Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous work demonstrated that the rabbit smooth muscle myosin heavy chain gene showed sequence divergence at the 25kDa/50kDa junction of the S1 subfragment when compared to chicken gizzard and chicken epithelial nonmuscle myosin. RNase protection analysis with a probe spanning this region detected two partially protected fragments which were not present in RNA from vascular tissue and only found in RNA from visceral tissue. The polymerase chain reaction was used to amplify a 162bp product from primers spanning the putative region of divergence and DNA sequence analysis revealed a seven amino acid insertion not previously detected in other characterised cDNA clones. RNase protection analysis using the PCR product as probe showed that the inserted sequence was expressed exclusively in RNA from visceral tissue. Similar RNA analysis showed that the visceral isoform was not expressed in 20 day fetal rabbit smooth muscle tissues. These results indicated that the new visceral isoform was expressed in a tissue-specific and developmentally regulated manner. Genomic DNA sequencing and mapping of the exon-intron boundaries showed that the visceral isoform was the product of cassette-type alternative splicing. The inclusion of a visceral-specific sequence near the Mg-ATPase domain and at the 25kDa/50kDa junction suggests that the visceral isoform may be important for myosin function in smooth muscle cells.
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PMID:Tissue-specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain. 846 39

Myosin heavy chain (MHC) expression was studied in rat soleus and tibialis anterior (TA) at the mRNA and protein levels following reinnervation 8 and 32 wk after sciatic nerve injury. A sciatic nerve crush or transection injury was produced in the midthigh region of adult female Sprague-Dawley rats. A ribonuclease protection assay was developed to measure four of the adult MHCs (I, IIa, IIx, IIb) in a single sample. MHC mRNA and protein were measured and compared in the same muscles. Eight and thirty-two weeks after a crush injury, the MHC mRNA profiles were similar to control with the exception of soleus MHC IIa and TA MHC IIb, which were significantly less than control at both time points. In contrast, reinnervation of the soleus following a sciatic nerve transection injury resulted in an MHC isoform shift characterized by increases in the relative amounts of fast myosin (IIa and IIx) and a decrease in slow myosin. As expected, significant changes first occurred at the mRNA level followed by changes in protein expression. Thirty-two weeks after transection injury and repair, the primary MHC mRNA isoform in the soleus was MHC IIx. Moreover, at 32 wk, MHC IIb mRNA was detected in 50% of the reinnervated soleus following a transection injury. Reinnervation of the TA following sciatic nerve transection led to replacement of the MHC IIb isoform with MHC IIx.
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PMID:Altered expression of myosin mRNA and protein in rat soleus and tibialis anterior following reinnervation. 899 4

The skeletal rat myoblast omega (RMo) cell line forms myotubes that exhibit spontaneous contractions under appropriate conditions in culture. We examined if the RMo cells would provide a model for studying atrophy and muscle contraction. To better understand how to obtain contractile cultures, we examined levels of contraction under different growing conditions. The proliferation medium and density of plating affected the subsequent proportion of spontaneously contracting myotubes. Using a ribonuclease protection assay, we found that exponentially growing RMo myoblasts contained no detectable myogenin or herculin mRNA, while differentiating myoblasts contained high levels of myogenin mRNA but no herculin mRNA. There was no increase in myogenin mRNA concentration in either primary chick or RMo myotubes whose contractions were inhibited by depolarizing concentrations of potassium (K+). Thus, altered myogenin mRNA concentrations are not involved in atrophy of chick myotubes. Depolarizing concentrations of potassium inhibited spontaneous contractions in both RMo cultures and primary chick myotube cultures. However, we found that the myosin concentration of 6-d-old contracting RMo cells fed medium plus AraC was 11 +/- 3 micrograms myosin/microgram DNA, not significantly different from 12 +/- 4 micrograms myosin/microgram DNA (n = 3), the myosin concentration of noncontracting RMo cells (treated with 12 mM K+ for 6 d). Resolving how RMo cells maintained their myosin content when contraction is inhibited may be important for understanding atrophy.
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PMID:Effect of atrophy and contractions on myogenin mRNA concentration in chick and rat myoblast omega muscle cells. 911 27

Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of approximately 86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.
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PMID:Skeletal muscle-specific myosin binding protein-H is expressed in Purkinje fibers of the cardiac conduction system. 913 Apr 47

Myosin heavy chains (hcs) are the major determinant in the speed of contraction of skeletal muscle, and various isoforms are differentially expressed depending on the functional activity of the muscle. Using the rapid amplification of cDNA ends (3' RACE) method, we have characterised the 3' end of the embryonic, perinatal, type 1, 2a, 2x, and 2b myosin hc genes in rabbit skeletal muscle and used them as probes in RNase protection assays to quantitatively monitor their expression in different type of skeletal muscles just before and after birth. SDS PAGE was used to study the changes in the expression level of their respective protein and to determine the relative abundance of each myosin hc isoform in the muscles studied. The results show that for each anatomical muscle, the developmental changes in myosin hc gene expression at the mRNA level correlate strongly to those observed at the protein level. By studying their developmental expression in four functionally diverse skeletal muscles (semimembranosus proprius, diaphragm, tibialis anterior, and semimembranosus accessorius), it was shown that all muscles express the embryonic, perinatal, and type 1 isoform during prenatal development up to the E27 stage. In the diaphragm, low levels of the type 2a and 2x transcripts, which are adult fast isoforms, were also detected at the E27 stage. During the first week of postnatal growth the myosin hc transition leading to the expression of the adult isoforms is complex, and as many as five different myosin heavy chains are concurrently expressed in some muscles at around birth. As the animal matures, individual muscles become adapted to perform highly specialised functions, and this is reflected in the myosin hc composition within these muscles. Accordingly, the expression of the type 1 isoform, and the sequence of appearance and the expression levels of the type 2 isoforms, were exclusively dependent on the muscle type and largely reflect the functional activity of each muscle during the postnatal growth period.
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PMID:Differential expression of myosin heavy chain mRNA and protein isoforms in four functionally diverse rabbit skeletal muscles during pre- and postnatal development. 952 Jan 7

To investigate specific effects of androgens on whole body metabolism, we studied six healthy lean men (mean +/- SEM age, 23.2 +/- 0.5 yr) before and after gonadal steroid suppression with a GnRH analog (Lupron), given twice, 3 weeks apart. Primed infusions of [13C]leucine, indirect calorimetry, isokinetic dynamometry, growth factor measurements, and percutaneous muscle biopsies were performed at baseline (D1) and after 10 weeks of treatment (D2); each subject served as his own control. Testosterone concentrations were markedly suppressed after 10 weeks of treatment (D1, 535 +/- 141 ng/dL; D2, 31 +/- 9). Leucine's rate of appearance (index of proteolysis) was markedly suppressed after 10 weeks of hypogonadism (-13%; P = 0.01) as well as the nonoxidative leucine disposal, an index of whole body protein synthesis (-13%; P = 0.01) without any changes in plasma amino acid concentrations. All subjects studied after 10 weeks showed a decrease in fat-free mass, as measured by skinfold calipers and dual emission x-ray absortiometry scans (D1, 56.5 +/- 2.9 kg; D2, 54.4 +/- 2.5; P = 0.005), and an increase in percent fat mass (D1, 19.2 +/- 2.5%; D2, 22.2 +/- 2.5; P = 0.001). Rates of lipid oxidation decreased (-31%; P = 0.05) after treatment, with parallel changes in resting energy expenditure (-9%; P = 0.05). Mean and peak GH concentrations (measured every 10 min for 6 h) and GH production rates did not decrease after testosterone deficiency, with an actual increase in basal secretion (P < 0.02). Plasma insulin-like growth factor I (IGF-I) concentrations did not change significantly after 10 weeks of treatment (D1, 227 +/- 44 micrograms/L; D2, 291 +/- 60; P = 0.08). Isokinetic dynamometry of leg extensors at 60 degrees and 180 degrees/s was also decreased after 10 weeks of hypogonadism. Total ribonucleic acid (RNA) was isolated from muscle biopsy samples, and ribonuclease protection assays were performed using human complementary DNA clones for IGF-I, IGF-binding protein-4, myosin, and actin. Ten weeks after Lupron treatment, messenger RNA (mRNA) concentrations of IGF-I decreased significantly, whereas there was a trend toward higher IGF-binding protein-4 concentrations, with no change in myosin or actin mRNA concentrations. In conclusion, testosterone deficiency in young men is associated with a marked decrease in measures of whole body protein anabolism, decreased strength, decreased fat oxidation, and increased adiposity. These effects of testosterone deficiency are independent of changes in peripheral GH production and IGF-I concentrations, even though im IGF-I mRNA concentrations decrease. These data suggest a direct effect of androgens on whole body lipid and protein metabolism.
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PMID:Testosterone deficiency in young men: marked alterations in whole body protein kinetics, strength, and adiposity. 962 14

Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms.
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PMID:Myosin heavy chain isoform expression in rat smooth muscle development. 968 13

The rates of transcription of several protein coding genes during Acanthamoeba differentiation have been examined by nuclear run-on and RNase protection assays. During early encystment, transcription by RNA polymerase II increases approximately 4-fold, whereas transcription by RNA polymerases I and III is decreased, as previously described. The rates of transcription from a wide variety of individual genes are only slightly affected during the first 16 h of encystment, although profilin gene expression is markedly increased. The levels of mRNAs encoding TPBF, TATA binding protein, cyclin-dependent kinase, protein disulfide isomerase, profilin, myosin II heavy chain, ubiquitin and extendin are stable during mature cyst formation, whereas mRNAs encoding actin, S-adenosyl methionine synthase and tubulin are substantially decreased in abundance within 16 h of starvation-induced encystment. We conclude that in contrast to the negative regulation of large rRNA and 5S rRNA synthesis during differentiation, the RNA polymerase II transcription apparatus is not negatively regulated. Control of Acanthamoeba differentiation is likely to be mediated by positive regulation of genes necessary for cyst maturation.
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PMID:Transcription by RNA polymerase II during Acanthamoeba differentiation. 987 98

1. Skeletal muscle is a major source of circulating insulin growth factor-1 (IGF-1), particularly during exercise. It expresses two main isoforms. One of the muscle IGF-1 isoforms (muscle L.IGF-1) is similar to the main liver IGF-1 and presumably has an endocrine action. The other muscle isoform as a result of alternative splicing has a different 3' exon sequence and is apparently designed for an autocrine/paracrine action (mechano-growth factor, MGF). Using RNase protection assays with a probe that distinguishes these differently spliced forms of IGF-1, their expression and also the expression of two structural genes was measured in rabbit extensor digitorum longus muscles subjected to different mechanical signals. 2. Within 4 days, stretch using plaster cast immobilization with the limb in the plantar flexed position resulted in marked upregulation of both forms of IGF-1 mRNA. Electrical stimulation at 10 Hz combined with stretch (overload) resulted in an even greater increase of both types of IGF-1 transcript, whereas electrical stimulation alone, i.e. without stretch, resulted in no significant increase over muscle from sham-operated controls. Previously, it was shown that stretch combined with electrical stimulation of the dorsiflexor muscles in the adult rabbit results in a marked increase in muscle mass involving increases in both length and girth, within a few days. The expression of both systemic and autocrine IGF-1 growth factors provides a link between the mechanical signal and the marked increase in the structural gene expression involved in tissue remodelling and repair. 3. The expression of the beta actin gene was seen to be markedly upregulated in the stretched and stretched/stimulated muscles. It was concluded that the increased expression of this cytoskeletal protein gene is an indication that the production of IGF-1 may initially be a response to local damage. 4. Switches in muscle fibre phenotype were studied using a specific gene probe for the 2X myosin heavy chain gene. Type 2X expression was found to decrease markedly with stimulation alone and when electrical stimulation was combined with stretch. Unlike the induction of IGF-1 and beta actin, the decreased expression of the 2X myosin mRNA was less marked in the 'stretch only' muscles. This indicates that the interconversion of fibre type 2X to 2A may in some situations be commensurate with, but not under the control of IGF-1.
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PMID:Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation. 1008 55

The ability of a localized conformational searching method to predict probe orientation was tested on model nucleic acid and protein structures and applied to the prediction of skeletal myosin integrity upon chemical modification of its reactive thiols. Double-stranded oligonucleotides were chemically labeled with donor and acceptor resonance energy transfer probes at each end for distance determinations. These measurements were made independently using a terbium chelate as a donor to each of four chemically and spectroscopically distinct acceptor probes from the xanthene and cyanine dye groups. The choice of acceptor significantly affected the separation distance measured. Conformational searching algorithms on the atomic model corrected for the differences to within 0.2 nm on average. Verifying its usefulness on proteins, the localized conformational searching method determined the orientation of a fluorescent probe on RNase A that corresponds closely to available crystallographic models of the labeled protein (RMS deviation = 0.1 nm). Also, analysis of the symmetry of the fluorophores' structures suggests why FRET orientation factors are often closer to their dynamic average value than might normally be expected. Furthermore, the computational method provides insights about FRET data that are important for assessing the stability of the alpha-helix separating the SH1 and SH2 reactive thiols in skeletal myosin.
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PMID:Determination of fluorescent probe orientations on biomolecules by conformational searching: algorithm testing and applications to the atomic model of myosin. 1047 14


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