ORCID Profile
0000-0002-4975-6189
Current Organisations
University of Western Australia
,
University of Nottingham
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Publisher: Wiley
Date: 19-11-2013
DOI: 10.1002/NAU.22519
Abstract: An age-related increase in prostatic smooth muscle tone is partly responsible for the lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH). Changes in the effectors of prostatic smooth muscle contraction with age may play a role in the development of these symptoms. Using a mouse model of prostate contractility, this study investigated the effect of age on the different components of contractility in the prostate gland. The isometric force developed in response to electrical field stimulation or exogenously applied agonists by mouse prostates mounted in organ baths, was evaluated to determine the effect of age on contractile mechanisms. Changes with age in the rate of ATP breakdown and levels of the P2rx1 gene and P2X1-purinoceptor expression in mouse prostate were measured by a modified luciferin-luciferase assay, RT-PCR and western blot, respectively. Nerve mediated contractile responses containing a component elicited by P2X1-purinoceptors were observed in prostates taken from aged mice, but not in prostates taken from young adult mice. Furthermore, the potency of the endogenous purinoceptor agonist ATP was 50-fold greater in aged mice, whereas the potency of its stable analogue α,β-metATP was unchanged. An age-related decrease in ATP metabolism was also observed. With age, a purinergic contractile response to nerve stimulation develops in the mouse prostate gland due to a decrease in the rate of ATP breakdown. This may contribute to the increase in muscular tone observed in BPH and suggests that P2X1-purinoceptors are an additional target for the treatment of BPH.
Publisher: Springer Science and Business Media LLC
Date: 24-02-2010
DOI: 10.1007/S00210-010-0492-Y
Abstract: This study characterised the inhibitory actions of prostaglandins on smooth muscle contractility in the rat prostate gland. Immunohistochemical studies were carried out to identify and localise the two isoforms of cyclooxygenase (COX) enzyme and the subtypes of prostanoid receptors present in the rat prostate. Isolated organ bath studies were carried out to pharmacologically characterise the subtype of prostanoid receptor mediating the inhibitory effects of prostanoids on the rat prostate. Immunohistochemical studies confirmed the presence of mainly COX-2 within the prostatic stroma. Isolated organ bath studies showed that prostaglandin E(2) (PGE(2) 10 nM-10 microM) but not prostaglandin D(2) (10 nM-10 microM), PGF2alpha (10 nM-10 microM), prostacyclin (10 nM-10 microM) or U46619 (10 nM-10 microM) inhibited nerve-mediated contractile responses to electrical field stimulation. Similarly, sulprostone (10 nM-10 microM) had no affect on the magnitude of the electrically evoked contractions. PGE(2) (0.1-10 microM) did not affect contractions elicited by noradrenaline or adenosine 5'-triphosphate. PGE(2)-mediated inhibition of electrical field stimulation induced contractions was attenuated by AH 6809 (10 microM) but not SC 19220 (10 microM) or AH 23848 (10 microM). It is concluded that prostaglandins can inhibit contractions of the rat prostate gland through a prostanoid receptor of the EP(2) subtype.
Publisher: Springer Science and Business Media LLC
Date: 02-10-2019
DOI: 10.1038/S41598-019-50733-9
Abstract: The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled s les allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.
Publisher: Cold Spring Harbor Laboratory
Date: 30-11-2018
DOI: 10.1101/482596
Abstract: The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR- knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled s les allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.
Publisher: Wiley
Date: 05-11-2015
DOI: 10.1111/BPH.13316
Publisher: Elsevier BV
Date: 2021
Publisher: Wiley
Date: 05-2021
DOI: 10.1002/PRP2.779
Abstract: Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand‐receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β 2 ‐adrenoceptor (β 2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β 2 AR (pK D .0) with good selectivity over the β 1 AR (pK D .0). The most potent and selective ligands being 8c (ICI 118,551‐Gly‐Ala‐BODIPY‐FL‐X β 2 AR pK D 7.48), 9c (ICI 118,551‐βAla‐βAla‐BODIPY‐FL‐X β 2 AR pK D 7.48), 12a (ICI 118,551‐PEG‐BODIPY‐X‐630/650 β 2 AR pK D 7.56), and 12b (ICI 118,551‐PEG‐BODIPY‐FL β 2 AR pK D 7.42). 9a (ICI 118,551‐βAla‐βAla‐BODIPY‐X‐630/650) had the highest affinity at recombinant β 2 ARs (pK D 7.57), but also exhibited significant binding affinity to the β 1 AR (pK D 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular β 2 ARs in HEK293 T cell expressing exogenous β 2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY‐X‐630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular β 2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome‐edited HEK293 T cells to undertake for the first time real‐time ligand binding to native HEK293 T β 2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand‐binding characteristics but also allowed real‐time visualization of the ligand‐binding interactions in genome‐edited cells using NanoBRET luminescence imaging.
Publisher: Springer Science and Business Media LLC
Date: 09-06-2017
DOI: 10.1038/S41598-017-03486-2
Abstract: Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated β-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.
Publisher: Proceedings of the National Academy of Sciences
Date: 02-12-2013
Abstract: The search for a viable male contraceptive target has been a medical challenge for many years. Most strategies have focused on hormonal or germ-line strategies to produce dysfunctional sperm that are incapable of fertilization. The problem with such approaches is that they have intolerable side effects such as affecting male sexual activity or causing long-term irreversible effects on fertility. In addition, some strategies may transmit detrimental changes to future offspring. This manuscript describes a male contraceptive target within the autonomic nervous system, which would not affect the long-term viability of sperm nor the sexual or general health of males. In addition, due to the nature of the target, the contraceptive has the potential to be orally administered.
Publisher: Springer Science and Business Media LLC
Date: 10-2018
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 19-08-2010
Publisher: Wiley
Date: 08-06-2011
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.EJPHAR.2013.09.012
Abstract: With age an increase in prostatic smooth muscle tone mediated by α1L-adrenoceptors contributes to the lower urinary tract symptoms associated with benign prostatic hyperplasia. Current treatments for benign prostatic hyperplasia include α1-adrenoceptor antagonists which inhibit smooth muscle contraction. However, muscarinic receptors also mediate prostatic smooth muscle contraction and targeting a convergent signalling pathway may be a more effective treatment strategy. This study determined signalling pathways involved in contraction by measuring isometric force developed by prostates from wild type, α1A-adrenoceptor and M3-muscarinic receptor knockout mice mounted in organ baths in response to, electrical field stimulation or exogenously applied agonists, in the presence or absence of signalling pathway inhibitors. Fluorescence immunohistochemistry was also used to confirm functional observations. Contractile responses mediated by carbachol were reduced by inhibitors of phospholipase C (U73122 3-10 µM), L-type Ca(2+) channels (nifedipine 1 µM), Rho kinase (Y-27632 10-30 µM), but not protein kinase C (GF109203 X µM). Nifedipine (1 µM), Y-27632 (10 µM), and GF109203 X (10 µM) inhibited nerve mediated contractile responses. Y-27632 (10-30 µM) inhibited noradrenaline mediated contractions. RhoA and ROCK2 were found to be immunolocalised with prostatic smooth-muscle. Contractions mediated by M3-muscarinic receptors in the mouse prostate involve the prototypical phospholipase C signalling pathway, as well as L-type Ca(2+) channels. Adrenergic and cholinergic components of smooth muscle contraction in the mouse prostate both involve the activation of the Rho-kinase pathway, which may be a suitable common pathway for more effective treatments of symptoms associated with benign prostatic hyperplasia.
Publisher: Informa UK Limited
Date: 07-2013
DOI: 10.4161/ORG.24843
Publisher: Wiley
Date: 12-2041
Publisher: Elsevier BV
Date: 05-2020
Publisher: Frontiers Media SA
Date: 26-03-2019
Publisher: Wiley
Date: 09-02-2021
DOI: 10.1111/FEBS.15729
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 09-2011
Abstract: Increased smooth muscle tone in the human prostate contributes to the symptoms associated with benign prostatic hyperplasia. In the mouse prostate gland, cholinergic innervation is responsible for a component of the nerve-mediated contractile response. This study investigates the muscarinic receptor subtype responsible for the cholinergic contractile response in the mouse prostate gland. To characterize the muscarinic receptor subtype, mouse prostates taken from wild-type or M(3) muscarinic receptor knockout mice were mounted in organ baths. The isometric force that tissues developed in response to electrical-field stimulation or exogenously applied cholinergic agonists in the presence or absence of a range of muscarinic receptor antagonists was evaluated. Carbachol elicited reproducible and concentration-dependent contractions of the isolated mouse prostate, which were antagonized by the presence of muscarinic receptor antagonists. Calculation of antagonist affinities (pA(2) values) indicated a rank order of antagonist potencies in the mouse prostate of: darifenacin (9.08) = atropine (9.07) = 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (9.02) > cyclohexyl-hydroxy-phenyl-(3-piperidin-1-ylpropyl)silane (7.85) > cyclohexyl-(4-fluorophenyl)-hydroxy-(3-piperidin-1-ylpropyl)silane (7.39) > himbacine (7.19) > pirenzipine (6.88) > methoctramine (6.20). Furthermore, genetic deletion of the M(3) muscarinic receptor inhibited prostatic contractions to electrical-field stimulation or exogenous administration of acetylcholine. In this study we identified that the cholinergic component of contraction in the mouse prostate is mediated by the M(3) muscarinic receptor subtype. Pharmacological antagonism of the M(3) muscarinic receptor may be a beneficial additional target for the treatment of benign prostatic hyperplasia in the human prostate gland.
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.CELLSIG.2018.11.018
Abstract: Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A
Publisher: Elsevier BV
Date: 04-2012
Publisher: The Endocrine Society
Date: 08-2016
DOI: 10.1210/ME.2016-1002
Publisher: Wiley
Date: 27-03-2019
DOI: 10.1111/BPH.14599
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2015
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2015
End Date: 2019
Funder: National Health and Medical Research Council
View Funded Activity