Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate

1. The ability of myo-inositol polyphosphates to inhibit iron-catalysed hydroxyl radical formation was studied in a hypoxanthine/xanthine oxidase system [Graf, Empson and Eaton (1987) J. Biol. Chem. 262, 11647-11650]. Fe3+ present in the assay reagents supported some radical formation, and a standard assay, with 5 microM Fe3+ added, was used to investigate the specificity of compounds which could inhibit radical generation. 2. InsP6 (phytic acid) was able to inhibit radical formation in this assay completely. In this respect it was similar to the effects of the high affinity Fe3+ chelator Desferral, and dissimilar to the effects of EDTA which, even at high concentrations, still allowed detectable radical formation to take place. 3. The six isomers of InsP5 were purified from an alkaline hydrolysate of InsP6 (four of them as two enantiomeric mixtures) and they were compared with InsP6 in this assay. Ins(1,2,3,4,6)P5 and D/L-Ins(1,2,3,4,5)P5 were similar to InsP6 in that they caused a complete inhibition of iron-catalysed radical formation at > 30 microM. Ins(1,3,4,5,6)P5 and D/L-Ins(1,2,4,5,6)P5, however, were markedly less potent than InsP6, and did not inhibit radical formation completely; even when Ins(1,3,4,5,6)P5 was added up to 600 microM, significant radical formation was still detected. Thus InsP5s lacking 2 or 1/3 phosphates are in this respect qualitatively different from InsP6 and the other InsP5s. 4. scyllo-Inositol hexakisphosphate was also tested, and although it caused a greater inhibition than Ins(1,3,4,5,6)P5, it too still allowed detectable free radical formation even at 600 microM. 5. We conclude that the 1,2,3 (equatorial-axial-equatorial) phosphate grouping in InsP6 has a conformation that uniquely provides a specific interaction with iron to inhibit totally its ability to catalyse hydroxyl radical formation; we suggest that a physiological function of InsP6 might be to act as a 'safe' binding site for iron during its transport through the cytosol or cellular organelles

Oxygen radical induced alterations in polyclonal IgG

In the sera and synovial fluid of patients with rheumatoid arthritis, part of the IgG fraction is found in an aggregated and fluorescent form. Oxygen-free radicals have been implicated in this denaturation, although the precise radical species responsible is unknown. In this work, oxygen-free radicals generated radiolytically were allowed to attack polyclonal IgG in solution. OH radicals induced aggregation of the monomer and a new fluorescence appeared in the visible region (Ex 360 nm, Em 454 nm). The superoxide radical anion was found to be inert in both these respects, whilst peroxy radicals induced autofluorescence without concomitant aggregation. The results suggest that OH.and/or peroxy radical attack may be an in vivo mechanism for IgG denaturation.

Free radical reactions in atherosclerosis; An EPR spectrometry study

The copper catalysed oxidation of homocysteine has been studied by electron paramagnetic resonance (EPR) spectroscopy and spin trapping techniques to determine the nature of free radical species formed under varying experimental conditions. Three radicals; thiyl, alkyl and hydroxyl were detected with hydroxyl being predominant. A reaction mechanism is proposed involving Fenton chemistry. Inclusion of catalase to test for intermediate generation of hydrogen peroxide showed a marked reduction in amount of hydroxyl radical generated. In contrast, the addition of superoxide dismutase showed no significant effect on the level of hydroxyl radical formed. Enhanced radical formation was observed at higher levels of oxygen, an effect which has consequences for differential oxygen levels in arterial and venous systems. Implications are drawn for a higher incidence of atherosclerotic plaque formation in arteries versus veins. © 2006 - IOS Press and the authors. All rights reserved.

Die Poetik des Extremen. Ausschreitungen einer Sprache des Radikalen:excesses of a language of radical

Gibt es eine Traditionslinie extremistischer Poetiken in der deutschsprachigen Literatur? Uwe Schütte untersucht anhand literarischer Texte ab dem späten 18. Jahrhundert den Konnex zwischen historischen Phänomenen wie Revolution, Krieg oder Terrorismus und extremen biografischen Umständen wie Schizophrenie für die Herausbildung radikaler Schreibweisen. Die Spannbreite der behandelten Autoren reicht dabei von der Klassikertrias Kleist, Hölderlin und Büchner über Schriftsteller des 20. Jahrhunderts wie Ernst Jünger oder Hans Henny Jahnn bis zu den Gegenwartsautoren Ernst Herbeck und Rainald Goetz. The study investigates aesthetic representations of extremism in German-language literature from around 1800 to the present. Its aim is to examine the interplay between three different areas: historical circumstances, (auto)biographical issues, and literary texts. Discussed are texts by both major and marginal writers from various genres, ranging from classics such as Heinrich von Kleist or Friedrich Hölderlin to the marginalised poet Ernst Herbeck or the contemporary writer Rainald Goetz. Subjects and factors considered include extremist phenomena in modern history (such as revolutions, wars, terrorism) and extreme individual experiences (such as suicide or schizophrenia) on the aesthetic domain(s) with regard to the production of literary discourses that could be considered as extremist. These manifest themselves in the development of what can be viewed as ‘radical poetics’, decidedly innovative styles of writing and moral or political transgression in fiction. Being the first critical attempt to trace the history of radical discourses in German literature, the study explores the validity of creating an aesthetic category of 'literary extremism'.

A radical agenda for local government

If we want to rebuild Labour's councillor base, we will need to repoliticise the role of local government.

Mass spectrometric analysis of HOCl- and free-radical-induced damage to lipids and proteins

In inflammatory diseases, release of oxidants leads to oxidative damage to biomolecules. HOCl (hypochlorous acid), released by the myeloperoxidase/H2O2/Cl- system, can cause formation of phospholipid chlorohydrins, or alpha-chloro-fatty aldehydes from plasmalogens. It can attack several amino acid residues in proteins, causing post-translational oxidative modifications of proteins, but the formation of 3-chlorotyrosine is one of the most stable markers of HOCl-induced damage. Soft-ionization MS has proved invaluable for detecting the occurrence of oxidative modifications to both phospholipids and proteins, and characterizing the products generated by HOCl-induced attack. For both phospholipids and proteins, the application of advanced mass spectrometric methods such as product or precursor ion scanning and neutral loss analysis can yield information both about the specific nature of the oxidative modification and the biomolecule modified. The ideal is to be able to apply these methods to complex biological or clinical samples, to determine the site-specific modifications of particular cellular components. This is important for understanding disease mechanisms and offers potential for development of novel biomarkers of inflammatory diseases. In the present paper, we review some of the progress that has been made towards this goal.

Sequence distributions in free-radical polymers

This thesis is concerned with demonstrating how the visual representation of the sequence distribution of individual monomer units, of a polymer, that would be observed upon polymerisation, may be utilised in designing and synthesizing polymers with relatively low cell adhesion characteristics, The initial part of this thesis is concerned with demonstrating the use of a computer simulation technique, in illustrating the sequence distribution that would be observed upon the polymerisation of a set of monomers. The power of the computer simulation technique has been demonstrated through the simulation of the sequence distributions of some generic contact lens materials. These generic contact lens materials were chosen simply because in the field of biomaterials their compositions are amongst the most systematically regulated and they present a wide range of compositions. The validity of the computer simulation technique has been assessed through the synthesis and analysis of linear free-radical polymers at different conversions. Two main parameters were examined, that of composition and the number-average sequence lengths of individual monomer units, at various conversions. The polymers were synthesized through the solution polymerisation process. The monomer composition was determined by elemental analysis and 13C nuclear magnetic analysis (NMR). Number-average sequence lengths were determined exclusively through 13C NMR. Although the computer simulation technique provides a visual representation of the monomer sequence distribution up to 100% conversion, these assessments were made on linear polymers at a reasonably high conversion (above 50%) but below 100% conversion of ease for analysis. The analyses proved that the computer simulation technique was reasonably accurate in predicting the sequence distribution of monomer units, upon polymerisation, in the polymer.An approach has been presented which allows one to manipulate the use of monomers, with their reactivity ratios, thereby enabling us to design polymers with controlled sequence distributions.Hydrogel membranes, with relatively controlled sequence distributions and polymerised to 100% conversion, were synthesized to represent prospective biomaterials. Cell adhesion studies were used as a biological probe to investigate the susceptibility of the surface of these membranes to cell adhesion. This was necessary in order to assess the surface biocompatibility or biotolerance of these prospective biomaterials.

Atom transfer radical polymerisation - towards the synthesis of a fully-functional photorefractive polymer

Atom transfer radical polymerisation (ATRP) of styrene in xylene solution initiated with 1-phenylethyl bromide and mediated by CuBr/N-propyl-2- pyridinemethanimine catalyst complex was studied. The polymerisation was ill-controlled, yielding polymers with broad molecular weight distributions and values of number average molecular weight considerably higher than the theoretical values calculated from 100% initiator efficiency. The degree of control afforded over the polymerisation was enhanced by use of a more soluble catalyst complex, CuBr/N-octyl-2-pyridinemethanimine. Furthermore, the use of a more polar solvent, diglyme, generated a homogeneous catalyst complex that facilitated the production of polymers having narrow molecular weight distributions (1.10 < PDi < 1.20). The kinetics of the atom transfer radical polymerisation of methyl methacrylate at 90°C in diglyme solution initiated with ethyl-2-bromoisobutyrate and mediated by CuBr/N-octyl-2-pyridinemethanimine was studied and the orders of the reaction were established. The effect on the rate of polymerisation of the ratio of CuBr:N-octyl-2-pyridinemethanimine was also determined. The temperature dependencies of the rate of polymerisation of methyl methacrylate in diglyme solution and xylene solution were studied, and were found to be non-linear and dependent upon the polarity of the solvent. The use of highly polar aprotic solvents, such as N,N-dimethylformamide and dimethylsulphoxide, was found to be detrimental to the degree of control afforded over the polymerisation of methyl methacrylate. This was circumvented by use of a 5-fold excess, over that conventionally used, of catalyst complex. The atom transfer radical polymerisation of (4-nitrophenyl)-[3-[N-[2- (methacryloyloxy)ethyl]carbazolyl]]diazene in dimethyl sulphoxide solution was studied. Although homopolymerisation yielded only oligomers, copolymerisation of this monomer with methyl methacrylate was found to be readily achievable. Keywords: ATRP, Styrene; Methyl methacrylate; Polar solvents; Fully-functional photorefractive polymer. 2

The mechanochemical modification of polyolefins using reactive modifier systems

The principal objective of this work was to improve the mechanical properties of glass fibre reinforced polypropylene (PP) composites by the mechanochemical modification of the PP. The modification of the PP was carried out by reactive processing of the PP with a modifier in a Buss Ko-Kneader. Two main types of modifier were evaluated one type based on N-substituted maleimides the others based on 2-allylamino-4,6-dichloro-1,3,5-triazine (ACCT). The modification of the PP was carried out in two stages. Firstly the PP was reactively processed with the modifier and a free radical initiator. The objective of this stage was to bind the modifier to the PP. In the second stage the modified PP was reactively processed with the glass fibre. The objective in this stage was to form a chemical bond between the bound modifier and the silane coupling agent on the surface of the glass. Two silane coupling agents were evaluated these had a aliphatic chloro group and an aliphatic amino group respectively available for reaction with the modifier. The modifiers synthesised for this work had two main functional groups. The first was a double bond for free radical addition to the PP. The second was an organic group chosen for its potential reactivity to the silane coupling agent. A preliminary investigation was carried out using maleic anhydride (MA) as the modifier, this is reactive to the amino silane coupled glass. Studies of a commercially available system were also carried out for comparison purposes. During the work it was found that the amino silane coupled glass fibres produced, without any modification being made to the PP, mechanical properties comparable to the commercial system. Further any modification added to the amino silane system failed to improve the mechanical performance and in some cases acted in the opposite fashion. This failure was evident even when a chemical bond between glass fibre and PP could be shown. In the case of the chloro silane coupled glass fibres the mechanical properties of the composite without modification were poorer than those of the commercial system. It was found that the mechanical properties of these systems could be enhanced by the modifiers, however, no system tested significantly out performed the commercial system. Of the two modifier systems tested those based on the n-substituted maleimides were more successful at enhancing mechanical properties than those based on ACCT. This was attributed to the Poor chemical binding of the ACCT based modifiers to the PP. During the work it was found that several of the modifiers improved the properties of the PP when no glass fibres were present, particularly the % elongation and impact strength. It is possible that these modifiers could be used to improve the impact performance of PP, this may be of particular interest in recycling. These modifiers have only been tested for improving the properties of glass fibre composites. The N-substituted maleimide based modifiers could be used as compatibleisers for alloys of PP and other polymers. These could function by the formation of the bond with PP via the double bond whilst the group attached to the nitrogen atom could react with the alloying polymer.

The role of stable nitroxyl radical precursors as antioxidants in polyolefins

Various 2,2,6,6-tetramethyl piperidines and their N-alkyl derivatives of stable nitroxyl radical precursors containing acrylic(s) and methacrylic(s) groups were reactively processed in the presence of a peroxide as bound-antioxidant masterbatches for polyolefin stabilisation. It was found that grafting of the antioxidant monomers onto the polymer backbone was inevitably in competition with homopolymerisation of the monomers as well as melt degradation of the polymer and other side reactions. As previously reported, binding efficiency of bisacrylic nitroxyl precursor was maximum due to formation of unextractable homopolymer of the antioxidant. On the other hand, the binding efficiency of monoacrylic derivatives was low and the homopolymers were found extractable, which suggests that the bound monoacrylic derivatives are entirely grafted onto the polyolefin backbone. Application of bis and tri-functional coagents gave improved binding efficiency of the monoacrylic monomers. This may be due to copolymerisation of the antioxidants with the coagents and grafting of the copolymers onto the polymer backbone. Comparison of photostabilising activity of the fully extracted bound antioxidants to those of the corresponding unbound analogous showed lower results for the former. However, thermal stabilising activity of the bound antioxidants was higher than that of the unbound analogous due to better substantivity. Analysis using physical techniques and GPC for molecular weight distribution of masterbatches containing the bound monoacrylic antioxidants showed formation of high molecular weight products. Model reaction of a secondary amine derivative in liquid hydrocarbon and analysis of the product using FTIR and NMR spectroscopy indicated a possibility of side reaction, i.e. involvement of the amine active group (>N-H) of the antioxidant in the binding process to form the high molecular weight product. Implementation of various N-alkylated derivatives did not inhibit the side reaction. The photostabilising activity of the bound-antioxidants can be improved when applied in conjunction with small amounts of a benzophenone uv-stabiliser. The synergistic stabilising activity, however, was diminished when the uv-stabiliser was removed from the system during the service time. Nitroxyl precursors containing methacrylic group(s) gave lower binding efficiency than the corresponding acrylic derivatives. Reversible deploymerisation of the grafted methacrylic antioxidants may be responsible for this. Bis and tris-acrylic coagents improved the binding efficiency, and the presence of methacrylic group improved stabilising activity of the antioxidants. N-methyl derivatives were found to exhibit better stabilising activity than their parent secondary amine derivatives.

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.

Domain and nucleotide dependence of the interaction between Saccharomyces cerevisiae translation elongation factors 3 and 1A

Eukaryotic translation elongation factor 3 (eEF3) is a fungal-specific ATPase proposed to catalyze the release of deacylated-tRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperature-sensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.

Approximating differentiable relationships between delay embedded dynamical systems with radical basis functions

This thesis is about the study of relationships between experimental dynamical systems. The basic approach is to fit radial basis function maps between time delay embeddings of manifolds. We have shown that under certain conditions these maps are generically diffeomorphisms, and can be analysed to determine whether or not the manifolds in question are diffeomorphically related to each other. If not, a study of the distribution of errors may provide information about the lack of equivalence between the two. The method has applications wherever two or more sensors are used to measure a single system, or where a single sensor can respond on more than one time scale: their respective time series can be tested to determine whether or not they are coupled, and to what degree. One application which we have explored is the determination of a minimum embedding dimension for dynamical system reconstruction. In this special case the diffeomorphism in question is closely related to the predictor for the time series itself. Linear transformations of delay embedded manifolds can also be shown to have nonlinear inverses under the right conditions, and we have used radial basis functions to approximate these inverse maps in a variety of contexts. This method is particularly useful when the linear transformation corresponds to the delay embedding of a finite impulse response filtered time series. One application of fitting an inverse to this linear map is the detection of periodic orbits in chaotic attractors, using suitably tuned filters. This method has also been used to separate signals with known bandwidths from deterministic noise, by tuning a filter to stop the signal and then recovering the chaos with the nonlinear inverse. The method may have applications to the cancellation of noise generated by mechanical or electrical systems. In the course of this research a sophisticated piece of software has been developed. The program allows the construction of a hierarchy of delay embeddings from scalar and multi-valued time series. The embedded objects can be analysed graphically, and radial basis function maps can be fitted between them asynchronously, in parallel, on a multi-processor machine. In addition to a graphical user interface, the program can be driven by a batch mode command language, incorporating the concept of parallel and sequential instruction groups and enabling complex sequences of experiments to be performed in parallel in a resource-efficient manner.

A spectroscope study of some semiquinone radical ions

The effect of substituents on the value of the oxidation potential of quinones is reviewed and attempts to prepare substituted diphenoquinones with high oxidation potentials are reported. Attempts to characterise the mechanism of addition and substitution in diphenoquinones by identifying the products of the Thiele acetylation of diphenoquinone are reported. The reaction proved most efficient when the incoming acetylinium ion is directed by substituents in the diphenoquinone. A 1,8-addition to diphenoquinone is reported and characterised by isolating the products of the reaction between acetyl chloride and diphenoquinone, with perchloric acid as catalyst. The alternating linewidth effects observed in e.s.r.spectra are discussed and applied to account for such effects observed in the e.s.r.spectra of diphenosemiquinone anion and cation radicals. The spectra are analysed and the intramolecular processes producing these effects are discussed. A dianion diradical where intramolecular rotation about the 1 - 1' bond is restricted is produced by the oxidation of 2,2' ,4,4' -tetra hydroxybiphenyl. Previous studies of diphenosemiquinone anions are reviewed and alkylated diphenosemiquinone anion are produced by the reduction of the parent quinone with potassium hydroxide solution, the resulting radical being stabilised by the presence of pyridine. A qualitative interpretation of the solvent-ion effect in alkylated diphenosemiquinone anions is given. Diphanosemiquinone cation radicals are reviewed and previous studies are re-examined.