7,150 research outputs found
Lecture: Author Susan Orlean
No full textShaker Library and the Shaker Schools Foundation present Susan Orlean, SHHS grad and author of The Library Book, who will speak about her love of libraries and the impact of books on her life.
Susan Orlean grew up in Shaker Heights and graduated from Shaker Heights High School in 1973, where she was editor in chief of the school’s yearbook, The Gristmill. She graduated with honors from the University of Michigan in 1976. She has written for the Boston Phoenix, the Boston Globe and has been a staff writer at The New Yorker since 1992. She is the author of seven books, including Rin Tin Tin, Saturday Night, and The Orchid Thief, which was made into the Academy Award–winning film, Adaptation. She lives with her family and her animals in upstate New York
Functional characterization of the RYR1 mutation p.Arg4737Trp associated with susceptibility to malignant hyperthermia
No full textAside from the in vitro contracture test, genetic screening for causative RYR1 mutations is the established procedure to diagnose susceptibility
to malignant hyperthermia (MH). However, currently only 34 out of more than 300 known RYR1 mutations have been confirmed to be causative
for MH by experimental studies addressing their functional impact on intracellular calcium homeostasis. The RYR1 mutation p.Arg4737Trp has
been recently detected in a German MH family. To evaluate the effects of that mutation on intracellular calcium handling, the response after
stimulation with the RYR1 agonist 4-chloro-m-cresol was investigated in immortalized B lymphocytes containing the p.Arg4737Trp mutation and
compared to the response of wild type RYR1 from unaffected family members and unrelated controls. Intracellular resting calcium was slightly
but significantly elevated in mutation positive cells. Calcium release following stimulation with 4-chloro-m-cresol was significantly increased in
B lymphocytes carrying the p.Arg4737Trp mutation compared to mutation negative controls. Hence, the functional properties of the RYR1
mutation p.Arg4737Trp are consistent with susceptibility to MH. Together with previously published data, the mutation has now been reported in
three independent MH positive families
Current and future therapeutic approaches to the congenitalmyopathies
No full texttThe congenital myopathies – including Central Core Disease (CCD), Multi-minicore Disease (MmD), Cen-tronuclear Myopathy (CNM), Nemaline Myopathy (NM) and Congenital Fibre Type Disproportion (CFTD)– are a genetically heterogeneous group of early-onset neuromuscular conditions characterized by dis-tinct histopathological features, and associated with a substantial individual and societal disease burden.Appropriate supportive management has substantially improved patient morbidity and mortality butthere is currently no cure.Recent years have seen an exponential increase in the genetic and molecular understanding of theseconditions, leading to the identification of underlying defects in proteins involved in calcium homeostasisand excitation-contraction coupling, thick/thin filament assembly and function, redox regulation, mem-brane trafficking and/or autophagic pathways. Based on these findings, specific therapies are currentlybeing developed, or are already approaching the clinical trial stage. Despite undeniable progress, therapydevelopment faces considerable challenges, considering the rarity and diversity of specific conditions,and the size and complexity of some of the genes and proteins involved.The present review will summarize the key genetic, histopathological and clinical features of specificcongenital myopathies, and outline therapies already available or currently being developed in the con-text of known pathogenic mechanisms. The relevance of newly discovered molecular mechanisms andnovel gene editing strategies for future therapy development will be discussed
Calcium homeostasis and role of ryanodine receptor type 1 (RyR1) in immune cells
No full textRyanodine receptors are intracellular Ca2+ release channels located in the membrane of the Endoplasmatic/Sarcoplasmatic Reticulum. Ryanodine receptor 1 isoform is preferentially expressed in skeletal muscle where it is responsible for release of Ca2+ from the SR, an event that leads to muscle contraction. Point mutations in the gene encoding ryanodine receptor 1 have been linked to disease such as Malignant Hyperthermia, Central core disease and Multi-minicore disease.
Malignant Hyperthermia is a pharmacogenetic disorder with autosomal dominant inheritance and abnormal Ca2+ homeostasis in skeletal muscle in response to triggering agents. In susceptible individuals, a malignant hyperthermia crisis may be triggered by commonly used halogenated anaesthetics (halothane, isoflurane) or muscle relaxants (succhinylcholine). The main symptoms are hypermetabolism and muscle rigidity. Without treatment, death would occur in more than 80% of cases. Although a genetic-chip based diagnostic approach is under development, the invasive in vitro contracture test remains the “gold standard” to diagnose this disorder.
Central core disease is a slowly progressive myopathy characterized by muscle weakness and hypotonia. Central core disease is characterized histologically by the presence of central cores running along longitudinal axis of the muscle fiber.
Multi-minicore disease disease is a more severe, rare, autosomal recessive myopathy characterized histologically by the presence of multi-minicores in only a small number of sarcomeres. So far, no effective therapy has been developed to treat muscle weakness in central core disease and multi-minicore disease patients and their diagnosis is difficult on the basis of clinical findings alone. Histological examination of muscle tissue in these diseases is essential.
Recent data has shown that ryanodine receptor 1 is also expressed in some areas of the central nervous system as well as in cells of the immune system, specifically B-lymphocytes and dendritic cells.
The first part of my thesis focuses on the role of the ryanodine receptor 1 in dendritic cell. We first show that both immature and mature in vitro derived dendritic cells as well as circulating plasmocytoid cells express the ryanodine receptor 1 Ca2+ release channel within the endoplasmatic reticulum. Pharmacological activation of the ryanodine receptor 1 leads to the rapid release of Ca2+ from intracellular stores, and in the presence of sub-optimal concentrations of microbial stimuli, provides synergistic signals resulting in dendritic cell maturation and stimulation of T cell function. Furthermore, we were interested in unravelling more direct roles of this receptor in dendritic cells function. Interestingly, ryanodine receptor 1 activation in dendritic cells causes a very rapid increase in surface expression of major histocompatibility complex II molecules. In order to dissect the physiological route of ryanodine receptor 1 activation in vivo we hypothesized that a possible functional partner of ryanodine receptor 1 in dendritic cells could be, an L-type Ca2+ channel. We were able to show that human dendritic cells express the cardiac isoform of the L-type Ca2+ channel, which acts as a ryanodine receptor 1 functional partner on the plasma membrane of dendritic cells. We show that depolarization of dendritic cells by the addition of potassium chloride activates L-type Ca2+ channels initiating Ca2+ influx and activation of Ca2+ release via ryanodine receptor 1 and that this process could be prevented by nifedipine or ryanodine. Physiologically potassium could be released from dying cells within an inflamed tissue or from T- cells into immunological synapse during dendritic cell T-cell engagement and these events could be possible routes for activation of L-type Ca2+ channel- ryanodine receptor 1 signalling in dendritic cells in vivo. Thus, in vivo, activation of the ryanodine receptor 1 signalling cascade may be important during the early stages of infection, providing the immune system with rapid mechanisms to initiate an early response, facilitating the presentation of antigens to T cells.
While continuing our investigation on Ca2+ homeostasis in dendritic cells we noticed that spontaneous Ca2+ oscillations occur in immature dendritic cells but not in dendritic cells stimulated to undergo maturation with lipopolysaccharide or other toll like-receptor agonists. We investigated the mechanism and role of spontaneous Ca2+ oscillations in immature dendritic cells and found that they are mediated by the inositol-1,4,5-trisphosphate receptor since they were blocked by pre-treatment of cells with the inositol-1,4,5-trisphosphate receptor antagonist Xestospongin C and 2-Aminoethoxydiphenyl borate. A component of the Ca2+ signal is also due to influx from the extracellular environment. As to the biological function of these high frequency oscillations, our results indicate that they are associated with the translocation of a Ca2+ dependent transcription factor (nuclear factor of activated T-cells) into the nucleus of immature dendritic cells. In fact, once the Ca2+ oscillations are blocked with the 2-aminoethoxydiphenyl borate or by treating cells with lipopolysaccharide, nuclear factor of activated T-cells remains cytoplasmic.
The results from the first part of my thesis provide novel insights into the physiology of dendritic cells, role of ryanodine receptor 1 signaling and Ca2+ as an important second messenger in these cells.
The second aim of my thesis deals with functional properties of ryanodine receptor 1 carrying mutations linked to neuromuscular disorders, which is important from diagnostic point of view but also to understand the basic pathophysiological mechanism leading to these different diseases.
Since functional ryanodine receptors 1 are expressed in B-lymphocytes we investigated Ca2+ homeostasis in B-lymphocytes transformed with Epstein Barr virus from patients carrying the mutation linked to malignant hyperthermia and healthy donors.
In the first study from the Swiss population we investigated four novel mutations found in malignant hyperthermia susceptible pedigrees: (p.D544Y, p.R2336H, p.E2404K and p.D2730G). We found that the resting Ca2+ levels were significantly higher in cells from all four mutations bearing individuals compared to controls. These four mutations were also found to significantly affect either 4-chloro-m-cresol or caffeine dose response curves suggesting higher sensitivity of ryanodine receptor 1 to pharmacological activation in patients carrying these mutations.
In the second study we examined patients from the Swedish population carrying five different novel mutations (p.E1058K, p.R1679H, p.H382N, p.K1393R and p.R2508G). The first 4 patients had serious malignant hyperthermia clinical reactions and thereafter have tested by the in vitro contracture test and classified as malignant hyperthermia susceptible; the patient with the fifth mutation, p.Arg2508Gly, had been diagnosed as a central core disease. In this study as well functional studies were performed on Epstein Barr virus transformed B-lymphocytes from patients carrying mutations and healthy donors. Our results from the Swedish population suggest that ryanodine receptor 1 mutations also lead to abnormal Ca2+ homeostasis. Results from these and other studies support the use of Epstein Barr virus transformed -B-lymphocytes as an alternative, non-invasive, protocol for the diagnosis and the functional proof that a mutation in the ryanodine receptor causes alterations in Ca2+ homeostasis. This is a pre-requisite for the molecular diagnosis of malignant hyperthermia. These results also provide new concepts for the treatment of muscular pathologies involving mutations in ryanodine receptor 1
TIRF: using evanescent waves for high resolution membrane fluorescent imaging
No full textChanges in the intracellular Ca2+ concentration ([Ca2+]i) accompany many diverse biological phenomena from neuronal excitability and muscle contraction to activation of transcription and cell death. Thanks to the introduction of fluorescent intracellular Ca2+ dyes our understanding of Ca2+ signalling has greatly advanced; it is established that depending on the signalling molecule and cell type, the global increase in cytoplasmic Ca2+ is mediated by the mobilisation of Ca2+ from intracellular stores and by the opening channels on the plasma membrane, or by a combination of both mechanisms. In this application note, we use TIRF to monitor intracellular free calcium ion concentration [Ca2+]i on or very close to the plasma membrane of muscle cells including human myotube
Study of calcium sparks in skeletal and smooth muscle cells in normal and pathological conditions
No full textmTOR signaling influence a wide range of cellular process including protein synthesis (Iadevaia et al., 2012; Ma and Blenis, 2009; Thoreen et al., 2012), lipids synthesis (Lamming and Sabatini, 2013), transcription (Dibble and Manning, 2013; Vazquez-Martin et al., 2011), nucleotides biosynthesis (Ben-Sahra et al., 2013; Robitaille et al., 2013) and cellular energetics (Albert and Hall, 2015; Duvel et al., 2010; Inoki et al., 2012). In muscle, suppression of mTORC1 signaling results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres (Bentzinger et al., 2008), however it is unclear what is its specific role in the excitation contraction (EC) coupling. Likewise, the ryanodine receptor (RyR), the calcium release channel of the sarcoplasmic reticulum, plays a fundamental role in calcium homeostasis in a variety of cells and particularly in muscle (Lanner et al., 2010). Mutations in the gene encoding this channel have been associated with a number of debilitating or life-threatening neuromuscular pathologies including malignant hyperthermia (Kolb et al., 1982; Rosenberg et al., 2015; Treves et al., 2005), but little or no knowledge is known about their pathophysiological influence in mild bleeding disorders. In this thesis we investigated in greater detail 1) the effect of the mTORC1 signalling pathway on the integrity of the protein participants in skeletal muscle EC coupling and calcium homeostasis by using a muscle specific Raptor KO mouse model. 2) The calcium homeostasis of vascular smooth muscle cells of an MH mouse model and its association to mild bleeding disorders as also observed in MH patients.
As far as the mTOR is concerned, we found that in raptor knockout (RamKO) mice, the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, radio ligand binding assay showed a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively, which was confirmed by Western Blot analysis. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. These findings together with previous reports should be taken into consideration in the clinical practice when rapamycin or its analogs (rapalogs) is administrated to patients.
As far as RYR1–mutations in human patients and its relationship to bleeding abnormlities is concerned, 8/20 mutation carriers revealed abnormal bleeding scores compared with their healthy relatives (0/11). Similarly, MHS RYR1Y522S knock in mice exhibited 3 times longer bleeding times compared to their wild type littermates. The bleeding defect of MHS mice could be reversed by pre-treatment with the ryanodine receptor 1 antagonist dantrolene. Primary vascular SMCs from RYR1Y522S knock-in mice exhibited a higher frequency of subplasmalemmal Ca2+ sparks leading to a more negative resting membrane potential. Furthermore, Ca2+ sparks were blocked by pre-treatment with ryanodine or dantrolene. These results stimulated us to generate a model that could explain how impaired calcium homeostasis addressed by RyR1 mutation could affect bleeding without influencing platelet or coagulation factor function. Our results on impaired calcium homeostasis caused by RyR1 mutations could extend to other tissues that functionally express this channel.
In conclusion, the present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation–contraction (EC) coupling is affected by mTORC1 signaling and that RYR1 mutations cause prolonged bleeding by altering vascular SMC function and emphasize the potential therapeutic value of dantrolene in the treatment of such bleeding abnormalities
Identification of the domain recognized by anti-(ryanodine receptor) antibodies which affect Ca2+-induced Ca2+ release
No full textIn the present paper we have defined putative functional domains of the ryanodine receptor Ca2+ channel. cDNA fragments of the skeletal muscle ryanodine receptor were fused in-frame with the Escherichia coli trpe protein and the resulting fusion proteins were evaluated for their ability to react with anti-(ryanodine receptor) antibodies, which are known to block Ca(2+)-dependent activation of the Ca(2+)-release channel. Anti-(ryanodine receptor) antibodies react with epitopes lying within a 245-amino-acid-long polypeptide which is located in a region (residues 4380-4625) encompassing most of myoplasmic loop 2, the predicted transmembrane segment M5 and part of the next lumenal loop (45 residues). Purification of the anti-(ryanodine receptor) antibodies by affinity chromatography led to the isolation of a population of antibodies which was capable of decreasing (by > 30%) the doxorubicin-induced Ca2+ release from isolated terminal cisternae. Polyclonal antibodies raised against a ryanodine receptor fusion encompassing part (198 out of 245 residues) of the immunopositive polypeptide decreased by 2-fold the first-order rate constant of Ca(2+)-induced 45Ca2+ efflux from isolated terminal cisternae. These results suggest strongly that the Ca(2+)-activating domain of the skeletal muscle Ca(2+)-release channel is close to, or associated with, myoplasmic loop
Identification of calreticulin isoforms in the central nervous system
No full textIn the present paper we report the cloning and sequencing of the cDNA encoding two calreticulin isoforms from Xenopus laevis central nervous system. The two isoforms display 93% identity at the amino acid level. The predicted amino acid sequences of the amphibian calreticulins are very similar (76%) to those of mammalian liver and skeletal muscle. Xenopus laevis calreticulins are characterized by a very acidic c-terminal domain endowed with the endoplasmic-reticulum retention signal KDEL. The cDNAs of both clones encode an N-glycosylation consensus sequence. A third clone of calreticulin was also identified. The restriction map of this clone was clearly distinct from that of the two sequenced clones. These results indicate the existence of multiple calreticulin isoforms in the central nervous system and open questions about their functional role in different cells and/or subcellular compartment
Functional Characterization of Endogenously Expressed Human RYR1 Variants
No full textMore than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients' cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant
Malignant hyperthermia domain in the regulation of Ca2+ release channel (Ryanodine receptor)
No full textMalignant hyperthermia (MH) is a potentially lethal condition that is manifested in humans as an acute increase of body temperature in response to stress and exposure to volatile anaesthetics (halothane, enflurane) and muscle relaxants. To date, eight point mutations in the ryanodine receptor gene, the Ca(2+) release channel of the skeletal muscle sarcoplasmic reticulum, segregate with the MH phenotype, yet direct evidence linking altered [Ca(2+)](i) homeostasis to mutation in recombinant RYR has been obtained only for one such mutation. Most of these mutations appear in an "MH domain" that is localized at the NH(2) terminus of the skeletal muscle ryanodine receptor Ca(2+) channel. In this review, we summarize the available data concerning the role of the MH domain in the altered functions of the ryanodine receptor Ca(2+) channel. (Trends Cardiovasc Med 1997;7:312-316
- …
