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

myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate

1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).

Fatigue crack growth behaviour in molten-metal processed SiC particle-reinforced aluminium alloys

Fatigue crack initiation and subsequent short crack growth behaviour of 2014-5wt%SiC aluminium alloy composites has been examined in 4-point bend loading using smooth bar specimens. The growth rates of long fatigue cracks have also been measured at different stress ratios using pre-cracked specimens. The distributions of SiC particles and of coarse constituent particles in the matrix (which arise as a result of the molten-metal processing and relatively slow cooling rate) have been investigated. Preferential crack initiation sites were found to be SiC-matrix interfaces, SiC particles associated with constituent particles and the coarse constituent particles themselves. For microstructurally short cracks the dispersed SiC particles also act as temporary crack arresters. In the long crack growth tests, higher fatigue crack growth rates were obtained than for monolithic alloys. This effect is attributed to the contribution of void formation, due to the decohesion of SiC particles, to the fatigue crack growth process in the composite. Above crack depths of about 200 μm 'short' crack growth rates were in good agreement with the long crack data, showing a Pris exponent, m = 4 in both cases. For the long crack and short crack growth tests little effect of specimen orientation and grain size was observed on fatigue crack growth rates, but, specimen orientation affected the toughness. No effect of stress ratio in the range R = 0.2-0.5 was seen for long crack data in the Paris region.

Optimization and characterization of femtosecond laser inscribed in-fiber microchannels for liquid sensing

In-fiber microchannels were fabricated directly in standard single mode fiber using the femtosecond laser inscribe and etch technique. This method of creating in-fiber microchannels offers great versatility since it allows complex three dimensional structures to be inscribed and then etched with hydrofluoric acid. Four in-fiber microchannel designs were experimentally investigated using this technique. Device characteristics were evaluated through monitoring the spectral change while inserting index matching oils into each microchannel - a R.I. sensitivity up to 1.55 dB/RIU was achieved. Furthermore, a simple Fabry-Pérot based refractometer with a R.I. sensitivity of 2.75 nm/RIU was also demonstrated. © 2014 SPIE.

Optimization and characterization of femtosecond laser inscribed in-fiber microchannels for liquid sensing

In-fiber microchannels were fabricated directly in standard single mode fiber using the femtosecond laser inscribe and etch technique. This method of creating in-fiber microchannels offers great versatility, since it allows complex three-dimensional structures to be inscribed and then preferentially etched with hydrofluoric acid. In addition, inscription does not require a photosensitive fiber; the modification is induced through nonlinear processes triggered by an ultrashort laser pulse. Four in-fiber microchannel designs were experimentally investigated using this technique - microhole, microslot channel along the core, microslot channel perpendicular to the core and helical channel around the core. Each device design was evaluated through monitoring the optical spectral change while inserting a range of index matching oils into each microchannel; an R.I. sensitivity up to 1.55 dB/RIU was achieved in these initial tests. Furthermore, an all femtosecond laser inscribed Fabry-Pérot-based refractometer with an R.I. sensitivity of 2.75 nm/RIU was also demonstrated. The Fabry-Pérot refractometer was formed by positioning a microchannel between two femtosecond laser inscribed point-by-point fiber Bragg gratings.

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Une histoire du sens:la sémantique linguistique depuis Bréal

Il y a plus d'un siècle, Michel Bréal institua l'analyse du sens dans le langage. À partir de là, le développement de la sémantique repose sur des paradigmes, qui constituent de véritables métaphores de la nature générale du langage. Cet ouvrage montre que, hors des modèles logiques, la sémantique s'est historiquement développée à travers quatre paradigmes. Le sens linguistique relève de la vie mentale des individus pour le paradigme psychologiste qui réunit Bréal, Brunot, Damourette et Pichon, Guillaume et Bally. La nature incertaine de cette vie mentale porte le structuralisme de Saussure, Greimas, Rastier et Wierzbicka à envisager le sens comme dérivant de l'équilibre des oppositions internes à la langue. Les déterminismes de cette organisation sont associés aux conditions de sa mise en oeuvre pour le paradigme de l'énonciation élaboré par Benveniste, Ducrot, Anscombre, Nølke et Culioli. Ils sont pour le cognitivisme de Kleiber, de Cadiot, de Fuchs et pour la Grammaire cognitive dérivés d'un esprit au fonctionnement mécaniste. Permettant de délimiter la forme et l'évolution des paradigmes scientifiques, l'analyse des théories donne à voir l'état actuel de la discipline et les questions qu'elle sera appelée à résoudre.

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1α(βS*, δS*), 2α, 6α, 8β-(R*), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1∝(ßS*,dS*),2∝,6a∝,8ß-(R*),8a∝]]-1,2,6,7,8,8a-hexahydro-ß,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 ± 1.9% versus 16.6 ± 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 ± 2.9% versus 23.4 ± 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC50, 6.37 ± 0.37) compared with both vehicle-treated (-logEC50, 5.81 ± 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 ± 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 ± 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.