Social and Behavioral Sciences Commons^™

The Two-Faced Nature Of Small Heat Shock Proteins: Amyloid Assembly And The Inhibition Of Fibril Formation. Relevance To Disease States, Heath W. Ecroyd, S Meehan, John A. Carver

Heath Ecroyd

The ability of small heat-shock proteins (sHsps) such as alphaB-crystallin to inhibit the amorphous (disordered) aggregation of varied target proteins in a chaperone-like manner has been well described. The mechanistic details of this action are not understood. Amyloid fibril formation is an alternative off-folding pathway that leads to highly ordered beta-sheet-containing aggregates. Amyloid fibril formation is associated with a broad range of protein conformational diseases such as Alzhiemer's, Parkinson's and Huntington's and sHsp expression is elevated in the protein deposits that are characteristic of these disease states. The ability of sHsps to prevent fibril formation has been less well characterised. …

Molecular Dynamics Analysis Of Apolipoprotein-D - Lipid Hydroperoxide Interactions: Mechanism For Selective Oxidation Of Met-93, Aaron J. Oakley, Surabhi Bhatia, Heath Ecroyd, Brett Garner

Heath Ecroyd

Background: Recent studies suggest reduction of radical-propagating fatty acid hydroperoxides to inert hydroxides by interaction with apolipoprotein-D (apoD) Met93 may represent an antioxidant function for apoD. The nature and structural consequences of this selective interaction are unknown. Methodology/Principal Findings: Herein we used molecular dynamics (MD) analysis to address these issues. Longtimescale simulations of apoD suggest lipid molecules are bound flexibly, with the molecules free to explore multiple conformations in a binding site at the entrance to the classical lipocalin ligand-binding pocket. Models of 5s- 12s- and 15s hydroperoxyeicosatetraenoic acids were created and the lipids found to wrap around Met93 thus …

Binding Of The Molecular Chaperone Alphab-Crystallin To Abeta Amyloid Fibrils Inhibits Fibril Elongation, Sarah L. Shammas, Christopher A. Waudby, Shuyu Wang, Alexander K. Buell, Tuomas P. Knowles, Heath W. Ecroyd, Mark E. Welland, John A. Carver, Christopher M. Dobson, Sarah Meehan

Heath Ecroyd

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ42 fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ42. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect …

Identification Of Phospholipids In Human Meibum By Nano-Electrospray Ionisation Tandem Mass Spectrometry, Jennifer T. Saville, Zhenjun Zhao, Mark Dp Willcox, Manjula A. Ariyavidana, Stephen J. Blanksby, Todd W. Mitchell

Stephen Blanksby

Meibum is believed to be the major source of tear !lm lipids, which are vital in the prevention of excessevaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established.While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometricstudies have not detected them. In this study we use electrospray ionisation tandem massspectrometry to investigate the presence of phospholipids in meibum and provide comparison to thephospholipid pro!le of tears.Lipids were extracted from human meibum and tear samples using standard biphasic methods andanalysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. …

Ozone Induced Dissociation: Elucidation Of Double Bond Position Within Mass-Selected Lipid Ions, Michael C. Thomas, Todd W. Mitchell, David G. Harman, Jane M. Deeley, Jessica R. Nealon, Stephen J. Blanksby

Stephen Blanksby

Ions formed from lipids during electrospray ionization of crude lipid extracts have been mass-selected within a quadrupole linear ion trap mass spectrometer and allowed to react with ozone vapor. Gas-phase ion−molecule reactions between unsaturated lipid ions and ozone are found to yield two primary product ions for each carbon−carbon double bond within the molecule. The mass-to-charge ratios of these chemically induced fragments are diagnostic of the position of unsaturation within the precursor ion. This novel analytical technique, dubbed ozone-induced dissociation (OzID), can be applied both in series and in parallel with conventional collision-induced dissociation (CID) to provide near-complete structural assignment …

Understanding The Α-Crystallin Cell Membrane Conjunction, Shih-Ping Su, Jason D. Mcarthur, Michael G. Friedrich, Roger J. W Truscott, J. A. Aquilina

J. A. Aquilina

PURPOSE. It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. METHODS. Fibre cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea and alkaline solutions. Protein constituents of the respective membrane isolates …

Tandem Mass Spectrometry Reveals The Quaternary Organization Of Macromolecular Assemblies, J L Benesch, Andrew Aquilina, Brandon T. Ruotolo, Frank Sobott, C V Robinson

J. A. Aquilina

The application of mass spectrometry (MS) to the study of progressively larger and more complex macromolecular assemblies is proving increasingly useful for structural biologists. The scope of this approach has recently been widened through the application of a tandem MS procedure. This two-step technique involves the selection of specific assemblies in the gas phase, and inducing their dissociation through collisions with argon atoms. Here we investigate the mechanism of this process and show that dissociation of subunits from a macromolecular assembly follows a sequential pathway, with the partitioning of charge between the dissociation products governed primarily by their relative surface …

The Major Toxin From The Australian Common Brown Snake Is A Hexamer With Unusual Gas-Phase Dissociation Properties, Andrew Aquilina

J. A. Aquilina

Asymmetric dissociation of multiply charged proteins assemblies has been frequently reported. This phenomenon, which relies on the dissociation of one or more highly charged monomers, has been shown to provide insights into the structure and organization of large monodisperse and polydisperse assemblies. Here, the process of asymmetric dissociation is investigated using the multi-subunit protein, textilotoxin, which has unusually high structural constraints on its monomers due to multiple disulfide linkages. Initially, it is shown that, contrary to previous reports, textilotoxin is made up of 6, rather than 5 subunits. Furthermore, the hexamer exists as two isoforms, one of which is substantially …

Air-Drying Of Banana: Influence Of Experimental Parameters, Slab Thickness, Banana Maturity And Harvesting Season, Minh-Hue Nguyen, William E. Price

William E. Price

Air-drying of banana slabs has been investigated and the influence of experimental parameters such as temperature, relative humidity and slab thickness has been studied. This was in part re-investigated because of inconsistencies in previous studies, particularly in relation to derived water diffusion coefficients. In addition, it is shown that harvest season and hence initial moisture content has a very marked influence on the drying kinetics. By contrast banana maturity (ripeness) has little influence on the kinetics despite there being significant differences in morphology and chemical composition between green and ripe bananas. The effect of these two variables on the drying …

Increased Probiotic Yogurt Or Resistant Starch Intake Does Not Affect Isoflavone Bioavailability In Subjects Consuming A High Soy Diet, Theresa A. Larkin, William E. Price, Lee B. Astheimer

William E. Price

Objective: Probiotics and prebiotics that affect gut microflora balance and its associated enzymeactivity may contribute to interindividual variation in isoflavone absorption after soy intake, possiblyenhancing isoflavone bioavailability. This study examined the effects of the consumption ofbioactive yogurt (a probiotic) or resistant starch (a known prebiotic) in combination with high soyintake on soy isoflavone bioavailability.Methods: Using a crossover design, chronic soy consumption was compared with soy plusprobiotic yogurt or resistant starch in older male and postmenopausal females (n 31). Isoflavonebioavailability was assessed at the beginning and end of each 5-wk dietary period by samplingplasma and urine after a standardized soy meal.Results: …

Fractionation Of Sedimentary Arsenic From Port Kembla Harbour, Nsw, Australia, Glennys A. O'Brien, William E. Price, Bryan E. Chenhall, Muhammad Damris

William E. Price

The binding of arsenic in sediments of the heavily industrialised Port Kembla Harbour, NSW, Australia, has been investigated. Both dredge and core samples have been used to develop a sieving/sequential extraction (SE) procedure. Dredge samples included oxic surficial and deeper anoxic sediment. The main core sample analysed was 18 cm deep, sliced at 2 cm intervals. Sediment was sieved to three size ranges (250 microm) and each of these was then subjected to a four step SE, sequentially solubilizing arsenic as ion exchangeable, 1 M HCl soluble, NH(2)OH.HCl soluble, and strong oxidising acid soluble. Concentrations of 50-500 mg As kg(-1) …

A Fragmentation Study Of Isoflavones In Negative Electrospray Ionization By Msn Ion Trap Mass Spectrometry And Triple Quadrupole Mass Spectrometry, Jinguo Kang, Larry A. Hick, William E. Price

William E. Price

This study has elucidated the fragmentation pathway for deprotonated isoflavones in electrospray ionization using MSn ion trap mass spectrometry and triple quadrupole mass spectrometry. Genistein-d4 and daidzein-d3 were used as references for the clarification of fragment structures. To confirm the relationship between precursor and product ions, some fragments were traced from MS2 to MS5. The previous literature for the structurally related flavones and flavanones located the loss of ketene (C2H2O) to ring C, whereas the present fragmentation study for isoflavones has shown that the loss of ketene occurs elsewhere at ring A. In the further fragmentation of the [M-H-CH3]•- radical …

Effect Of Organic Solvents On The Separation Of Benzoic Acids By Capillary Electrophoresis, Young J. Lee, William E. Price, Margaret Sheil

William E. Price

The effect of organic modifiers on the separation of a number of closely related isomeric benzoic acids by capillary electrophoresis is described. It is shown that while a single modifier concentration cannot help resolve the entire electropherogram, organic modifiers do significantly enhance the resolution of parts of the separation system by comparison with 40 mmol l-1 phosphate buffer. The effects on separation and retention times are discussed in terms of the effects on electroosmotic flow and the electrophoretic mobilities of the charged solutes. The effects were found to be modifier specific, although the trends were in the same direction (ie., …

Occurrence Of Phytoestrogens In Municipal Wastewater And Surface Waters, Jinguo Kang, William E. Price

William E. Price

Phytoestrogens (isoflavones, enterolignans and coumestrol) in wastewater samples and surface water samples have been analysed by LC-ESI-MSn. In wastewater samples, high levels of enterolactone (581 – 2111 ng/L), daidzein (341 – 1688 ng/L) and enterodiol (60 – 834 ng/L) were detected in raw sewage, but the vast majority of the analysed phytoestrogens were removed effectively in treatment process. The removal rates of the analysed phytoestrogens in the two advanced tertiary treatment plants were > 99%; a case study in one of the treatment plants showed that most of the residual phytoestrogens were removed by biological treatment using activated sludge. In surface …

Corrigendum To ‘‘The Chaperone Action Of Bovine Milk As1- And As2-Caseins And Their Associated Form As-Casein’’ [Arch. Biochem. Biophys. 510 (2011) 42–52], Teresa M. Treweek, David C. Thorn, William E. Price, John A. Carver

William E. Price

No abstract provided.

The Key Importance Of Soy Isoflavone Bioavailability To Understanding Health Benefits, T. A. Larkin, William E. Price, L. Astheimer

William E. Price

Research over the past two decades has provided significant epidemiological and other evidence for the health benefits of the consumption of soy-based foods. A large number of dietary intervention studies have examined the effects of soy isoflavones on risk factors for cardiovascular disease and hormone-dependent cancers. However, these report large variability in outcome measures, very limited reproducibility between studies and in some cases, controversy between results of clinical trials using dietary soy or soy protein and isoflavone supplementation. This highlights a major gap in our understanding of soy isoflavone uptake, metabolism, distribution, and overall bioavailability. There are many potential factors …

Using Calibration Approaches To Compensate For Remaining Matrix Effects In Quantitative Liquid Chromatography Electrospray Ionization Multistage Mass Spectrometric Analysis Of Phytoestrogens In Aqueous Environmental Samples, Jinguo Kang, Larry A. Hick, William E. Price

William E. Price

Signal suppression is a common problem in quantitative LC-ESI-MSn analysis in environment samples, especially in highly loaded wastewater samples with highly complex matrix. Optimization of sample preparation and improvement of chromatographic separation are prerequisite to improve reproducibility and selectivity. Matrix components may be reduced if not eliminated by a series of sample preparation steps. However, extensive sample preparation can be time-consuming and risk the significant loss of some trace analytes. The best way to further compensate matrix effects is the use of internal standard for each analyte. However, in a multi-component analysis, finding appropriate internal standards for every analyte is …

Localization Of Low Molecular Weight Crystallin Peptides In The Aging Human Lens Using A Maldi Mass Spectrometry Imaging Approach, S. P. Su, Jason D. Mcarthur, J. A. Aquilina

J. A. Aquilina

Low molecular weight (LMW) peptides, derived from the breakdown of the major eye lens proteins, the crystallins, accumulate in the human lens with age. These LMW peptides are associated with age-related lens opacity and cataract, with some shown to inhibit the chaperone activity of α-crystallin. However, the mechanism(s) giving rise to the production of these peptides, as well as their distribution within the lens, are not well understood. In this study, we have mapped the distribution of these crystallin-derived peptides present in human lenses of different ages using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Our data showed that most of …

Defining The Structural Basis Of Human Plasminogen Binding By Streptococcal Surface Enolase, Amanda J. Cork, Slobodan Jergic, Sven Hammerschmidt, Bostjan Kobe, Vijay Pancholi, Justin L.P. Benesch, Carol V, Robinson, Nicholas E. Dixon, J Andrew Aquilina, Mark J. Walker

J. A. Aquilina

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of S. pneumoniae -enolase. Site-directed mutagenesis of surface-located lysine residues (SENK252+255A, SENK304A, SENK334A, SENK344E, SENK435L and SEN434-435) was used to examine their roles in maintaining structural integrity, enzymatic function and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SENK344E, as …

Mimicking Phosphorylation Of Alphab-Crystallin Affects Its Chaperone Activity, Heath W. Ecroyd, Sarah Meehan, J Horwitz, Andrew Aquilina, J L Benesch, C V Robinson, Cait Macphee, John Carver

J. A. Aquilina

No abstract provided.

The Role Of Streptokinase As A Virulence Determinant Of Streptococcus Pyogenes - Potential For Therapeutic Targeting, Jason D. Mcarthur, Simon M. Cook, Carola Venturini, Mark J. Walker

Faculty of Science - Papers (Archive)

Streptococcus pyogenes is a major human pathogen responsible for numerous diseases ranging from uncomplicated skin and throat infections to severe, life threatening invasive disease such as necrotising fasciitis and streptococcal toxic shock syndrome. These severe invasive infections progress rapidly and produce high rates of morbidity and mortality despite the implementation of aggressive treatment plans. The activation of plasminogen and the acquisition of plasmin activity at the bacterial cell surface is critical for the invasive pathogenesis of this organism. To facilitate this process, S. pyogenes secrete streptokinase, a potent plasminogen activating protein. Here, we describe the role of streptokinase in invasive …

Optimization Of Colorimetric Det Technique For The In Situ, Two-Dimensional Measurement Of Iron(Ii) Distributions In Sediment Porewaters, William W. Bennett, Peter R. Teasdale, David T. Welsh, Jarad G. Panther, Dianne F. Jolley

Faculty of Science - Papers (Archive)

No abstract provided.

Inorganic Arsenic And Iron(Ii) Distributions In Sediment Porewaters Investigated By A Combined Dgt - Colourimetric Det Technique, William Bennett, Peter R. Teasdale, David T. Welsh, Jarad Panther, Ryan R. Stewart, Helen L. Price, Dianne F. Jolley

Faculty of Science - Papers (Archive)

No abstract provided.

Different Radiolabelling Methods Alter The Pharmacokinetic And Biodistribution Properties Of Plasminogen Activator Inhibitor Type 2 (Pai-2) Forms, Marie Ranson, Paula Berghofer, Kara L. Vine, Ivan Greguric, Rachael Shepherd, Andrew Katsifis

Faculty of Science - Papers (Archive)

Introduction: Tumour-associated urokinase plasminogen activator (uPA) is a critical marker of invasion and metastasis, and it is recognised as having strong prognostic relevance as well as being a therapeutic target. The specific uPA inhibitor plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) specifically targets cell bound uPA and is internalised. Furthermore, preclinical studies have established the “proof-of-principle” of uPA-targeting by PAI-2-cytotoxin conjugates in human carcinoma models. However, these studies also suggest that PAI-2 is rapidly cleared via the renal system with low total dose reaching the tumour. In this study, a comparative single photon emission computed tomography (SPECT) and biodistribution (BD) analysis …

Does Cytotoxicity Of Metallointercalators Correlate With Cellular Uptake Or Dna Affinity?, Kimberley J. Davis, Judith A. Carrall, Barry Lai, Janice R. Aldrich-Wright, Stephen F. Ralph, Carolyn T. Dillon

Faculty of Science - Papers (Archive)

The cytotoxicity of the metallointercalators, [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1R,2Rdiaminocyclohexane)] 2+ ([56MERR]) and [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1S,2Sdiaminocyclohexane)] 2+ ([56MESS]), towards A549 human lung cancer cells was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 value obtained following exposure of A549 cells to [56MESS] for 4 h was approximately three times smaller than that obtained when [56MERR] was administered under the same conditions, indicating that the former complex displayed greater cytotoxicity. Both IC50 values were greater than that obtained after exposure of A549 cells to cisplatin, demonstrating that the latter compound was the most cytotoxic of the three examined. Microprobe synchrotron radiation X-ray fluorescence (SR-XRF) analyses of …

Antimalarial Alkaloids From A Bhutanese Traditional Medicinal Plant Corydalis Dubia, Phurpa Wangchuk, Paul A. Keller, Stephen G. Pyne, Anthony C. Willis, Sumalee Kamchonwongpaisan

Faculty of Science - Papers (Archive)

Ethnopharmacological relevance: Corydalis dubia is used in Bhutanese traditional medicine as a febrifuge and for treating infections in the blood, liver and bile which correlate to the signs and symptoms of malarial and microbial infections.

Aim of the study: To validate the ethnopharmacological uses of the plant and to discover potential new therapeutic drug leads. Materials and methods C. dubia was collected from Bhutan and the alkaloids were obtained using acid–base fractionation and separation by repeated column and preparative plate chromatography. The alkaloids were identified from analysis of their physiochemical and spectroscopic data and were tested for antiplasmodial, antimicrobial and …

Binaphthyl-Anchored Antibacterial Tripeptide Derivatives With Hydrophobic C-Terminal Amino Acid Variations, John Bremner, Paul A. Keller, Stephen Pyne, Mark Robertson, Kandasamy Sakthivel, Kittiya Somphol, Dean Baylis, Jonathon A Coates, John Deadman, Dharshini Jeevarajah, David I. Rhodes

Faculty of Science - Papers (Archive)

The facile synthesis of seven new dicationic tripeptide benzyl ester derivatives, with hydrophobic group variations in the C-terminal amino acid component, is described. Moderate to good activity was seen against Gram-positive bacteria in vitro. One cyclohexyl-substituted compound 2c was tested more widely and showed good potency (MIC values ranging from 2–4 μg/mL) against antibiotic-resistant strains of Staphylococcus aureus and Enterococci (VRE, VSE), and against Staphylococcus epidermidis.

High-Yield Cell-Free Protein Synthesis For Site-Specific Incorporation Of Unnatural Amino Acids At Two Sites, Kiyoshi Ozawa, Karin V. Loscha, Kekini V. Kuppan, Choy Theng Loh, Nicholas E. Dixon, Gottfried Otting

Faculty of Science - Papers (Archive)

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA_CUA opt and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyanophenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.

Dgt-Induced Copper Flux Predicts Bioaccumulation And Toxicity To Bivalves In Sediments With Varying Properties, Stuart L. Simpson, Heloise Yverneau, Anne Cremazy, Chad V. Jarolimek, Helen Price, Dianne F. Jolley

Faculty of Science - Papers (Archive)

Many regulatory frameworks for sediment quality assessment include consideration of contaminant bioavailability. However, the “snap-shots” of metal bioavailability provided by analyses of porewaters or acid-volatile sulfidesimultaneously extractable metal (AVS-SEM) relationships do not always contribute sufficient information. The use of inappropriate or inadequate information for assessing metal bioavailability in sediments may result in incorrect assessment decisions. The technique of diffusive gradients in thin films (DGT) enables the in situ measurement of metal concentrations in waters and fluxes from sediment porewaters. We utilized the DGT technique to interpret the bioavailability of copper to the benthic bivalve Tellina deltoidalis in sediments of varying …

Molecular Characterization Of A 21.4 Kilobase Antibiotic Resistance Plasmid From An Hemolytic Escherichia Coli O108:H-Human Clinical Isolate, Fay E. Dawes, Dieter M. Bulach, Alexander Kuzevski, Karl A. Bettelheim, Carola Venturini, Steven P. Djordjevic, Mark J. Walker

Faculty of Science - Papers (Archive)

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, …