ORCID Profile
0000-0002-5405-8422
Current Organisation
University of Queensland
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Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.EXPNEUROL.2012.06.034
Abstract: In myasthenia gravis muscle weakness is caused by autoantibodies against components of the neuromuscular junction. Patient autoantibodies against muscle specific kinase (MuSK) deplete MuSK from the postsynaptic membrane and reproduce signs of myasthenia gravis when injected into mice. Here we have examined the time-course of structural and functional changes that lead up to synaptic failure. C57Bl6J mice received daily injections of anti-MuSK patient IgG for 15 days. Mice began to lose weight from day 12 and demonstrated whole-body weakness by day 14. Electromyography indicated synaptic impairment from day 6 in the gastrocnemius muscle and from day 10 in the diaphragm muscle. Confocal microscopy revealed linear declines in the area and density of postsynaptic acetylcholine receptors (3-5% per day) from day 1 through day 15 of the injection series in all five muscles examined. Intracellular recordings from the diaphragm muscle revealed comparable progressive declines in the litude of the endplate potential and miniature endplate potential of 3-4% per day. Neither quantal content nor the postsynaptic action potential threshold changed significantly over the injection series. The inverse relationship between the quantal litude of a synapse and its quantal content disappeared only late in the injection series (day 10). Our results suggest that the primary myasthenogenic action of anti-MuSK IgG is to cause wastage of postsynaptic acetylcholine receptor density. Consequent reductions in endplate potential litudes culminated in failure of neuromuscular transmission.
Publisher: Wiley
Date: 12-2015
DOI: 10.14814/PHY2.12658
Publisher: The Company of Biologists
Date: 2012
DOI: 10.1242/JCS.095109
Abstract: At neuromuscular synapses, neural agrin (n-agrin) stabilizes embryonic postsynaptic acetylcholine receptor (AChR) clusters by signaling through the Muscle Specific Kinase (MuSK) complex. Live imaging of cultured myotubes showed that the formation and disassembly of primitive AChR clusters is a dynamic and reversible process favoured by n-agrin, and possibly other synaptic signals. Neuregulin-1 is a growth factor that can act via muscle ErbB receptor kinases to enhance synaptic gene transcription. Recent studies suggest that neuregulin-1-ErbB signaling can modulate n-agrin-induced AChR clustering independent of its effects on transcription. Here we report that when injected into muscles of embryonic mice, neuregulin-1increased the size of developing AChR clusters. We investigated this phenomenon using cultured myotubes, and found thatin the ongoing presence of n-agrin,neuregulin-1 potentiates AChR clustering by increasing the tyrosine phosphorylation of MuSK. Thispotentiation could be blocked by inhibiting Shp2, a postsynaptic tyrosine phosphatase known to modulate the activity of MuSK. Our results provide new evidence that neuregulin-1 modulates the signaling activity of MuSK and hence may function as a second order regulator of postsynaptic AChR clustering at the neuromuscular synapse. Thus two classic synaptic signaling systems (neuregulin-1 and n-agrin) converge upon MuSK to regulate postsynaptic differentiation.
Publisher: Wiley
Date: 2001
DOI: 10.1002/SYN.1094
Abstract: P2X1-type purinoceptors have been shown to mediate fast transmission between sympathetic varicosities and smooth muscle cells in the mouse vas deferens but the spatial organization of these receptors on the smooth muscle cells remains inconclusive. Voltage cl techniques were used to estimate the litudes of spontaneous excitatory junction currents (SEJCs) in cells of the vas deferens longitudinal smooth muscle layer. These currents involved the activation of about 6% of the P2X-type channels present on the cell, as compared to whole cell currents produced when isolated smooth muscle cells were exposed to maximal concentrations of either ATP or alpha,beta-MeATP. Immunofluorescence staining of the vas deferens with antibodies against P2X1 receptor showed a diffuse, grainy distribution over the entire membrane of each smooth muscle cell. Anti-P2X1 staining was not markedly clustered beneath anti-SV2-stained sympathetic varicosities. Similar results were obtained for cells in the urinary bladder. During development, P2X1 mRNA was detected as early as embryonic day 15 (E15). Increasing intensities of diffuse immunostaining for P2X1 were observed in the walls of the bladder, tail artery, and aorta from E15 until 6 weeks postnatal. The vas deferens showed increasing intensities of diffuse staining of its smooth muscle layers between 2 and 6 weeks postnatal, consistent with the time-course of development of fast purinergic transmission described previously. Together, the results suggest that the response of smooth muscle of the vas deferens to ATP released from sympathetic varicosities relies on rapidly desensitizing P2X1 receptors, distributed diffusely across the smooth muscle cell surface.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-2004
DOI: 10.1097/00001756-200411150-00014
Abstract: Agrin and neuregulin (HRG-beta1) play complementary roles in synapse formation. While HRG-beta1 induces transcriptional up-regulation of postsynaptic proteins, here we present evidence that it can potentiate agrin-induced acetylcholine receptor (AChR) clustering in C2 myotubes. Agrin induced maximal AChR clustering in 4 h. HRG-beta1 treatment for 4 h produced no increase over basal AChR cluster numbers. When myotubes were treated for 4 h with 100 pM agrin, HRG-beta1 augmented AChR cluster numbers by 2-fold compared to myotubes treated with 100 pM agrin alone. Thus, HRG-beta1 can potentiate agrin-induced AChR clustering.
Publisher: Wiley
Date: 29-04-2016
DOI: 10.1111/BJH.13470
Publisher: The Company of Biologists
Date: 11-2011
DOI: 10.1242/JCS.084376
Abstract: Caveolae are invaginations of the plasma membrane that are formed by caveolins. Caveolar membranes are also enriched in cholesterol, glycosphingolipids and signaling enzymes such as Src kinase. Here we investigate the effect of cell stretch upon caveolar dynamics and signaling. Transfection of C2 myoblasts with caveolin-3–YFP led to the formation of caveolae-like membrane pits 50–100 nm in diameter. Glycosphingolipids became immobilized and tightly packed together within caveolin-rich regions of the plasma membrane. Fluorescence resonance energy transfer (FRET) was used to assess the degree of glycosphingolipid packing. Myoblasts were subjected to a brief (1 minute) stretch on an elastic substratum. Stretch caused a reduction in glycosphingolipid FRET, consistent with a reversible unfolding of caveolar pits in response to membrane tension. Cells expressing caveolin-3–YFP also displayed an enhanced stretch-induced activation of Src kinase, as assessed by immunofluorescence. Repeated stretches resulted in the trafficking and remodeling of caveolin-3-rich membrane domains and accelerated turnover of membrane glycosphingolipids. The stretch-induced unfolding of caveolae, activation of Src and redistribution of caveolin and glycosphingolipids might reflect mechanisms of the cellular adaptation to mechanical stresses.
Publisher: Elsevier BV
Date: 2007
DOI: 10.1016/J.BIOCEL.2006.10.012
Abstract: Neural agrin is a heparan sulphate proteoglycan first defined by its ability to induce the clustering of acetylcholine receptors (AChRs) on cultured muscle cells. Neural agrin activates the transmembrane Muscle Specific Kinase (MuSK) on the postsynaptic muscle cell to stabilise the developing neuromuscular synapse. Three biological mechanisms for agrin/MuSK signalling are briefly discussed: selective transcription of synaptic genes such as MuSK itself, to reinforce developing postsynaptic clusters of AChRs initiation of second messenger signalling pathways that can induce the formation of AChR clusters and retrograde signalling downstream of agrin/MuSK that may transform the growth cone of the motor axon into a stable differentiated nerve terminal, specialised for regulated exocytosis of neurotransmitter. Here we briefly review some key mechanisms through which neural agrin acts to foster the formation of mature neuromuscular synapses.
Publisher: Wiley
Date: 31-08-2010
Publisher: Wiley
Date: 10-1992
Abstract: The developing neuromuscular junction has provided an important paradigm for studying synapse formation. An outstanding feature of neuromuscular differentiation is the aggregation of acetylcholine receptors (AChRs) at high density in the postsynaptic membrane. While AChR aggregation is generally believed to be induced by the nerve, the mechanisms underlying aggregation remain to be clarified. A 43-kD protein (43k) normally associated with the cytoplasmic aspect of AChR clusters has long been suspected of immobilizing AChRs by linking them to the cytoskeleton. In recent studies, the AChR clustering activity of 43k has, at last, been demonstrated by expressing recombinant AChR and 43k in non-muscle cells. Mutagenesis of 43k has revealed distinct domains within the primary structure which may be responsible for plasma membrane targeting and AChR binding. Other lines of study have provided clues as to how nerve-derived (extracellular) AChR-cluster inducing factors such as agrin might activate 43k-driven postsynaptic membrane specialization.
Publisher: The University of Kansas
Date: 29-08-2023
DOI: 10.17161/RRNMF.V4I3.19543
Abstract: In myasthenia gravis autoantibodies attack the postsynaptic membrane of the neuromuscular junction causing fatiguing weakness that can wax and wane. Weakness occurs when the safety factor for neuromuscular transmission becomes marginal, meaning that the (postsynaptic) endplate potential is no longer sufficient to reliably trigger action potentials in the muscle fiber. Cholinesterase inhibitor drugs provide temporary relief by increasing the endplate potential litude, but additional symptomatic treatment options are needed. Here we discuss our recent experience in early preclinical testing of candidate compounds. Using an ex vivo mouse nerve-muscle contraction assay, followed up by endplate potential recordings, we examined the effects of cannabinoids. Our findings highlighted the potentially confounding effects of dimethylsulfoxide (DMSO) when used as a solubilizing agent. DMSO produced a dose-dependent restoration of force to curarized muscle and enhanced miniature endplate potential litude, thus enhancing the safety factor for neuromuscular transmission at concentrations as low as 0.1% v/v. When examined in the absence of curare, the DMSO-induced increase in quantal litude was opposed by a homeostatic reduction in the number of quanta of acetylcholine released by the nerve terminal. In contrast to DMSO, cannabinoids appear to work via cannabinoid receptor type 1 to reduce quantal number, thereby weakening the safety factor. Our results highlight the need to consider the effects of solubilizing agents per se when screening new therapeutics for neurological diseases. They also demonstrate the need to take synaptic homeostasis into account, which can otherwise distort or mask the effects of bioactive agents upon neurotransmission.
Publisher: Springer Science and Business Media LLC
Date: 1999
Abstract: Rapsyn is a protein on the cytoplasmic face of the postsynaptic membrane of skeletal muscle that is essential for clustering acetylcholine receptors (AChR). Here we show that transfection of rapsyn cDNA can restore AChR clustering function to muscle cells cultured from rapsyn deficient (KORAP) mice. KORAP myotubes displayed no AChR aggregates before or after treatment with neural agrin. After transfection with rapsyn expression plasmid, some KORAP myotubes expressed rapsyn at physiological levels. These formed large AChR-rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed rapsyn formed only scattered AChR-rapsyn microaggregates, irrespective of agrin treatment. KORAP cells were then transfected with mutant forms of rapsyn. A deletion mutant lacking residues 16-254 formed rapsyn microaggregates, but failed to aggregate AChRs. Substitution mutation to the C-terminal serine phosphorylation site of rapsyn (M43(D405,D406)) did not impair the response to agrin, showing that differential phosphorylation of this site is unlikely to mediate agrin-induced clustering. The results indicate that rapsyn expression is essential for agrin-induced AChR clustering but that its overexpression inhibits this pathway. The approach of using rapsyn-deficient muscle cells opens the way for defining the role of rapsyn in agrin-induced AChR clustering.
Publisher: Wiley
Date: 2000
DOI: 10.1002/1098-2396(20000915)37:4<283::AID-SYN5>3.0.CO;2-W
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.EXPNEUROL.2015.01.007
Abstract: Study of the electrophysiological function of the neuromuscular junction (NMJ) is instrumental in the understanding of the symptoms and pathophysiology of myasthenia gravis (MG), an autoimmune disorder characterized by fluctuating and fatigable muscle weakness. Most patients have autoantibodies to the acetylcholine receptor at the NMJ. However, in recent years autoantibodies to other crucial postsynaptic membrane proteins have been found in previously 'seronegative' MG patients. Electromyographical recording of compound and single-fibre muscle action potentials provides a crucial in vivo method to determine neuromuscular transmission failure while ex vivo (miniature) endplate potential recordings can reveal the precise synaptic impairment. Here we will review these electrophysiological methods used to assess NMJ function and discuss their application and typical results found in the diagnostic and experimental study of patients and animal models of the several forms of MG.
Publisher: Elsevier BV
Date: 2002
DOI: 10.1016/S0896-6273(01)00569-4
Abstract: Coyle et al. (2002), in this issue of Neuron, reveal the crystal structure for the GABA(A) receptor binding protein, GABARAP. They show GABARAP can switch from a monomer to an extended linear polymer form that may function to assemble microtubules during the intracellular trafficking or postsynaptic clustering of GABA(A) receptors.
Publisher: Wiley
Date: 07-2014
Publisher: Wiley
Date: 20-01-2005
Publisher: Wiley
Date: 12-1995
DOI: 10.1111/J.1440-1681.1995.TB02333.X
Abstract: 1. The post-synaptic membranes of neurons and muscle cells are characterized by clusters of transmitter receptors, the number and type of which help to determine synaptic efficacy. Here I briefly review what is known of the mechanism of clustering of nicotinic acetylcholine receptors (AChR) at neuromuscular synapses. 2. The extracellular protein agrin is thought to be secreted by the motor nerve terminal and trigger localized clustering of AChR in the post-synaptic membrane of the skeletal muscle cell. 3. Binding of agrin to its receptor, alpha-dystroglycan, is followed by rearrangements of the muscle membrane cytoskeleton with localized replacement of dystrophin by utrophin. It remains unclear how these changes relate to the clustering of AChR. 4. In separate studies, RAPsyn/43k protein, a protein associated with the inner face of the post-synaptic membrane was shown to be able to cluster AChR and link them to the cytoskeleton when both proteins were co-transfected into fibroblasts. 5. Mutational studies on RAPsyn identified putative binding domains for AChR and for the cytoskeleton within the RAPsyn primary structure. Targeted disruption of the RAPsyn gene in mice prevented post-synaptic AChR clustering and led to neonatal lethality. Thus RAPsyn might be the final link in the pathway that leads to AChR immobilization in the post-synaptic membrane. 6. The recent observation that active forms of agrin are not restricted to cholinergic regions of the brain suggests that analogous pathways may exist for clustering other receptor types.
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.BIOCEL.2010.10.008
Abstract: Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ.
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.YDBIO.2007.02.008
Abstract: Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, rapsyn. When 1-month old muscle was denervated, postsynaptic rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of rapsyn-EGFP into muscle fibers increased postsynaptic rapsyn levels. Those synapses with higher ratios of rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.
Publisher: Wiley
Date: 25-03-2013
Publisher: F1000 Research Ltd
Date: 27-06-2016
DOI: 10.12688/F1000RESEARCH.8206.1
Abstract: Myasthenia gravis is an autoimmune disease of the neuromuscular junction (NMJ) caused by antibodies that attack components of the postsynaptic membrane, impair neuromuscular transmission, and lead to weakness and fatigue of skeletal muscle. This can be generalised or localised to certain muscle groups, and involvement of the bulbar and respiratory muscles can be life threatening. The pathogenesis of myasthenia gravis depends upon the target and isotype of the autoantibodies. Most cases are caused by immunoglobulin (Ig)G1 and IgG3 antibodies to the acetylcholine receptor (AChR). They produce complement-mediated damage and increase the rate of AChR turnover, both mechanisms causing loss of AChR from the postsynaptic membrane. The thymus gland is involved in many patients, and there are experimental and genetic approaches to understand the failure of immune tolerance to the AChR. In a proportion of those patients without AChR antibodies, antibodies to muscle-specific kinase (MuSK), or related proteins such as agrin and low-density lipoprotein receptor-related protein 4 (LRP4), are present. MuSK antibodies are predominantly IgG4 and cause disassembly of the neuromuscular junction by disrupting the physiological function of MuSK in synapse maintenance and adaptation. Here we discuss how knowledge of neuromuscular junction structure and function has fed into understanding the mechanisms of AChR and MuSK antibodies. Myasthenia gravis remains a paradigm for autoantibody-mediated conditions and these observations show how much there is still to learn about synaptic function and pathological mechanisms.
Publisher: The Company of Biologists
Date: 07-2008
DOI: 10.1242/JCS.032003
Abstract: Transient receptor potential canonical 1 (TRPC1), a widely expressed calcium (Ca2+)-permeable channel, is potentially involved in the pathogenesis of Duchenne muscular dystrophy (DMD). Ca2+ influx through stretch-activated channels, possibly formed by TRPC1, induces muscle-cell damage in the mdx mouse, an animal model of DMD. In this study, we showed that TRPC1, caveolin-3 and Src-kinase protein levels are increased in mdx muscle compared with wild type. TRPC1 and caveolin-3 colocalised and co-immunoprecipitated. Direct binding of TRPC1-CFP to caveolin-3–YFP was confirmed in C2 myoblasts by fluorescence energy resonance transfer (FRET). Caveolin-3–YFP targeted TRPC1-CFP to the plasma membrane. Hydrogen peroxide, a reactive oxygen species (ROS), increased Src activity and enhanced Ca2+ influx, but only in C2 myoblasts co-expressing TRPC1 and caveolin-3. In mdx muscle, Tiron, a ROS scavenger, and PP2, a Src inhibitor, reduced stretch-induced Ca2+ entry and increased force recovery. Because ROS production is increased in mdx/DMD, these results suggest that a ROS-Src-TRPC1/caveolin-3 pathway contributes to the pathogenesis of mdx/DMD.
Publisher: Springer Science and Business Media LLC
Date: 06-1986
DOI: 10.1007/BF01611441
Abstract: Changes in antioxidant capacity in the body as a result of oxidative stress play an important role in the development of diabetic complications. The aim of this study was to evaluate the effect of aqueous extract of Artemisia afra Jacq. ex Willd. on antioxidant defense systems in the liver and kidney of streptozotocin-induced diabetic rats. Administration of the extract to diabetic rats for 21 days significantly reduced blood glucose levels and increased body weight. The diabetic animals exhibited decreased levels of glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD) and reduced glutathione (GSH) in the liver and kidney, which were restored to near normal levels following treatment with the herb. The increased levels of lipid peroxidation observed in the tissues of diabetic rats were also reverted back to near normalcy after administering the extract. These findings revealed the protective role of A. afra on tissues by reducing oxidative stress which could be attributed to its flavonoids content. The efficacy of the plant compared favourably well with glibenclamide, a standard hypoglycemic drug.
Publisher: MyJove Corporation
Date: 26-12-2014
DOI: 10.3791/52220
Publisher: Springer Science and Business Media LLC
Date: 16-03-2018
DOI: 10.1038/S41598-018-22888-4
Abstract: Cannabinoids exert dynamic control over many physiological processes including memory formation, cognition and pain perception. In the central nervous system endocannabinoids mediate negative feedback of quantal transmitter release following postsynaptic depolarization. The influence of cannabinoids in the peripheral nervous system is less clear and might have broad implications for the therapeutic application of cannabinoids. We report a novel cannabinoid effect upon the mouse neuromuscular synapse: acutely increasing synaptic vesicle volume and raising the quantal litudes. In a mouse model of myasthenia gravis the cannabinoid receptor agonist WIN 55,212 reversed fatiguing failure of neuromuscular transmission, suggesting future therapeutic potential. Our data suggest an endogenous pathway by which cannabinoids might help to regulate transmitter release at the neuromuscular junction.
Publisher: S. Karger AG
Date: 2001
DOI: 10.1159/000056923
Abstract: P2X sub /sub receptors are ATP-gated cation channels that mediate the fast, purinergic component of sympathetic nerve–smooth muscle neurotransmission in the mouse vas deferens and may serve comparable functions in the urinary bladder and the arteries. The gene for mouse P2X sub /sub ( i P2rx1 /i ) was cloned and its genomic structure defined by sequencing. The gene spans about 10 kb and consists of 12 exons. All splice sites conformed to the GT-AG motif and the exon-intron boundaries were largely conserved with other members of the P2X gene family so far cloned. A single transcription-starting site was identified by 5′ RACE analysis, 233 bp upstream of the translation start site. The P2X sub /sub gene maps to the central region of mouse chromosome 11.
Publisher: Wiley
Date: 18-08-2019
DOI: 10.1113/JP277839
Publisher: Springer Science and Business Media LLC
Date: 02-1987
DOI: 10.1007/BF02456693
Abstract: Two primary areas of damage have been implicated in apraxia of speech (AOS) based on the time post-stroke: (1) the left inferior frontal gyrus (IFG) in acute patients, and (2) the left anterior insula (aIns) in chronic patients. While AOS is widely characterized as a disorder in motor speech planning, little is known about the specific contributions of each of these regions in speech. The purpose of this study was to investigate cortical activation during speech production with a specific focus on the aIns and the IFG in normal adults. While undergoing sparse fMRI, 30 normal adults completed a 30-minute speech-repetition task consisting of three-syllable nonwords that contained either (a) English (native) syllables or (b) non-English (novel) syllables. When the novel syllable productions were compared to the native syllable productions, greater neural activation was observed in the aIns and IFG, particularly during the first 10 min of the task when novelty was the greatest. Although activation in the aIns remained high throughout the task for novel productions, greater activation was clearly demonstrated when the initial 10 min was compared to the final 10 min of the task. These results suggest increased activity within an extensive neural network, including the aIns and IFG, when the motor speech system is taxed, such as during the production of novel speech. We speculate that the amount of left aIns recruitment during speech production may be related to the internal construction of the motor speech unit such that the degree of novelty/automaticity would result in more or less demands respectively. The role of the IFG as a storehouse and integrative processor for previously acquired routines is also discussed.
Publisher: Springer Science and Business Media LLC
Date: 12-1987
DOI: 10.1007/BF01611986
Abstract: Chromatin remodeling is an essential part of transcription initiation. We show that at heat shock gene promoters functional interactions between in idual ATP-dependent chromatin remodeling complexes play critical role in both nucleosome displacement and Pol II recruitment. Using HSP12, HSP82 and SSA4 gene promoters as reporters, we demonstrated that while inactivation of SNF2, a critical ATPase of the SWI/SNF complex, primarily affects the HSP12 promoter, depletion of STH1- a SNF2 homolog from the RSC complex reduces histone displacement and abolishes the Pol II recruitment at all three promoters. From these results, we conclude that redundancy between SWI/SNF and RSC complexes is only partial and likely is affecting different chromatin remodeling steps. While inactivation of other in idual ATP-dependent chromatin remodeling complexes negligibly affects reporter promoters, combinatorial inactivation of SNF2 and ISW1 has a synergistic effect by diminishing histone loss during heat induction and eliminating Pol II recruitment. Importantly, it also eliminates preloading of HSF on HSP82 and SSA4 promoters before heat shock and diminishes HSF binding during heat shock. These observations suggest that prior action of chromatin remodeling complexes is necessary for the activator binding.
Publisher: Rockefeller University Press
Date: 11-1993
Abstract: Recombinant acetylcholine receptors (AChRs) expressed on the surface of cultured fibroblasts become organized into discrete membrane domains when the 43-kD postsynaptic protein (43k) is co-expressed in the same cells (Froehner, S.C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410 Phillips, W. D., M. C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we show that AChRs present on the fibroblast cell surface prior to transfection of 43k are recruited into 43k-rich membrane domains. Aggregated AChRs show increased resistance to extraction with Triton X-100, suggesting a 43k-dependent linkage to the cytoskeleton. Myotubes of the mouse cell line C2 spontaneously display occasional AChR/43k-rich membrane domains that ranged in diameter up to 15 microns, but expressed many more when 43k was overexpressed following transfection of 43k cDNA. However, the membrane domains induced by recombinant 43k were predominantly small (& or = 2 microns). We were then interested in whether the cytoskeletal component, dystrophin related protein (DRP Tinsley, J. M., D. J. Blake, A. Roche, U. Fairbrother, J. Riss, B. C. Byth, A. E. Knight, J. Kendrick-Jones, G. K. Suthers, D. R. Love, Y. H. Edwards, and K. E. Davis, 1992. Nature (Lond.). 360:591-593) contributed to the development of AChR clusters. Immunofluorescent anti-DRP staining was present at the earliest stages of AChR clustering at the neuromuscular synapse in mouse embryos and was also concentrated at the large AChR-rich domains on nontransfected C2 myotubes. Surprisingly, anti-DRP staining was concentrated mainly at the large, but not the small AChR clusters on C2 myotubes suggesting that DRP may be principally involved in permitting the growth of AChR clusters.
Publisher: Elsevier BV
Date: 04-2020
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 10-2014
Publisher: Springer Science and Business Media LLC
Date: 04-1985
DOI: 10.1007/BF01258455
Abstract: Purpose. The purpose of this study was to evaluate the feasibility of detecting pulmonary emboli utilizing noncontrast magnetic resonance imaging techniques in patients with known pulmonary embolism. Materials and Methods. Eleven patients were enrolled in a study to evaluate right ventricular function by cardiac MRI in patients diagnosed with acute pulmonary embolism on CT pulmonary angiogram. Cardiac MRI was performed as soon as possible following pulmonary embolism detection. Two independent observers reviewed the precontrast portion of each MRI, scoring right, left, and lobar arteries as positive or negative for PE. The CTs were reviewed and interpreted in the same manner. Results. MRI was obtained on average of 40 hours after the CT. Forty-eight vessels were affected by PE on CT, 69% of which were identified on MRI. All eight pulmonary emboli located in the right or left pulmonary arteries were detected on MRI. Of the 15 pulmonary emboli that were not detected on MRI, 7 were subsegmental, 6 were segmental, and 2 were located in a branch not included in the MRI field of view. Conclusions. Most pulmonary emboli detected on CT were identified on noncontrast MRI, even though our MRI protocol was not optimized for pulmonary artery visualization.
Publisher: Elsevier BV
Date: 12-1984
DOI: 10.1016/0012-1606(84)90245-8
Abstract: The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a s le muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.
Publisher: Wiley
Date: 23-06-2021
DOI: 10.1113/EP089439
Abstract: What is the central question of this study? The (dystrophin‐deficient) muscles of mdx mice generate less contractile force per cross‐sectional area (specific force) than those of healthy wild‐type mice: what is the influence of muscle specific kinase (MuSK) upon the properties of the tibialis anterior (TA) muscle in mdx mice? What is the main finding and its importance? Injection of adeno‐associated viral vector encoding MuSK into the TA muscle of young mdx mice increased the specific force of the muscle, suggesting the MuSK signalling system has the potential to restore healthy growth to dystrophin‐deficient muscles. In the mdx mouse model of Duchenne muscular dystrophy, muscle fibres are fragile and prone to injury and degeneration. Compared to wild‐type mice, muscles of mdx mice also develop less specific force (contractile force/cross‐sectional area). We recently reported that injecting adeno‐associated viral vector encoding muscle specific kinase (AAV‐MuSK) into muscles of mdx mice increased utrophin expression and made the muscles more resistant to acute stretch‐induced injury. Here we injected AAV‐MuSK unilaterally into the tibialis anterior muscle of mdx mice at a younger age (4 weeks), and recorded contraction force from the muscles in situ at 12 weeks of age. Compared to contralateral empty‐vector control muscles, muscles injected with AAV‐MuSK produced 28% greater specific force ( P = 0.0005). They did not undergo the compensatory hypertrophy that normally occurs in muscles of mdx mice. Injection of AAV encoding rapsyn (a downstream effector of MuSK signalling) caused no such improvement in muscle strength. Muscles injected with AAV‐MuSK displayed a 10% reduction in the number of fibres with centralized nuclei ( P = 0.0015). Our results in mdx mice suggest that elevating the expression of MuSK can reduce the incidence of muscle fibre regeneration and improve the strength of dystrophin‐deficient muscles.
Publisher: Elsevier BV
Date: 1997
Abstract: Rapsyn is a 43-kDa cytoplasmic protein that clusters nicotinic acetylcholine receptors (AChR) in the postsynaptic membrane. Here we examine the effect of rapsynmediated AChR clustering on the metabolic stability of the AChR. When transfected into QT-6 fibroblasts, cell surface AChRs (alpha, beta, epsilon, and delta subunit combination) pulse labeled with 125I-alpha-bungarotoxin were degraded with a half-life of 16.4 +/- 1.1 h (mean +/- SEM). Cotransfection of rapsyn with AChR caused extensive AChR clustering and increased AChR half-life to 20.5 +/- 1.0 h. Anti-AChR antibodies such as mab 35 cause an increased AChR degradation often associated with myasthenia gravis: 80.8 +/- 2.5% of AChRs labeled at zero time were degraded over a 12-h period. Contransfection of rapsyn reduced this AChR loss to 66.4 +/- 3.8%. Rapsyn also reduced normal AChR degradation, from 53.2 +/- 2.1 to 44.2 +/- 2.2%. Muscle cell lines from wild-type myotubes displayed few AChR clusters, but treatment with neural agrin increased the number of AChR clusters 30-fold. Clustering was accompanied by reductions in AChR degradation (both in the presence and absence of mab 35) similar in magnitude to those produced by overexpression of rapsyn in QT-6 cells. In rapsyn-deficient myotubes, treatment with neural agrin neither caused AChR clustering nor reduced AChR degradation. Thus neural agrin may slow AChR degradation by inducing the rapsyn-dependent clustering of AChRs.
Publisher: Wiley
Date: 05-12-2013
DOI: 10.1002/AR.22633
Abstract: Ischemic heart disease is a major cause of morbidity and mortality worldwide. Stem cell-based therapy, which aims to restore cardiac structure and function by regeneration of functional myocardium, has recently been proposed as a novel alternative treatment modality. Resident cardiac stem cells (CSCs) in adult hearts are a key cell type under investigation. CSCs have been shown to be able to repair damaged myocardium and improve myocardial function in both human and animal studies. This approach relies not only on the proliferation of the CSCs, but also upon their migration to the site of injury within the heart. Here, we briefly review reported CSC populations and discuss signaling factors and pathways required for the migration of CSCs.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-1991
Abstract: Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.
Publisher: Elsevier BV
Date: 1993
Publisher: Wiley
Date: 10-11-2017
DOI: 10.1111/NYAS.13513
Abstract: While the majority of myasthenia gravis patients express antibodies targeting the acetylcholine receptor, the second most common cohort instead displays autoantibodies against muscle-specific kinase (MuSK). MuSK is a transmembrane tyrosine kinase found in the postsynaptic membrane of the neuromuscular junction. During development, MuSK serves as a signaling hub, coordinating the alignment of the pre- and postsynaptic components of the synapse. Adult mice that received repeated daily injections of IgG from anti-MuSK
Publisher: Wiley
Date: 08-2008
DOI: 10.1002/DNEU.20654
Abstract: Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.EXPNEUROL.2014.12.013
Abstract: Muscle-specific tyrosine kinase (MuSK) autoantibodies are the hallmark of a form of myasthenia gravis (MG) that can challenge the neurologist and the experimentalist. The clinical disease can be difficult to treat effectively. MuSK autoantibodies affect the neuromuscular junction in several ways. When added to muscle cells in culture, MuSK antibodies disperse acetylcholine receptor clusters. Experimental animals actively immunized with MuSK develop MuSK autoantibodies and muscle weakness. Weakness is associated with reduced postsynaptic acetylcholine receptor numbers, reduced litudes of miniature endplate potentials and endplate potentials, and failure of neuromuscular transmission. Similar impairments have been found in mice injected with IgG from MG patients positive for MuSK autoantibody (MuSK-MG). The active and passive models have begun to reveal the mechanisms by which MuSK antibodies disrupt synaptic function at the neuromuscular junction, and should be valuable in developing therapies for MuSK-MG. However, translation into new and improved treatments for patients requires procedures that are not too cumbersome but suitable for examining different aspects of MuSK function and the effects of potential therapies. Study design, conduct and analysis should be carefully considered and transparently reported. Here we review what has been learnt from animal and culture models of MuSK-MG, and offer guidelines for experimental design and conduct of studies, including s le size determination, randomization, outcome parameters and precautions for objective data analysis. These principles may also be relevant to the increasing number of other antibody-mediated diseases that are now recognized.
Publisher: Elsevier BV
Date: 1997
Abstract: Rapsyn, a 43-kDa protein on the cytoplasmic face of the postsynaptic membrane, is essential for clustering acetylcholine receptors (AChR) at the neuromuscular junction. When transfected into nonmuscle cells (QT-6), rapsyn forms discrete membrane domains and can cluster AChR into these same domains. Here we examined whether rapsyn can cluster other ion channels as well. When expressed in QT-6 cells, the GABAA receptor (human alpha 1, beta 1, and gamma 2 subunits) and the skeletal muscle sodium channel were each diffusely scattered across the cell surface. Rapsyn, when co-expressed, clustered the GABAA receptor as effectively as it clustered AChR in previous studies. Rapsyn did not cluster co-transfected sodium channel, confirming that it does not cluster ion channels indiscriminately. Rapsyn mRNA was detected at low levels in the brain by polymerase chain reaction lification of reverse-transcribed RNA, raising the possibility of a broader role for rapsyn.
Publisher: Rockefeller University Press
Date: 15-12-1991
Abstract: The postsynaptic membrane of the neuromuscular junction contains a myristoylated 43-kD protein (43k) that is closely associated with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR)-rich plasma membrane. Previously, we described fibroblast cell lines expressing recombinant AChRs. Transfection of these cell lines with 43k was necessary and sufficient for reorganization of AChR into discrete 43k-rich plasma membrane domains (Phillips, W. D., C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we demonstrate the utility of this expression system for the study of 43k function by site-directed mutagenesis. Substitution of a termination codon for Asp254 produced a truncated (28-kD) protein that associated poorly with the cell membrane. The conversion of Gly2 to Ala2, to preclude NH2-terminal myristoylation, reduced the frequency with which 43k formed plasma membrane domains by threefold, but did not eliminate the aggregation of AChRs at these domains. Since both NH2 and COOH-termini seemed important for association of 43k with the plasma membrane, a deletion mutant was constructed in which the codon Gln15 was fused in-frame to Ile255 to create a 19-kD protein. This mutated protein formed 43k-rich plasma membrane domains at wild-type frequency, but the domains failed to aggregate AChRs, suggesting that the central part of the 43k polypeptide may be involved in AChR aggregation. Our results suggest that membrane association and AChR interactions are separable functions of the 43k molecule.
Publisher: Public Library of Science (PLoS)
Date: 05-02-2014
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.IJCARD.2016.11.047
Abstract: Side-population (SP) cells, identified by their capacity to efflux Hoechst dye, are highly enriched for stem rogenitor cell activity. They are found in many mammalian tissues, including mouse heart. Studies suggest that cardiac SP (CSP) cells can be ided into SCA1 Using fluorescence-activated cell sorting, reverse transcriptase olymerase chain reaction, assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction we show that SCA1 Based on their gene expression profile, localization and ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we conclude that SCA1
Publisher: Bentham Science Publishers Ltd.
Date: 30-04-2015
DOI: 10.2174/1381612821666150316123233
Abstract: Myasthenia gravis is a muscle weakness disease characterized by autoantibodies that target components of the neuromuscular junction, impairing synaptic transmission. The most common form of myasthenia gravis involves antibodies that bind the nicotinic acetylcholine receptors in the postsynaptic membrane. Many of the remaining cases are due to antibodies against muscle specific tyrosine kinase (MuSK). Recently, autoantibodies against LRP4 (another component of the MuSK signaling complex in the postsynaptic membrane) were identified as the likely cause of myasthenia gravis in some patients. Fatiguing weakness is the common symptom in all forms of myasthenia gravis, but muscles of the body are differentially affected, for reasons that are not fully understood. Much of what we have learnt about the immunological and neurobiological aspects of the pathogenesis derives from mouse models. The most widely used mouse models involve either passive transfer of autoantibodies, or active immunization of the mouse with acetylcholine receptors or MuSK protein. These models can provide a robust replication of many of the features of the human disease. Depending upon the protocol, acute fatiguing weakness develops 2 - 14 days after the start of autoantibody injections (passive transfer) or might require repeated immunizations over several weeks (active models). Here we review mouse models of myasthenia gravis, including what they have contributed to current understanding of the pathogenic mechanisms and their current application to the testing of therapeutics.
Publisher: Spandidos Publications
Date: 08-01-2019
Publisher: Springer Science and Business Media LLC
Date: 04-1989
DOI: 10.1007/BF01206665
Abstract: To examine how adolescent physical activity (PA) and screen-based media sedentary behaviours (SBM) relate to psychological and social health and identify cross-national differences in these relationships. Associations were examined in five regions using two Health Behaviour in School-Aged Children (HBSC) countries from each. Self-reported psychological and social health indices such as self-image, perceived health status, and Life Satisfaction were positively related to PA in all five regions but, with a few exceptions, negatively related to SBM. Negative health indices such as health complaints and tobacco use were negatively related to PA but, with exceptions, positively related to SBM. Significant regional differences were present. Regional differences in correlates of PA and SBM suggest cultural differences in potential effects of PA and SBM and the need to tailor school and public health efforts to the different meanings of PA and SBM for positive and negative health consequences.
Publisher: Public Library of Science (PLoS)
Date: 03-07-2013
Publisher: Wiley
Date: 02-04-2008
DOI: 10.1002/ANA.21371
Abstract: A subset of myasthenia gravis patients that are seronegative for anti-acetylcholine receptor (anti-AChR) antibodies are instead seropositive for antibodies against the muscle-specific kinase (anti-MuSK-positive). Here, we test whether transfer of IgG from anti-MuSK-positive patients to mice confers impairment of the neuromuscular junction and muscle weakness. IgG from anti-MuSK-positive myasthenia gravis patients or control IgG (seronegative for AChR and MuSK) was injected intraperitoneally (45 mg daily for 14 days) into 6-week-old female FVB/NJ and C57BL/6J mice. Changes at neuromuscular junctions in the tibialis anterior and diaphragm muscles were assessed by confocal fluorescent imaging of AChRs stained with fluorescent-alpha-bungarotoxin. Loss of function was assessed by electromyography. In experimental mice injected with anti-MuSK-positive patient IgG, postsynaptic AChR staining was reduced to as little as 22% of that seen in control mice. Experimental mice showed reduced apposition of the nerve terminal (labeled with antibodies against synaptophysin and neurofilament) and the postsynaptic AChR cluster (labeled with fluorescent-alpha-bungarotoxin). Mice injected with IgG from two of three anti-MuSK-positive patients lost weight and developed muscle weakness associated with a decremental electromyographic trace on repetitive nerve stimulation. IgG from anti-MuSK-positive patients can cause myasthenia gravis when injected into mice. This may be explained by a progressive reduction in the density of postsynaptic AChR combined with changes in the nerve terminal and its relation to the postsynaptic structure.
Publisher: Wiley
Date: 19-12-2003
DOI: 10.1002/SYN.10119
Abstract: Contraction of the smooth muscle in the mouse vas deferens is elicited by sympathetic nerves releasing at least two neurotransmitters, adenosine triphosphate (ATP) and noradrenaline (NA). Several studies have indicated the presence of regional variation in the purinergic and noradrenergic contributions to sympathetic nerve-evoked contractions in rodent vasa deferentia. We examined the relative contribution of ATP and NA to neurotransmission and contraction at the prostatic and epididymal ends of the mouse vas deferens. The success rate of recording excitatory junction currents (EJCs, extracellular indication of ATP release) from varicosities at the prostatic end of the vas deferens was eight times greater than for varicosities located at the epididymal end. Both regions of the vas deferens responded similarly to focal application of NA and ATP. Furthermore, the relative density and distribution of P2X(1)-receptor mRNA and anti-P2X(1) immunostaining did not differ between the two regions. Our results suggest that most varicosities located at the epididymal end of the vas deferens are releasing an insufficient amount of ATP to evoke detectable EJCs.
Location: United States of America
Location: No location found
No related grants have been discovered for William Phillips.