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).

Lipoidal species in ocular spoilation processes

Tear component deposition onto contact lenses is termed `spoilation' and occurs due to the interaction of synthetic polymers with their biological fluid environment. Spoilation phenomena alter the physico-chemical properties of hydrophilic contact lenses, diminishing the optical properties of the lens; causing discomfort and complications for the wearer. Eventually these alterations render the lens unwearable. The primary aim of this interdisciplinary study was to develop analytical techniques capable of analysing the minute quantities of biological deposition involved, in particular the lipid fraction. Prior to this work such techniques were unavailable for single contact lenses. It is envisaged that these investigations will further the understanding of this biological interfacial conversion. Two main analytical techniques were developed: a high performance liquid chromatography (HPLC) technique and fluorescence spectrofluorimetry. The HPLC method allows analysis of a single contact lens and provided previously unavailable valuable information about variations in the lipid profiles of deposited contact lenses and patient tear films. Fluorescence spectrophotofluorimetry is a sensitive non-destructive technique for observing changes in the fluorescence intensity of biological components on contact lenses. The progression and deposition of tear materials can be monitored and assessed for both in vivo and in vitro spoiled lenses using this technique. An improved in vitro model which is comparable to tears and chemically mimics ocular spoilation was also developed. This model allows the controlled study of extrinsic factors and hydrogel compositions. These studies show that unsaturated tear lipids, probably unsaturated fatty acids, are involved in the interfacial conversion of hydrogel lenses, rendering them incompatible with the ocular microenvironment. Lipid interaction with the lens surface then facilitates secondary deposition of other tear components. Interaction, exchange and immobilisation (by polymerisation) of the lipid layer appears to occur before the final and rapid growth of more complex, insoluble discrete deposits, sometimes called `white spots'.

Ecological studies on:(a) the use of colonisation samplers in relation to biological surveillance of river water quality (b) the requirements of freshwater gastropoda

Some of the factors affecting colonisation of a colonisation sampler, the Standard Aufwuchs Unit (S. Auf. U.) were investigated, namely immersion period, whether anchored on the bottom or suspended, and the influence of riffles. It was concluded that a four-week immersion period was best. S. Auf. U. anchored on the bottom collected both more taxa and individuals than suspended ones. Fewer taxa but more individuals colonised S. Auf. U. in the potamon zone compared to the rhithron zone with a consequent reduction in the values of pollution indexes and diversity. It was concluded that a completely different scoring system was necessary for lowland rivers. Macroinvertebrates colonising S. Auf. U. in simulated streams, lowland rivers and the R. Churnet reflected water quality. A variety of pollution and diversity indexes were applied to results from lowland river sites. Instead of these, it was recommended that an abbreviated species - relative abundance list be used to summarise biological data for use in lowland river surveillance. An intensive study of gastropod populations was made in simulated streams. Lynnaea peregra increased in abundance whereas Potamopyrgas jenkinsi decreased with increasing sewage effluent concentration. No clear-cut differences in reproduction were observed. The presence/absence of eight gastropod taxa was compared with concentrations of various pollutants in lowland rivers. On the basis of all field work it appeared that ammonia, nitrite, copper and zinc were the toxicants most likely to be detrimental to gastropods and that P. jenkinsi and Theodoxus fluviatilis were the least tolerant taxa. 96h acute toxicity tests of P. jenkinsi using ammonia and copper were carried out in a flow-through system after a variety of static range finding tests. P. jenkinsi was intolerant to both toxicants compared to reports on other taxa and the results suggested that these toxicants would affect distribution of this species in the field.

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

Reporter ion-based mass spectrometry approaches for the detection of non-enzymatic protein modifications in biological samples

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

Reactions of copper macrocycles with antioxidants and HOCl:potential for biological redox sensing

A series of simple copper N(2)S(2) macrocycles were examined for their potential as biological redox sensors, following previous characterization of their redox potentials and crystal structures. The divalent species were reduced by glutathione or ascorbate at a biologically relevant pH in aqueous buffer. A less efficient reduction was also achieved by vitamin E in DMSO. Oxidation of the corresponding univalent copper species by sodium hypochlorite resulted in only partial (~65 %) recovery of the divalent form. This was concluded to be due to competition between metal oxidation and ligand oxidation, which is believed to contribute to macrocycle demetallation. Electrospray mass spectrometry confirmed that ligand oxidation had occurred. Moreover, the macrocyclic complexes could be demetallated by incubation with EDTA and bovine serum albumin, demonstrating that they would be inappropriate for use in biological systems. The susceptibility to oxidation and demetallation was hypothesized to be due to oxidation of the secondary amines. Consequently these were modified to incorporate additional oxygen donor atoms. This modification led to greater resistance to demetallation and ligand oxidation, providing a better platform for further development of copper macrocycles as redox sensors for use in biological systems.

Chemical specificity in REDOX-responsive materials:the diverse effects of different Reactive Oxygen Species (ROS) on polysulfide nanoparticles

REDOX responsive (nano)materials typically exhibit chemical changes in response to the presence and concentration of oxidants/reductants. Due to the complexity of biological environments, it is critical to ascertain whether the chemical response may depend on the chemical details of the stimulus, in addition to its REDOX potential, and whether chemically different responses can determine a different overall performance of the material. Here, we have used oxidation-sensitive materials, although these considerations can be extended also to reducible ones. In particular, we have used poly(propylene sulfide) (PPS) nanoparticles coated with a PEGylated emulsifier (Pluronic F127); inter alia, we here present also an improved preparative method. The nanoparticles were exposed to two Reactive Oxygen Species (ROS) typically encountered in inflammatory reactions, hydrogen peroxide (H2O2) and hypochlorite (ClO−); their response was evaluated with a variety of techniques, including diffusion NMR spectroscopy that allowed to separately characterize the chemically different colloidal species produced. The two oxidants triggered a different chemical response: H2O2 converted sulfides to sulfoxides, while ClO− partially oxidized them further to sulfones. The different chemistry correlated to a different material response: H2O2 increased the polarity of the nanoparticles, causing them to swell in water and to release the surface PEGylated emulsifier; the uncoated oxidized particles still exhibited very low toxicity. On the contrary, ClO− rapidly converted the nanoparticles into water-soluble, depolymerized fragments with a significantly higher toxicity. The take-home message is that it is more correct to discuss ‘smart’ materials in terms of an environmentally specific response to (REDOX) stimuli. Far from being a problem, this could open the way to more sophisticated and precisely targeted applications.

Observing population changes of thrips (Thysanoptera) species damaging forced pepper and their natural enemies

By the strengthening of environmental protection and food safety efforts in Hungary, integrated and especially biological pest control methods should increasingly put forward, for which a solid knowledge on the life course and efficiency of natural enemies applied against certain pests is necessary. Pepper has distinguished significance in domestic vegetable forcing, and the profitability of production is determined primarily by the efficiency of the control of thrips pests. This is why we attached great importance to study what results may be expected by introducing arthropod predators (Amblyseius cucumeris, Orius laevigatus) to control thrips species under domestic conditions on rock wool in a long vegetation period pepper culture. We also liked to find out what kind of role the cultivars play in the change of phytophagous and zoophagous populations. The A. cucumeris predatory mite introduced in late January proved to be effective in controlling thrips pests until mid-April. Despite repeated introductions, the predatory bug O. laevigatus (Heteroptera: Anthocoridae) did not proliferate. Among the three pepper cultivars (Hó, Keceli, Titán) grown at Ráckeve, thrips species proliferated in the highest number on cultivar ‘Hó’, while the population of predatory mites was lowest on the cultivar ‘Titán’, compared to the other two cultivars.

Biological Activity of Seed Proteins in Malesian Legumes

In a study of the effects on animals of seed protein extracts of 15 Malesian members of the Leguminosae (including 11 rain forest tree species), most of the taxa agglutinated red blood cells, induced mitosis, and inhibited amylases. These results are consistent with the hypothesis that these proteins interact with other organisms, most probably in defense mechanisms against predation by animals. The functions of these proteins are most profitably studied in rain forest environments where their activity is so marked, and where biological interactions are particularly important.