Illawarra Health and Medical Research Institute

Sydney, Australia

Illawarra Health and Medical Research Institute

Sydney, Australia
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Sellorsm K.,Prince of Wales Hospital | Jones A.,University of Wollongong | Jones A.,Illawarra Health and Medical Research Institute | Chan B.,Prince of Wales Hospital
Medical Journal of Australia | Year: 2014

Intoxication with synthetic cathinones (psychoactive designer drugs) can involve cardiovascular, autonomic, neuromuscular and neuropsychiatric features. We report a case of cardiac arrest and subsequent death in a 44-year-old man after intravenous use of one such drug — α-pyrrolidinopentiophenone. We believe this is the first death associated with this drug to be reported in Australia. Currently, no specific antidote exists for cathinone exposure.


Sluyter R.,University of Wollongong | Sluyter R.,Illawarra Health and Medical Research Institute | Stokes L.,University of Sydney
Recent Patents on DNA and Gene Sequences | Year: 2011

The human P2X7 receptor is a trimeric ligand-gated cation channel coded by the P2XR7 gene located at chromosome position 12q24. P2X7 is expressed in a wide variety of normal and disease-associated cell types. Activation of this receptor by extracellular adenosine 5'-triphosphate results in numerous downstream events including the release of pro-inflammatory mediators, cell proliferation or death, and killing of intracellular pathogens. As a result, P2X7 plays important roles in inflammation, immunity, bone homeostasis, neurological function and neoplasia. The P2XR7 gene encodes a P2X7 subunit 595 amino acids in length, however splice isoforms that can alter receptor expression and function, and modify the signaling properties downstream of receptor activation also exist. Moreover, the relative amount of P2X7 function varies between human individuals due to numerous single nucleotide polymorphisms resulting in either loss- or gain-of-function. Combinations of these polymorphisms give rise to various haplotypes that can also modify P2X7 function. Collectively, P2X7, and its splice and polymorphic variants are attracting considerable interest in relation to human health and disease, including the development and publication of a number of patents. © 2011 Bentham Science Publishers Ltd.


Wang B.,University of Wollongong | Wang B.,Illawarra Health and Medical Research Institute | Wang B.,University of New South Wales | Sluyter R.,University of Wollongong | Sluyter R.,Illawarra Health and Medical Research Institute
Purinergic Signalling | Year: 2013

The presence of P2X7 on erythroid cells is well established, but its physiological role remains unclear. The current study aimed to determine if P2X7 activation induces reactive oxygen species (ROS) formation in murine erythroleukaemia (MEL) cells, a commonly used erythroid cell line. ATP induced ROS formation in a time- and concentration-dependent fashion. The most potent P2X7 agonist, 2′(3′)-O-(4-benzoylbenzoyl)ATP, but not UTP or ADP, also induced ROS formation. The P2X7 antagonist, A-438079, impaired ATP-induced ROS formation. The ROS scavenger, N-acetyl-l-cysteine, and the ROS inhibitor, diphenyleneiodonium, also impaired P2X7-induced ROS formation, but use of enzyme-specific ROS inhibitors failed to identify the intracellular source of P2X7-induced ROS formation. P2X7-induced ROS formation was impaired partly by physiological concentrations of Ca2+ and Mg2+ and almost completely in cells in N-methyl-d-glucamine chloride medium. The p38 MAPK inhibitors SB202190 and SB203580, and the caspase inhibitor Z-VAD-FMK, but not N-acetyl-l-cysteine, impaired P2X7-induced MEL cell apoptosis. ATP also stimulated p38 MAPK and caspase activation, both of which could be impaired by A-438079. In conclusion, these findings indicate that P2X7 activation induces ROS formation in MEL cells and that this process may be involved in events downstream of P2X7 activation, other than apoptosis, in erythroid cells. © 2012 Springer Science+Business Media Dordrecht.


Bartlett R.,University of Wollongong | Bartlett R.,Illawarra Health and Medical Research Institute | Stokes L.,RMIT University | Sluyter R.,University of Wollongong | Sluyter R.,Illawarra Health and Medical Research Institute
Pharmacological Reviews | Year: 2014

The P2X7 receptor is a trimeric ATP-gated cation channel found predominantly, but not exclusively, on immune cells. P2X7 activation results in a number of downstream events, including the release of proinflam-matory mediators and cell death and proliferation. As such, P2X7 plays important roles in various inflammatory, immune, neurologic and musculoskeletal disorders. This review focuses on the use of P2X7 antagonists in rodent models of neurologic disease and injury, inflammation, and musculoskeletal and other disorders. The cloning and characterization of human, rat, mouse, guinea pig, dog, and Rhesus macaque P2X7, as well as recent observations regarding the gating and permeability of P2X7, are discussed. Furthermore, this review discusses polymorphic and splice variants of P2X7, as well as the generation and use of P2X7 knockout mice. Recent evidence for emerging signaling pathways downstream of P2X7 activation and the growing list of negative and positive modulators of P2X7 activation and expression are also described. In addition, the use of P2X7 antagonists in numerous rodent models of disease is extensively summarized. Finally, the use of P2X7 antagonists in clinical trials in humans and future directions exploring P2X7 as a therapeutic target are described. © 2014 by The American Society for Pharmacology and Experimental Therapeutics.


Pupovac A.,University of Wollongong | Pupovac A.,Illawarra Health and Medical Research Institute | Foster C.M.,University of Wollongong | Sluyter R.,University of Wollongong | Sluyter R.,Illawarra Health and Medical Research Institute
Biochemical and Biophysical Research Communications | Year: 2013

Activation of the purinergic P2X7 receptor by extracellular ATP induces the shedding of cell-surface molecules including the low-affinity IgE receptor, CD23 from leukocytes. CD23 is a known substrate of a disintegrin and metalloprotease (ADAM)10. The aim of the current study was to determine if P2X7 activation induced the shedding of the chemokine CXCL16, an ADAM10 substrate. Using immunolabelling and flow cytometry we demonstrate that human RPMI 8226 multiple myeloma B cells, which have been previously shown to express P2X7, also express CXCL16. Flow cytometric and ELISA measurements of ATP-induced loss of cell-surface CXCL16 showed that ATP treatment of RPMI 8226 cells induced the rapid shedding of CXCL16. Treatment of RPMI 8226 cells with the specific P2X7 antagonists, AZ10606120 and KN-62 impaired ATP-induced CXCL16 shedding by ∼86% and ∼90% respectively. RT-PCR demonstrated that ADAM10 is expressed in these cells and treatment of cells with the ADAM10 inhibitor, GI254023X, impaired ATP-induced CXCL16 shedding by ∼87%. GI254023X also impaired P2X7-induced CD23 shedding by ∼57%. This data indicates that human P2X7 activation induces the rapid shedding of CXCL16 and that this process involves ADAM10. © 2013 Elsevier Inc.


Bartlett R.,University of Wollongong | Bartlett R.,Illawarra Health and Medical Research Institute | Yerbury J.J.,University of Wollongong | Yerbury J.J.,Illawarra Health and Medical Research Institute | And 2 more authors.
Mediators of Inflammation | Year: 2013

The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5′- triphosphate (ATP) and 2′(3′)-O-(4-benzoylbenzoyl) ATP induced ethidium+ or YO-PRO-12+ uptake into both cell lines. ATP induced ethidium+ uptake into EOC13 cells in a concentration- dependent manner, with an ECof 130 M. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75-100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca2+ influx and K+ efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia. © 2013 Rachael Bartlett et al.


Pupovac A.,University of Wollongong | Pupovac A.,Illawarra Health and Medical Research Institute | Stokes L.,University of Sydney | Stokes L.,RMIT University | And 2 more authors.
Purinergic Signalling | Year: 2013

The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the 'low affinity' IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium+ uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself. © 2013 Springer Science+Business Media Dordrecht.


Van Oijen A.M.,University of Wollongong | Van Oijen A.M.,Illawarra Health and Medical Research Institute | Dixon N.E.,University of Wollongong | Dixon N.E.,Illawarra Health and Medical Research Institute
Nature Structural and Molecular Biology | Year: 2015

Single-molecule approaches are having a dramatic impact on views of how proteins work. The ability to observe molecular properties at the single-molecule level allows characterization of subpopulations and acquisition of detailed kinetic information that would otherwise be hidden in the averaging over an ensemble of molecules. In this Perspective, we discuss how such approaches have successfully been applied to in vitro-reconstituted systems of increasing complexity. © 2015 Nature America, Inc. All rights reserved.


Treweek T.M.,University of Wollongong | Treweek T.M.,Illawarra Health and Medical Research Institute | Meehan S.,University of Cambridge | Ecroyd H.,Illawarra Health and Medical Research Institute | And 2 more authors.
Cellular and Molecular Life Sciences | Year: 2015

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease. © 2014 Springer Basel.


Butson E.T.,The Illawarra Grammar School | Cheung T.,City University of Hong Kong | Yu P.K.N.,City University of Hong Kong | Butson M.J.,City University of Hong Kong | Butson M.J.,Illawarra Health and Medical Research Institute
Physics in Medicine and Biology | Year: 2010

Ultraviolet radiation dosimetry has been performed with the use of a radiochromic film dosimeter called Gafchromic EBT for solar radiation exposure. The film changes from a clear colour to blue colour when exposed to ultraviolet radiation and results have shown that the colour change is reproducible within ±10% at 5 kJ m-2 UV exposure under various conditions of solar radiation. Parameters tested included changes in season (summer versus winter exposure), time of day, as well as sky conditions such as cloudy skies versus clear skies. As the radiochromic films' permanent colour change occurs in the visible wavelengths the film can be analysed with a desktop scanner with the most sensitive channel for analysis being the red component of the signal. Results showed that an exposure of 5 kJ m-2 (approximately 1 h exposure in full sun during summer) produced an approximate 0.28 change in the net OD when analysed in reflection mode on the desktop scanner which is significant darkening. The main advantages of this film type, and thus the new EBT2 film which has replaced EBT for measurement of UV exposure, is the visible colour change and thus easy analysis using a desktop scanner, its uniformity in response and its robust physical strength for use in outside exposure situations. © 2010 Institute of Physics and Engineering in Medicine.

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