Involvement of histidines 11, 15 and 19 in the binding of zinc to the fusogenic H5WYG peptide

The histidine‐rich GLFHAIAHFIHGGWHGLIHGWYG peptide (H5WYG) coordinates a Zn²⁺ ion and forms a stable peptide–metal complex promoting membrane fusion at physiologic pH. In our previous article titled ‘Histidine‐rich peptide: evidence for a single zinc‐binding site on H5WYG peptide that promotes membrane fusion at neutral pH’ in Journal of Mass Spectrometry (2009, 44, 81–89), tandem mass spectrometry experiments have provided evidence for the binding of a single Zn²⁺ ion to H5WYG and suggested that this binding is shared between H¹¹, H¹⁹ and probably H¹⁵ residues. To clarify the involvement of these histidine residues in the binding to the Zn²⁺ ion and especially to remove the doubt about the implication of the H¹⁵ residue, here we have used three H5WYG mutants termed H5WYGH11A, H5WYGH15A and H5WYGH19A, whose H¹¹, H¹⁵ and H¹⁹ residues were replaced with an alanine residue. The novelty introduced by these new tandem mass spectrometry experiments performed with the mutants is the demonstration that H¹⁵ is involved in the binding of the single Zn²⁺ ion and that it may even favour the setting of another Zn²⁺ ion. Thus, the three histidines H¹¹, H¹⁵ and H¹⁹ could lead to a specific structuring of H5WYG that can promote membrane fusion upon the binding of zinc. Copyright © 2009 John Wiley & Sons, Ltd.     

Is there any 12C/13C fractionation during starch remobilisation and sucrose export in potato tubers?

The δ¹³C (carbon isotope composition) variations in respired CO₂, total organic matter, proteins, sucrose and starch have been measured during tuber sprouting of potato (Solanum tuberosum) in darkness. Measurements were carried out both on tubers and on their growing sprouts for 23 days after the start of sprout development. Sucrose was slightly ¹³C‐depleted compared with starch in tubers, suggesting that starch breakdown was associated with a small isotope fractionation. In sprouts, all biochemical fractions including sucrose were ¹³C‐enriched compared with source tuber‐sucrose, suggesting that sucrose translocation from tuber to sprouts fractionated against ¹²C. However, both apparent fractionations were explained by the consumption of ¹³C‐depleted carbon for respiration or growth that enriched in the ¹³C sucrose molecules left behind. In addition, whole tuber sucrose is constantly composed of recent sucrose from starch breakdown and old sucrose associated with an inherited, slightly ¹³C‐depleted pool. We therefore conclude that any fractionation at either the starch breakdown or the sucrose translocation level is unlikely under our conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of cobaltocenium salts for use as redox labels and their incorporation into Nafion films

A cobaltocenium label was covalently attached to two antidepressants, nortriptyline and desipramine, via an amide linkage, and also to the hydrazine derivative of the biologically important compound biotin (vitamin H), again via an amide linkage. Analytically pure samples of these new cobaltocenium salts could be obtained by chromatography on silica gel followed by elution with aqueous acetone solutions containing sodium chloride (NaCl). These positively charged cobaltocenium ions preconcentrate in a polyanionic Nafion film coated on a glassy carbon surface, albeit at different concentration levels. One factor which seems to influence the amount of cobaltocenium ion that enters the film is polarity since the cobaltocenium ion containing the rather polar biotin preconcentrates at the lowest level in the relatively hydrophobic Nafion. Square-wave voltammograms of Nafion films containing these cobaltocenium cations exhibit a one-electron, reversible, reduction wave at approximately ?1.1 V (vs Ag/AgCl) with peak currents that are sufficiently large to permit detection of 10^?8 M quantities of these substances in the bulk solution.     

Design and synthesis of europium luminescent bio-probes featuring sulfobetaine moieties

Herein we report the straightforward preparation of chromophore-functionalized TACN ligand via Cu-free cross-coupling reactions using a common halogenated platform. This versatile methodology allows the preparation of original macrocyclic ligand featuring both optimized antenna for the sensitization of europium luminescence and sulfobetaine zwitterionic groups to ensure water solubility of the complex. In addition preliminary two-photon excited microscopy imaging experiments of fixed cells reveal that sulfobetaine groups are able to limit undesirable non specific interactions with biological surrounding.     

Sensitization of Eu(III) luminescence by donor-phenylethynyl-functionalized DTPA and DO3A macrocycles

The synthesis and complexation to Eu(III) of two macrocyclic ligands functionalized donor-phenylethynyl-moieties as sensitizer is presented. The two ligands based on DTPA bis-amide ([Eu(L¹)]) and DO3A ([Na][Eu(L²)]) have been chosen because of their increased stability in water for potential applications as luminescent bioprobes for one- or two-photon excited scanning microscopy. The DTPA and DO3A ligands possess oxygen or sulfur donor atoms, respectively, connected to a triethyleneglycol (PEG) chain to ensure the water solubility. The optical properties were studied and reveal that ([Eu(L¹)]) present a lower brightness than the ([Na][Eu(L²)]) counterpart because of its inner sphere water molecule coordination that quenches the emission. On the other hand, [Na][Eu(L²)] exhibits excellent spectroscopic properties with high quantum yield ? = 0.284, and brightness B = 10,220 M^?1cm^?1(defined as ?.?). Unfortunately, the two-photon cross-section of this last complex was measured to be only 4 GM limiting its potential applications to linear microscopy.     

Photoelectrochemical immunosensor for label-free detection and quantification of anti-cholera toxin antibody

We demonstrate herein a newly developed photoelectrochemical immunosensor for the determination of anti-cholera toxin antibody by using a photosensitive biotinylated polypyrrole film. The latter was generated by electro-oxidation of a biotinylated tris(bipyridyl) ruthenium(II) complex bearing pyrrole groups. The photoexcitation of this modified electrode potentiostated at 0.5 V vs SCE, in the presence of an oxidative quencher, pentaaminechloro cobalt(III) chloride (15 mM), led to a cathodic photocurrent. As a result of the affinity interactions, a layer of biotinylated cholera toxin was firmly bound to the functionalized polypyrrole film via avidin bridges. The resulting modified electrodes were tested as immunosensors for the detection of the corresponding antibody from 0 to 200 microg mL(-)(1). The antibody concentration was measured through the decrease in photocurrent intensity resulting from its specific binding onto the polymeric coating, the detection limit being 0.5 microg mL(-)(1).     

Selective Michael additions of primary and secondary amines to perfluoroalkylated sulfoxides and sulfones as a tool for fluorous tagging

Vinyl tridecafluorohexyl sulfoxide 1 and sulfone 2 are shown to be highly reactive towards Michael addition with a broad range of secondary and primary amines. Full selectivity for monofunctionalization of primary amines can be achieved by a proper choice of reagent 1 or 2 depending on amine basicity.     

How energy funnels from the phycoerythrin antenna complex to photosystem I and photosystem II in cryptophyte Rhodomonas CS24 cells

We report an investigation of energy migration dynamics in intact cells of the photosynthetic cryptophyte Rhodomonas CS24 using analyses of steady-state and time-resolved fluorescence anisotropy measurements. By fitting a specific model to the fluorescence data, we obtain three time scales (17, 58, and 113 ps) by which the energy is transferred from phycoerythrin 545 (PE545) to the membrane-associated chlorophylls (Chls). We propose that these time scales reflect both an angular distribution of PE545 around the photosystems and the relative orientations of the donor dihydrobiliverdin (DBV) bilin and the acceptor Chl. Contrary to investigations of the isolated antenna complex, it is demonstrated that energy transfer from PE545 does not occur from a single-emitting bilin, but rather both the peripheral dihydrobiliverdin (DBV) chromophores in PE545 appear to be viable donors of excitation energy to the membrane-bound proteins. The model shows an almost equal distribution of excitation energy from PE545 to both photosystem I (PSI) and photosystem II (PSII), whose trap times correspond well to those obtained from experiments on isolated photosystems.     

Laser processing for bio-microfluidics applications (part II)

This paper reviews applications of laser-based techniques to the fabrication of microfluidic devices for biochips and addresses some of the challenges associated with the manufacture of these devices. Special emphasis is placed on the use of lasers for the rapid prototyping and production of biochips, in particular for applications in which silicon is not the preferred material base. This review addresses applications and devices based on ablation using femtosecond lasers, infrared lasers as well as laser-induced micro-joining, and the laser-assisted generation of micro-replication tools, for subsequent replication of polymeric chips with a technique like laser LIGA.     

Detection of polycyclic aromatic hydrocarbon (PAH) compounds in artificial sea-water using surface-enhanced Raman scattering (SERS)

This paper reports an accurate synthesis of surface-enhanced Raman scattering (SERS) active substrates, based on gold colloidal monolayer, suitable for in situ environmental analysis. Quartz substrates were functionalized by silanization with (3-mercaptopropyl)trimethoxysilane (MPMS) or (3-aminopropyl)trimethoxysilane (APTMS) and they subsequently reacted with colloidal suspension of gold metal nanoparticles: respectively, the functional groups SH and NH₂ bound gold nanoparticles. Gold nanoparticles were prepared by the chemical reduction of HAuCl₄ using sodium tricitrate and immobilized onto silanized quartz substrates. Active substrate surface morphology was characterized with scanning electron microscopy (SEM) measurements and gold nanoparticles presented a diameter in the range 40-100 nm. Colloidal hydrophobic films, allowing nonpolar molecule pre-concentration, were obtained. The surfaces exhibit strong enhancement of Raman scattering from molecules adsorbed on the films. Spectra were recorded for two PAHs, naphthalene and pyrene, in artificial sea-water (ASW) with limits of detection (LODs) of 10 ppb for both on MPMS silanized substrates.