ORCID Profile
0000-0001-9506-5998
Current Organisation
Florey Institute of Neuroscience and Mental Health
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Clinical Pharmacology And Therapeutics | Medical Biochemistry: Proteins And Peptides | Law | Biochemistry and Cell Biology | Analytical Biochemistry | Cellular Immunology | Intellectual Property | Public Policy
Understanding legal processes | Immune system and allergy | Prevention—biologicals (e.g. vaccines) | Health policy economic outcomes |
Publisher: Elsevier BV
Date: 12-2000
Publisher: Mary Ann Liebert Inc
Date: 2018
DOI: 10.1089/ADT.2017.799
Abstract: Protease-activated receptor 2 (PAR
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.MOLMED.2017.07.007
Abstract: Mechanical stress from blood flow has a significant effect on endothelial physiology, with a key role in initiating vasoregulatory signals. Disturbances in blood flow, such as in regions of disease-associated stenosis, arterial branch points, and sharp turns, can induce proatherogenic phenotypes in endothelial cells. The disruption of vascular homeostasis as a result of endothelial dysfunction may contribute to early and late stages of atherosclerosis, the underlying cause of coronary artery disease. In-depth knowledge of the mechanobiology of endothelial cells is essential to identifying mechanosensory complexes involved in the pathogenesis of atherosclerosis. In this review, we describe different blood flow patterns and summarize current knowledge on mechanosensory molecules regulating endothelial vasoregulatory functions, with clinical implications. Such information may help in the search for novel therapeutic approaches.
Publisher: Wiley
Date: 05-1998
Publisher: Elsevier BV
Date: 02-2013
Publisher: Wiley
Date: 08-2002
DOI: 10.1002/ART.10421
Abstract: To investigate the functional implications of CD44 splice variant expression in fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA). FLS were isolated from synovial tissue obtained from both diseased and nondiseased joints. The expression of splice variants containing exons v3 and v6 was analyzed using immunocytochemistry with exon-specific antibodies and reverse transcription-polymerase chain reaction followed by Southern blotting. The invasive capacity of the cells was studied in a transwell invasion assay. FLS obtained from RA joints expressed various CD44 splicing combinations containing the variant exons v3 and/or v6. These cells were highly invasive, whereas cells from normal tissues, which lacked expression of CD44 splice variants, were not. Variant exons CD44v3 and CD44v6 were instrumental in matrix invasion in vitro, with cells enriched for CD44v3 and v6 exhibiting greater invasion and antibodies that specifically recognize CD44v3 and v6 abrogating this capacity to invade. Invasive cells showed a reduced expression of CD44v7/8, and antibodies against this epitope had no significant effect on cellular infiltration of the matrix. The antibodies had no effect on cell migration into the porous section of the transwell. FLS obtained from patients with RA express CD44 splice variants and are highly invasive, whereas cells obtained from healthy tissue do not express these variants and are not invasive. Expression of the epitopes CD44v3 and CD44v6 is instrumental in the invasive capacity but not in cell migration. This finding highlights a functional implication for the expression of CD44 splice variants at the level of matrix degradation.
Publisher: SPIE
Date: 07-12-2013
DOI: 10.1117/12.2033734
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.BIOCEL.2016.07.012
Abstract: Transient receptor potential ion channels (TRP) are a superfamily of non-selective ion channels which are opened in response to a erse range of stimuli. The TRP vanilloid 4 (TRPV4) ion channel is opened in response to heat, mechanical stimuli, hypo-osmolarity and arachidonic acid metabolites. However, recently TRPV4 has been identified as an ion channel that is modulated by, and opened by intracellular signalling cascades from other receptors and signalling pathways. Although TRPV4 knockout mice show relatively mild phenotypes, some mutations in TRPV4 cause severe developmental abnormalities, such as the skeletal dyplasia and arthropathy. Regulated TRPV4 function is also essential for healthy cardiovascular system function as a potent agonist compromises endothelial cell function, leading to vascular collapse. A better understanding of the signalling mechanisms that modulate TRPV4 function is necessary to understand its physiological roles. Post translational modification of TRPV4 by kinases and other signalling molecules can modulate TRPV4 opening in response to stimuli such as mechanical and hyposmolarity and there is an emerging area of research implicating TRPV4 as a transducer of these signals as opposed to a direct sensor of the stimuli. Due to its wide expression profile, TRPV4 is implicated in multiple pathophysiological states. TRPV4 contributes to the sensation of pain due to hypo-osmotic stimuli and inflammatory mechanical hyperalsgesia, where TRPV4 sensitizaton by intracellular signalling leads to pain behaviors in mice. In the vasculature, TRPV4 is a regulator of vessel tone and is implicated in hypertension and diabetes due to endothelial dysfunction. TRPV4 is a key regulator of epithelial and endothelial barrier function and signalling to and opening of TRPV4 can disrupt these critical protective barriers. In respiratory function, TRPV4 is involved in cystic fibrosis, cilary beat frequency, bronchoconstriction, chronic obstructive pulmonary disease, pulmonary hypertension, acute lung injury, acute respiratory distress syndrome and cough.In this review we highlight how modulation of TRPV4 opening is a vital signalling component in a range of tissues and why understanding of TRPV4 regulation in the body may lead to novel therapeutic approaches to treating a range of disease states.
Publisher: Elsevier BV
Date: 02-2008
DOI: 10.1016/J.TOXICON.2007.10.012
Abstract: Many plant and animal toxins cause aversive behaviors in animals due to their pungent or unpleasant taste or because they cause other unpleasant senstations like pain. This article reviews the current state of knowledge of toxins that act at the TRPV1 ion channel, which is expressed in primary sensory neurons, is activated by multiple painful stimuli and is thought to be a key pain sensor and integrator. The recent finding that painful peptide "vanillotoxin" components of tarantula toxin activate the TRPV1 ion channel to cause pain led us to survey what is known about toxins that act at this receptor. Toxins from plants, spiders and jellyfish are considered. Where possible, structural information about sites of interaction is considered in relation to toxin-binding sites on the Kv ion channel, for which more structural information exists. We discuss a developing model where toxin agonists such as resiniferatoxin and vanillotoxins are proposed to interact with a region of TRPV1 that is homologous to the "voltage sensor" in the Kv1.2 ion channel, to open the channel and activate primary sensory nerves, causing pain.
Publisher: Springer Science and Business Media LLC
Date: 08-06-2014
DOI: 10.1007/S00424-014-1539-6
Abstract: Proteolytic activation of protease-activated receptor 2 (PAR2) may represent a major mechanism of regulating the transient receptor potential vanilloid 4 (TRPV4) non-selective cation channel in pathophysiological conditions associated with protease activation (e.g. during inflammation). To provide electrophysiological evidence for PAR2-mediated TRPV4 regulation, we characterised the properties of human TRPV4 heterologously expressed in Xenopus laevis oocytes in the presence and absence of co-expressed human PAR2. In outside-out patches from TRPV4 expressing oocytes, we detected single-channel activity typical for TRPV4 with a single-channel conductance of about 100 pS for outward and 55 pS for inward currents. The synthetic TRPV4 activator GSK1016790A stimulated TRPV4 mainly by converting previously silent channels into active channels with an open probability of nearly one. In oocytes co-expressing TRPV4 and PAR2, PAR2 activation by trypsin or by specific PAR2 agonist SLIGRL-NH2 potentiated the GSK1016790A-stimulated TRPV4 whole-cell currents several fold, indicative of channel sensitisation. Pre-incubation of oocytes with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM did not reduce the stimulatory effect of PAR2 activation on TRPV4, which indicates that the effect is independent of intracellular calcium signalling. Neutrophil elastase, a biased agonist of PAR2 that does not induce intracellular calcium signalling, also caused a PAR2-dependent sensitisation of TRPV4. The Rho-kinase inhibitor Y27362 abolished elastase-stimulated sensitisation of TRPV4, which indicates that Rho-kinase signalling plays a critical role in PAR2-mediated TRPV4 sensitisation by the biased agonist neutrophil elastase. During acute inflammation, neutrophil elastase may sensitise TRPV4 by a mechanism involving biased agonism of PAR2 and activation of Rho-kinase.
Publisher: Springer Science and Business Media LLC
Date: 02-10-2011
DOI: 10.1038/NG.945
Abstract: Familial digital arthropathy-brachydactyly (FDAB) is a dominantly inherited condition that is characterized by aggressive osteoarthropathy of the fingers and toes and consequent shortening of the middle and distal phalanges. Here we show in three unrelated families that FDAB is caused by mutations encoding p.Gly270Val, p.Arg271Pro and p.Phe273Leu substitutions in the intracellular ankyrin-repeat domain of the cation channel TRPV4. Functional testing of mutant TRPV4 in HEK-293 cells showed that the mutant proteins have poor cell-surface localization. Calcium influx in response to the synthetic TRPV4 agonists GSK1016790A and 4αPDD was significantly reduced, and mutant channels did not respond to hypotonic stress. Others have shown that gain-of-function TRPV4 mutations cause skeletal dysplasias and peripheral neuropathies. Our data indicate that TRPV4 mutations that reduce channel activity cause a third phenotype, inherited osteoarthropathy, and show the importance of TRPV4 activity in articular cartilage homeostasis. Our data raise the possibility that TRPV4 may also have a role in age- or injury-related osteoarthritis.
Publisher: Wiley
Date: 25-07-2014
DOI: 10.1111/BPH.12750
Publisher: AIP Publishing
Date: 07-2014
DOI: 10.1063/1.4893272
Abstract: Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca2+]i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca2+]i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca2+]i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.
Publisher: Wiley
Date: 08-1984
DOI: 10.1111/J.1432-1033.1984.TB08357.X
Abstract: A cDNA clone corresponding to the mature form of ornithine transcarbamylase (OTCase) was selected from a rat liver cDNA library constructed in bacteriophage lambda gt10. OTCase clones were selected using a synthetic DNA probe of 15 bases corresponding to the 3' end of the OTCase mRNA [Horwich, A. L., Kraus, J.P., Williams, K., Kalousek, F., Königsberg, W. & Rosenberg, L.E. (1983) Proc. Natl Acad. Sci. USA, 80, 4258-4262]. Putative OTCase clones were subcloned into the expression vector, pUC9, and the identity of inserts confirmed by colony immunoassay and by electrophoretic transfer of cloned proteins from sodium dodecyl sulphate olyacrylamide gels to nitrocellulose filters followed by probing with monospecific anti-OTCase antibodies and 125I-labelled protein A. A clone corresponding to the full-length mature form of rat liver OTCase (plus 15 amino acids from Escherichia coli beta-galactosidase) was obtained and the identity of the clone was confirmed by comparison of the 5' sequence with a limited N-terminal amino acid sequence [Lusty, C., Jilka, R. L. & Nietsch, E. H. (1979) J. Biol. Chem. 254, 10030-10036]. A sequence discrepancy between the published sequence (Lusty et al.) and the sequence predicted from the cDNA structure is noted.
Publisher: Elsevier BV
Date: 03-2002
DOI: 10.1016/S0092-8674(02)00652-9
Abstract: A distinct subset of sensory neurons are thought to directly sense changes in thermal energy through their termini in the skin. Very little is known about the molecules that mediate thermoreception by these neurons. Vanilloid Receptor 1 (VR1), a member of the TRP family of channels, is activated by noxious heat. Here we describe the cloning and characterization of TRPM8, a distant relative of VR1. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neurons. Cells overexpressing the TRPM8 channel can be activated by cold temperatures and by a cooling agent, menthol. Our identification of a cold-sensing TRP channel in a distinct subpopulation of sensory neurons implicates an expanded role for this family of ion channels in somatic sensory detection.
Publisher: Wiley
Date: 09-11-1992
DOI: 10.1016/0014-5793(92)80934-9
Abstract: Using a PKC-epsilon cDNA probe a cDNA for PKC-eta has been cloned from a rat lung cDNA library. When expressed in COS cells, rat PKC-eta appeared as an 84 kDa protein. PKC-eta expressed in COS cells, was solubilized by 1% Triton X-100 and purified away from the endogenous PKC-alpha by ammonium sulphate fractionation. The activity of this PKC-eta preparation was characterized with respect to cofactor dependence and substrate specificity. Various PKC pseudosubstrate peptides are phosphorylated by PKC-eta in a phospholipid and TPA-dependent but calcium-independent manner. The polypeptide histone IIIS is a poor substrate.
Publisher: American Chemical Society (ACS)
Date: 20-12-2005
DOI: 10.1021/JM051058X
Abstract: Vanilloid receptor 1 (VR1, TRPV1) is a cation-selective ion channel that is expressed on primary afferent neurons and is upregulated following inflammation and nerve damage. Blockers of this channel may have utility in the treatment of chronic nociceptive and neuropathic pain. Here, we describe the optimization from a high throughput screening hit, of a series of 6-aryl-7-isopropylquinazolinones that are TRPV1 antagonists in vitro. We also demonstrate that one compound is active in vivo against capsaicin-induced hyperalgesia and in models of neuropathic and nociceptive pain in the rat.
Publisher: Mary Ann Liebert Inc
Date: 04-1985
Abstract: Ornithine transcarbamylase, one of the enzymes of the urea cycle in ureotelic organisms, is synthesized in the cytoplasm of hepatocytes as a precursor larger than the mature form found in the mitochondrial matrix. We deduced the amino acid sequence of the precursor of ornithine transcarbamylase from rat liver from the nucleotide sequence of overlapping cDNA clones spanning the complete coding region, 3' untranslated region, and most of the 5' untranslated region of the mRNA. The mature enzyme consists of 322 amino acids and is derived from the larger precursor by proteolytic removal of 32 amino acids from the amino-terminus. The amino-terminal extension contains eight basic and no acidic residues. This highly basic character appears to be a feature of presequences on cytoplasmically synthesized mitochondrial proteins. Comparison of the amino acid sequence determined for the enzyme from rat with that from human liver (Horwich et al., 1984) shows that there is a high degree of homology between the sequences of the mature protein (93%) and relatively less homology between the sequences of the amino-terminal extension (72%). The ornithine transcarbamylase from rat liver also shows a considerable degree of amino acid homology (44%) with the enzyme from Escherichia coli (Van Vliet et al., 1984) and leads to suggestions about residues involved in substrate binding and catalysis. An analysis of levels of RNA in fetal and neonatal liver shows that ornithine transcarbamylase mRNA levels increase from about 40% of adult levels at day 14 of gestation to a peak at day 20 of gestation, and, after a drop around the time of birth, rises to adult levels during the second week after birth.
Publisher: Elsevier BV
Date: 03-2003
DOI: 10.1016/S0092-8674(03)00158-2
Abstract: Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.
Publisher: Elsevier BV
Date: 06-2000
DOI: 10.1016/S0014-2999(00)00323-X
Abstract: In WI-38 human fibroblasts, interleukin-1 beta and tumour necrosis factor-alpha (TNF-alpha) increased bradykinin B(1) receptor mRNA, which peaked between 2 and 4 h, remaining elevated for 20 h. Binding of the bradykinin B(1) receptor selective ligand [3H]des-Arg(10)-kallidin, also increased, peaking at 4 h and remaining elevated for 20 h. The B(max) value for [3H]des-Arg(10)-kallidin rose from 280+/-102 fmol/mg (n=3) to 701+/-147 fmol/mg (n=3), but the K(D) value remained unaltered (control, 1.04+/-0.33 nM (n=3) interleukin-1 beta, 0.88+/-0.41 nM (n=3)). The interleukin-1 beta-induced [3H]des-Arg(10)-kallidin binding sites were functional receptors, as bradykinin B(1) receptor agonist-induced responses increased in treated cells. Bradykinin B(2) receptor mRNA and [3H]bradykinin binding were upregulated by interleukin-1 beta, but not TNF-alpha. The effect of interleukin-1 beta on bradykinin B(2) receptors was smaller than for bradykinin B(1) receptors. Cycloheximide prevented interleukin-1 beta-mediated increases in B(1) and B(2) binding, but not mRNA suggesting that de novo synthesis of a transcriptional activator was unnecessary.
Publisher: No publisher found
Date: 2014
DOI: 10.1016/J.BIOTECHADV.2013.11.008
Abstract: Microfluidics has facilitated immunological studies by enhancing speed, efficiency and sensitivity of current analysis methods. It offers miniaturization of current laboratory equipment, and enables analysis of clinical s les without the need for sophisticated infrastructure. More importantly, microfluidics offers unique capabilities including conducting multiple serial or parallel tasks as well as providing complex and precisely controlled environmental conditions that are not achievable using conventional laboratory equipment. Microfluidics is a promising technology for fundamental and applied immunological studies, allowing generation of high throughput, robust and portable platforms, opening a new area of automation in immunology.
Publisher: Elsevier BV
Date: 05-1992
DOI: 10.1016/0165-1110(92)90008-W
Abstract: Treatment of Syrian hamster kidney cells with the demethylating agent 5-aza-2'-deoxycytidine (azadC) increased both the frequency and the rate of gene lification appreciably. AzadC caused substantial DNA demethylation, which is likely to be responsible. The magnitude of the increases depended on the concentrations of both azadC and the drug used for selection. A transient stress response is not responsible since the increases were not dependent on cytotoxicity and were still apparent after several weeks. We discuss mechanisms by which azadC treatment may induce lification by rendering DNA more prone to this process or by increasing the transcription of genes whose protein products stimulate lification.
Publisher: Wiley
Date: 07-1997
Abstract: CD44 is a broadly expressed cell surface glycoprotein which is the major cell surface receptor for the glycosaminoglycan, hyaluronan. In humans, alternative splicing of up to 9 variant exons (v2-v10) into CD44 mRNA, together with post-translational modification via glycosylation and chondroitin sulfate attachment has the potential of generating a large number of CD44 isoforms. Insertion of these various exons has the potential to change the functional capacities of the molecule and has implications in disease. We have analyzed CD44 splice variant expression in cultured VCAM-1-positive synovial fibroblasts isolated from patients with osteo- or rheumatoid arthritis and from normal synovium. Rheumatoid and osteoarthritic tissue express CD44 splice variants at the cell surface level. At the mRNA level exons v3, v6, v7, v8, v9 and v10 were detected in different splicing combinations. Rheumatoid tissue showed high expression, osteoarthritic tissues showed great variation. In contrast, non-inflamed tissue showed no splicing events. Our results indicate that the nature of CD44 splice variant expression may be linked to the inflammatory state of the synovial joint.
Publisher: Springer Science and Business Media LLC
Date: 23-07-2015
DOI: 10.1038/SREP11973
Abstract: Immobilisation of cells is an important feature of many cellular assays, as it enables the physical/chemical stimulation of cells whilst, monitoring cellular processes using microscopic techniques. Current approaches for immobilising cells, however, are h ered by time-consuming processes, the need for specific antibodies or coatings and adverse effects on cell integrity. Here, we present a dielectrophoresis-based approach for the robust immobilisation of cells and analysis of their responses under high shear flows. This approach is quick and label-free and more importantly, minimises the adverse effects of electric field on the cell integrity, by activating the field for a short duration of 120 s, just long enough to immobilise the cells, after which cell culture media (such as HEPES) is flushed through the platform. In optimal conditions, at least 90% of the cells remained stably immobilised, when exposed to a shear stress of 63 dyn/cm 2 . This approach was used to examine the shear-induced calcium signalling of HEK-293 cells expressing a mechanosensitive ion channel, transient receptor potential vaniloid type 4 (TRPV4), when exposed to the full physiological range of shear stress.
Publisher: Canadian Science Publishing
Date: 07-1995
DOI: 10.1139/Y95-105
Abstract: We describe the results of functional studies on DNA clones encoding functional bradykinin receptors derived from human, rat, and mouse sources and including both genomic and complementary DNA clones. In both the Xenopus oocyte and the COS cell expression systems, the receptors from human and rat showed the pharmacological properties of B 2 receptors, but receptors from mouse displayed both B 1 - and B 2 -like pharmacological properties. We further investigated the molecular relationship between the B 1 and B 2 receptor subtypes expressed by a human fibroblast cell line, and we demonstrate that these two receptor subtypes are encoded by distinct mRNA species.Key words: B 1 receptor, antisense, Xenopus oocyte.
Publisher: Elsevier BV
Date: 06-2012
Publisher: Wiley
Date: 18-03-2015
DOI: 10.1111/BPH.13072
Publisher: Elsevier BV
Date: 05-2014
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.PHRS.2018.05.009
Abstract: The aim of this study was to better understand the role of TRPV4 in the regulation of blood vessel dilatation by blood flow and activation of GPCRs. Using pressure myography, the dilator responses to the TRPV4 agonist GSK1016790A and to acetylcholine, were examined in rat cremaster arterioles exposed to either no shear stress or to 200 μl/min flow for 6 min. In control vessels GSK1016709A caused vasodilatation (pEC
Publisher: OSA
Date: 2010
Publisher: Elsevier BV
Date: 08-2016
DOI: 10.1016/J.JOCA.2016.03.019
Abstract: Scottish fold cats, named for their unique ear shape, have a dominantly inherited osteochondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and progressive joint destruction. This study aimed to identify the gene and the underlying variant responsible for the osteochondrodysplasia. DNA s les from 44 Scottish fold and 54 control cats were genotyped using a feline DNA array and a case-control genome-wide association analysis conducted. The gene encoding a calcium permeable ion channel, transient receptor potential cation channel, subfamily V, member 4 (TRPV4) was identified as a candidate within the associated region and sequenced. Stably transfected HEK293 cells were used to compare wild-type and mutant TRPV4 expression, cell surface localisation and responses to activation with a synthetic agonist GSK1016709A, hypo-osmolarity, and protease-activated receptor 2 stimulation. The dominantly inherited folded ear and osteochondrodysplasia in Scottish fold cats is associated with a p.V342F substitution (c.1024G>T) in TRPV4. The change was not found in 648 unaffected cats. Functional analysis in HEK293 cells showed V342F mutant TRPV4 was poorly expressed at the cell surface compared to wild-type TRPV4 and as a consequence the maximum response to a synthetic agonist was reduced. Mutant TRPV4 channels had a higher basal activity and an increased response to hypotonic conditions. Access to a naturally-occurring TRPV4 mutation in the Scottish fold cat will allow further functional studies to identify how and why the mutations affect cartilage and bone development.
Publisher: Springer Science and Business Media LLC
Date: 27-02-2014
DOI: 10.1038/SREP04217
Publisher: PeerJ
Date: 28-01-2014
DOI: 10.7717/PEERJ.248
Publisher: Springer Berlin Heidelberg
Date: 2007
Publisher: Wiley
Date: 06-2007
DOI: 10.1111/J.1460-9568.2007.05562.X
Abstract: The choroid plexuses secrete cerebrospinal fluid (CSF) and regulate the brain's internal environment via the blood-CSF barrier. The permeability properties of the blood-CSF interface have been studied previously in adult and immature brains, however, little is known about the development of CSF secretion and its modulation. ATP influences secretion in other epithelia via ionotropic P2X or metabotropic P2Y receptors. P2 receptors have frequently been found to be down-regulated in the postnatal period, suggesting a developmental role for purinergic and pyrimidine signalling. The present study investigated the expression of P2 receptors in lateral ventricular choroid plexus in relation to recent studies of aquaporin-1 expression and rapid expansion of the lateral ventricles in rat embryos. In the present study mRNAs for all known mammalian nucleotide receptor subtypes, except P2X(7), were identified from as early as E15. P2X(7) mRNA was detected from E18. Indications of differential expression patterns were observed for the different subtypes during development: an apparent increase in expression for P2Y(2) and P2X(7), a decline in P2X(1-2,4), no detectable difference in expression levels for P2X(6) and P2Y(12-13) and transient expression peaks for P2X(3,5) and P2Y(1,4,6,14). P2X(4,5,7) and P2Y(1,4) receptor proteins were detected immunohistochemically in the choroidal epithelium from early in development (E15 or E18). Their differing developmental profiles suggest specific roles in the development of CSF secretion that may have particular relevance for the rapid expansion of the ventricles that occurs in the embryo. P2X(5) and P2Y(6) were also detected in the developing neuropendyma from P0 and P9, respectively.
Publisher: Elsevier BV
Date: 12-2006
Publisher: Proceedings of the National Academy of Sciences
Date: 11-04-2011
Abstract: The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-cl ing of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.
Publisher: Elsevier BV
Date: 2005
DOI: 10.1016/J.NEUROSCIENCE.2005.06.009
Abstract: We have localized cannabinoid receptor 2 protein in rat and mouse somatic sensory nervous system, using an antibody that recognizes mouse cannabinoid receptor 2. Little or no cannabinoid receptor 2 immunoreactivity was found in sections of naive rat or mouse dorsal root ganglia or spinal cord. This was in accord with the lack of detectable cannabinoid receptor 2 mRNA in (dorsal root ganglion) neurons by in situ hybridization experiments described in the literature. However, we could detect cannabinoid receptor 2 immunoreactivity following unilateral nerve damage-either by sciatic nerve section, or by spinal nerve ligation. It was localized to the superficial laminae of the dorsal horn of the spinal cord, ipsilateral to the nerve damage, coincident with the area of termination of damaged afferents which was marked by loss of isolectin B4 binding. This upregulation was not seen in cannabinoid receptor 2 null mice. The cannabinoid receptor 2 protein in spinal cord appeared to be expressed on sensory neuron afferent terminals as it colocalized with two markers of damaged afferents, namely growth associated protein-43 and the neuropeptide galanin. Moreover, it did not colocalize with markers of activated microglial cells (OX-42) or astroglial cells (glial fibrillary acidic protein) in rat spinal cord. In the peripheral nerve, accumulation of cannabinoid receptor 2 immunoreactivity was seen in nerve sections proximal, but not distal, to the ligation site, suggesting transport down the nerve from the cell bodies. Although convincing cannabinoid receptor 2 immunoreactivity was seen in neither uninjured nor injured dorsal root ganglion neuron cell bodies in tissue sections, expression was detectable in isolated, cultured neurons that had received a prior axotomy in vivo. This clear demonstration of CB(2) receptors on sensory neurons suggests an additional cellular target for CB(2) agonist induced analgesia, at least in neuropathic models.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 31-10-2014
Abstract: Sensory nerves are equipped with receptors and ion channels that allow them to detect and respond to erse chemical, mechanical, and thermal stimuli. These sensory proteins include G protein-coupled receptors (GPCRs) and transient receptor potential (TRP) ion channels. A subclass of peptidergic sensory nerves express GPCRs and TRP channels that detect noxious, irritant, and inflammatory stimuli. Activation of these nerves triggers protective mechanisms that lead to withdrawal from danger (pain), removal of irritants (itch, cough), and resolution of infection (neurogenic inflammation). The GPCR-TRP axis is central to these mechanisms. Signals that emanate from the GPCR superfamily converge on the small TRP family, leading to channel sensitization and activation, which lify pain, itch, cough, and neurogenic inflammation. Herein we discuss how GPCRs and TRP channels function independently and synergistically to excite sensory nerves that mediate noxious and irritant responses and inflammation in the skin and the gastrointestinal and respiratory systems. We discuss the signaling mechanisms that underlie the GPCR-TRP axis and evaluate how new information about the structure of GPCRs and TRP channels provides insights into their functional interactions. We propose that a deeper understanding of the GPCR-TRP axis may facilitate the development of more selective and effective therapies to treat dysregulated processes that underlie chronic pain, itch, cough, and inflammation.
Publisher: Elsevier BV
Date: 04-1999
DOI: 10.1016/S0002-9440(10)65367-9
Abstract: Rheumatoid arthritis is characterized by hyperplasia of the synovial lining and invasion of cartilage and bone by a subset of resident synovial cells named fibroblast-like synoviocytes. They are characterized by elevated expression of the vascular cell adhesion molecule-1 (VCAM-1). The intensity of VCAM-1 expression correlates with the degree of inflammation of the synovial joint. Differential VCAM-1 expression may determine inflammatory cell accumulation through its interaction with leukocytes that express the counterreceptor integrins alpha4beta1 and alpha4beta7. Elevated levels of VCAM-1 expression are thought to be a consequence of the presence of inflammatory mediators, in particular IL-1beta and TNF-alpha. Fibroblast-like synoviocytes rapidly up-regulate VCAM-1 expression in response to IL-1beta and TNF-alpha, but also to IL-4. However, we now show that the response to IL-1beta or TNF-alpha is of a brief transient nature, even when applied continuously over a period of 12 days, whereas the response to IL-4 or IL-13 is sustained. Great synergy is obtained by combining either IL-4 or IL-13 with TNF-alpha, which results in a highly elevated but also sustained expression of VCAM-1. The mechanism by which IL-4 or IL-13 prolongs VCAM-1 expression can be explained by a dramatic increase in the half-life of VCAM-1 mRNA.
Publisher: American Physiological Society
Date: 15-10-2012
DOI: 10.1152/AJPHEART.00418.2012
Abstract: Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations times their EC 50 determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.
Publisher: Springer Science and Business Media LLC
Date: 21-11-2017
DOI: 10.1038/S41598-017-16276-7
Abstract: Localized Ca 2+ influx via TRPV4 on the surface of endothelial cells greatly influences endothelial adaptation to blood flow, but how mechanical stress from blood flow controls TRPV4 integration into this physiological function is not fully understood. Here, we studied the spatial organization of TRPV4 and its relationship to the adherens junction component β-catenin using single- and dual-color direct stochastic optical reconstruction microscopy (dSTORM). In non-stimulated endothelial cells, TRPV4 is clustered in small protein islands, as is β-catenin. Using dual-color imaging, we found that TRPV4 and β-catenin reside in similar islands and can be found at both the basolateral and basal membranes. Following shear stress stimulation, TRPV4 molecules formed smaller clusters, with the majority residing outside of clusters. Further shear stress stimulation changed the molecular distribution of TRPV4 molecules, limiting them to the basal membrane. This redistribution and the smaller clusters resulted in the segregation of TRPV4 from β-catenin. Furthermore, TRPV4 trafficking was controlled by focal adhesion kinase and activation of the α5ß1 integrin. These highly differentiated spatial redistributions suggest that mechanotransduction of blood flow is controlled via a more complex hierarchy than previously thought.
Publisher: Frontiers Media SA
Date: 23-01-2019
Publisher: Elsevier BV
Date: 06-1999
DOI: 10.1016/S0014-2999(99)00315-5
Abstract: This report describes the characterisation of cloned rat and human bradykinin B1 receptors in African green monkey kidney fibroblast (Cos-7) cells. A ligand binding assay with [3H]des-Arg10-kallidin was used to compare their pharmacology with respect to known bradykinin B1 and B2 receptor ligands. In addition, the pharmacology of T-kinin and its' derivative des-Arg11-T-kinin was investigated. The cloned rat receptor had a similar pharmacology to that of the recently described mouse receptor and differs from that described for the human receptor. The rat receptor had a higher affinity for des-Arg11-T-kinin than the human receptor. These differences in pharmacological properties may relate to the presence of T-kinin, bradykinin and their des-Arg derivatives as the major physiological peptides in rat and the predominance of kallidin and its derivatives in human. We confirm that the rat bradykinin B1 receptor gene is organised in a two exon structure and differs from the human gene which has a three exon structure and we further examine the inducible expression of this gene in a wide range of tissues using Northern blotting.
Publisher: Wiley
Date: 04-07-2016
Abstract: Studies have utilised immortalised mouse cerebral endothelial cells (bEnd.3) exposed to oxygen glucose deprivation (OGD) to study blood-brain barrier (BBB) disruption after ischaemia. However, there is a paucity of literature describing the duration of OGD (and reoxygenation [RO]) required to best simulate BBB disruption in vivo. In this study we assessed BBB disruption in bEnd.3 cells after exposure to a range of OGD periods, and also after OGD + RO. Exposure of bEnd.3 monolayers to 4, 6, 16, or 24 hours of OGD resulted in a significant increase in permeability. The hyperpermeability after 16 or 24 hours was associated with decreased expression of tight junction proteins (occludin and claudin-5). Furthermore, there was a decrease in cell viability and increased expression of the pro-apoptotic protein, cleaved caspase-3. Exposure of bEnd.3 monolayers to 1 hour OGD+ 23 hours RO exacerbated hyperpermeability relative to 1 hour OGD, which was associated with decreased expression levels of occludin and ZO-1, but no change in cell viability or caspase-3. 4 hours OGD + 23 hours RO exacerbated hyperpermeability, decreased expression levels of tight junction proteins, decreased cell viability, and increased caspase-3 expression. Thus, bEnd.3 cells exhibit hyperpermeability, a loss of tight junction proteins, and undergo cell death, after exposure to prolonged periods of OGD. Moreover, they exhibit exacerbated hyperpermeability, a loss of tight junction proteins, and increased expression of caspase-3 after OGD + RO. These findings will facilitate the use of this cell line in studies of BBB disruption and for the testing of therapeutics.
Publisher: Elsevier BV
Date: 08-2020
Publisher: Springer Science and Business Media LLC
Date: 07-04-2017
Publisher: AIP Publishing
Date: 03-2016
DOI: 10.1063/1.4945309
Abstract: Microfluidic platforms enable a variety of physical or chemical stimulation of single or multiple cells to be examined and monitored in real-time. To date, intracellular calcium signalling research is, however, predominantly focused on observing the response of cells to a single mode of stimulation consequently, the sensitising/desensitising of cell responses under concurrent stimuli is not well studied. In this paper, we provide an extended Discontinuous Dielectrophoresis procedure to investigate the sensitising of chemical stimulation, over an extensive range of shear stress, up to 63 dyn/cm2, which encompasses shear stresses experienced in the arterial and venus systems (10 to 60 dyn/cm2). Furthermore, the TRPV4-selective agonist GSK1016790A, a form of chemical stimulation, did not influence the ability of the cells' to remain immobilised under high levels of shear stress thus, enabling us to investigate shear stress stimulation on agonism. Our experiments revealed that shear stress sensitises GSK1016790A-evoked intracellular calcium signalling of cells in a shear-stimulus dependent manner, as observed through a reduction in the cellular response time and an increase in the pharmacological efficacy. Consequently, suggesting that the role of TRPV4 may be underestimated in endothelial cells—which experience high levels of shear stress. This study highlights the importance of conducting studies at high levels of shear stress. Additionally, our approach will be valuable for examining the effect of high levels of shear on different cell types under different conditions, as presented here for agonist activation.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 07-05-2012
Abstract: The therapeutic potential of transient receptor potential vanilloid type 1 (TRPV1) antagonists for chronic pain has been recognized for more than a decade. However, preclinical and clinical data revealed that acute pharmacological blockade of TRPV1 perturbs thermoregulation, resulting in hyperthermia, which is a major hurdle for the clinical development of these drugs. Here, we describe the properties of 7-tert-butyl-6-(4-chloro-phenyl)-2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (BCTP), a TRPV1 antagonist with excellent analgesic properties that does not induce significant hyperthermia in rodents at doses providing maximal analgesia. BCTP is a classic polymodal inhibitor of TRPV1, blocking activation of the human channel by capsaicin and low pH with IC(50) values of 65.4 and 26.4 nM, respectively. Similar activity was observed with rat TRPV1, and the inhibition by BCTP was competitive and reversible. BCTP also blocked heat-induced activation of TRPV1. In rats, the inhibition of capsaicin-induced mechanical hyperalgesia was observed with a D(50) value of 2 mg/kg p.o. BCTP also reversed visceral hypersensitivity and somatic inflammatory pain, and using a model of neuropathic pain in TRPV1 null mice we confirmed that its analgesic properties were solely through the inhibition of TRPV1. We were surprised to find that BCTP administered orally induced only a maximal 0.6°C increase in core body temperature at the highest tested doses (30 and 100 mg/kg), contrasting markedly with N-[4-({6-[4-(trifluoromethyl)phenyl]pyrimidin-4-yl}oxy)-1,3-benzothiazol-2-yl]acetamide (AMG517), a clinically tested TRPV1 antagonist, which induced marked hyperthermia (>1°C) at doses eliciting submaximal reversal of capsaicin-induced hyperalgesia. The combined data indicate that TRPV1 antagonists with a classic polymodal inhibition profile can be identified where the analgesic action is separated from the effects on body temperature.
Publisher: Elsevier BV
Date: 07-1998
DOI: 10.1016/S0304-3940(98)00475-3
Abstract: A vanilloid receptor (VR1) has recently been cloned and shown to be a target for capsaicin, the excitotoxic component of capsicum peppers (Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D. and Julius, D., Nature, 389 (1997) 816-824). The effects of capsaicin appear to be selective for a subset of sensory neurones which includes polymodal nociceptors. The present study describes the distribution of VR1 mRNA, together with measurements of capsaicin sensitivity, in sensory nerve ganglia of different embryological origins and a single sympathetic ganglion, the superior cervical ganglion (SCG). In situ hybridisation revealed the expression of VR1 mRNA in small-to-medium-sized neurones of the dorsal root, trigeminal and vagal ganglia. No hybridisation signal was observed in the SCG neurones. This pattern of expression correlated with capsaicin sensitivity measured by whole-cell voltage cl where, in similar sized cells, over 80% of neurones from dorsal root and vagal ganglia were capsaicin sensitive, but all SCG neurones were insensitive to capsaicin.
Publisher: Wiley
Date: 21-02-1984
DOI: 10.1007/BF01801764
Abstract: The growing demand for personal healthcare monitoring requires a challenging combination of performance, size, power, and cost that is difficult to achieve with existing gas sensor technologies. This paper presents a new CMOS monolithic gas sensor microsystem that meets these requirements through a unique combination of electrochemical readout circuits, post-CMOS planar electrodes, and room temperature ionic liquid (RTIL) sensing materials. The architecture and design of the CMOS-RTIL-based monolithic gas sensor are described. The monolithic device occupies less than 0.5mm
Publisher: Bioscientifica
Date: 07-2020
DOI: 10.1530/ERC-20-0092
Abstract: Identifying the factors stimulating prostate cancer cells migration and invasion has the potential to bring new therapeutic targets to the clinic. Cysteine-rich secretory protein 3 (CRISP3) is one of the most highly upregulated proteins during the transition of a healthy human prostatic epithelium to prostate cancer. Here we show using a genetically engineered mouse model of prostate cancer that CRISP3 production greatly facilitates disease progression from carcinoma in situ to invasive prostate cancer in vivo . This interpretation was confirmed using both human and mouse prostate cancer cell lines, which showed that exposure to CRISP3 enhanced cell motility and invasion. Further, using mass spectrometry, we show that CRISP3 induces changes in abundance of a subset of cell-cell adhesion proteins, including LASP1 and TJP1 both in vivo and in vitro . Collectively, these data identify CRISP3 as being pro-tumorigenic in the prostate and validate it as a potential target for therapeutic intervention.
Publisher: Wiley
Date: 23-08-1993
DOI: 10.1016/0014-5793(93)80208-C
Abstract: Three protein kinase C (PKC)-eta mutants were constructed in which the whole or part of the pseudosubstrate site was replaced with corresponding parts of the PKC-alpha pseudosubstrate site. The resulting chimaeric kinases were compared with wild-type PKC-eta in their ability to phosphorylate a PKC-eta peptide substrate or histone. Changes in the pseudosubstrate site of PKC-eta are accompanied by changes in substrate selectivity, indicating that the substrate selectivity observed for PKC-eta is at least in part due to its pseudosubstrate site.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 2003
Abstract: Vanilloid receptor type 1 (VR1) (TRPV1) is a ligand-gated ion channel expressed on sensory nerves that responds to noxious heat, protons, and chemical stimuli such as capsaicin. Herein, we have examined the activity of the VR1 antagonist capsazepine in models of inflammatory and neuropathic pain in the rat, mouse, and guinea pig. In naïve animals, subcutaneous administration of capsazepine (10-100 mg/kg s.c.) did not affect withdrawal thresholds to noxious thermal or mechanical stimuli. However, pretreatment with capsazepine prevented the development of mechanical hyperalgesia induced by intraplantar injection of capsaicin, with a similar potency in all three species. Capsazepine (up to 100 mg/kg s.c.) did not affect mechanical hyperalgesia in the Freund's complete adjuvant (FCA)-inflamed hind paw of the rat or mouse. Strikingly, capsazepine (3-30 mg/kg s.c.) produced up to 44% reversal of FCA-induced mechanical hyperalgesia in the guinea pig. Capsazepine also produced significant reversal of carageenan-induced thermal hyperalgesia in the guinea pig at 30 mg/kg s.c., but was ineffective in the rat. Similarly, in the partial sciatic nerve ligation model of neuropathic pain, capsazepine was surprisingly effective in the guinea pig, producing up to 80% reversal of mechanical hyperalgesia (1-30 mg/kg s.c.) but had no effect in the rat or mouse. These data show that VR1 antagonists have antihyperalgesic activity in animal models of chronic inflammatory and neuropathic pain, and illustrate species differences in the in vivo pharmacology of VR1 that correlate with differences in pharmacology previously seen in vitro.
Publisher: Springer Berlin Heidelberg
Date: 2013
Publisher: Public Library of Science (PLoS)
Date: 05-04-2013
Publisher: Elsevier BV
Date: 04-2004
Publisher: Wiley
Date: 04-1994
DOI: 10.1046/J.1471-4159.1994.62041247.X
Abstract: Bradykinin receptors have been sub ided into at least two major pharmacological subtypes, B1 and B2. The cDNAs encoding functional B2 receptors have recently been cloned, but no molecular information exists at present on the B1 receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B1 and B2 types of receptor. We showed previously that the human fibroblast cell line WI38 expresses both B1 and B2 receptors. In this report, we describe oocyte expression experiments showing that the B1 receptor in WI38 human fibroblast cells is encoded by a distinct mRNA approximately 2 kb shorter than that encoding the B2 receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B2 sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B1-type pharmacology may arise from two independent molecular mechanisms.
Publisher: Elsevier BV
Date: 03-1992
DOI: 10.1016/0378-1119(92)90384-2
Abstract: We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75% homologous to human and mouse p53, respectively. Immunoprecipitations of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.
Publisher: Wiley
Date: 1992
DOI: 10.1111/J.1471-4159.1992.TB09302.X
Abstract: mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage cl ed and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.
Publisher: Elsevier BV
Date: 02-1987
DOI: 10.1016/0166-6851(87)90191-5
Abstract: A cDNA clone expressing an antigen of Plasmodium falciparum, selected by screening an expression library cloned in Escherichia coli, encodes a portion of the protein identified as a glycophorin-binding protein [Kochan et al. (1986) Cell 44, 689-696]. Human antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting. This protein was present in all isolates tested, restricted to mature trophozoites and schizonts. It was abundant in culture supernatants at the time of merozoite release but present in minor amounts if at all in merozoites. The pattern of antigen distribution over schizont-infected cells observed by immunoelectron microscopy differed from that of the precursor of the major merozoite surface antigens in that most of the antigen appeared to be located over the erythrocyte cytoplasm without any obvious association with organelles. It thus appears unlikely that this antigen is present on the merozoite surface prior to schizont rupture.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 14-06-2002
Abstract: Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.
Publisher: Elsevier BV
Date: 09-2002
DOI: 10.1016/S0028-3908(02)00122-3
Abstract: We have cloned a guinea pig Vanilloid receptor 1 (VR1) from a dorsal root ganglion cDNA library and expressed it in CHO cells. The receptor has been functionally characterized by measuring changes in intracellular calcium produced by capsaicin, low pH and noxious heat. Capsaicin produced a concentration-dependent increase in intracellular calcium in guinea pig VR1-CHO cells with an estimated EC(50) of 0.17 +/- 0.0065 micro M, similar to that previously reported for rat and human VR1. Olvanil and resiniferatoxin were also effective agonists (EC(50) values of 0.0087 +/- 0.0035 micro M and 0.067 +/- 0.014 micro M, respectively), but 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) and anandamide showed little agonist activity up to 10 micro M. As with human and rat VR1, guinea pig VR1 was also activated by pH below 6.0 and by noxious heat (>42 degrees C). Capsazepine acted as an antagonist of capsaicin responses in guinea pig VR1-CHO cells (IC(50) of 0.324 +/- 0.041 micro M ), as seen at rat VR1. However, in contrast to its lack of activity against pH and heat responses at rat VR1, capsazepine was an effective antagonist of these responses at guinea pig VR1. Capsazepine displayed an IC(50) of 0.355 +/- 25 micro M against pH 5.5, and provided complete blockade of heat responses at 1 micro M. Thus, capsazepine can significantly inhibit calcium influx due to heat and pH 5.5 at guinea pig VR1 and human VR1 but is inactive against these activators at rat VR1.
Publisher: Springer Science and Business Media LLC
Date: 20-08-2016
DOI: 10.1007/S00018-015-2018-8
Abstract: Mechanosensitive ion channels are implicated in the biology of touch, pain, hearing and vascular reactivity however, the identity of these ion channels and the molecular basis of their activation is poorly understood. We previously found that transient receptor potential vanilloid 4 (TRPV4) is a receptor operated ion channel that is sensitised and activated by mechanical stress. Here, we investigated the effects of mechanical stimulation on TRPV4 localisation and activation in native and recombinant TRPV4-expressing cells. We used a combination of total internal reflection fluorescence microscopy, cell surface biotinylation assay and Ca(2+) imaging with laser scanning confocal microscope to show that TRPV4 is expressed in primary vascular endothelial cells and that shear stress sensitises the response of TRPV4 to its agonist, GSK1016790A. The sensitisation was attributed to the recruitment of intracellular pools of TRPV4 to the plasma membrane, through the clathrin and dynamin-mediated exocytosis. The translocation was dependent on ILK/Akt signalling pathway, release of Ca(2+) from intracellular stores and we demonstrated that shear stress stimulated phosphorylation of TRPV4 at tyrosine Y110. Our findings implicate calcium-sensitive TRPV4 translocation in the regulation of endothelial responses to mechanical stimulation.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-1999
DOI: 10.1097/00001756-199907130-00021
Abstract: Many nociceptive dorsal root ganglion (DRG) sensory neurons express the high affinity nerve growth factor (NGF) receptor TRKA, and respond to NGF. However, many do not express TRKA but are thought to respond to glial cell-derived neurotrophic factor (GDNF) and related molecules. We therefore cultured DRG neurons in the presence of GDNF, and looked at the expression of substance P and of the capsaicin receptor, VR1, two nociceptive properties already known to be NGF regulated. Using several different approaches we demonstrated that GDNF produced clear increases in expression of both properties, comparable in magnitude to increases seen with NGF. Following axotomy, aberrant expression of substance P in A fibres may be involved in the generation of neuropathic pain. Factors regulating substance P and other properties in the absence of retrogradely transported NGF may therefore be of significance in neuropathic pain states.
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.PHARMTHERA.2017.02.019
Abstract: Transient Receptor Potential Vanilloid 4 (TRPV4) is a broadly expressed, polymodally gated ion channel that plays an important role in many physiological and pathophysiological processes. TRPV4 knockout mice and several synthetic pharmacological compounds that selectively target TRPV4 are now available, which has allowed detailed investigation in to the therapeutic potential of this ion channel. Results from animal studies suggest that TRPV4 antagonism has therapeutic potential in oedema, pain, gastrointestinal disorders, and lung diseases such as cough, bronchoconstriction, pulmonary hypertension, and acute lung injury. A lack of observed side-effects in vivo has prompted a first-in-human trial for a TRPV4 antagonist in healthy participants and stable heart failure patients. If successful, this would open up an exciting new area of research for a multitude of TRPV4-related pathologies. This review will discuss the known roles of TRPV4 in disease, and highlight the possible implications of targeting this important cation channel for therapy.
Publisher: Royal Society of Chemistry
Date: 2015
Location: Australia
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2018
End Date: 2021
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2011
End Date: 2014
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2009
End Date: 2011
Funder: Australian Research Council
View Funded ActivityStart Date: 2008
End Date: 2008
Funder: Australian Research Council
View Funded ActivityStart Date: 2012
End Date: 2014
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2012
End Date: 2014
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2009
End Date: 12-2016
Amount: $290,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 05-2008
End Date: 12-2008
Amount: $300,000.00
Funder: Australian Research Council
View Funded Activity