Model peptides based on the binding loop of the copper metallochaperone Atx1: selectivity of the consensus sequence MxCxxC for metal ions Hg(II), Cu(I), Cd(II), Pb(II), and Zn(II)

The amino acid sequence MxCxxC is conserved in many soft-metal transporters that are involved in the control of the intracellular concentration of ions such as Cu(I), Hg(II), Zn(II), Cd(II), and Pb(II). A relevant task is thus the selectivity of the motif MxCxxC for these different metal ions. To analyze the coordination properties and the selectivity of this consensus sequence, we have designed two model peptides that mimic the binding loop of the copper chaperone Atx1: the cyclic peptide P(C) c(GMTCSGCSRP) and its linear analogue P(L) (Ac-MTCSGCSRPG-NH2). By using complementary analytical and spectroscopic methods, we have demonstrated that 1:1 complexes are obtained with Cu(I) and Hg(II), whereas 1:1 and 1:2 (M:P) species are successively formed with Zn(II), Cd(II), and Pb(II). The complexation properties of the cyclic and linear peptides are very close, but the cyclic compound provides systematically higher affinity constants than its unstructured analogue. The introduction of a xPGx motif that forms a type II beta turn in P(C) induces a preorganization of the binding loop of the peptide that enhances the stabilities of the complexes (up to 2 orders of magnitude difference for the Hg complexes). The affinity constants were measured in the absence of any reducing agent that would compete with the peptides and range in the order Hg(II) > Cu(I) > Cd(II) > Pb(II) > Zn(II). This sequence is thus highly selective for Cu(I) compared to the essential ion Zn(II) that could compete in vivo or compared to the toxic ions Cd(II) and Pb(II). Only Hg(II) may be an efficient competitor of Cu(I) for binding to the MxCxxC motif in metalloproteins.     

Ion-pair, anion-exchange and ligand-exchange high-performance liquid chromatography of tenuazonic acid and 3-acetyl 5-substituted pyrrolidine-2,4-diones

The ion-pair, ligand-exchange and anion-exchange chromatography of the fungal metabolic tenuazonic acid (TA) and its related 3-acetyl 5-substituted pyrrolidine-2,4-diones were studied. Ion-pair chromatography was performed on a C18 column with a mobile phase composed of cetrimide, phosphate buffer in water-methanol and a metal complexant (ethylenediamine) to improve the peak sharpness. Addition of the same metal complexant to the mobile phase of the anion-exchange chromatographic system also improved its efficiency. TA and its 5-substituted analogues derived from valine and leucine were separated with the ion-pair and anion-exchange chromatographic systems. With ligand-exchange chromatography, TA could only be separated from its valine analogue. These chromatographic systems were used for the detection of TA in the culture filtrates of the fungus Pyricularia oryzae and in infected rice leaves. Deproteinated culture filtrates could be rapidly analysed for their TA content by anion-exchange chromatography. However, this system was not suitable for the detection of TA in the infected rice leaf as interfering compounds were coeluted with TA. Ion-pair and ligand-exchange chromatographic systems allowed the efficient quantification of TA in infected leaves.     

Lanthanide-binding peptides with two pendant aminodiacetate arms: impact of the sequence on chelation

Lanthanide complexes with a series of hexapeptides-incorporating two unnatural chelating amino acids with aminodiacetate groups, Ada(1) and Ada(2)-have been examined in terms of their speciation, structure, stability and luminescence properties. Whereas Ada(2) acts as a tridentate donor in all cases, Ada(1) may act as a tetradentate donor thanks to the coordination of the amide carbonyl function assisted by the formation of a six-membered chelate ring. The position of the Ada(1) residue in the sequence is demonstrated to be critical for the lanthanide complex speciation and structure. Ada(1) promotes the coordination of the backbone amide function to afford a highly dehydrated Ln complex and an S-shape structure of the peptide backbone, only when found in position 2.     

New labdane diterpenes from the stem bark of Larix laricina

Two new labdane diterpenes, cis-19-hydroxyabienol (1) and 8alpha-hydroxy-12Z,14-labdadien-19-al (2), along with another labdane described for the first time in the genus Larix, 19-acetoxy-13S-hydroxy-8(17),14-labdadiene (3) and a stilbene, 3-methoxy-3,3',5'-trihydroxystilbene (4), were isolated from the stem bark of Larch (Larix laricina). Their structures were established by standard chemical and spectroscopic methods. Compounds 2 and 3 were shown to be moderately cytotoxic against A-549, DLD-1 and normal skin fibroblast cell lines, WS1. Compound 1 was found to be selectively active against colon carcinoma cell lines, DLD-1.     

Hepatocyte Targeting and Intracellular Copper Chelation by a Thiol-Containing Glycocyclopeptide

Metal overload plays an important role in several diseases or intoxications, like in Wilson's disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P(3). Two simple cyclodecapeptides modeling the reduced and complexing form of P(3) in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P(3) becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P(3) a good candidate to fight copper overload in the liver.     

A concise benzotriazolyl-mediated synthesis of 9-methoxycepharanone A

A concise synthesis of 9-methoxycepharanone A is described. The key step is the benzotriazolyl-assisted assemblage of an arylmethyleneisoindolinone ring system comprising the enol ether unit. Radical cyclization followed by deprotections and ultimate formation of the methylenedioxy group complete the total synthesis of the title compound.     

Single shot phase contrast imaging using laser-produced Betatron x-ray beams

Development of x-ray phase contrast imaging applications with a laboratory scale source have been limited by the long exposure time needed to obtain one image. We demonstrate, using the Betatron x-ray radiation produced when electrons are accelerated and wiggled in the laser-wakefield cavity, that a high-quality phase contrast image of a complex object (here, a bee), located in air, can be obtained with a single laser shot. The Betatron x-ray source used in this proof of principle experiment has a source diameter of 1.7 μm and produces a synchrotron spectrum with critical energy E(c)=12.3±2.5 keV and 10? photons per shot in the whole spectrum.     

Particle field characterization by digital in-line holography: 3D location and sizing

Recent developments have shown the potential of digital in-line holography for diagnostics in fluids. This new method provides a low-cost and easy access method for measuring both size and velocity of small particles in a volume. Here it is shown that by applying traditional image processing tools on the particle images digitally reconstructed, statistically reliable results on particles size and location are provided. The method is experimentally illustrated by glass microspheres that are moving in a turbulent flow generated by an annular jet. A comparison with the histogram diameters provided by a common diffraction particle sizer are presented.