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C C Liew

Publications and source records attributed to C C Liew.

At least 91 records · Page 5Linked to original sources
PubMed1993

Phosphorylation of an acid-soluble nuclear protein in response to catecholamine in cultured rat cardiocytes.

Effect of norepinephrine on the phosphorylation of nuclear proteins was examined in cultured rat cardiocytes. Differential effects of phorbol esters, endothelin and platelet activating factor on the phosphorylation of a nuclear acid soluble fraction were evaluated. A specific phosphorylated nuclear acid soluble protein was identified in response to norepinephrine. The increase in phosphorylation in the norepinephrine-treated cardiocytes is significantly greater than 8 fold (p < 0.001) as compared to the control group. This nuclear acid soluble protein has a high content of serine (> 12%) as well as acidic amino acids (> 20%) which is distinctively different from the histones as analyzed by amino acid composition. The specific phosphorylation of this nuclear protein (Mr 31 Kda) can be induced by norepinephrine within 5 minutes, indicating of an early gene activation event which modulates nucleosomal structure by phosphorylation.

Amino Acids↗
PubMed1993

Characterization of the GArC motif. A novel cis-acting element of the human cardiac myosin heavy chain genes.

A positive element between positions -924 and -851 and a negative element between -851 and -762 of the 5'-upstream region of the alpha-myosin heavy chain gene were identified through transient transfection assays in primary cultures of neonatal rat heart cells. Subsequent DNase I protection analysis revealed almost identical footprints at two positions (GAAAAATCT at -904 to -896 and GAAAATCT at -823 to -816). We have designated this sequence the GArC motif (for G,AT-rich,C). Gel mobility shift assays demonstrated the formation of specific complexes with GArC oligomers when either rat heart, rat liver, or HeLa cell nuclear extracts were used. Competition studies with unlabeled GArC oligomers resulted in a loss of binding. Oligomers were also made to the Xenopus cytoskeletal actin serum response element and to a segment of the alpha-MyHC gene (AT-core), each with a similar AT-rich core sequence. No detectable loss of binding resulted from the addition of an excess of either of these unlabeled oligomers. Southwestern blot analysis identified several proteins which interacted with the GArC element, suggesting the presence of a group of related trans-acting factors. Analysis of a sequence in the beta-MyHC gene with the same AT-rich core was negative, suggesting a role for the bases surrounding the protected area in binding. We propose that the GArC motif, together with its associated trans-acting factor(s), provides a novel mechanism of transcriptional control in addition to those previously reported for the cardiac myosin heavy chain genes.

Animals↗
PubMed1993

A human heart cDNA library--the development of an efficient and simple method for automated DNA sequencing.

A direct polymerase chain reaction (PCR) amplification of the human heart cDNA clones was used to generate lambda PCR product. By the use of the first set of primers derived from lambda gt11, each cDNA insert can readily be obtained. Using millipore filters, primers and nucleotides are removed and this purified PCR product can then be subjected to a second set of fluorescent primers in the generation of nucleotides in the auto-cycle reactions for automated DNA sequencing.

Base Sequence↗
PubMed1993

Structural organization of the human cardiac alpha-myosin heavy chain gene (MYH6).

The human myocardium expresses two cardiac myosin heavy chain (MyHC) isoforms, alpha and beta, that exist in tandem array on chromosome 14q12. We have previously sequenced the entire human cardiac beta-MyHC gene and now report the complete nucleotide sequence of the human cardiac alpha-MyHC, encompassing 26,159 bp as well as the entire 4484-bp 5'-flanking intergenic region. The gene (MYH6) consists of 39 exons, 37 of which contain coding information. The 5'-untranslated region is split into 3 exons, with the third exon containing the AUG translation initiation codon. With the exception of the 13th intron of the human cardiac beta-MyHC, which is not present within the alpha-isogene, all exon/intron boundaries are conserved. Conspicuous sequence motifs contained within the alpha-MyHC gene include four Alu repeats, a single (GT)n element, and a homopurine-homopyrimidine tract containing 23 GAA repeating units followed by 10 GAG repeating units. Comparison of the encoded amino acid sequence with a previously reported human alpha-MyHC cDNA sequence reveals several potential polymorphisms.

Amino Acid Sequence↗
PubMed1993

The v-raf oncogene enhances tumorigenicity and suppresses differentiation in vivo in a rat hepatocyte cell line.

raf oncogenes have been implicated in hepatic carcinogenesis. We studied the effects of the v-raf of murine retrovirus 3611-MSV on the growth and differentiation of a simian virus 40 (SV40)-immortalized rat liver cell line (ALB-8) which maintained many of characteristics of differentiated hepatocytes. Cells were co-transfected with v-raf and the neo gene followed by selection with G418 for transfectants. In culture, the expression of v-raf stimulated cell proliferation without altering cell morphology or expression of liver-specific genes: albumin, fibrinogen, alpha-1-antitrypsin and alpha-1-acid glycoprotein. The v-raf-transfected cells induced rapidly growing tumors in 100% of nude mice, while control DNA-transfected cells were only weakly tumorigenic, producing slowly growing tumors in 2/7 mice after a long latency. These slowly growing tumors were histologically moderately to well-differentiated hepatocellular carcinomas in which the liver-specific genes were highly expressed. In contrast, v-raf-induced tumors were histologically poorly differentiated and showed a dramatic decline in the expression of the liver-specific genes. In a tumor cell culture established from a v-raf-induced tumor, however, expression of the liver-specific genes was coordinately recovered. These observations indicate that v-raf is capable of inducing progression of SV40-immortalized hepatocytes into highly malignant cells and the progression is accompanied by loss, in vivo, of the hepatic differentiation.

Albumins↗
PubMed1993

Quantitation of enteroviral RNA by competitive polymerase chain reaction.

The polymerase chain reaction (PCR) is a new diagnostic technique for the detection of enteroviral infection; however, it currently provides only qualitative results. The aim of this study was to adapt PCR for the accurate quantitation of enteroviral RNA in clinical specimens. For this purpose, we designed a standard RNA which was homologous to sequences at the 5' end of the coxsackie B3 enterovirus genome but contained a single-base-pair mutation which created a novel internal restriction site. Serial dilutions of this standard template RNA were mixed with a fixed concentration of coxsackie B3 enterovirus RNA. The viral and standard templates were reversed transcribed to cDNA and coamplified by PCR, and a comparison of the radioactive PCR products was made. Since the templates were both present in a single reaction tube and competed for the same primers, the ratio of products remained proportional throughout the amplification process. By this approach, a fourfold-difference in viral titer was clearly distinguishable. Moreover, we were able to accurately quantitate as few as 15 50% tissue culture infectious doses, which reflects common clinical viral titers. This study lays the foundation for quantitation of enteroviral RNA in clinical specimens and establishes a technique that can readily be applied to the diagnosis of enteroviral infection.

Base Sequence↗
PubMed1992

Regulation of human cardiac myosin heavy chain genes: the effect of catecholamine.

The 5'-flanking regions of the alpha- and beta-cardiac myosin heavy chain (MyHC) genes were excised from the cosmid human genomic clones using Hind III and Xbal for the alpha-MyHC gene, and the Hind III and Hind III sites for the beta-MyHC gene. These fragments were linked to chloramphenicol acetyl transferase (CAT) vector to generate a chimeric fusion gene. These fusion genes were subsequently transfected to neonatal rat cardiac cultured cells to analyze the CAT activity. The alpha-MyHC gene is preferentially expressed as compared to the beta-MyHC. In the presence of norepinephrine (NE) the beta-MyHC gene is remarkably induced (within 24 hours following the addition of norepinephrine to the cardiocyte culture). However, the alpha-MyHC is also induced. Specific alpha andrenergic antagonists such as terazosin (Tz) partially suppressed both the alpha- and beta-MyHC genes as revealed by the CAT activity. These findings suggest that catecholamine does activate the human cardiac MyHC genes but does not differentiate the specific expression of either the alpha- or beta-MyHC genes.

Animals↗
PubMed1992

Characterization of a strong positive cis-acting element of the human beta-myosin heavy chain gene in fetal rat heart cells.

A strong positive element within the proximal promoter region of the human beta-myosin heavy chain (beta-MHC) gene that is required for high level expression in primary cultures of fetal rat heart cells was localized by transient assays and DNase I footprinting to positions- 277/-298. Using gel shift studies, this sequence was found to bind specifically at high affinity (Kd approximately 4 x 10(-9) M) to a transcriptional factor (beta F1) found in nuclear extracts from rabbit heart. Dimethyl sulfate interference studies suggested that beta F1 may bind as a dimer to two hexameric imperfect direct repeats containing the consensus sequence 5'-(C/G)-T-G-(T/A)-G-G-3'. Gel shift analyses suggested that beta F1 is related to the M-CAT factor, which is known to control muscle-specific expression of the cardiac troponin T gene. A clustered mutation of the region between the putative binding half-sites and within the "M-CAT"-like domain abolished beta-MHC promoter activity. The sequence of the positive element also contains binding motifs for several transcriptional factors that regulate viral and cellular genes, including AP4, AP5, TEF-1, and MyoD-like proteins. When multiple copies of the beta-MHC element were inserted downstream from the transcriptional initiation site of the thymidine kinase gene, it did not act as a classical enhancer, showing some dependence upon orientation.

Animals↗
PubMed1992

Endothelin stimulates cardiac alpha- and beta- myosin heavy chain gene expression.

The effect of endothelin-1 on cardiac myosin heavy chain gene expression was examined using an isolated neonatal rat myocardial cell culture system. The effects of endothelin-1 on the expression of alpha- and beta- myosin heavy chain genes in the primary rat myocardial cell culture system were examined by S1 nuclease protection analysis. Endothelin-1 was found to stimulate both alpha- and beta- myosin heavy chain gene expression. The 5' flanking regions of both the alpha- and beta- myosin heavy chain gene promoters ligated to a reporter gene, chloramphenicol acetyltransferase, were used to study the effect of endothelin-1 on transcription. Myocardial cells treated with endothelin-1 increased the transcription rate of alpha- and beta- myosin heavy chain genes in a dose-dependent manner. Thus, the hypertrophic effect of endothelin-1 on cardiac myocytes involves augmentation of alpha- and beta- myosin heavy chain gene expression by increasing gene transcription.

Animals↗
PubMed1992

Regulation of basal and luminal cell-specific cytokeratin expression in rat accessory sex organs. Evidence for a new class of androgen-repressed genes and insight into their pairwise control.

Co-expression of cytokeratin (CK) pairs has been found to be associated with specific epithelial cell types whose expressions are developmentally regulated. In the prostate, CK 8 and 18 have been identified as luminal cell-specific markers, and CK 5 and 15 have been identified as basal cell-specific markers. In this study, we report the cloning and sequencing of a full-length CK 8 cDNA (1.9 kilobases) from a rat ventral prostate (VP) cDNA library. Although the open reading frame shares 90% homology with mouse CK 8 sequences, nucleotide comparison revealed that rat CK 8 cDNA comprises a species-specific sequence on both 5' and 3' ends. The steady-state levels of CK 8 mRNA were elevated in VP, seminal vesicle (SV), and liver of a castrated rat but not in the other organs such as the coagulating gland, bladder, and thymus. Unlike the other androgen-repressed genes, elevated CK 8 mRNA levels persisted even after the glandular involution was completed, indicating that CK 8 is a new class of androgen-repressed gene. The regression of CK 8 expression may be androgen receptor-mediated, since androgen but not estrogen administration to castrated hosts repressed the CK 8 mRNA levels, and this effect can be antagonized by the simultaneous administration of an antiandrogen (4-hydroxyflutamide). Immunohistochemical staining of prostatic tissues reveals that the CK 8 filamentous structure is shifted reversibly from a uniform distribution to a predominantly basal surface upon androgen deprivation. We noted that the steady-state levels of CK 8 protein remain rather constant throughout the various hormonal treatment, and the steady-state levels of CK 8 mRNA and the rate of CK 8 protein synthesis are consistently elevated. These results suggest that the turnover rate of CK 8 protein may be elevated in the prostatic epithelium from the castrated host. Similarly, the steady-state levels of CK 15 and 18 mRNA in VP and SV are also repressed in an androgen-dependent manner. These data, taken together, indicate that pairwise control of luminal (and possibly basal) specific cytokeratin gene expression remains intact in both VP and SV tissues and that the levels of CK mRNAs expression are negatively regulated by androgen.

Amino Acid Sequence↗
PubMed1992

No evidence for linkage of familial hypertrophic cardiomyopathy and chromosome 14q1 locus D14S26 in a Chinese family: evidence for genetic heterogeneity.

To understand the molecular basis of familial hypertrophic cardiomyopathy (FHC) in the Chinese population, a family with FHC was investigated. Nineteen family members who were 16 years of age or older were examined by M-mode or two-dimensional echocardiography. Eight members were diagnosed to be affected echocardiographically or clinically. Lymphocytes isolated from 20 family members were successfully transformed into permanent lymphoblastoid cell lines by Epstein-Barr virus. Three genomic DNA probes (CRI-L436, CRI-L329, and pSC14) that were derived from chromosome 14q1 loci and demonstrated to be linked closely to FHC were used to probe this family. Using the techniques of restriction fragment length polymorphism (RFLP) and linkage analysis, the probe CRI-L436, which recognized locus D14S26, was found informative in this family. The lod scores were -2.0 at theta = 0.025 and -1.49 at theta = 0.05. Thus, there was no evidence of linkage between the locus D14S26 and the gene for FHC in the pedigree studied. In addition, polymerase chain reaction (PCR) amplification did not indicate a mutation on exon 13 of the beta cardiac myosin heavy chain gene as previously reported. Our data suggest that FHC is a genetically heterogeneous disease.

Adult↗
PubMed1992

The genetic basis of hypertrophic cardiomyopathy.

In this article we review the techniques of molecular biology as they apply to the elucidation of the genetic basis of hypertrophic cardiomyopathy. We review the evidence for linkage to chromosome 14 and the specific mutations described to date. The evidence for genetic heterogeneity is presented. We speculate on the pathophysiology of the disease from the perspective of the known molecular defects and review the clinical implications the evolving information may have.

Cardiomyopathy, Hypertrophic↗
PubMed1992

Persistence of viral genome into late stages of murine myocarditis detected by polymerase chain reaction.

BACKGROUND: Enteroviruses have been considered as the most common etiologic agents in clinical myocarditis and dilated cardiomyopathy; however, their pathogenetic role remains unknown. Hence, the relation of viral replication and development of cardiomyopathy has been determined in a murine model of myocarditis by evaluating the persistence of viral genome during acute and chronic stages of myocarditis by means of Northern blot hybridization and polymerase chain reaction (PCR). METHODS AND RESULTS: DBA/2 mice (n = 146) were injected peritoneally with 10 plaque-forming units of encephalomyocarditis (EMC) virus, and the control mice (n = 33) were injected with normal saline. Animals were randomly killed at 4, 7, 10, 14, 21, 28, 35, and 42 days after infection. Histology revealed acute myocardial necrosis with massive inflammatory cell infiltrate peaking on day 14 followed by increasing fibrosis and declining chronic inflammation features compatible with dilated cardiomyopathy between days 21 and 42. Northern blot analysis of control and infected hearts showed detectable viral RNA in the infected hearts initially at day 4, peaking by day 7, diminishing between day 7 and day 14, and absent at day 21 and day 28. However, potential viral remnants present in low quantities and undetectable by Northern blot were further detected by PCR followed by confirmation with an internal oligonucleotide probe after day 14 up to day 42. CONCLUSIONS: Viral RNA signals on Northern blot showed a strong correlation with massive myocyte necrosis on day 14, but the viral RNA fragment was consistently detectable into late stages of cardiomyopathy on days 21, 28, 35, and 42 by PCR. This indicated that the mature virions are fully developed early in infection and are capable of persisting in the myocardium after virus-mediated myocytolysis stage. Therefore, PCR is an extremely sensitive method for detecting residual viral genome and viral persistence in the myocardium and may offer insights into the pathogenesis of chronic myocarditis leading to dilated cardiomyopathy.

Animals↗
PubMed1991

Isolation and characterization of a previously unrecognized myosin heavy chain gene present in the Syrian hamster.

A full length (25,000 base-pair) myosin heavy chain gene completely contained within a single cosmid clone was isolated from a Syrian hamster cosmid genomic library. Sequence comparison of the 3' untranslated region indicated the presence of a 75% homology with the rat embryonic myosin heavy chain gene. Extensive 5' flanking region regulatory element conservation was also found when the sequence was compared to the rat myosin heavy chain gene. S1 nuclease digestion analysis, however, indicated that the Syrian hamster myosin heavy chain gene exhibited expression in adult Syrian hamster ventricular tissue, as well as the adult vastus medialis, a fast twitch skeletal muscle. Expression also appears to be enhanced in myopathic relative to control hearts. This myosin heavy chain gene is neither the alpha nor beta cardiac myosin heavy chain gene, but is a unique, previously unrecognized, myosin heavy chain gene present in both myocardial and skeletal muscle tissues.

Animals↗
PubMed1991

The DNA-binding properties of a rat nuclear phosphoprotein.

The non-histone nuclear phosphoprotein B2 (Mr 68,000; pI 6.5-8.2) was found to bind specifically defined fragments of DNA. With the use of monoclonal IgG2A antibodies prepared against this nuclear antigen, nucleosomal DNA fragments associated with phosphoprotein B2 were isolated and cloned. Nine cloned fragments were sequenced and analysed for similarity. The clone having the most similarity to the others was chosen to serve as a model in gel shift and footprinting assays. Subsequently, the DNA binding site was found to reside within a 30 bp region. Synthetic oligonucleotides corresponding to this site confirmed the specificity of DNA binding exhibited by the nuclear antigen as demonstrated in competition assays. Moreover, a 5'-TATTAG/C-3' motif was found to exist within the binding site and in the other sequenced clones, possibly implicating the involvement of this motif in protein binding.

Animals↗
PubMed1991

RNA transcription and translation in the hearts of normal and cardiomyopathic Syrian hamsters.

The cardiomyopathic Syrian hamster has an autosomal recessive defect that results in the development of an early onset cardiac myopathy leading to cardiac dysfunction and, eventually, complete heart failure. To assess the regulatory mechanisms modulating gene expression in the normal and myopathic myocardium, we investigated both RNA transcription and translation. Our results indicated that the incorporation of [3H]UMP into myocardial cell nuclear RNA decreased 10-fold from 7 to 210 days of age in the normal Syrian hamster. The incorporation of [3H]UMP was approximately 50% lower in the cardiomyopathic as compared with the normal Syrian hamster. RNA translation, as assessed by rabbit reticulocyte lysate in vitro translation, indicated that a coordinated 50% decrease in RNA translation occurred in normal Syrian hamster from 7 to 210 days of age. A further reduction of 20% in translation was found in cardiomyopathic Syrian hamster ventricular RNA translation as compared with matched random bred control groups. Two-dimensional polyacrylamide gel analysis of cell-free translated protein products demonstrated two myocardial peptides that were found to be consistently altered when the normal and cardiomyopathic Syrian hamsters were compared. These results indicate that transcription and translation decrease with age and that these processes are further downregulated, in an additive manner, with the genesis of the disease process.

Aging↗
PubMed1990

Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction.

Recent molecular studies have suggested that viral myocarditis frequently underlies human congestive cardiomyopathy; however, only moderately sensitive and specific techniques were used. Polymerase chain reaction (PCR) gene amplification is a sensitive, specific technique ideally suited for the diagnosis of viral disease in small tissue samples where low copy numbers of the viral genome may be present. Using PCR and high stringency condition, we screened biopsies taken from 48 patients with clinically suspected myocarditis or dilated cardiomyopathy. Five patients demonstrated positive enteroviral signals by PCR; two of them had myocarditis by pathology, whereas the other three had changes consistent with cardiomyopathy. Four other patients had myocarditis diagnosed by pathology from 3 months to 1 year earlier but were now negative by both PCR and pathology. Both pathology and PCR were negative for active myocarditis in all other patients. Ventricular samples taken from left ventricular myectomy in four additional patients with hypertrophic cardiomyopathy, normal human ventricle samples, and uninfected monkey kidney cells were also negative by PCR. This study supports a link between viral infection and dilated cardiomyopathy in some patients. PCR gene amplification provides a new diagnostic approach to patients with suspected myocarditis.

Biopsy↗