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Biomedical subjects

C C Liew

Publications and source records attributed to C C Liew.

At least 109 records · Page 6Linked to original sources
PubMed1989

Immunological evidence for the role of phosphoprotein p68/pI = 7.3 in premessenger RNA splicing.

A 68 kDa (pI = 7.3) nuclear phosphoprotein has been previously characterized as a component of transcriptionally active chromatin. Two-dimensional PAGE Western blotting and radioimmunoassay with monoclonal antibodies have identified this protein in nuclear extracts used for in vitro RNA splicing. In vitro splicing activity could be quantitatively inhibited by preincubating nuclear extracts with the antibodies, but the assembly of 60 S spliceosomes could not.

Antibodies, Monoclonal↗
PubMed1989

Characterization of human cardiac myosin heavy chain genes.

We have isolated and analyzed the structure of the genes coding for the alpha and beta forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the alpha-MYHC and beta-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The beta-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the alpha-MYHC-encoding gene, which is predominantly expressed in normal human atrium. We have determined the nucleotide sequences of the beta form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac beta-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (pSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same beta form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both alpha- and beta-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5'-flanking region of the alpha- and beta-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the alpha- and beta-MYHC genes is independently regulated.

Amino Acid Sequence↗
PubMed1989

Rat brain regional preproenkephalin A messenger RNA levels are altered in genetic hypertension.

Considerable neuroanatomical and pharmacological evidence suggests that preproenkephalin A-derived peptides, particularly methionine-enkephalin, are involved in regulation of the cardiovascular system in both physiological and pathological states. In this study, we used a rat preproenkephalin A complementary DNA to determine whether proenkephalin A-derived peptides participate in the pathogenesis of hypertension as reflected by brain regional messenger RNA levels. Complementary DNA clones of the rat preproenkephalin A mRNA and rat small myelin basic protein mRNA were hybridized to total RNA extracted from hypothalamus, pons-medulla, thoracic cord, midbrain, and cerebellum of 3 1/2-week-old and 12-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. In 3 1/2-week and 12-week animals there were no differences in the levels of myelin basic protein messenger RNA between the two groups in any brain region. At 3 1/2 weeks, preproenkephalin A mRNA levels did not differ between normotensive and hypertensive strains. In contrast, at 12 weeks preproenkephalin A mRNA levels were increased in hypothalamus, midbrain, thoracic cord, and cerebellum of SHR relative to WKY. Preproenkephalin A mRNA was significantly reduced in the pons-medulla of SHR relative to WKY. Our findings provide evidence that alterations in brain regional preproenkephalin A mRNA levels are associated with the development of spontaneous hypertension in the rat.

Aging↗
PubMed1988

RNA transcription in myocardial-cell nuclei during postnatal development. A study establishing an assay system for transcription in vitro.

A system for RNA transcription in vitro was established in order to determine the relative rate of RNA synthesis in neonatal and adult rat myocardial cells. This assay system optimizes the incorporation of [3H]UMP into RNA by using 3.5 x 10(7) myocardial-cell nuclei, and minimizes RNA degradation for at least 1 h in transcription in vitro, by the addition of human placental RNAase inhibitor. A 100% increase in the incorporation of [3H]UMP into myocardial-cell RNA was found on addition of this inhibitor. Myocardial-cell nuclei derived from 5-, 10-, 15-, 20-, and greater than 100-day-old rat hearts indicated that there is a progressive decrease in RNA synthesis with age. A 3-fold increase in RNA synthesis in 5-day-old myocardial cell nuclei as compared with 20-day-old rat heart was found. RNA synthesis in the adult myocardial cell nuclei decreased more than 10-fold in comparison with the 5-day-old newborn. The incorporation of [3H]UMP into rat liver nuclear RNA was 3-fold greater than in the myocardial-cell nuclear RNA, even when compared with the highly active transcription of 12-day-old heart nuclei. In order to determine the relationship between total RNA synthesis and the extent of specific gene expression in myocardial-cell nuclei during development, two distinct cDNA probes were used for Northern-blot analysis. Our results indicate that myosin-heavy-chain gene expression is remarkably decreased with age, whereas the 'housekeeping' gene is continually expressed independently of age.

Animals↗
PubMed1988

Catecholamines, calcium and cardiomyopathy.

The cardiomyopathic Syrian hamster has a genetically transmitted form of dilated cardiomyopathy and is an important paradigm of myocardial disease, particularly for studies addressing the earliest stages of myocardial dysfunction. This model exhibits an increase in cardiac sympathetic tone in the presence of an altered expression of sarcolemmal calcium channels or of alpha 1 receptors, and a defective handling of calcium by both cardiomyocytes and vascular smooth muscle cells. Increased expression of the oncogene c-myc is evident in cardiomyocytes before any overt evidence of heart disease. Alterations in a nuclear phosphoprotein, which appears to be important in the regulation of gene expression, have also been identified. The disease becomes phenotypically manifest by the development of microvascular spasm, reperfusion injury and myocyte loss. Myocyte loss, in turn, burdens the remaining cells with an increasing load, increasing sympathetic stimulation, myocyte hypertrophy and further cell loss--a continuing vicious spiral that culminates in the development of myocardial failure. All of the features of hamster cardiomyopathy may be prevented by the administration of verapamil or prazosin to juvenile hamsters before the phenotypic onset of their heart disease. This understanding has led to the study of new imaging agents that promise the detection of such forms of cardiomyopathy in their earliest stages and a means by which the effects of therapy can be assessed. If such mechanisms are applicable to human cardiomyopathy, early treatment of patients with adrenergic antagonists or calcium antagonists should be beneficial.

Animals↗
PubMed1988

Structure and function of nonhistone phosphoproteins.

Many nonhistone nuclear proteins have been shown to be phosphorylated and associated with a diverse range of cellular activities. The aim of this present review is to update the most recent developments in this field with respect to the traditional roles that these proteins have been postulated to play as enzymes, DNA-binding proteins, hormonal receptors, nucleosome associated proteins, and nucleolar associated proteins. In addition, evidence is presented suggesting that these proteins may also function as nuclear oncogene protein products, structural constituents of nuclear organization (e.g., lamina-nuclear matrix associated proteins), and RNA processing factors.

Animals↗
PubMed1987

Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies.

Previous work in this laboratory has established that a rat liver nuclear phosphoprotein (B2:Mr 68,000, pI 6.5-8.2) is associated with actively transcribed nucleosomes, as demonstrated by its preferential release after mild treatment with micrococcal nuclease. In the present report we provide further immunological evidence ('Western Blot' analysis, solid-phase radioimmunoassay and indirect immunofluorescence) that in addition establishes the presence of this phosphoprotein in the nuclear-matrix protein fraction. This paradoxical localization suggests that this phosphoprotein may function in two separate and distinct roles within the realm of nuclear organization.

Animals↗
PubMed1987

Two different forms of beta myosin heavy chain are expressed in human striated muscle.

We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3' untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5' end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3' untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.

Base Sequence↗
PubMed1987

Construction of a human ventricular cDNA library and characterization of a beta myosin heavy chain cDNA clone.

We have constructed and characterized for the first time a complementary DNA (cDNA) clone, pHMC3, which codes for a cardiac myosin heavy chain mRNA from human heart. This clone contains a 1.7 kb DNA segment and specifies 543 amino acids of the carboxyl portion of the myosin heavy chain. The DNA sequence and encoded amino acid sequence were compared to the hamster alpha (pVHC1) and beta (pVHC2/pVHC3) cardiac myosin heavy chain cDNA and amino acid sequences and the rat cardiac myosin heavy chain sequences as well. The myosin heavy chain mRNAs are highly conserved and this is reflected in our cDNA clone. The pHMC3 clone is 87.9% homologous to the hamster alpha cDNA and 92.2% homologous to the hamster beta cDNA clones. The 3' untranslated region of pHMC3 is 64.1% homologous to the hamster beta clone while the hamster alpha myosin heavy chain shows only 25% homology to pHMC3 and exhibits extensive diversity. Similar results were obtained when pHMC3 was compared to the rat cardiac myosin heavy chain cDNA sequences. The comparisons showed that pHMC3 is a beta cardiac myosin heavy chain cDNA clone.

Amino Acid Sequence↗
PubMed1987

Construction of cosmid genomic libraries for the normal and myopathic Syrian hamsters.

Cosmid genomic libraries from both normal and myopathic Syrian hamsters have been constructed. MboI was used to generate 35- to 50-kilobase DNA fragments which were isolated from a 5-25% NaCl gradient. The 35- to 50-kilobase DNA fragments were ligated to the cosmid vector pCV108 and packaged into Escherichia coli DK1. Approximately 3 X 10(5) - 4 X 10(5) clones were obtained per microgram of ligated DNA. Thirteen clones have been isolated from 2 X 10(5) colonies using a cardiac myosin heavy chain clone as a probe. Restriction maps of two of these clones are presented here.

Animals↗
PubMed1986

Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequences, the carboxyl-terminal peptide and the 3'-untranslated region are highly divergent and specific for this cDNA clone. There appears to be an amino acid deletion in the 3' end of the hamster alpha myosin heavy chain as compared to the rat alpha myosin heavy chain. S1 nuclease mapping experiments have shown that the mRNA represented by this cDNA clone is scarcely expressed in neonatal development, but its expression increases with age and reaches maximal levels in adult life. This cDNA clone provides a useful tool to follow the myosin heavy chain mRNA changes during development and during the genesis of a cardiomyopathy, an autosomal recessive defect carried by the Syrian hamster.

Animals↗
PubMed1985

Monoclonal antibodies to a phosphoprotein from chromatin of rat liver.

Three monoclonal antibody subclasses (IgG1, IgG2a, and IgM) were raised to the phosphoprotein B2 (Mr 68000, pI6.5-8.2) which has been shown previously to be associated with the nucleosomes of rat liver nuclei. These antibodies do not show any significant cross reactivity with CM-cellulose 'unbound' non-histone chromosomal proteins, bovine serum albumin or histones. Further verification of the specificity of these antibodies to this phosphoprotein was carried out using both 'dot' blot and immunological transfer analysis ('Western blot'). The monoclonal antibodies (IgG1 and IgG2a) could also be used to semi-quantify the phosphoprotein B2 in rat liver nuclei. The high specificity and unlimited availability of this type of probe provides a means to study the role(s) of this phosphoprotein in the overall scheme of actively transcribed chromatin.

Animals↗
PubMed1985

Partial characterization of chromosomal proteins tightly bound to chicken erythroid DNA.

Extraction of chicken reticulocyte and erythrocyte chromatins with 2 M NaCl yields a small fraction (about 5%) of the total DNA which is very tightly bound to a class of nonhistone chromatin proteins (DNA-P). This DNA fraction has previously been shown to be significantly enriched in active gene sequences. The proteins associated with reticulocyte and erythrocyte DNA-P were analyzed by two-dimensional gel electrophoresis. Reticulocyte DNA-P yield predominantly three major proteins, designated G1, G2, and G3 with relative masses of 80 000, 50 000, and 58 000, respectively. Erythrocyte DNA-P show only two proteins which appear to be similar to the reticulocyte G1 and G2 proteins, except in much reduced quantities as revealed by two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis of the three reticulocyte proteins revealed that the ratio of acidic to basic amino acid residues increased in the order G1 less than G2 less than G3, while the respective isoelectric points also increased in that order.

Amino Acids↗
PubMed1984

A chromosomal phosphoprotein is preferentially released by mild micrococcal-nuclease digestion.

[32P]Pi was administered to rats (5mCi/rat) 2h before the isolation of liver nuclei. The isolated nuclei were subjected to mild micrococcal-nuclease digestion for 2.5, 5 and 10 min at 37 degrees C, and the mononucleosomal fraction was subsequently isolated by sucrose-density-gradient centrifugation. The specific radioactivity of 32P-labelled mononucleosomal fractions decreased with increased digestion times. A phosphorylated chromosomal protein, B2 (Mr 68000, pI6.5-8.2), was demonstrated immunologically in the mononucleosomal fraction by using an antibody specific to this electrophoretically purified phosphoprotein. The incorporation of 32P into this phosphoprotein, previously shown to be mainly through covalent linkage, was revealed by antibody precipitation followed by gel electrophoresis. The rate of release of acid-soluble nucleotides by micrococcal-nuclease digestion of liver nuclei from partially hepatectomized rats 16 h after operation was strikingly higher than that for sham-operated controls. After partial hepatectomy, an increase in 32P incorporation into phosphoprotein in the monomer fractions specifically precipitated by this antibody was also found. This suggests that the phosphorylated non-histone chromatin protein B2 is preferentially associated with the transcriptionally active chromatin.

Animals↗
PubMed1984

Characterization of neonatal cardiac myosin heavy chain mRNA and synthesis of complementary DNA.

A procedure for the extraction of intact RNA from whole heart tissue using guanidine hydrochloride has been accomplished. Subsequent chromatography on oligo(dT)-cellulose of the total RNA and translation of the poly(A)+ RNA in a cell-free reticulocyte translation system has shown that the myosin heavy chain mRNA from rat heart tissue was present. The myosin heavy chain mRNA was identified by several criteria: (i) sizing by sucrose density gradient centrifugation, (ii) migration of a polypeptide translation product coincided with a myosin heavy chain marker in one-dimensional sodium dodecyl sulfate-- polyacrylamide gel electrophoresis, and (iii) immunoprecipitation of the mRNA translation product with a myosin heavy chain specific antibody. A myosin heavy chain mRNA enriched fraction was used to direct cDNA synthesis in an in vitro system. An optimal condition has been elucidated in which large molecular weight cDNA strands are produced. Evidence is presented here for the production of cDNA strands in excess of 4000 nucleotides which includes the heavy chain myosin in a high potassium--low sodium transcription assay, as revealed by dot blot hybridization with a cDNA clone of the myosin heavy chain.

Animals↗
PubMed1984

Immunofluorescent microscopy for the identification of human necrotic myocardium.

Human postmortem cardiac muscle was studied by immunofluorescent microscopy. Necrotic cells in acute myocardial infarctions were first identified with the hematoxylin-eosin stain as showing hypereosinophilia and autofluorescence. The results of the immunofluorescence staining showed a marked decrease if not absence of labeling for the Ca+ and Mg+ adenosine triphosphatase (ATPase) and tropomyosin in all necrotic muscle cells within a myocardial infarction. Myocytolytic cells located at the border of the infarct showed a labeling intensity similar to that of normal muscle cells. The use of immunofluorescence localization of muscle-specific proteins can be used as a reliable method to detect myocardial cell necrosis.

Adenosine Triphosphatases↗
PubMed1983

Changes in non-histone nuclear proteins during postnatal myocardial development.

Myocardial-cell nuclei isolated from ventricles of rats between 4 days and 15 months of age showed a progressive loss of DNA-synthetic activity in vitro. Correlated with this loss is the differential appearance of three major groups of non-histone nuclear proteins. Group a, with pI5.7-6.5 and Mr 67000, appeared in detectable amounts in rats more than 10 days of age, whereas group b, consisting of proteins with pI6.8-7.2 and Mr 45000, appeared in rats older than 33 days. The third group (group c), with pI6.5-8.5 and Mr 32000-42000, was present throughout postnatal development, but the amount of these proteins appeared to be greater in the 10-day-old rat than in the 15-month-old rat. All groups of these specific proteins were localized within the nucleus and did not reflect cytoplasmic constituents. Furthermore, the amino acid compositions of these polypeptides were also analysed and compared with those of actin and tubulin.

Aging↗