainly composed of allylamine to introduce primary amino groups for postconjugation of fluorescein isothiocyanate (FITC), was grafted in situ on the core–MIP, whose binding cavities were still bound and protected by the immobilized trypsin. The resulting core–shell MIP was endowed with a homogeneous population of high-affinity binding sites, all having the same orientation. The MIP has no or little cross-reactivity with other serine proteases and unrelated proteins. Our MIP-based FIA system was successfully applied to detect low trypsin concentrations spiked into nondiluted human serum with a low limit of quantification of 50 pM, which indicates the significant potential of this assay for analytical and biomedical diagnosis applications

Nanoparticles in Biomedical Applications

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Programmable bioelectronics in a stimuli-encoded 3D graphene interfaces

The ability to program and mimic the dynamic microenvironment of living organisms is a crucial step towards the engineering of advanced bioelectronics. Here, we report for the first time a design for programmable bioelectronics, with ‘built-in’ switchable and tunable bio-catalytic performance that responds simultaneously to appropriate stimuli. The designed bio-electrodes comprise light and temperature responsive compartments, which allow the building of Boolean logic gates (i.e. “OR” and “AND”) based on enzymatic communications to deliver logic operations.Funding agencies: Swedish Research Council [VR- 2011-6058357]; European Commission [NANODRUG: MCITN-2011-289554]</p