Publications by authors named "Stephen J Fuller"

49 Publications

MAP4K4 expression in cardiomyocytes: multiple isoforms, multiple phosphorylations and interactions with striatins.

Biochem J 2021 Jun;478(11):2121-2143

School of Biological Sciences, University of Reading, Whiteknights Campus, Reading RG6 2AS, U.K.

The Ser/Thr kinase MAP4K4, like other GCKIV kinases, has N-terminal kinase and C-terminal citron homology (CNH) domains. MAP4K4 can activate c-Jun N-terminal kinases (JNKs), and studies in the heart suggest it links oxidative stress to JNKs and heart failure. In other systems, MAP4K4 is regulated in striatin-interacting phosphatase and kinase (STRIPAK) complexes, in which one of three striatins tethers PP2A adjacent to a kinase to keep it dephosphorylated and inactive. Our aim was to understand how MAP4K4 is regulated in cardiomyocytes. The rat MAP4K4 gene was not properly defined. We identified the first coding exon of the rat gene using 5'-RACE, we cloned the full-length sequence and confirmed alternative-splicing of MAP4K4 in rat cardiomyocytes. We identified an additional α-helix C-terminal to the kinase domain important for kinase activity. In further studies, FLAG-MAP4K4 was expressed in HEK293 cells or cardiomyocytes. The Ser/Thr protein phosphatase inhibitor calyculin A (CalA) induced MAP4K4 hyperphosphorylation, with phosphorylation of the activation loop and extensive phosphorylation of the linker between the kinase and CNH domains. This required kinase activity. MAP4K4 associated with myosin in untreated cardiomyocytes, and this was lost with CalA-treatment. FLAG-MAP4K4 associated with all three striatins in cardiomyocytes, indicative of regulation within STRIPAK complexes and consistent with activation by CalA. Computational analysis suggested the interaction was direct and mediated via coiled-coil domains. Surprisingly, FLAG-MAP4K4 inhibited JNK activation by H2O2 in cardiomyocytes and increased myofibrillar organisation. Our data identify MAP4K4 as a STRIPAK-regulated kinase in cardiomyocytes, and suggest it regulates the cytoskeleton rather than activates JNKs.
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http://dx.doi.org/10.1042/BCJ20210003DOI Listing
June 2021

The insulin receptor family and protein kinase B (Akt) are activated in the heart by alkaline pH and α1-adrenergic receptors.

Biochem J 2021 Jun;478(11):2059-2079

School of Biological Sciences, University of Reading, Reading RG6 6AS, U.K.

Insulin and insulin-like growth factor stimulate protein synthesis and cardioprotection in the heart, acting through their receptors (INSRs, IGF1Rs) and signalling via protein kinase B (PKB, also known as Akt). Protein synthesis is increased in hearts perfused at alkaline pHo to the same extent as with insulin. Moreover, α1-adrenergic receptor (α1-AR) agonists (e.g. phenylephrine) increase protein synthesis in cardiomyocytes, activating PKB/Akt. In both cases, the mechanisms are not understood. Our aim was to determine if insulin receptor-related receptors (INSRRs, activated in kidney by alkaline pH) may account for the effects of alkaline pHo on cardiac protein synthesis, and establish if α1-ARs signal through the insulin receptor family. Alkaline pHo activated PKB/Akt signalling to the same degree as insulin in perfused adult male rat hearts. INSRRs were expressed in rat hearts and, by immunoblotting for phosphorylation (activation) of INSRRs/INSRs/IGF1Rs, we established that INSRRs, together with INSRs/IGF1Rs, are activated by alkaline pHo. The INSRR/INSR/IGF1R kinase inhibitor, linsitinib, prevented PKB/Akt activation by alkaline pHo, indicating that INSRRs/INSRs/IGF1Rs are required. Activation of PKB/Akt in cardiomyocytes by α1-AR agonists was also inhibited by linsitinib. Furthermore, linsitinib inhibited cardiomyocyte hypertrophy induced by α1-ARs in cultured cells, reduced the initial cardiac adaptation (24 h) to phenylephrine in vivo (assessed by echocardiography) and increased cardiac fibrosis over 4 days. We conclude that INSRRs are expressed in the heart and, together with INSRs/IGF1Rs, the insulin receptor family provide a potent system for promoting protein synthesis and cardioprotection. Moreover, this system is required for adaptive hypertrophy induced by α1-ARs.
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http://dx.doi.org/10.1042/BCJ20210144DOI Listing
June 2021

Redox Regulation of Cardiac ASK1 (Apoptosis Signal-Regulating Kinase 1) Controls p38-MAPK (Mitogen-Activated Protein Kinase) and Orchestrates Cardiac Remodeling to Hypertension.

Hypertension 2020 10 9;76(4):1208-1218. Epub 2020 Sep 9.

School of Biological Sciences, University of Reading, United Kingdom (D.N.M., J.J.C., T.M., S.J.F., P.H.S., A.C.), St. George's Healthcare NHS Trust, London, United Kingdom.

Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of HO activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1β activated an alternative kinase, TAK1 (transforming growth factor-activated kinase 1). ASK1 was not activated by IL1β in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition of mRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.14556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480944PMC
October 2020

A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function.

FASEB J 2020 03 31;34(3):3884-3901. Epub 2020 Jan 31.

Department of Medicine, Sydney Medical School Nepean, Faculty of Medicine and Health, The University of Sydney, Kingswood, NSW, Australia.

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.
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http://dx.doi.org/10.1096/fj.201901198RRDOI Listing
March 2020

Increased P2X7 expression in the gastrointestinal tract and skin in a humanised mouse model of graft-versus-host disease.

Clin Sci (Lond) 2020 01;134(2):207-223

Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.

Background: Allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for blood cancers; but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen-presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of the present study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs.

Methods: NOD-scid IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or phosphate-buffered saline (PBS, control). Leucocytes were assessed by flow cytometry; gene expression was measured by quantitative polymerase chain reaction (qPCR), and tissue sections examined by histology.

Results: Compared with control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared with control mice, hu-PBMC-NSG mice displayed increased ear swelling.

Conclusion: Combined data revealed that P2rx7 is up-regulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leucocyte infiltration. Data also reveal increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early-stage gut GVHD and skin GVHD, respectively.
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http://dx.doi.org/10.1042/CS20191086DOI Listing
January 2020

Scurvy: An Unrecognized and Emerging Public Health Issue in Developed Economies.

Mayo Clin Proc 2019 12;94(12):2594-2597

Department of Medicine, Faculty of Medicine and Health, The University of Sydney, Nepean Hospital, Kingswood, Australia.

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http://dx.doi.org/10.1016/j.mayocp.2019.10.005DOI Listing
December 2019

The Effect of Antidepressants on Mesenchymal Stem Cell Differentiation.

J Bone Metab 2018 Feb 28;25(1):43-51. Epub 2018 Feb 28.

Sydney Medical School Nepean, The University of Sydney, Penrith, Australia.

Background: Use of antidepressant medications has been linked to detrimental impacts on bone mineral density and osteoporosis; however, the cellular basis behind these observations remains poorly understood. The effect does not appear to be homogeneous across the whole class of drugs and may be linked to affinity for the serotonin transporter system. In this study, we hypothesized that antidepressants have a class- and dose-dependent effect on mesenchymal stem cell (MSC) differentiation, which may affect bone metabolism.

Methods: Human MSCs (hMSCs) were committed to differentiate when either adipogenic or osteogenic media was added, supplemented with five increasing concentrations of amitriptyline (0.001-10 µM), venlafaxine (0.01-25 µM), or fluoxetine (0.001-10 µM). Alizarin red staining (mineralization), alkaline phosphatase (osteoblastogenesis), and oil red O (adipogenesis) assays were performed at timed intervals. In addition, cell viability was assessed using a MTT.

Results: We found that fluoxetine had a significant inhibitory effect on mineralization. Furthermore, adipogenic differentiation of hMSC was affected by the addition of amitriptyline, venlafaxine, and fluoxetine to the media. Finally, none of the tested medications significantly affected cell survival.

Conclusions: This study showed a divergent effect of three antidepressants on hMSC differentiation, which appears to be independent of class and dose. As fluoxetine and amitriptyline, but not venlafaxine, affected both osteoblastogenesis and adipogenesis, this inhibitory effect could be associated to the high affinity of fluoxetine to the serotonin transporter system.
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http://dx.doi.org/10.11005/jbm.2018.25.1.43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854822PMC
February 2018

The P2X7 receptor is not essential for development of imiquimod-induced psoriasis-like inflammation in mice.

Purinergic Signal 2017 12 8;13(4):405-415. Epub 2017 Jun 8.

School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia.

Psoriasis is a chronic inflammatory skin disorder, characterised by epidermal hyperplasia (acanthosis) and leukocyte infiltration of the skin. Current therapies are inadequate, highlighting the need for new therapeutic targets. The P2X7 receptor is implicated in the pathogenesis of psoriasis. This study investigated the role of P2X7 in imiquimod (IMQ)-induced psoriasis-like inflammation. Topically applied IMQ caused twofold greater ear swelling in BALB/c mice compared to C57BL/6 mice, which encode a partial loss-of-function missense mutation in the P2RX7 gene. However, there was no difference in histological skin pathology (acanthosis and leukocyte infiltration) between the two strains. IMQ treatment up-regulated P2X7 expression in skin from both mouse strains. Additionally, IMQ induced ATP release from cultured human keratinocytes, a process independent of cell death. Injection of the P2X7 antagonist Brilliant Blue G (BBG) but not A-804598 partly reduced ear swelling compared to vehicle-injected control mice. Neither antagonist altered skin pathology. Moreover, no difference in ear swelling or skin pathology was observed between C57BL/6 and P2X7 knock-out (KO) mice. Flow cytometric analysis of IMQ-treated skin from C57BL/6 and P2X7 KO mice demonstrated similar leukocyte infiltration, including neutrophils, macrophages and T cells. In conclusion, this study demonstrates that P2X7 is not essential for development of IMQ-induced psoriasis-like inflammation but does not exclude a role for this receptor in psoriasis development in humans or other mouse models of this disease.
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http://dx.doi.org/10.1007/s11302-017-9569-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714832PMC
December 2017

IL-10 Potentiates Differentiation of Human Induced Regulatory T Cells via STAT3 and Foxo1.

J Immunol 2015 Oct 11;195(8):3665-74. Epub 2015 Sep 11.

Charles Perkins Centre Nepean, The University of Sydney, Kingswood, New South Wales 2751, Australia;

Foxp3(+) regulatory T cells (Tregs) play essential roles in maintaining the immune balance. Although the majority of Tregs are formed in the thymus, increasing evidence suggests that induced Tregs (iTregs) may be generated in the periphery from naive cells. However, unlike in the murine system, significant controversy exists regarding the suppressive capacity of these iTregs in humans, especially those generated in vitro in the presence of TGF-β. Although it is well known that IL-10 is an important mediator of Treg suppression, the action of IL-10 on Tregs themselves is less well characterized. In this article, we show that the presence of IL-10, in addition to TGF-β, leads to increased expansion of Foxp3(+) iTregs with enhanced CTLA-4 expression and suppressive capability, comparable to that of natural Tregs. This process is dependent on IL-10R-mediated STAT3 signaling, as supported by the lack of an IL-10 effect in patients with IL-10R deficiency and dominant-negative STAT3 mutation. Additionally, IL-10-induced inhibition of Akt phosphorylation and subsequent preservation of Foxo1 function are critical. These results highlight a previously unrecognized function of IL-10 in human iTreg generation, with potential therapeutic implications for the treatment of immune diseases, such as autoimmunity and allergy.
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http://dx.doi.org/10.4049/jimmunol.1402898DOI Listing
October 2015

Paroxetine suppresses recombinant human P2X7 responses.

Purinergic Signal 2015 Dec 5;11(4):481-90. Epub 2015 Sep 5.

Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Level 5 South Block, Derby Street, Penrith, NSW, 2750, Australia.

P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC(50) of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC(50) of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14(+) monocytes was suppressed with trifluoperazine and paroxetine.
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http://dx.doi.org/10.1007/s11302-015-9467-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648795PMC
December 2015

Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts.

Cardiovasc Res 2015 Oct 10;108(1):87-98. Epub 2015 Aug 10.

School of Biological Sciences, University of Reading, Whiteknights Campus, Reading, Berkshire RG6 6AS, UK

Aims: Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts.

Methods And Results: Expression profiling (Affymetrix microarrays) detected >400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly up-regulated or down-regulated (greater than two-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified >180 cardiomyocyte protein kinases, with high relative expression of mitogen-activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k, and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were up-regulated in AVMs vs. NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were up-regulated (e.g. NRK, JAK2, STK38L) or down-regulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts.

Conclusion: This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets.
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http://dx.doi.org/10.1093/cvr/cvv210DOI Listing
October 2015

Lack of a Functioning P2X7 Receptor Leads to Increased Susceptibility to Toxoplasmic Ileitis.

PLoS One 2015 8;10(6):e0129048. Epub 2015 Jun 8.

Queensland Tropical Health Alliance Research Laboratory, Australian Institute of Tropical Health and Medicine, James Cook University, McGregor Rd, Smithfield, Queensland, 4878, Australia.

Background: Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis.

Principal Findings: Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This susceptibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen intermediates and altered regulation of elements of NFκB activation in P2X7R-deficient mice.

Conclusions: Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129048PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460092PMC
March 2016

Probenecid blocks human P2X7 receptor-induced dye uptake via a pannexin-1 independent mechanism.

PLoS One 2014 26;9(3):e93058. Epub 2014 Mar 26.

Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Penrith, New South Wales, Australia; Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093058PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966854PMC
June 2015

Quantitative real-time PCR eliminates false-positives in colony screening PCR.

J Microbiol Methods 2014 Jan 1;96:99-100. Epub 2013 Dec 1.

Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Penrith, New South Wales, Australia. Electronic address:

We report an alternative approach to colony screening using real-time PCR (qPCR) which can be used instead of the traditional end-point PCR to eliminate false-positives and reduce processing times. False-positive transformants can easily be distinguished from true-positives by comparing Ct values derived from qPCR amplification curves. In addition, the use of qPCR allows for more efficient processing since a gel electrophoresis step is not required and the screening process is no longer limited by the capacity of the gel apparatus.
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http://dx.doi.org/10.1016/j.mimet.2013.11.011DOI Listing
January 2014

A rare functional haplotype of the P2RX4 and P2RX7 genes leads to loss of innate phagocytosis and confers increased risk of age-related macular degeneration.

FASEB J 2013 Apr 9;27(4):1479-87. Epub 2013 Jan 9.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia.

Age-related macular degeneration (AMD) is a leading cause of blindness in Western countries and is diagnosed by the clinical appearance of yellow subretinal deposits called drusen. Genetic changes in immune components are clearly implicated in the pathology of this disease. We have previously shown that the purinergic receptor P2X7 can act as a scavenger receptor, mediating phagocytosis of apoptotic cells and insoluble debris. We performed a genetic association study of functional polymorphisms in the P2RX7 and P2RX4 genes in a cohort of 744 patients with AMD and 557 age-matched Caucasian control subjects. The P2X4 Tyr315Cys variant was 2-fold more frequent in patients with AMD compared to control subjects, with the minor allele predicting susceptibility to disease. Pairwise linkage disequilibrium was observed between Tyr315Cys in the P2RX4 gene and Gly150Arg in the P2RX7 gene, and these two minor alleles formed a rare haplotype that was overrepresented in patients with AMD (n=17) compared with control subjects (n=3) (odds ratio 4.05, P=0.026). Expression of P2X7 (wild type or variant 150Arg) in HEK293 cells conferred robust phagocytosis toward latex beads, whereas coexpression of the P2X7 150Arg with P2X4 315Cys variants almost completely inhibited phagocytic capacity. Fresh human monocytes harboring this heterozygous 150Arg-315Cys haplotype showed 40% reduction in bead phagocytosis. In the primate eye, immunohistochemistry indicated that P2X7 and P2X4 receptors were coexpressed on microglia and macrophages, but neither receptor was seen on retinal pigment epithelial cells. These results demonstrate that a haplotype including two rare variants in P2RX7 and P2RX4 confers a functional interaction between these two variant receptors that impairs the normal scavenger function of macrophages and microglia. Failure of this P2X7-mediated phagocytic pathway may impair removal of subretinal deposits and predispose individuals toward AMD.
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http://dx.doi.org/10.1096/fj.12-215368DOI Listing
April 2013

p90 ribosomal S6 kinases play a significant role in early gene regulation in the cardiomyocyte response to G(q)-protein-coupled receptor stimuli, endothelin-1 and α(1)-adrenergic receptor agonists.

Biochem J 2013 Mar;450(2):351-63

School of Biological Sciences, University of Reading, Whiteknights, Reading RG6 6BX, UK.

ERK1/2 (extracellular-signal-regulated kinase 1/2) and their substrates RSKs (p90 ribosomal S6 kinases) phosphorylate different transcription factors, contributing differentially to transcriptomic profiles. In cardiomyocytes ERK1/2 are required for >70% of the transcriptomic response to endothelin-1. In the present study we investigated the role of RSKs in the transcriptomic responses to the G(q)-protein-coupled receptor agonists endothelin-1, phenylephrine (a generic α(1)-adrenergic receptor agonist) and A61603 (α(1A)-adrenergic receptor selective). Phospho-ERK1/2 and phospho-RSKs appeared in cardiomyocyte nuclei within 2-3 min of stimulation (endothelin-1>A61603≈phenylephrine). All agonists increased nuclear RSK2, but only endothelin-1 increased the nuclear RSK1 content. PD184352 (inhibits ERK1/2 activation) and BI-D1870 (inhibits RSKs) were used to dissect the contribution of RSKs to the endothelin-1-responsive transcriptome. Of the 213 RNAs up-regulated after 1 h, 51% required RSKs for their up-regulation, whereas 29% required ERK1/2 but not RSKs. The transcriptomic response to phenylephrine overlapped with, but was not identical with, endothelin-1. As with endothelin-1, PD184352 inhibited the up-regulation of most phenylephrine-responsive transcripts, but the greater variation in the effects of BI-D1870 suggests that differential RSK signalling influences global gene expression. A61603 induced similar changes in RNA expression in cardiomyocytes as phenylephrine, indicating that the signal was mediated largely through α(1A)-adrenergic receptors. A61603 also increased expression of immediate early genes in perfused adult rat hearts and, as in cardiomyocytes, up-regulation of the majority of genes was inhibited by PD184352. PD184352 or BI-D1870 prevented the increased surface area induced by endothelin-1 in cardiomyocytes. Thus RSKs play a significant role in regulating cardiomyocyte gene expression and hypertrophy in response to G(q)-protein-coupled receptor stimulation.
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http://dx.doi.org/10.1042/BJ20121371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573779PMC
March 2013

New insights into the pathogenesis, diagnosis, and management of mastocytosis.

Authors:
Stephen J Fuller

Hematol Oncol Clin North Am 2012 Dec 9;26(6):1143-68. Epub 2012 Oct 9.

Department of Medicine, Sydney Medical School Nepean, The University of Sydney, Nepean Hospital, Penrith, New South Wales 2751, Australia.

This review describes developments in understanding normal mast cell function and genetic changes that predispose to malignant transformation to mastocytosis. Most mastocytosis cases are associated with somatically acquired activating mutations in the KIT receptor. The role these mutations play in the development of mastocytosis is discussed. Mastocytosis is classified into cutaneous mastocytosis and systemic mastocytosis. The classification of mastocytosis and clinical presentation of each variant is detailed in this report. Progress has been made in developing drugs that target the wild-type and mutated KIT receptor, and these and other new therapeutic strategies for the treatment of mastocytosis are reviewed.
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http://dx.doi.org/10.1016/j.hoc.2012.08.008DOI Listing
December 2012

Direct surface analysis of time-resolved aerosol impactor samples with ultrahigh-resolution mass spectrometry.

Anal Chem 2012 Nov 30;84(22):9858-64. Epub 2012 Oct 30.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.

Aerosol particles in the atmosphere strongly influence the Earth's climate and human health, but the quantification of their effects is highly uncertain. The complex and variable composition of atmospheric particles is a main reason for this uncertainty. About half of the particle mass is organic material, which is very poorly characterized on a molecular level, and therefore it is challenging to identify sources and atmospheric transformation processes. We present here a new combination of techniques for highly time-resolved aerosol sampling using a rotating drum impactor (RDI) and organic chemical analysis using direct liquid extraction surface analysis (LESA) combined with ultrahigh-resolution mass spectrometry. This minimizes sample preparation time and potential artifacts during sample workup compared to conventional off-line filter or impactor sampling. Due to the high time resolution of about 2.5 h intensity correlations of compounds detected in the high-resolution mass spectra were used to identify groups of compounds with likely common sources or atmospheric history.
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http://dx.doi.org/10.1021/ac3020615DOI Listing
November 2012

The effect of humidity on the ozonolysis of unsaturated compounds in aerosol particles.

Phys Chem Chem Phys 2012 Jun 25;14(22):8023-31. Epub 2012 Apr 25.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

Atmospheric aerosol particles are important in many atmospheric processes such as: light scattering, light absorption, and cloud formation. Oxidation reactions continuously change the chemical composition of aerosol particles, especially the organic mass component, which is often the dominant fraction. These ageing processes are poorly understood but are known to significantly affect the cloud formation potential of aerosol particles. In this study we investigate the effect of humidity and ozone on the chemical composition of two model organic aerosol systems: oleic acid and arachidonic acid. These two acids are also compared to maleic acid an aerosol system we have previously studied using the same techniques. The role of relative humidity in the oxidation scheme of the three carboxylic acids is very compound specific. Relative humidity was observed to have a major influence on the oxidation scheme of maleic acid and arachidonic acid, whereas no dependence was observed for the oxidation of oleic acid. In both, maleic acid and arachidonic acid, an evaporation of volatile oxidation products could only be observed when the particle was exposed to high relative humidities. The particle phase has a strong effect on the particle processing and the effect of water on the oxidation processes. Oleic acid is liquid under all conditions at room temperature (dry or elevated humidity, pure or oxidized particle). Thus ozone can easily diffuse into the bulk of the particle irrespective of the oxidation conditions. In addition, water does not influence the oxidation reactions of oleic acid particles, which is partly explained by the structure of oxidation intermediates. The low water solubility of oleic acid and its ozonolysis products limits the effect of water. This is very different for maleic and arachidonic acid, which change their phase from liquid to solid upon oxidation or upon changes in humidity. In a solid particle the reactions of ozone and water with the organic particle are restricted to the particle surface and hence different regimes of reactivity are dictated by particle phase. The potential relevance of these three model systems to mimic ambient atmospheric processes is discussed.
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http://dx.doi.org/10.1039/c2cp24094gDOI Listing
June 2012

Feedback regulation by Atf3 in the endothelin-1-responsive transcriptome of cardiomyocytes: Egr1 is a principal Atf3 target.

Biochem J 2012 Jun;444(2):343-55

Institute of Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Whiteknights, UK.

Endothelin-1 promotes cardiomyocyte hypertrophy by inducing changes in gene expression. Immediate early genes including Atf3 (activating transcription factor 3), Egr1 (early growth response 1) and Ptgs2 (prostaglandin-endoperoxide synthase 2) are rapidly and transiently up-regulated by endothelin-1 in cardiomyocytes. Atf3 regulates the expression of downstream genes and is implicated in negative feedback regulation of other immediate early genes. To identify Atf3-regulated genes, we knocked down Atf3 expression in cardiomyocytes exposed to endothelin-1 and used microarrays to interrogate the transcriptomic effects. The expression of 23 mRNAs (including Egr1 and Ptgs2) was enhanced and the expression of 25 mRNAs was inhibited by Atf3 knockdown. Using quantitative PCR, we determined that knockdown of Atf3 had little effect on up-regulation of Egr1 mRNA over 30 min, but abolished the subsequent decline, causing sustained Egr1 mRNA expression and enhanced protein expression. This resulted from direct binding of Atf3 to the Egr1 promoter. Mathematical modelling established that Atf3 can suffice to suppress Egr1 expression. Given the widespread co-regulation of Atf3 with Egr1, we suggest that the Atf3-Egr1 negative feedback loop is of general significance. Loss of Atf3 caused abnormal cardiomyocyte growth, presumably resulting from the dysregulation of target genes. The results of the present study therefore identify Atf3 as a nexus in cardiomyocyte hypertrophy required to facilitate the full and proper growth response.
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http://dx.doi.org/10.1042/BJ20120125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365354PMC
June 2012

A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3).

Biochem J 2012 Mar;442(3):595-610

School of Biological Sciences, University of Reading, Whiteknights Campus, UK.

The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr(178)), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative 'non-canonical' pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein GOLGA2/gm130 (golgin A2/Golgi matrix protein 130). Activation of MST3 by calyculin A (a protein serine/threonine phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr(178)) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr(328)) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr(178)) phosphorylation increased MST3 kinase activity, but this activity was independent of MST3(Thr(328)) phosphorylation. Interestingly, MST3(Thr(328)) lies immediately C-terminal to a STRAD (Sterile20-related adaptor) pseudokinase-like site identified recently as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr(178)/Thr(328)) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr(328)) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.
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http://dx.doi.org/10.1042/BJ20112000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286863PMC
March 2012

The role of the P2X₇ receptor in infectious diseases.

PLoS Pathog 2011 Nov 10;7(11):e1002212. Epub 2011 Nov 10.

Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, New South Wales, Australia.

ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X₇ receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X₇ receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1β and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X₇ receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X₇ receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X₇ receptor, underscoring its importance in infectious disease.
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http://dx.doi.org/10.1371/journal.ppat.1002212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3213081PMC
November 2011

MICAL-1 is a negative regulator of MST-NDR kinase signaling and apoptosis.

Mol Cell Biol 2011 Sep 5;31(17):3603-15. Epub 2011 Jul 5.

Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

MICALs (molecules interacting with CasL) are atypical multidomain flavoenzymes with diverse cellular functions. The molecular pathways employed by MICAL proteins to exert their cellular effects remain largely uncharacterized. Via an unbiased proteomics approach, we identify MICAL-1 as a binding partner of NDR (nuclear Dbf2-related) kinases. NDR1/2 kinases are known to mediate apoptosis downstream of the mammalian Ste-20-like kinase MST1, and ablation of NDR1 in mice predisposes the mice to cancer as a result of compromised apoptosis. MST1 phosphorylates NDR1/2 kinases at their hydrophobic motif, thereby facilitating full NDR kinase activity and function. However, if and how this key phosphorylation event is regulated are unknown. Here we show that MICAL-1 interacts with the hydrophobic motif of NDR1/2 and that overexpression or knockdown of MICAL-1 reduces or augments NDR kinase activation or activity, respectively. Surprisingly, MICAL-1 is a phosphoprotein but not an NDR or MST1 substrate. Rather, MICAL-1 competes with MST1 for NDR binding and thereby antagonizes MST1-induced NDR activation. In line with this inhibitory effect, overexpression or knockdown of MICAL-1 inhibits or enhances, respectively, NDR-dependent proapoptotic signaling induced by extrinsic stimuli. Our findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling.
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http://dx.doi.org/10.1128/MCB.01389-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165550PMC
September 2011

Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active.

Cell Signal 2011 Feb 30;23(2):468-77. Epub 2010 Oct 30.

Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Whiteknights, Reading RG6 6UB, UK.

ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their k(cat) values were estimated to be minimally ~30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.
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http://dx.doi.org/10.1016/j.cellsig.2010.10.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038257PMC
February 2011

Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice.

Int J Parasitol 2011 Mar 31;41(3-4):301-8. Epub 2010 Oct 31.

Institute for Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, NSW 2007, Australia.

The P2X(7) receptor (P2X(7)R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X(7)R gene knockout (P2X(7)R(-/-); on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X(7)R(-/-) mice lost significantly more weight than C57Bl/6J mice from day 8p.i. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10p.i., P2X(7)R(-/-) mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12p.i., P2X(7)R(-/-) mice had lost 15.1±0.6%, C57Bl/6J had lost 10.1±0.8% and BALB/c had lost 4.8 ± 0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X(7)R(-/-) mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X(7)R(-/-) and C57Bl/6J mice produced more IL-1β and TNF than BALB/c mice. There was one important point of differentiation between the P2X(7)R(-/-) and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X(7)R-deficient mice.
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http://dx.doi.org/10.1016/j.ijpara.2010.10.001DOI Listing
March 2011

P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages.

J Immunol 2010 Jun 19;184(12):7040-6. Epub 2010 May 19.

Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, NSW, Australia.

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R-/-) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.
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http://dx.doi.org/10.4049/jimmunol.1000012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931343PMC
June 2010

Two haplotypes of the P2X(7) receptor containing the Ala-348 to Thr polymorphism exhibit a gain-of-function effect and enhanced interleukin-1beta secretion.

FASEB J 2010 Aug 1;24(8):2916-27. Epub 2010 Apr 1.

Department of Medicine, University of Sydney, Nepean Clinical School, Nepean Hospital, Penrith, New South Wales, Australia.

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.
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http://dx.doi.org/10.1096/fj.09-150862DOI Listing
August 2010

Genetics of the P2X7 receptor and human disease.

Purinergic Signal 2009 Jun 25;5(2):257-62. Epub 2009 Mar 25.

Nepean Clinical School, Nepean Hospital, University of Sydney, Penrith, NSW, 2750, Australia.

The P2RX7 gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) underlie the wide variation observed in P2X7 receptor responses. We review the discovery of those non-synonymous SNPs that affect receptor function and compare their frequencies in different ethnic populations. Analysis of pairwise linkage disequilibrium (LD) predicts a limited range of haplotypes. The strong LD between certain functional SNPs provides insight into published studies of the association between SNPs and human disease.
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http://dx.doi.org/10.1007/s11302-009-9136-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2686826PMC
June 2009

Endothelin-1 regulation of immediate early gene expression in cardiac myocytes: negative feedback regulation of interleukin 6 by Atf3 and Klf2.

Adv Enzyme Regul 2009 31;49(1):30-42. Epub 2008 Dec 31.

NHLI Division (Cardiovascular Sciences), Faculty of Medicine, Imperial College London, Flowers Building, Armstrong Road, London SW72AZ, UK.

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http://dx.doi.org/10.1016/j.advenzreg.2008.12.007DOI Listing
September 2009