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

Time controlled photo-oxidation of polyolefins by polymer bound additives

A variety of iron compounds containing vinyl or thiol functional groups (used as photoactivators) have been synthesised and some of these were successfully bound to both polyethylene and polypropylene backbones during processing in the presence of peroxide and interlinking agent. Concentrates (masterbatches) of the photoactivators in PP and PE were prepared and the pro-oxidant effect of the diluted masterbatches in absence and presence of an antioxidant was evaluated. An antioxidant photoactivator (FeDNC ) was found to sensitise the photoactivity of pro-oxidants (Metone A / Metone M) whereas an antioxidant (ZnDNC) was found to stabilise the polymer (PP and PE) containing both of these combinations. It was observed that the lower concentration of FeDNC sensitises the stability of the polymer containing very small concentration of NiDNC whereas higher concentration of FeDNC stabilises the polymer (LDPE) containing same amount of NiDNC compared to FeDNC alone. The photostability of unstabilised PP containing FeAc could be varied by varying the concentration of ZnDEC. Both the induction period and the UV - life time of the polymer increased by increasing concentration of ZnDEC. It is suggested that ligand exchange reaction may take place between FeAc and ZnDNC. A polymer bound UV stabiliser (HAEB) and a thermal stabiliser (DBBA) were used with a non extractable photoactivator (FeAc) in PP. Small concentrations of the stabilisers (HAEB and DBBA) in combination with the photoactivator (FeAc) sensitise the polymer. The antioxidant present in commercial polymer (LDPE and PP) was found to be of a hindered phenol type, which was found to antagonise with ZnDNC when used in combination with the photoactivators.

The resistance of intra-amoebal grown legionella pneumophila

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellulary in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), 5-chloro-N-methylisothiazolone (CMIT) and tetradecyltrimethyl ammonium bromide (TTAB). Susceptibilities were also determined for L.pneumophila grown under nutrient sufficient and iron-, nitrogen- and phosphate-depleted conditions, in a chemically defined medium. BIT was relatively ineffective against cells grown under iron-depletion; in contrast iron-depleted conditions increased the susceptibilities of cells to PHMB, TTAB and CMIT. Cells grown under phosphate-depletion showed a marked increase in sensitivity towards all the biocides. Conversely, the activities of all four biocides were greatly reduced against L.pneumophila grown in amoebae. To study the physiological basis for the increased resistance of intra-amoebal grown legionella, the surface properties of the cells were examined by studying outer membrane proteins (OMs), lipopolysaccharides and cellular fatty acids. Intra-amoebal grown legionella were found to differ in several respects compared to cells grown in vitro; they contained a novel 15-kDal OM protein and a monosaturated straight-chain fatty acid (18:19). These compounds were also found in abundant quantities in the host amoeba. Intra-amoebal grown legionella contained more LPS bands than did in vitro grown organisms and were less susceptible to protease K digestion. Cells grown under phosphate depletion were markedly sensitive to protease K digestion and contained lower levels of LPS. Immunoblot analysis of intra-amoebal grown legionella with anti-acanthamoebal serum revealed that both the surface of the bacteria and sarkosyl extracted OMs contained amoebal proteins. These findings suggest that the 15-kDal OM protein is likely to be of amoebal origin and binds tightly to the OM of the bacterium. It is proposed that disruption of amoebal membranes, as a result of intra-amoebal infection liberates macromolecules, including a 15-kDal polypeptide, a major constituent of the membrane, which associates closely with the surface of the legionellae. Thus L.pneumophila which have extraneous membrane material bound to their surface may respond differently to biocide inactivation, as these macromolecules may act as a penetration barrier to such agents. This phenomenon could contribute to the recalcitrance of legionellae in water systems.

Linking gene expression in the intestine to production of gametes through the phosphate transporter PITR-1 in Caenorhabditis elegans

Inorganic phosphate is an essential mineral for both prokaryotic and eukaryotic cell metabolism and structure. Its uptake into the cell is mediated by membrane bound transporters and coupled to Na+ transport. Mammalian sodium-dependent Pi co-transporters have been grouped into three families NaPi-I, NaPi-II, and NaPi-III. Despite being discovered more than 2 decades ago, very little is known about requirements for NaPi-III transporters in vivo, in the context of intact animal models. Here we find that impaired function of the C. elegans NaPi-III transporter, pitr-1, results in decreased brood size and dramatically increased expression of vitellogenin by the worm intestine. Unexpectedly, we found that the effects of pitr-1 mutation on vitellogenin expression in the intestine could only be rescued by expression of pitr-1 in the germline, and not by expression of pitr-1 in the intestine itself. Our results indicate the existence of a signal from the germline that regulates gene expression in the intestine, perhaps linking nutrient export from the intestine to production of gametes by the germline.