Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the last decade, as a result of molecular cloning and the reverse-transcriptase polymerase chain reaction, numerous isoforms of the contractile protein myosin have been discovered. What lags behind their discovery is knowledge of their functions. This review focuses on some of my recent work on the structure, function and regulation of isoforms of the heavy chain of vertebrate smooth muscle and nonmuscle myosin II. Reference to related work in the field is included where appropriate. The particular isoforms discussed are those that are generated by alternative splicing near the 5' end of the pre-mRNA, resulting in either an insertion or a deletion of a cassette of amino acids near the amino-terminus of the myosin heavy chain (MHC) protein. In both the smooth muscle and nonmuscle MHCs, this splicing occurs in the exact same region, which begins at amino acid 212 in the primary sequence. In the three-dimensional structure of the molecule, these inserts are located near the ATP-binding pocket in a region of the MHC that was not resolved in the crystal structure and therefore is believed to represent a flexible loop. In the smooth muscle MHC, the insertion of seven amino acids in this loop confers a higher enzymatic activity on the myosin. The potential mechanism by which this occurs and the significance to smooth muscle contractile diversity is discussed. In the nonmuscle MHC, the insert in this region is a different size and sequence of amino acids than that in the smooth muscle MHC. A serine residue (Ser-214) in the nonmuscle loop is phosphorylated by p34cdc2 kinase in Xenopus during meiotic maturation of oocytes to eggs and is dephosphorylated in interphase egg extracts that are equivalent to the interphase after fertilization of the egg. Thus, MHC-B phosphorylation by cdc2 kinase correlates with the cortical reorganization that occurs during meiosis, and dephosphorylation correlates with the cortical contraction that occurs at fertilization, which aids in pronuclear fusion. In summary, these inserts in the MHC molecule, in a flexible loop near the ATP-binding pocket, appear to be important in determining differences in function or regulation among myosin II isoforms.
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PMID:Characterization of isoform diversity among smooth muscle and nonmuscle myosin heavy chains. 918 13

A highly sensitive method of reverse-transcriptase polymerase chain reaction (RT-PCR) was established to study myosin heavy-chain (MHC) mRNA isoform expression in single fibers of rabbit limb muscles. In combination with myofibrillar adenosine triphosphatase histochemistry and electrophoretic separation of MHC protein isoforms in fragments of the same fibers, the direct RT-PCR method identified the pMHC20-40 and pMHC24-79 cDNA sequences as being specific to MHCIIb and MHCIId/x isoforms, respectively. In addition, a direct RT-PCR was established for determining relative amounts of MHC mRNA isoforms by using a sequence specific to alpha-skeletal actin as an endogenous reference. Analyses of large amounts of single fibers revealed an unexpected heterogeneity of the fast fiber population with regard to numerous fibers coexpressing MHCIIb and MHCIId/x. Based on quantitative RT-PCR, the percentages of MHCIIb/MHCIId hybrid fibers amounted to approximately 55% in the deep portion of gastrocnemius, to 43% in the adductor magnus, and to 12% in psoas muscle. Moreover, the two MHC mRNA isoforms were nonuniformly distributed along the fiber length. Qualitative RT-PCR detected even higher amounts of hybrid fibers in the three muscles. The percentages of hybrid fibers identified at the protein level were smaller in adductor magnus muscle (25%) and psoas muscle (5%), but equaled that of the mRNA analysis in gastrocnemius muscle (61%). The detection of high amounts of IIBD and IIDB fibers suggested that hybrid fibers represent functional elements within the fiber spectrum of normal muscles. Our observations on hybrid fibers reveal a heterogeneity within the fiber population of normal muscles that has not been realized to date.
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PMID:Quantitative analyses of myosin heavy-chain mRNA and protein isoforms in single fibers reveal a pronounced fiber heterogeneity in normal rabbit muscles. 924 5

A highly sensitive method of reverse-transcriptase polymerase chain reaction (RT-PCR) was established to quantify transcript levels of the myogenic regulatory factors MyoD, myogenin and MRF4 (muscle regulatory factor 4) and for Id-1 (inhibitor of differentiation), a putative negative regulator of myogenesis. The method was sensitive enough to detect mRNA amounts as low as 20 molecules. Measurements in 10 different skeletal muscles of the rat revealed that the amounts of the four factors differ by almost three orders of magnitude. Id-1 is expressed at lowest levels (approximately 4x10(5) molecules/microg RNA) and MRF4 at highest levels (approximately 9x10(7) molecules/microg RNA). In general, myogenin and MyoD mRNAs were inversely distributed in slow and fast muscles. A correlation seemed to exist between the levels of MyoD and myosin heavy chain (MHC) IIb, the fastest MHC isoform. However, as revealed by changes in the expression levels of these two regulatory factors under conditions of hypothyroidism and chronic low-frequency stimulation (CLFS), MyoD and myogenin did not seem to be strictly correlated with fast and slow myosins, respectively. Hypothyroidism led to pronounced depressions of MyoD, but only to small increases in myogenin mRNA in fast muscles. These changes were only slightly increased by CLFS. However, as previously shown, CLFS in combination with hypothyroidism induces in rat muscle pronounced fast to slow transitions in myosin expression [Kirschbaum, B. J., Kucher. H.-B., Termin, A., Kelly, A. M. & Pette, D. (1990) J. Biol. Chem. 265, 13974-13980]. These findings suggest that MyoD and myogenin may not be causally related to the development and maintenance of fiber-type diversities.
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PMID:Quantification of MyoD, myogenin, MRF4 and Id-1 by reverse-transcriptase polymerase chain reaction in rat muscles--effects of hypothyroidism and chronic low-frequency stimulation. 924 14

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >>+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.
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PMID:HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. 950 Dec 1

We report the identification and cloning of a unique chick myosin heavy chain (CMHC1) that is expressed exclusively in the heart during embryogenesis. Using primers specific to myosin heavy chains, we used reverse transcriptase-polymerase chain reaction to clone and isolate CMHC1 from embryonic day 10 chicken heart RNA. Sequence analysis indicated that CMHC1 was a novel member of the myosin heavy chain family. Expression of the CMHC1 transcripts was detected in Hamburger Hamilton stage 10 chick embryos in the fusing myocardium. Expression of CMHC1 was maintained at high levels throughout the tubular heart of later stage embryos. Reverse transcriptase-polymerase chain reaction and in situ hybridizations failed to detect CMHC1 transcripts in the developing somites, limb buds, or skeletal musculature at any stage of chick development. Genomic CMHC1 clones have been isolated that contain sequences approximately 5.2 kilobase upstream of the presumptive CMHC1 transcription start site. Portions of the upstream regulatory region induced a 21-fold increase in reporter gene expression in primary cardiomyocytes. Because of its unique cardiac-restricted expression, CMHC1 will provide an excellent model system to study the molecular mechanisms required for the early developmental regulation of heart-specific genes.
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PMID:Identification and genomic cloning of CMHC1. A unique myosin heavy chain expressed exclusively in the developing chicken heart. 1063 96

Genetic responses that characterize experimental autoimmune myocarditis (EAM) have not yet been determined. To investigate gene expression in the myocardium of EAM, absolute copy numbers of 44 mRNA species [calcium-handling proteins, contractile proteins, natriuretic peptides (NPs), cytokines, chemokines, growth factors, renin-angiotensin-aldosterone (RAA) system, endothelins (ETs) and extracellular matrix] in synthesized cDNA from a fixed quantity of total heart RNA were assessed using real-time reverse-transcriptase PCR at days 0, 14, 21 and 28 after immunization. alpha-Cardiac myosin showed a 26.3-fold decrease and beta-cardiac myosin a 3.75-fold increase at day 14. Atrial NP and brain NP increased 47.7- and 6.35-fold at days 21 and 14 respectively. Angiotensin II type 1 receptor, angiotensin-converting enzyme and ET1 increased 22.3-fold at day 21, 6.30-fold at day 21 and 16.8-fold at day 14 respectively. Aldosterone receptor decreased 2.15-fold at day 14, but aldosterone synthetase was detected only at days 14 and 21. Interleukin (IL)-2, IL-10, interferon-gamma and monocyte chemo-attractant protein-1 increased 9.08-fold at day 14, 398-fold at day 21, 43.1-fold at day 14 and 142-fold at day 14 respectively. Collagen type 3, collagen type 1 and fibronectin increased 34.6-, 1.74- and 44.4-fold respectively at day 21. Interestingly, osteopontin showed a 4540-fold increase and it was the highest mRNA of all at day 14. An isoform of cardiac myosin and NP are dramatically changed in EAM. RAA system and ET expressions are changed differently during the EAM time course. Cytokine, chemokine and extracellular matrix greatly increase and, in particular, large numbers of osteopontin mRNA are expressed in early EAM.
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PMID:Time course of gene expression in rat experimental autoimmune myocarditis. 1244 15

Satellite cells were isolated from biopsies of the biceps femoris of adult dogs. Virtually all cells expressed muscle-specific proteins. Proliferation of satellite cells increased as the concentration of fetal calf serum (FCS) was increased from 1 to 10% of the basal medium. The addition of mitogenic growth factors resulted in greater proliferation than that of cells cultured in basal medium alone. Maximum proliferation was obtained when fibroblast growth factor-basic (FGF2) was added to the medium, but differences existed between sources or types. Proliferation did not plateau when the concentration of recombinant human FGF2 was 75 ng/ml but reached maximum levels when 50 ng/ml of bovine FGF2 or 10 ng/ml of growth hormone or insulin-like growth factor-1 were added to the medium. Proliferation of satellite cells decreased when more than 5 ng/ml of transforming growth factor-alpha was included in the medium. Exposure of canine satellite cells to chemically defined media induced greater fusion of total nuclei (ODM-34%; 4F, ITT-CF, and SFG-23%) than exposure to other treatments, such as basal medium plus 2 mg/ml of 1-beta-d-arabinofuranosylcytosine, 5% chick embryo extract, 1% horse serum (average 9% fused nuclei), or 1% FCS (2% fused nuclei). Actin, myosin, desmin, neural cell adhesion molecule, MyoD1, and myogenin were expressed by canine satellite cells, but expression of major histocompatibility complex class II antigen was not detected. Reverse transcriptase-polymerase chain reaction detected expression of messenger ribonucleic acid for interleukin-6 (IL-6), IL-15, and leukemia inhibitory factor by canine satellite cells. Collectively, these data suggest that isolated canine satellite cells display properties of other types of myogenic cells and may be useful for further study of the regulation of postnatal myogenesis.
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PMID:Isolation and characterization of canine satellite cells. 1260 41

Two smooth muscle myosin heavy chain isoforms that differ by the presence ([+]insert) or the absence ([-]insert) of a 7-amino acid insert in the motor domain have a 2-fold difference in their in vitro actin filament velocity. We hypothesized that a preferential expression of the fast (+)insert isoform in airway smooth muscle would increase the rate of bronchoconstriction. To verify our hypothesis we measured the time course of bronchoconstriction following a bolus injection of methacholine (160 microg/kg) in (+)insert isoform knockout (KO) and corresponding wild-type (WT) mice. Neither baseline airway resistance (Raw) (0.424 +/- 0.04 for WT and 0.374 +/- 0.01 cm H(2)O.s.ml(-1) for KO) nor peak Raw (4.1 +/- 0.9 for WT and 4.0 +/- 0.5 cm H(2)O.s.ml(-1) for KO) differed between groups. However, the time to peak Raw was significantly longer in the KO (17.2 +/- 0.6 s) compared with the WT (14.6 +/- 0.8 s) mice (P < 0.05). Differentiating Raw with respect to time revealed a greater rate of bronchoconstriction for the WT during the initial 4 s, presumably reflecting the faster shortening velocities under these relatively unloaded conditions. Reverse transcriptase-polymerase chain reaction analysis revealed that the (+)insert myosin isoform mRNA content in the WT airways was 47.8 +/- 5.6%. We conclude that the presence of the (+)insert myosin isoform in the airways increases the rate of bronchoconstriction.
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PMID:Time course of airway mechanics of the (+)insert myosin isoform knockout mouse. 1295 48