Mucoadhesive interactions of amphiphilic cationic copolymers based on [2-(methacryloyloxy)ethyl]trimethylammonium chloride

Three series of water-soluble cationic copolymers have been synthesised by free-radical copolymerisation of [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (MADQUAT) with methyl acrylate (MA), butyl acrylate (BA) and butyl methacrylate (BMA). The interactions between these copolymers and porcine stomach mucin have been studied in aqueous solutions using dynamic light scattering, zeta-potential measurements, turbidimetric titration and transmission electron microscopy (TEM). It was demonstrated that mixing aqueous dispersions of mucin with solutions of the cationic copolymers results in significant changes in size distribution and zeta-potential of its particles. It was found that an increase in the content of hydrophobic groups in copolymers leads to more efficient adsorption of macromolecules on the surface of mucin particles, which evidences the importance of hydrophobic effects in mucoadhesion. The efficiency of mucoadhesive interactions was found to be significantly dependent on pH, which affects the surface charge and aggregation stability of mucin. (C) 2007 Elsevier B.V. All rights reserved.

Rhodium cationic complexes using dithioethers as chiral ligands. Application in styrene hydroformylation

Addition of the dithioethers (−)-DIOSR2 (R=Me, iPr) (2,3-O-isopropylidene-1,4-dimethyl (and diisopropyl) thioether-L-threitol) to a dichloromethane solution of [Rh(COD)2]ClO4 (COD=1,5-cyclooctadiene) yielded the mononuclear complexes [Rh(COD)(DIOSR2)]ClO4. X-ray diffraction methods showed that the [Rh(COD)(DIOSiPr2)]ClO4 complex had an square-planar coordination geometry at the rhodium atom with the iPr groups in anti position. Cyclooctadiene complexes react with carbon monoxide to form dinuclear tetracarbonylated complexes [(CO)2Rh(μ-DIOSR2)2(CO)2](ClO4)2. [Rh(COD)(DIOSR2)]ClO4 are active catalyst precursors in styrene hydroformylation at 30 atm and 65°C which give conversions of up to 99% with a regioselectivity in 2-phenylpropanal as high as 74%. In all cases enantioselectivities are low.

Recovery of gallic acid with colloidal gas aphrons generated from a cationic surfactant

In a previous study we have demonstrated that gallic acid (GA) in its anionic form can be recovered from aqueous solutions using colloidal gas aphrons (CGA) generated from the cationic surfactant cetyltrimethylammonium bromide (CTAB). The aim of the present work is to get a better understanding of the separation mechanism in order to determine the optimum operating conditions to maximise the recovery of GA while preserving its antioxidant properties. Zeta potential measurements were carried out to characterise the surface charge of GA, CTAB and their mixtures at three different pH conditions (both in buffers and in aqueous solutions). GA interacted strongly with CTAB at pH higher than its pKa 3.14 where it is ionised and negatively charged. However, at pH higher than 7 GA becomes oxidised and loses its antioxidant power. GA recovery was mainly affected by pH, ionic strength, surfactant/GA molar ratio, mixing conditions and contact time. Scale-up of the separation using a flotation column resulted in both higher recovery and reproducibility. Preliminary experiments with grape marc extracts confirmed the potential application of this separation for the recovery of polyphenols from complex feedstocks

Predicting metal-binding site residues in low-resolution structural models

The accurate prediction of the biochemical function of a protein is becoming increasingly important, given the unprecedented growth of both structural and sequence databanks. Consequently, computational methods are required to analyse such data in an automated manner to ensure genomes are annotated accurately. Protein structure prediction methods, for example, are capable of generating approximate structural models on a genome-wide scale. However, the detection of functionally important regions in such crude models, as well as structural genomics targets, remains an extremely important problem. The method described in the current study, MetSite, represents a fully automatic approach for the detection of metal-binding residue clusters applicable to protein models of moderate quality. The method involves using sequence profile information in combination with approximate structural data. Several neural network classifiers are shown to be able to distinguish metal sites from non-sites with a mean accuracy of 94.5%. The method was demonstrated to identify metal-binding sites correctly in LiveBench targets where no obvious metal-binding sequence motifs were detectable using InterPro. Accurate detection of metal sites was shown to be feasible for low-resolution predicted structures generated using mGenTHREADER where no side-chain information was available. High-scoring predictions were observed for a recently solved hypothetical protein from Haemophilus influenzae, indicating a putative metal-binding site.

pH-tunable hydrogelators for water purification: structural optimisation and evaluation

A focused library of potential hydrogelators each containing two substituted aromatic residues separated by a urea or thiourea linkage have been synthesised and characterized. Six of these novel compounds are highly efficient hydrogelators, forming gels in aqueous solution at low concentrations (0.03–0.60 wt %). Gels were formed through a pH switching methodology, by acidification of a basic solution (pH 14 to ≈4) either by addition of HCl or via the slow hydrolysis of glucono-δ-lactone. Frequently, gelation was accompanied by a dramatic switch in the absorption spectra of the gelators, resulting in a significant change in colour, typically from a vibrant orange to pale yellow. Each of the gels was capable of sequestering significant quantities of the aromatic cationic dye, methylene blue, from aqueous solution (up to 1.02 g of dye per gram of dry gelator). Cryo-transmission electron microscopy of two of the gels revealed an extensive network of high aspect ratio fibers. The structure of the fibers altered dramatically upon addition of 20 wt % of the dye, resulting in aggregation and significant shortening of the fibrils. This study demonstrates the feasibility for these novel gels finding application as inexpensive and effective water purification platforms.

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Selected wheat seed defense proteins exhibit competitive binding to model microbial lipid interfaces

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. We have examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and β-purothionin (β-Pth) model anionic lipid layers, using a combination of surface pressure measurements, external reflection FTIR spectroscopy and neutron reflectometry. Results highlighted differences in the protein binding mechanisms, and in the competitive binding and penetration of lipid layers between respective Pins and β-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of β-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with β-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of β-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity.

Interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity

We investigate the properties of an antimicrobial surfactant-like peptide (Ala)6(Arg), A6R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A6R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A6R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A6R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A6R exhibits antimicrobial activity without membrane lysis.

Efficient masking of corrosion and fission products such as Ni(II) and Pd(II) in the presence of the minor actinide Am(III) using hydrophilic anionic or cationic bis-triazines

Water soluble anionic and cationic bis-triazine ligands are able to suppress (mask) the extraction of corrosion and fission products such as Ni(II) and Pd(II) that are found in PUREX raffinates. Thus it is possible to separate these elements from the minor actinide Am(III). Although some masking agents have previously been developed that retard the extraction of Pd(II), this is the first time a masking agent has been developed for Ni(II).