The antioxidant role of vitamin E in polyolefins

Polymers are subject to oxidation throughout their lifecycle. Antioxidants are generally incorporated in polymers to inhibit or minimise oxidative degradation. Hindered phenolic antioxidants are important stabilisers for polyolefins. However, hindered phenols undergo chemical transformations while performing their antioxidant function during processing and fabrication. In addition, antioxidants are subject to loss from polymers during processing, or subsequently in-service. Migration of antioxidants is a major concern in applications involving polymers in direct contact with food and human environment. This concern is compounded by the realisation that very little is known about the nature and the migration behaviour of antioxidant transformation products. In this work, the antioxidant role of the biological antioxidant -tocopherol (Vitamin E) , which is structurally similar to many synthetic hindered phenols, is investigated in low density polyethylene (LDPE) and polypropylene (PP). The melt stabilising effectiveness of -tocopherol (Toc) was found to be very high, higher than that of commercial hindered phenol antioxidants, such as Irganox 1076 (Irg 1076) and Irganox 1010 (Irg 1010), after multiple extrusions, especially at very low concentrations. The high antioxidant activity of Toc was shown to be due, at least in part, to the formation of transformation products during processing. The main products formed are stereoisomers of dimers and trimers, as well as aldehydes and a quinone - the relative concentration of each was shown to depend on the processing severity, the initial antioxidant concentration and oxygen availability. These transformation products are shown to impart better, similar or lower melt stability to the polymer than the parent antioxidant. The nature of the products formed from Toc during processing was compared with those formed during processing of Irg 1076 and Irg 1010 with LDPE and a mechanism for the melt stabilisation of Toc was proposed and compared with the stabilisation mechanisms of the synthetic antioxidants Irg 1076 and Irg 1010.

Investigation and development of techniques for the characterisation of the synthetic/biological interface

The purpose of this study is to increase our knowledge of the nature of the surface properties of polymeric materials and improve our understanding of how these factors influence the deposition of proteins to form a reactive biological/synthetic interface. A number of surface analytical techniques were identified as being of potential benefit to this investigation and included in a multidisciplinary research program. Cell adhesion in culture was the primary biological sensor of surface properties, and it showed that the cell response to different materials can be modified by adhesion promoting protein layers: cell adhesion is a protein-mediated event. A range of surface rugosity can be produced on polystyrene, and the results presented here show that surface rugosity does not play a major role in determining a material's cell adhesiveness. Contact angle measurements showed that surface energy (specifically the polar fraction) is important in promoting cell spreading on surfaces. The immunogold labelling technique indicated that there were small, but noticeable differences, between the distribution of proteins on a range of surfaces. This study has shown that surface analysis techniques have different sensitivities in terms of detection limits and depth probed, and these are important in determining the usefulness of the information obtained. The techniques provide information on differing aspects of the biological/synthetic interface, and the consequence of this is that a range of techniques is needed in any full study of such a complex field as the biomaterials area.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

The role of audiometry in the prevention of occupational deafness

The research set out to test three main hypotheses derived from a summary of literature relevant to the use of audiometry in industry. These hypotheses were: (1) performing audiometry increases the probability that hearing protectors, once issued, will be worn; (2) audiometry is considered by workers to be evidence of their employer's concern for their welfare; (3) audiometry is associated with common law claims by workers against employers for alleged occupational deafness. Six subsidiary hypotheses were also developed. Four methods of data collection were used: (1) attitude questionnaires were administered to samples of workers drawn from an industrial company performing audiometry and two industrial companies not performing audiometry; (2) a postal questionnaire was sent out to industrial medical officers; (3) surveys were undertaken to assess the proportion of the workforce in each of eight industrial companies that was wearing personal hearing protectors that had been provided; (4) structured interviews were carried out with relevant management level personnel in each of five industrial companies. Factor analysis was the main statistical analytic technique used. The data supported all three main hypotheses. Audiometry was also examined as an example of medical screening procedure. It was argued that the validation of medical screening procedures requires the satisfaction of attitudinal or motivational validation criteria in addition to the biological and economic criteria currently used. It was concluded that industrial audiometry failed to satisfy such attitudinal or motivational criteria and so should not be part of a programme of screening for occupational deafness. It was also concluded that industrial audiometry may be useful in creating awareness, amongst workers, of occupational deafness. It was argued that the only profitable approach to investigating the role of audiometry in preventing occupational deafness is to study the attitudes and perceptions of everyone involved.

Mapping the yeast host cell response to recombinant membrane protein production:relieving the biological bottlenecks

Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production.

Assessment of physical and hydrological properties of biological soil crusts using x-ray microtomography and modelling

Biological soil crusts (BSCs) are formed by aggregates of soil particles and communities of microbial organisms and are common in all drylands. The role of BSCs on infiltration remains uncertain due to the lack of data on their role in affecting soil physical properties such as porosity and structure. Quantitative assessment of these properties is primarily hindered by the fragile nature of the crusts. Here we show how the use of a combination of non-destructive imaging X-ray microtomography (XMT) and Lattice Boltzmann method (LBM) enables quantification of key soil physical parameters and the modeling of water flow through BSCs samples from Kalahari Sands, Botswana. We quantify porosity and flow changes as a result of mechanical disturbance of such a fragile cyanobacteria-dominated crust. Results show significant variations in porosity between different types of crusts and how they affect the flow and that disturbance of a cyanobacteria-dominated crust results in the breakdown of larger pore spaces and reduces flow rates through the surface layer. We conclude that the XMT–LBM approach is well suited for study of fragile surface crust samples where physical and hydraulic properties cannot be easily quantified using conventional methods.

Role of fluctuations in membrane models:thermal versus nonthermal

We study the comparative importance of thermal to nonthermal fluctuations for membrane-based models in the linear regime. Our results, both in 1+1 and 2+1 dimensions, suggest that nonthermal fluctuations dominate thermal ones only when the relaxation time τ is large. For moderate to small values of τ, the dynamics is defined by a competition between these two forces. The results are expected to act as a quantitative benchmark for biological modeling in systems involving cytoskeletal and other nonthermal fluctuations. © 2011 American Physical Society.

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.

Learning from mother nature:exploiting a biological antioxidant for the melt stabilisation of polymers

An overview of the antioxidant role of the biologically active form of vitamin E, α-tocopherol, in polyolefins is discussed. The effect of the vitamin antioxidant on the melt and colour stability of polyethylene (PE) and polypropylene (PP) is highlighted. It is shown that tocopherol is a highly effective antioxidant that results in superior melt stabilisation of polyolefins particularly when used at much lower concentration than that needed for conventional synthetic hindered phenol processing stabilisers. As with other hindered phenols,α-tocopherol imparts also some colour to the polymer but this is shown to be reduced drastically in the presence of other antioxidants, such as phosphites, or other additives, such as polyhydric alcohols.

Temporal dynamics in an immunological synapse:role of thermal fluctuations in signaling

The article analyzes the contribution of stochastic thermal fluctuations in the attachment times of the immature T-cell receptor TCR: peptide-major-histocompatibility-complex pMHC immunological synapse bond. The key question addressed here is the following: how does a synapse bond remain stabilized in the presence of high-frequency thermal noise that potentially equates to a strong detaching force? Focusing on the average time persistence of an immature synapse, we show that the high-frequency nodes accompanying large fluctuations are counterbalanced by low-frequency nodes that evolve over longer time periods, eventually leading to signaling of the immunological synapse bond primarily decided by nodes of the latter type. Our analysis shows that such a counterintuitive behavior could be easily explained from the fact that the survival probability distribution is governed by two distinct phases, corresponding to two separate time exponents, for the two different time regimes. The relatively shorter timescales correspond to the cohesion:adhesion induced immature bond formation whereas the larger time reciprocates the association:dissociation regime leading to TCR:pMHC signaling. From an estimate of the bond survival probability, we show that, at shorter timescales, this probability PΔ(τ) scales with time τ as a universal function of a rescaled noise amplitude DΔ2, such that PΔ(τ)∼τ-(ΔD+12),Δ being the distance from the mean intermembrane (T cell:Antigen Presenting Cell) separation distance. The crossover from this shorter to a longer time regime leads to a universality in the dynamics, at which point the survival probability shows a different power-law scaling compared to the one at shorter timescales. In biological terms, such a crossover indicates that the TCR:pMHC bond has a survival probability with a slower decay rate than the longer LFA-1:ICAM-1 bond justifying its stability.

Why Leaves Turn Red in Autumn. The Role of Anthocyanins in Senescing Leaves of Red-Osier Dogwood

Why the leaves of many woody species accumulate anthocyanins prior to being shed has long puzzled biologists because it is unclear what effects anthocyanins may have on leaf function. Here, we provide evidence for red-osier dogwood (Cornus stolonifera) that anthocyanins form a pigment layer in the palisade mesophyll layer that decreases light capture by chloroplasts. Measurements of leaf absorbance demonstrated that red-senescing leaves absorbed more light of blue-green to orange wavelengths (495–644 nm) compared with yellow-senescing leaves. Using chlorophyll a fluorescence measurements, we observed that maximum photosystem II (PSII) photon yield of red-senescing leaves recovered from a high-light stress treatment, whereas yellow-senescing leaves failed to recover after 6 h of dark adaptation, which suggests photo-oxidative damage. Because no differences were observed in light response curves of effective PSII photon yield for red- and yellow-senescing leaves, differences between red- and yellow-senescing cannot be explained by differences in the capacities for photochemical and non-photochemical light energy dissipation. A role of anthocyanins as screening pigments was explored further by measuring the responses PSII photon yield to blue light, which is preferentially absorbed by anthocyanins, versus red light, which is poorly absorbed. We found that dark-adapted PSII photon yield of red-senescing leaves recovered rapidly following illumination with blue light. However, red light induced a similar, prolonged decrease in PSII photon yield in both red- and yellow-senescing leaves. We suggest that optical masking of chlorophyll by anthocyanins reduces risk of photo-oxidative damage to leaf cells as they senesce, which otherwise may lower the efficiency of nutrient retrieval from senescing autumn leaves.

Biological activities of compounds from Piper species

It is well established that secondary metabolites play an important role in plant chemical defense. In an effort to find natural herbicides research on plant growth regulatory activity of secondary metabolites has received more and more attention recently. The genus Piper has been an important source for useful secondary metabolites.^ Crude extracts from Piper species inhibited gram-positive bacteria and higher plant growth under laboratory conditions. Bioassay-guided isolation and purification lead to the identification of safrole, a phenylpropene, as the responsible agent for the inhibitory activity. Safrole was found to leach from naturally fallen leaves with water. Mechanisms of plant growth inhibition by safrole were investigated. Disassociation of cell membrane from cell walls was determined to be a major cause.^ Phenylpropenes structurally similar to safrole had similar phytogrowth inhibitory activity. It is postulated that phenylpropanoids are an important group of naturally occurring secondary metabolites in plant-plant interactions. ^

Interactions of different arsenic species with thols: Chemical and biological implications

Arsenic has been classified as a group I carcinogen. It has been ranked number one in the CERCLA priority list of hazardous substances due to its frequency, toxicity and potential for human exposure. Paradoxically, arsenic has been employed as a successful chemotherapeutic agent for acute promyelocytic leukemia and has found some success in multiple myeloma. Since arsenic toxicity and efficacy is species dependent, a speciation method, based on the complementary use of reverse phase and cation exchange chromatography, was developed. Inductively coupled plasma mass spectrometer (ICP-MS), as an element specific detector, and electrospray ionization mass spectrometer (ESI-MS), as a molecule specific detector, were employed. Low detection limits in the µg. L−1 range on the ICP-MS and mg. L−1 range on the ESI-MS were obtained. The developed methods were validated against each other through the use of a Deming plot. With the developed speciation method, the effects of both pH on the stability of As species and reduced glutathione (GSH) concentration on the formation and stability of arsenic glutathione complexes were studied. To identify arsenicals in multiple myeloma (MM) cell lines post arsenic trioxide (ATO) and darinaparsin (DAR) incubation, an extraction method based on the use of ultrasonic probe was developed. Extraction tools and solvents were evaluated and the effect of GSH concentration on the quantitation of arsenic glutathione (As-GSH) complexes in MM cell extracts was studied. The developed method was employed for the identification of metabolites in DAR incubated cell lines where the effect of extraction pH, DAR incubation concentration and incubation time on the relative distribution of the As metabolites was assessed. A new arsenic species, dimethyarsinothioyl glutathione (DMMTA V-GS), a pentavalent thiolated arsenical, was identified in the cell extracts through the use of liquid chromatography tandem mass spectrometry. The formation of the new metabolite in the extracts was dependent on the decomposition of s-dimethylarsino glutathione (DMA(GS)). These results have major implications in both the medical and toxicological fields of As because they involve the metabolism of a chemotherapeutic agent and the role sulfur compounds play in this mechanism.

Role of Pseudomonas aeruginosa mifSR Two-Component System in Antibiotic Resistance and Biofilm Formation

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments. It is one of the leading causes of morbidity and mortality in cystic fibrosis patients, and one of the main sources of nosocomial infections in the United States. One of the most prominent features of this pathogen is its wide resistance to antibiotics. P. aeruginosa employs a variety of mechanisms including efflux pumps and the expression of B-lactamases to overcome antibiotic treatment. Two chromosomally encoded lactamases, ampC and poxB, have been identified in P. aeruginosa. Sequence analyses have shown the presence of a two-component system (TCS) called MifSR (MifS-Sensor and MifR-Response Regulator), immediately upstream of the poxAB operon. It is hypothesized that the MifSR TCS is involved in B-lactam resistance via the regulation of poxB. Recently, the response regulator MifR has been reported to play a crucial role in biofilm formation, a major characteristic of chronic infections and increased antibiotic resistance. In this study, mifR and mifSR deletion mutants were constructed, and compared to the wild type parent strain PAOl for differences in growth and B-lactam sensitivity. Results obtained thus far indicate that mifR and mifSR are not essential for growth, and do not confer B-lactam resistance under the conditions tested. This study is significant because biofilm formation and antibiotic resistance are two hallmarks of P. aeruginosa infections, and finding a link between these two may lead to the development of improved treatment strategies.

Role of Pseudomonas aeruginosa PA4157in Quorum Sensing and Iron Transport Regulation

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is a pnmary contributing factor responsible for the morbidity and mortality in patients with cystic fibrosis. One of the trademarks of P. aeruginosa is its ability to resist antibiotics. P. aeruginosa does so in part through the LysR-type transcription factor, AmpR. To identify additional members of the AmpR regulon, a new algorithm called iterative enhancement of motifs was used to identify putative AmpR binding sites upstream of open reading frames in the P. aeruginosa genome. The surprising primary hit of this analysis was the promoter of an uncharacterized open reading frame, P A 415 7. P A 415 7 is located upstream ofthefep operon, which is known to be involved in iron acquisition. PA4157 shares high homology to the IclR family of transcriptional regulators which are known to regulate quorum sensing (QS), an elaborate cell-cell communication signaling system that uses quoromones. We postulated two hypotheses: 1) AmpR regulation of QS genes is mediated by PA4157, and 2) PA4157 may be involved in iron acquisition. To address the role of P A 415 7 we generated an in-frame chromosomal deletion of P A 415 7 in P. aeruginosa PA01 (PA0 PA4157). We compared PA0 PA4157 with its parent strain P A0 1 for its ability to produce quoromones using Chromobacterium violaceum as an indicator strain and LasA proteases using Staphylococcus aureus. We also tested its role in virulence using a Caenorhabditis elegans killing assay. Growth in iron-deficient media was also examined to determine if P A4157 has a potential role in iron uptake regulation. Our preliminary results suggest that P A 415 7 is not involved in quorum sensing regulation but does seem to exert a negative regulatory effect on iron uptake in P. aeruginosa P A0 1.