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Robert S Adelstein - One of the best experts on this subject based on the ideXlab platform.
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characterization of isoform diversity in smooth muscle Myosin Heavy Chains
Canadian Journal of Physiology and Pharmacology, 1994Co-Authors: Christine A Kelley, Robert S AdelsteinAbstract:Nous revisons une partie de nos recents travaux sur la caracterisation biochimique et structurale des isoformes de la chaine lourde de Myosine II du muscle lisse des vertebres. Il existe des isoformes alternativement epissees en terminaison aminoterminale et carboxyle-terminale de la chaine lourde de Myosine du muscle lisse. L'epissage de l'ARN m a l'extremite 3' produit deux chaines lourdes de Myosine de longueurs differentes et une sequence d'acides amines en terminaison carboxyle. Nous appellerons la plus longue isoforme (de 204 kDa) MHC 204 , et la plus courte (de 200 kDa) MHC 200 . Nous constatons que MHC 204 , mais non pas MHC 200 , peut etre phosphorylee par la kinase II de la caseine sur une serine pres de l'extremite carboxyle, suggerant que ces isoformes pourraient etre regulees de maniere differente. L'importance physiologique de cette phosphorylation est inconnue. Toutefois, tel que demontre dans cet article, la phosphorylation ne semble pas affecter ni la formation des filaments et la vitesse de mouvement des filaments d'actine par la Myosine dans un test de motilite in vitro, ni l'activite de Mg 2+ ATPase induite par l'actine ou la conformation de Myosine. Nos resultats montrent aussi que MHC 204 et MHC 200 forment des homodimeres, mais pas des heterodimeres. Les homodimeres purifies de MHC 204 et MHC 200 ne sont pas enzymatiquement differents, du moins lorsqu'ils sont mesures en utilisant un test de motilite in vitro. Les isoformes MHC 204 et MHC 200 epissees a l'extremite amino-terminale sont le resultat d'une insertion ou d'une deletion specifique de sept acides amines pres de la region de fixation de l'ATP dans la tete de Myosine. Nous considerons ces isoformes comme etant, respectivement, inseres (MHC 204 -I; MHC 200 -I) ou non inseres (MHC 204 ; MHC 200 ). Contrairement aux isoformes epissees a l'extremite carboxyle-terminale, les isoformes non inserees et inserees epissees a l'extremite amino-terminale de la chaine lourde de Myosine sont enzymatiquement differentes. L'isoforme inseree, qui est exprimee dans le muscle lisse de type phasique intestinal, a une plus grande activite de Mg ATPase induite par l'actine et deplace les filaments d'actine a une plus grande vitesse dans un test de motilite in vitro que l'isoforme MHC non inseree qui est exprimee dans le muscle lisse vasculaire de type tonique. Ces resultats suggerent que l'epissage alternatif de l'ARN m du muscle lisse se produit dans au moins 4 isoformes differentes de la molecule de la chaine lourde de:Myosine. On discute de l'utilite potentielle de ces isoformes moleculaires pour la fonction du muscle lisse
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phosphorylation of vertebrate nonmuscle and smooth muscle Myosin Heavy Chains and light Chains
Molecular and Cellular Biochemistry, 1993Co-Authors: Robabeh S Moussavi, Christine A Kelley, Robert S AdelsteinAbstract:In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle Myosin that can undergo phosphorylation. The sites for phosphorylation in the 20kD Myosin light chain include serine-19 and threonine-18 which are substrates for Myosin light chain kinase and serine-1 and/or -2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle Myosin Heavy Chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.
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human nonmuscle Myosin Heavy Chains are encoded by two genes located on different chromosomes
Circulation Research, 1991Co-Authors: Michael Simons, Sachiyo Kawamoto, Robert S Adelstein, Mingyi Wang, O W Mcbride, K Yamakawa, D Gdula, L WeirAbstract:We report the cloning of cDNAs encoding two different human nonmuscle Myosin Heavy Chains designated NMMHC-A and NMMHC-B. The mRNAs encoding NMMHC-A and NMMHC-B are both 7.5 kb in size but are shown to be the products of different genes, which are localized to chromosome 22q11.2 and chromosome 17q13, respectively. In aggreement with previously reported results using avian tissues, we show that the mRNAs encoding the two Myosin Heavy chain isoforms are differentially expressed in rat nonmuscle and muscle tissues as well as in a number of human cell lines. The cDNA sequence encoding the 5' portion of the NMMHC-A isoform completes the previously published 3' cDNA sequence encoding a human Myosin Heavy chain, thus providing the cDNA sequence encoding the entire NMMHC-A amino acid sequence. Comparison of this sequence to cDNA clones encoding the amino-terminal one third of the NMMHC-B sequence (amino acids 58-718) shows them to be 89% identical at the amino acid level and 74% identical at the nucleotide level.
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chicken nonmuscle Myosin Heavy Chains differential expression of two mrnas and evidence for two different polypeptides
Journal of Cell Biology, 1991Co-Authors: Sachiyo Kawamoto, Robert S AdelsteinAbstract:Two different mRNAs encoding two different nonmuscle Myosin Heavy Chains (MHCs) of approximately 200 kD have been identified in chicken nonmuscle cells, in agreement with the results of Katsuragawa et al. (Katsuragawa, Y., M. Yanagisawa, A. Inoue, and T. Masaki. 1989. Eur. J. Biochem. 184:611-616). In this paper, we quantitate the content of mRNA encoding the two MHCs in a number of different tissues using RNA blot analysis with two specific oligonucleotide probes. Our results show that the relative content of mRNA encoding MHC-A and MHC-B differs in a tissue-dependent manner. Thus the ratio of mRNA encoding MHC-A versus MHC-B varies from greater than 9:1 in spleen and intestinal epithelial cells, to 6:4 in kidney and 2:8 in brain. The effect of serum on MHC mRNA expression was studied in serum-starved cultures of chick embryo fibroblasts. Serum stimulation results in a threefold increase in the mRNA encoding MHC-A and a threefold decrease in mRNA encoding MHC-B. Using SDS polyacrylamide gels, we have separated two nonmuscle MHC isoforms (198 and 196 kD) that can be distinguished from each other by two-dimensional peptide mapping of chymotryptic digests. We provide preliminary evidence that the MHC-A mRNA encodes the 196-kD polypeptide and that the MHC-B mRNA encodes the 198-kD polypeptide.
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identification of the serine residue phosphorylated by protein kinase c in vertebrate nonmuscle Myosin Heavy Chains
Biochemistry, 1991Co-Authors: Mary Anne Conti, Robert S Adelstein, James R Sellers, Marshall ElzingaAbstract:Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the Myosin Heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell Myosin Heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the Myosin rod. Although other vertebrate nonmuscle Myosin Heavy Chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle Myosin Heavy Chains (both 204 and 200 kDa) sequenced to date.
Richard L Moss - One of the best experts on this subject based on the ideXlab platform.
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pulse electrophoresis of muscle Myosin Heavy Chains in sodium dodecyl sulfate polyacrylamide gels
Analytical Biochemistry, 2001Co-Authors: Jose Santana A A Pereira, Marion L Greaser, Richard L MossAbstract:Abstract We have developed a new method that provides enhanced resolution of Myosin Heavy chain (MHC) isoforms by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.
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cardiac Myosin Heavy Chains lacking the light chain binding domain cause hypertrophic cardiomyopathy in mice
American Journal of Physiology-heart and Circulatory Physiology, 1999Co-Authors: Robert E Welikson, Stephen M Factor, Karen L Vikstrom, Richard L Moss, Scott H Buck, Jitandrakumar R Patel, Setsuya Miyata, Howard D Weinberger, Leslie A LeinwandAbstract:Myosin is a chemomechanical motor that converts chemical energy into the mechanical work of muscle contraction. More than 40 missense mutations in the cardiac Myosin Heavy chain (MHC) gene and seve...
Leslie A Leinwand - One of the best experts on this subject based on the ideXlab platform.
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cardiac Myosin Heavy Chains lacking the light chain binding domain cause hypertrophic cardiomyopathy in mice
American Journal of Physiology-heart and Circulatory Physiology, 1999Co-Authors: Robert E Welikson, Stephen M Factor, Karen L Vikstrom, Richard L Moss, Scott H Buck, Jitandrakumar R Patel, Setsuya Miyata, Howard D Weinberger, Leslie A LeinwandAbstract:Myosin is a chemomechanical motor that converts chemical energy into the mechanical work of muscle contraction. More than 40 missense mutations in the cardiac Myosin Heavy chain (MHC) gene and seve...
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Myosin Heavy Chains iia and iid are functionally distinct in the mouse
Journal of Cell Biology, 1998Co-Authors: Carol A Sartorius, Leslie Acakposatchivi, Renee P Jacobsen, William C Byrnes, Leslie A LeinwandAbstract:Myosin in adult murine skeletal muscle is composed primarily of three adult fast Myosin Heavy chain (MyHC) isoforms. These isoforms, MyHC-IIa, -IId, and -IIb, are >93% identical at the amino acid level and are broadly expressed in numerous muscles, and their genes are tightly linked. Mice with a null mutation in the MyHC-IId gene have phenotypes that include growth inhibition, muscle weakness, histological abnormalities, kyphosis (spinal curvature), and aberrant kinetics of muscle contraction and relaxation. Despite the lack of MyHC-IId, IId null mice have normal amounts of Myosin in their muscles because of compensation by the MyHC-IIa gene. In each muscle examined from IId null mice, there was an increase in MyHC-IIa– containing fibers. MyHC-IIb content was unaffected in all muscles except the masseter, where its expression was extinguished in the IId null mice. Cross-sectional fiber areas, total muscle cross-sectional area, and total fiber number were affected in ways particular to each muscle. Developmental expression of adult MyHC genes remained unchanged in IId null mice. Despite this universal compensation of MyHC-IIa expression, IId null mice have severe phenotypes. We conclude that despite the similarity in sequence, MyHC-IIa and -IId have unique roles in the development and function of skeletal muscle.
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mice expressing mutant Myosin Heavy Chains are a model for familial hypertrophic cardiomyopathy
Molecular Medicine, 1996Co-Authors: Karen L Vikstrom, Stephen M Factor, Leslie A LeinwandAbstract:Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myocellular disarray, arrhythmias, and sudden death. Mutations in several contractile proteins, including cardiac Myosin Heavy Chains, have been described in families with this disease, leading to the hypothesis that HCM is a disease of the sarcomere. A mutation in the Myosin Heavy chain (Myh) predicted to interfere strongly with Myosin’s binding to actin was designed and used to create an animal model for HCM. Five independent lines of transgenic mice were produced with cardiac-specific expression of the mutant Myh. Although the mutant Myh represents a small proportion (1–12%) of the heart’s Myosin, the mice exhibit the cardiac histopathology seen in HCM patients. Histopathology is absent from the atria and primarily restricted to the left ventricle. The line exhibiting the highest level of mutant Myh expression demonstrates ventricular hypertrophy by 12 weeks of age, but the further course of the disease is strongly affected by the sex of the animal. Hypertrophy increases with age in female animals while the hearts of male show severe dilation by 8 months of age, in the absence of increased mass. The low levels of the transgene protein in the presence of the phenotypic features of HCM suggest that the mutant protein acts as a dominant negative. In addition, the distinct phenotypes developed by aging male or female transgenic mice suggest that extragenic factors strongly influence the development of the disease phenotype.
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fast Myosin Heavy Chains expressed in secondary mammalian muscle fibers at the time of their inception
Journal of Cell Science, 1994Co-Authors: Mildred K Cho, Leslie A Leinwand, Simon M Hughes, Ilene Karschmizrachi, Marilyn Travis, Helen M BlauAbstract:Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast Myosin Heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, monoclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development.
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cardiac alpha Myosin Heavy Chains differ in their induction of myocarditis identification of pathogenic epitopes
Journal of Clinical Investigation, 1993Co-Authors: Li Liao, Rajeev Sindhwani, Leslie A Leinwand, Betty Diamond, Stephen M FactorAbstract:BALB/c mice develop autoimmune myocarditis after immunization with mouse cardiac Myosin, whereas C57B/6 mice do not. To define the immunogenicity and pathogenicity of cardiac Myosin in BALB/c mice, we immunized mice with different forms of cardiac Myosin. These studies demonstrate the discordance of immunogenicity and pathogenicity of Myosin Heavy Chains. The cardiac alpha-Myosin Heavy Chains of BALB/c and C57B/6 mice differ by two residues that are near the junction of the head and rod in the S2 fragment of Myosin. Myosin preparations from both strains are immunogenic in susceptible BALB/c as well as in nonsusceptible C57B/6 mice; however, BALB/c Myosin induces a greater incidence of disease. To further delineate epitopes of Myosin Heavy chain responsible for immunogenicity and disease, mice were immunized with fragments of genetically engineered rat alpha cardiac Myosin. Epitopes in the region of difference between BALB/c and C57B/6 (residues 735-1032) induce disease in both susceptible and nonsusceptible mice. The data presented here demonstrate that pathogenic epitopes of both mouse and rat Myosin residue in the polymorphic region of the S2 subunit. In addition, these studies suggest that polymorphisms in the autoantigen may be part of the genetic basis for autoimmune myocarditis.
Ian A. Johnston - One of the best experts on this subject based on the ideXlab platform.
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Duplication of a Single myhz1.1 Gene Facilitated the Ability of Goldfish (Carassius auratus) to Alter Fast Muscle Contractile Properties With Seasonal Temperature Change.
Frontiers in physiology, 2018Co-Authors: Daniel Garcia De La Serrana, Kristin M. Wreggelsworth, Ian A. JohnstonAbstract:Seasonal temperature changes markedly effect the swimming performance of some cyprinid fish acutely tested at different temperatures, involving a restructuring of skeletal muscle phenotype including changes in contractile properties and Myosin Heavy chain expression. We analyzed the transcriptome of fast myotomal muscle from goldfish (Carassius auratus L.) acclimated to either 8 or 25°C for 4 weeks (12 h light: 12 h dark) and identified 10 Myosin Heavy Chains (myh) and 13 Myosin light chain (myl) transcripts. Goldfish orthologs were classified based on zebrafish nomenclature as myhz1.1α, myhz1.1β, myhz1.1γ, myha, myhb, embryo_myh1, myh9b, smyh2, symh3, and myh11 (Myosin Heavy Chains) and myl1a, myl1b, myl2, myl9a, myl9b, myl3, myl13, myl6, myl12.1a, myl12.1b, myl12.2a, myl12.2b, and myl10 (Myosin light Chains). The most abundantly expressed transcripts myhz1.1α, myhz1.1β, myhz1.1γ, myha, myl1a, myl1b, myl2, and myl3) were further investigated in fast skeletal muscle of goldfish acclimated to either 4, 8, 15, or 30°C for 12 weeks (12 h light:12 h dark). Total copy number for the Myosin Heavy Chains showed a distinct optimum at 15°C (P < 0.01). Together myhz1.1α and myhz1.1β comprised 90 to 97% of myhc transcripts below 15°C, but only 62% at 30°C. Whereas myhz1.1α and myhz1.1β were equally abundant at 4 and 8°C, myhz1.1β transcripts were 17 and 12 times higher than myhz1.1α at 15 and 30°C, respectively, (P < 0.01). Myhz1.1γ expression was at least nine-fold higher at 30°C than at cooler temperatures (P < 0.01). In contrast, the expression of myha and Myosin light Chains showed no consistent pattern with acclimation temperature. A phylogenetic analysis indicated that the previously reported ability of goldfish and common carp to alter contractile properties and myofibrillar ATPase activity with temperature acclimation was related to the duplication of a single myhz1.1 fast muscle Myosin Heavy chain found in basal cyprinids such as the zebrafish (Danio rerio).
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Table_4_Duplication of a Single myhz1.1 Gene Facilitated the Ability of Goldfish (Carassius auratus) to Alter Fast Muscle Contractile Properties With Seasonal Temperature Change.DOCX
2018Co-Authors: Daniel Garcia De La Serrana, Kristin M. Wreggelsworth, Ian A. JohnstonAbstract:Seasonal temperature changes markedly effect the swimming performance of some cyprinid fish acutely tested at different temperatures, involving a restructuring of skeletal muscle phenotype including changes in contractile properties and Myosin Heavy chain expression. We analyzed the transcriptome of fast myotomal muscle from goldfish (Carassius auratus L.) acclimated to either 8 or 25°C for 4 weeks (12 h light: 12 h dark) and identified 10 Myosin Heavy Chains (myh) and 13 Myosin light chain (myl) transcripts. Goldfish orthologs were classified based on zebrafish nomenclature as myhz1.1α, myhz1.1β, myhz1.1γ, myha, myhb, embryo_myh1, myh9b, smyh2, symh3, and myh11 (Myosin Heavy Chains) and myl1a, myl1b, myl2, myl9a, myl9b, myl3, myl13, myl6, myl12.1a, myl12.1b, myl12.2a, myl12.2b, and myl10 (Myosin light Chains). The most abundantly expressed transcripts myhz1.1α, myhz1.1β, myhz1.1γ, myha, myl1a, myl1b, myl2, and myl3) were further investigated in fast skeletal muscle of goldfish acclimated to either 4, 8, 15, or 30°C for 12 weeks (12 h light:12 h dark). Total copy number for the Myosin Heavy Chains showed a distinct optimum at 15°C (P < 0.01). Together myhz1.1α and myhz1.1β comprised 90 to 97% of myhc transcripts below 15°C, but only 62% at 30°C. Whereas myhz1.1α and myhz1.1β were equally abundant at 4 and 8°C, myhz1.1β transcripts were 17 and 12 times higher than myhz1.1α at 15 and 30°C, respectively, (P < 0.01). Myhz1.1γ expression was at least nine-fold higher at 30°C than at cooler temperatures (P < 0.01). In contrast, the expression of myha and Myosin light Chains showed no consistent pattern with acclimation temperature. A phylogenetic analysis indicated that the previously reported ability of goldfish and common carp to alter contractile properties and myofibrillar ATPase activity with temperature acclimation was related to the duplication of a single myhz1.1 fast muscle Myosin Heavy chain found in basal cyprinids such as the zebrafish (Danio rerio).
Ugo Carraro - One of the best experts on this subject based on the ideXlab platform.
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high resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunochemical identification of the 2x and embryonic Myosin Heavy Chains in complex mixtures of isoMyosins
Electrophoresis, 1995Co-Authors: Katia Rossini, Corrado Rizzi, Marco Sandri, A Bruson, Ugo CarraroAbstract:In mammals Myosin Heavy Chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemistry. Electrophoretic analysis is the only method for quantitating MHC profiles in single myofibers and/or cryostat sections of biopsied muscle. We present a method for SDS-PAGE of adult rat skeletal muscle which resolves MHC into four bands: 1, 2B, 2X, and 2A from the faster to the slower migrating band. Furthermore, embryonic MHC can be also resolved in a complex mixture of isoMyosins, e.g. developing or regenerating muscles. The method does not involve preparation of gradient gels or electrophoresis at low temperature. Improved reproducibility is obtained by: (i) modification of the sample buffer; (ii) use of 7% polyacrylamide in the separating gel; (iii) control of pH of running buffer by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, silver and immunoblot staining. Resolution is sufficient to permit transblotting of separated MHC after SDS-PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides an improved method to separate MHC and quantitate MHC2X and MHCemb in complex mixtures of MHC from a few cryostat sections of normal and diseased muscle.
