Evidence for a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as a-lipoic acid and alpha-tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with alpha-lipoic acid and alpha-tocopherol, alone or in combination, prior to incubation with H2O2 or staurosporine. alpha-lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H2O2 and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with a-lipoic acid and/or a-tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of BcI-2, Bax, HSP70 or pERK1/2 or pJNK. alpha-lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with alpha-lipoic acid and alpha-tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of alpha-lipoic acid as an antioxidant.

5,6-Dihydroxyindole-2-carboxylic acid: a first principles density functional study

We report first principles density functional calculations for 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and several oxidised forms. DHICA and 5,6-dihydroxyindole (DHI) are believed to be the basic building blocks of the eumelanins. Our results show that carboxylation has a significant effect on the physical properties of the molecules. In particular, the relative stabilities and the highest occupied molecular orbital-lowest unoccupied molecular orbital gaps (calculated with the DeltaSCF method) of the various redox forms are strongly affected. We predict that, in contrast to DHI, the density of unpaired electrons, and hence the ESR signal, in DHICA is negligibly small. (C) 2004 Elsevier B.V. All rights reserved.

The effect of high temperature on the insecticidal properties of Bt Cotton

Bt transgenic cotton has not shown the same level of resistance to bollworm in China, as in other major Bt cotton growing areas of the world. The objective of this study was to investigate the effects of high temperature on the CryIA insecticidal protein content and nitrogen metabolism, in the leaf of Bt transgenic cotton. The study was undertaken on two transgenic cotton cultivars, one conventional (Xinyang 822) and the other a hybrid (Kumian No. 1), during the 2001 and 2002 growing seasons at the Yangzhou University Farm, Yangzhou, China. In the 2001 study, potted cotton plants were exposed to 37 C for 24 h under glasshouse conditions at three growth stages peak square, peak flowering and peak boll developing periods. Based on the 2001 results, in 2002 the same two cultivars were exposed to the same temperature for 48 h at two growth stages-peak flowering and boll developing periods. The results of the study indicated that the insecticidal protein content of the leaf was not significantly affected by the stress during the square and flowering periods. However, exposure to high temperature for 24h during the boll period reduced the CryIA protein content by approximately 51% in the cultivar Kumian No 1, and 30% in Xinyang 822 in the 2001 study, and by approximately 73 and 63% for 48 h with the same cultivars, respectively, in the 2002 study. Glutamic-pyruvic transaminase (GPT) activity, total free amino acid and soluble protein content, and the activity of protease in the leaf, showed relatively little change in response to high temperature in the flowering period. However, exposure to high temperature in the boll period resulted in the following changes - a reduction of GPT activity, a sharp increase in free amino acid content, a significant decrease in soluble protein content, and significant increases in the activity of protease. The results suggest that high temperature may result in the degradation of soluble protein in the leaf, with a resulting decline in the level of the toxin CryIA. It is believed that this may be the cause of the reduced efficacy of Bt cotton in growing conditions in China, where temperatures during the boll period often reach 36-40° C. © 2004 Elsevier B.V All rights reserved.

Rhizodeposition and the enhanced mineralization of 2,4-dichlorophenoxyacetic acid in soil from the Trifolium pratense rhizosphere

Enhanced biodegradation of organic xenobiotic compounds in the rhizosphere is frequently recorded although the specific mechanisms are poorly understood. We have shown that the mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) is enhanced in soil collected from the rhizosphere of Trifolium pratense[e.g. maximum mineralization rate = 7.9 days(-1) and time at maximum rate (t(1)) = 16.7 days for 12-day-old T. pratense soil in comparison with 4.7 days(-1) and 25.4 days, respectively, for non-planted controls). The purpose of this study was to gain a better understanding of the plant-microbe interactions involved in rhizosphere-enhanced biodegradation by narrowing down the identity of the T. pratense rhizodeposit responsible for stimulating the microbial mineralization of 2,4-D. Specifically, we investigated the distribution of the stimulatory component(s) among rhizodeposit fractions (exudates or root debris) and the influence of soil properties and plant species on its production. Production of the stimulatory rhizodeposit was dependent on soil pH (e.g. t(1) for roots grown at pH 6.5 was significantly lower than for those grown at pH 4.4) but independent of soil inorganic N concentration. Most strikingly, the stimulatory rhizodeposit was only produced by T. pratense grown in non-sterile soil and was present in both exudates and root debris. Comparison of the effect of root debris from plant species (three each) from the classes monocotyledon, dicotyledon (non-legume) and dicotyledon (legume) revealed that legumes had by far the greatest positive impact on 2,4-D mineralization kinetics. We discuss the significance of these findings with respect to legume-rhizobia interactions in the rhizosphere.

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MITI, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pia ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 mu M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. (c) 2005 Elsevier Inc. All rights reserved.

Comparative Analysis of Structural and Morphological Properties of Large-Pore Periodic Mesoporous Organosilicas and Pure Silicas

A systematic study on the structural properties and external morphologies of large-pore mesoporous organosilicas synthesized using triblock copolymer EO20PO70EO20 as a template under low-acid conditions was carried out. By employing the characterization techniques of SAXS, FE-SEM, and physical adsorption of N-2 in combination with alpha(s)-plot method, the structural properties and external morphologies of large-pore mesoporous organosilicas were critically examined and compared with that of their pure-silica counterparts synthesized under similar conditions. It has been observed that unlike mesoporous pure silicas, the structural and morphological properties of mesoporous organosilicas are highly acid-sensitive. High-quality mesoporous organosilicas can only be obtained from synthesis gels with the molar ratios of HCl/H2O between 7.08 x 10(-4) and 6.33 x 10(-3), whereas mesoporous pure silicas with well-ordered structure can be obtained in a wider range of acid concentration. Simply by adjusting the HCl/H2O molar ratios, the micro-, meso-, and macroporosities of the organosilica materials can be finely tuned without obvious effect on their structural order. Such a behavior is closely related to their acid-controlled morphological evolution: from necklacelike fibers to cobweb-supported pearl-like particles and to nanosized particulates.

The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies-therm ally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 mu m. (C) 2005 Elsevier Ltd. All rights reserved.

Heterogeneity of physico-chemical properties in structured soils and its consequences

Structured soils are characterized by the presence of inter- and intra-aggregate pore systems and aggregates, which show varying chemical, physical, and biological properties depending on the aggregate type and land use system. How far these aspects also affect the ion exchange processes and to what extent the interaction between the carbon distribution and kind of organic substances affect the internal soil strength as well as hydraulic properties like wettability are still under discussion. Thus, the objective of this research was to clarify the effect of soil aggregation on physical and chemical properties of structured soils at two scales: homogenized material and single aggregates. Data obtained by sequentially peeling off soil aggregates layers revealed gradients in the chemical composition from the aggregate surface to the aggregate core. In aggregates from long term untreated forest soils we found lower amounts of carbon in the external layer, while in arable soils the differentiation was not pronounced. However, soil aggregates originating from these sites exhibited a higher concentration of microbial activity in the outer aggregate layer and declined towards the interior. Furthermore, soil depth and the vegetation type affected the wettability. Aggregate strength depended. on water suction and differences in tillage treatments.

Amino acid substitutions around the chromophore of the chromoprotein Rtms5 influence polypeptide cleavage

Extension of the conjugated pi-system of many all-protein chromophores with an acylimine bond is the basis for their red-shifted optical properties. The presence of this post-translational modification is evident in crystal structures of these proteins. Harsh denaturation of proteins containing an acylimine bond results in partial polypeptide cleavage. For the red fluorescent protein DsRed, the extent of cleavage is quantitative. However, this is not the case for the blue non-fluorescent chromoprotein Rtms5, even though all chromophores in tetrameric Rtms5 contain an acylimine bond. We have identified two positions around the chromophore of Rtms5 where substitutions can promote or suppress the extent of cleavage on harsh denaturation. We propose a model in which cleavage of Rtms5 is facilitated by a trans to cis isomerisation of the chromophore. (c) 2006 Elsevier Inc. All rights reserved.

Reversible active switching of the mechanical properties of a peptide film at a fluid-fluid interface

Designer peptides have recently been developed as building blocks for novel self-assembled materials with stimuli-responsive properties. To date, such materials have been based on self-assembly in bulk aqueous solution or at solid-fluid interfaces. We have designed a 21-residue peptide, AM1, as a stimuli-responsive surfactant that switches molecular architectures at a fluid-fluid interface in response to changes in bulk aqueous solution composition. In the presence of divalent zinc at neutral pH, the peptide forms a mechanically strong 'film state'. In the absence of metal ions or at acid pH, the peptide adsorbs to form a mobile 'detergent state'. The two interfacial states can be actively and reversibly switched. Switching between the two states by a change in pH or the addition of a chelating agent leads to rapid emulsion coalescence or foam collapse. This work introduces a new class of surfactants that offer an environmentally friendly approach to control the stability of interfaces in foams, emulsions and fluid-fluid interfaces more generally.

STAR: predicting recombination sites from amino acid sequence

Background: Designing novel proteins with site-directed recombination has enormous prospects. By locating effective recombination sites for swapping sequence parts, the probability that hybrid sequences have the desired properties is increased dramatically. The prohibitive requirements for applying current tools led us to investigate machine learning to assist in finding useful recombination sites from amino acid sequence alone. Results: We present STAR, Site Targeted Amino acid Recombination predictor, which produces a score indicating the structural disruption caused by recombination, for each position in an amino acid sequence. Example predictions contrasted with those of alternative tools, illustrate STAR'S utility to assist in determining useful recombination sites. Overall, the correlation coefficient between the output of the experimentally validated protein design algorithm SCHEMA and the prediction of STAR is very high (0.89). Conclusion: STAR allows the user to explore useful recombination sites in amino acid sequences with unknown structure and unknown evolutionary origin. The predictor service is available from http://pprowler.itee.uq.edu.au/star.

Tailoring surface properties to build colloidal diagnostic devices: Controlling interparticle associations

The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized. The interparticle association must withstand a harsh solvent environment multiple synthetic and washing procedures, and biological screening buffers. The encoded support beads were exposed to different solvents used for peptide synthesis, and different solutions used for biological screening including phosphate buffered saline (PBS), 2-[N-morpholino]ethane sulfonic acid (VIES) and a mixture of MES and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC). The number of reporters remaining adhered to the support bead was quantified after each step. The nature of the associations were explored and tested to optimize the efficiency of these phenomena. Results presented illustrate the influence of the surface functionality and polyelectrolyte modification of the reporters. These parameters were investigated through zeta potential and X-ray photoelectron spectroscopy.

The physical and chemical properties of eumelanin

In this article, we review the current state of knowledge concerning the physical and chemical properties of the eumelanin pigment. We examine properties related to its photoprotective functionality, and draw the crucial link between fundamental molecular structure and observable macroscopic behaviour. Where necessary, we also briefly review certain aspects of the pheomelanin literature to draw relevant comparison. A full understanding of melanin function, and indeed its role in retarding or promoting the disease state, can only be obtained through a full mapping of key structure-property relationships in the main pigment types. We are engaged in such an endeavor for the case of eumelanin.

A blank slate? Layer-by-layer deposition of hyaluronic acid and chitosan onto various surfaces

Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called blank slate: essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

A pore-lining glutamic acid in the olfactory cyclic nucleotide-gated channel controls external spermine block

Spermine is a potent, voltage-dependent blocker of the olfactory cyclic nucleotide-gated channel from both the intracellular and extracellular sides. However, its sites of action are unknown. This study investigated the external spermine binding site in the rat CNC alpha3 subunit. Neutralization of a glutamic acid residue (E342Q) in the P-loop region eliminated voltage-dependence of block by externally applied spermine. The charge-conservative E342D mutation had little effect on spermine block. Thus, E342 forms the binding site for externally applied spermine. However, spermine remained a potent voltage-independent blocker of the E342Q mutant channel, suggesting that the mutation either created a novel binding site outside the membrane electrical field or that it dramatically changed the properties of the existing pore site. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.