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Biomedical subjects

S K Tsui

Publications and source records attributed to S K Tsui.

At least 19 recordsLinked to original sources
PubMed2001

Translocation of a human focal adhesion LIM-only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization.

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle.

Actins↗
PubMed2001

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET).

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.

Base Sequence↗
PubMed2000

Differential gene expression of rat neonatal heart analyzed by suppression subtractive hybridization and expressed sequence tag sequencing.

Heart diseases have been one of the major killers among the human population worldwide. Because the vast majority of cardiomyocytes cannot regenerate once they cease to proliferate shortly after birth, functionally significant myocardial regeneration is not observed clinically. Whether these cells are terminally differentiated and permanently withdrawn from the cell cycle is controversial, but broadening our understanding of the rapid switch from hyperplastic to hypertrophic growth of cardiomyocytes during neonatal myocardial development may shed light on novel cardiovascular therapies. By suppression subtractive hybridization (SSH) and expressed sequence tag (EST) sequencing, we analyzed the differential gene expression of rat neonatal heart. SSH yielded subtracted and normalized cDNA libraries and enhanced the probability of detecting ESTs, which represent genes pertinent to signal transduction/cell regulation and replication/transcription/translation machinery, as compared to the traditional EST sequencing of heart cDNA libraries.

Animals↗
PubMed2000

Expression of replication factor C 40-kDa subunit is down-regulated during neonatal development in rat ventricular myocardium.

During neonatal development, cardiac myocytes undergo a transition from hyperplastic to hypertrophic growth. Whether these cells are terminally differentiated and permanently withdrawn from the cell cycle shortly after birth is controversial. Nevertheless, the clinical observation that functionally significant myocardial regeneration has not been documented in cardiovascular disease or injury during adulthood seems to support the notion that the vast majority of cardiac myocytes do not proliferate once they differentiate. Regardless of the controversy, the elucidation on how mitosis is blocked in cardiac myocytes may facilitate development of new cardiovascular therapies, based on the regeneration of the adult myocardium. To better understand postnatal myocardial development, we performed suppression subtractive hybridization to isolate genes that are differentially expressed in day one or day seven postnatal rat ventricular myocardium. Here we report the down-regulated mRNA expression of the 40-kDa subunit of replication factor C (RFC p40 or RFC2), which is an essential processive factor for proliferating cellular nuclear antigen-dependent DNA replication during neonatal myocardial development.

Age Factors↗
PubMed2000

Interaction of hCLIM1, an enigma family protein, with alpha-actinin 2.

Enigma proteins are proteins that possess a PDZ domain at the amino terminal and one to three LIM domains at the carboxyl terminal. They are cytoplasmic proteins that are involved with the cytoskeleton and signal transduction pathway. By virtue of the two protein interacting domains, they are capable of protein-protein interactions. Here we report a study on a human Enigma protein hCLIM1, in particular. Our study describes the interaction of the human 36 kDa carboxyl terminal LIM domain protein (hCLIM1), the human homologue of CLP36 in rat, with alpha-actinin 2, the skeletal muscle isoform of alpha-actinin. hCLIM1 protein was shown to interact with alpha-actinin 2 by yeast two-hybrid screening and immunochemical analyses. Yeast two-hybrid analyses also demonstrated that the LIM domain of hCLIM1 binds to the EF-hand region of alpha-actinin 2, defining a new mode of LIM domain interactions. Immunofluorescent study demonstrates that hCLIM1 colocalizes with alpha-actinin at the Z-disks in human myocardium. Taken together, our experimental results suggest that hCLIM1is a novel cytoskeletal protein and may act as an adapter that brings other proteins to the cytoskeleton.

Actinin↗
PubMed2000

Modulation of the Ca(2+)-activated Cl(-) channel by 14-3-3epsilon.

We have previously reported an association of 14-3-3epsilon isoform with calmodulin. Using the voltage-clamp technique, the present study investigated the potential role of 14-3-3 in modulating the Ca(2+)-activated Cl(-) channel (CaCC) endogenously expressed in Xenopus oocytes. Injection of 14-3-3epsilon antisense oligodeoxynucleotides resulted in potentiation of the ionomycin-induced Cl(-) current, while 14-3-3 peptide and calmodulin inhibitor, W13, suppressed the antisense-potentiated current. The data suggest that 14-3-3epsilon plays an inhibitory role in modulating the CaCC by interacting with the calmodulin-dependent pathway. The potential role of 14-3-3epsilon in other tissues and its therapeutic potential for cystic fibrosis are discussed.

14-3-3 Proteins↗
PubMed2000

Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation.

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.

Adult↗
PubMed2000

Identification and characterization of Podophyllum emodi by API-LC/MS/MS.

An API-LC/MS/MS method was developed for the identification of the medicinal herb Podophyllum emodi based on the profile of its aryltetrahydronapthalene and related lignan marker compounds. This was done by matching the structural information from the tandem mass spectrometric data with those lignan marker compounds already reported for the herb. The method could be employed in the absence of reference standards for the markers and was particularly useful in view of the scarcity of supply of these chemical standards. It has been used successfully to differentiate Podophyllum emodi from two commonly used medicinal herbs of a different genus but having similar appearance, Radix clematidis and Radix gentiana, as well as a closely related herb, Podophyllum peltatum.

Biomarkers↗
PubMed2000

In silico studies of energy metabolism of normal and diseased heart.

Biotechnology research is developing into genomic analyses that involve the simultaneous monitoring of thousands of genes. The development of various bioinformatics resources that provide efficient access to information is necessary. We have used single-pass sequencing of randomly selected cDNA clones to generate expressed sequence tags (ESTs). These ESTs data has been widely used to study gene expression in a variety of heart libraries [1, 21]. Data annotation on our recent finding allows us to construct the profiles of genes in the energy metabolizing pathways (glycolysis and glycogen metabolism) that are expressed in heart cDNA libraries. In silico studies of genes of energy metabolism yields data that are consistent with results derived from conventional metabolic experiments. The change in gene profiles describing the metabolism of diseased hearts is also presented here.

Cardiomegaly↗
PubMed2000

Protein-protein interaction of FHL2, a LIM domain protein preferentially expressed in human heart, with hCDC47.

In the yeast two-hybrid library screening, the heart-specific FHL2 protein was found to interact with hCDC47. In vitro interaction study between FHL2 protein and hCDC47 was demonstrated. From the results of domain studies by the yeast two-hybrid assay, the second and third LIM domains in conjunction with the first half LIM domain of FHL2 were identified to be important in binding with hCDC47. Besides, in Northern blot hybridization of human cancer cell lines, the highest FHL2 mRNA expression was detected in colorectal adenocarcinoma SW480 and HeLa cell S3. Our results imply that FHL2 protein may associate with cancer development and may act as a molecular adapter to form a multicomplex with hCDC47 in the nucleus, thus it plays an important role in the specification or maintenance of the terminal differentiated phenotype of heart muscle cells.

Base Sequence↗
PubMed1999

Characterization of a brain-specific nuclear LIM domain protein (FHL1B) which is an alternatively spliced variant of FHL1.

We have amplified and sequenced a novel, alternatively spliced variant of a human gene coding for the four-and-a-half LIM domain protein 1 (FHL1). This gene is located at chromosome Xq27 and the spliced variant is named FHL1B. The ORF of FHL1B cDNA codes for a LIM-only protein that possesses a zinc finger and three tandem repeats of LIM domains at the N-terminus with an active bipartite nuclear localization signal (NLS) motif and a possible RBP-J binding region at the C-terminus. FHL1B and FHL1 have the same N-terminal three-and-a-half LIM domains but different C-terminal protein sequences, due to the presence of an additional alternative exon 4b in FHL1B causing a frame-shift in the 3'coding region. RT-PCR results revealed that the expression of FHL1 is not restricted in skeletal muscle and heart, but is widely distributed in other tissues, including brain, placenta, lung, liver, kidney and pancreas, albeit as a low abundance transcript. In contrast, FHL1B is specifically expressed in brain. The C-terminal alternative region in FHL1B is sufficient to localize FHL1B in the nucleus of mammalian cell. FHL1B is probably related to neural differentiation and certain fragile X syndrome.

Alternative Splicing↗
PubMed1999

In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation.

Using a yeast two-hybrid screen, human 14-3-3 epsilon protein was found to interact with human calmodulin. In vitro binding assay between human 14-3-3 epsilon protein/peptide and calmodulin was demonstrated by native gel electrophoresis, and the interaction was shown to be calcium dependent. Our results, along with the association of the 14-3-3 epsilon protein with other signaling proteins, suggest that the 14-3-3 protein could provide a link between signal transduction and cell proliferation.

14-3-3 Proteins↗
PubMed1999

Cloning and characterization of a cDNA encoding a novel fibroblast growth factor preferentially expressed in human heart.

A novel human fibroblast growth factor (hFGF), which shows 75% sequence homology with fibroblast growth factor-9, was isolated in random sequencing of a human heart cDNA library. The full-length sequence is 928 bp, the encoded protein is composed of 168 amino acid residues, and its pI value and molecular weight were estimated to be 8.13 and 19.1 kDa, respectively. RT-PCR using Marathon human heart cDNA shows that the coding region is approximately 507 bp. Southern hybridization showed a single band which indicates that this is a single copy gene. Northern hybridization done on a human multiple tissues blot showed that the gene is preferentially expressed in human heart, very weakly detectable in human brain and not detectable in 18 other different human tissues.

Amino Acid Sequence↗
PubMed1999

Characterization of the human 36-kDa carboxyl terminal LIM domain protein (hCLIM1).

We characterized a human cDNA clone encoding a 36-kDa carboxyl terminal LIM domain protein with a PDZ domain at the amino terminal. This full-length cDNA clone has a predicted open reading frame (ORF) of 329 amino-acid residues. The ORF of this cDNA encodes the human homolog of rat CLP36, and the putative protein is named human 36-kDa carboxyl terminal LIM domain protein (hCLIM1, nomenclature approved by the HUGO/GDB Nomenclature Committee). The hCLIM1 probe was used to hybridize with poly(A)+ RNA of various human tissues. Strong signals were detected in heart and skeletal muscle; moderate signals were detected in spleen, small intestine, colon, placenta, and lung; weaker levels were detected in liver, thymus, kidney, prostate, and pancreas; and no observable signals were detected in brain, testis, ovary, and peripheral blood leukocytes. The hCLIM1 gene was studied by fluorescence in situ hybridization (FISH), somatic cell hybrid analysis, and radiation hybrid mapping, and it is located at the human chromosome 10q26.

Amino Acid Sequence↗
PubMed1999

Radiation hybrid mapping of human cytosolic malate dehydrogenase (hcMDH) to the short arm of chromosome 2.

Compartmentalization of human cytosolic malate dehydrogenase, hcMDH, together with its isozyme partner-mitochondrial form, hmMDH, plays an important role in the aerobic metabolism of the malate-aspartate shuttle and the citric acid cycle. However, they share few structural homology at the molecular level. The pseudogenes of mMDH has been reported in mice but hcMDH has no pseudogenes as shown by Southern blot analysis. A single band only was detected for the EcoRI digestion with 9.4 kb long of human genomic DNA and HindIII cutting with 2.8 kb long. hcMDH gene was mapped to chromosome 2 by somatic cell hybrid analysis and further localised to 268.72 cR from the top telomere of Chromosome 2 (near 2p15) by radiation hybrid mapping. The genes falling into this region may be related to dilated cardiomyopathy (DCM), several types of cancers and immunoregulation mechanism of cancers.

Chromosome Mapping↗
PubMed1998

Chromosomal mapping, tissue distribution and cDNA sequence of four-and-a-half LIM domain protein 1 (FHL1).

We have isolated and sequenced a human heart cDNA clone encoding a novel LIM-only protein. This full-length cDNA clone has a predicted open reading frame (ORF) encoding 280 amino acids. The ORF of this cDNA codes for a LIM-only protein that possesses four repeats of LIM domain and an extra zinc finger and this putative protein is named four-and-a-half LIM domain protein 1 (FHL1). FHL1 is unique when compared with other LIM-only proteins because it possesses an odd number of zinc fingers. When the FHL1 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, a moderate one in the heart; only weak signals were associated with the placenta, ovary, prostate, testis, small intestine, colon and spleen, and virtually no signal could be detected in brain, lung, liver, kidney, pancreas, thymus and peripheral blood leukocytes. The FHL1 gene was located to human chromosome at Xq27.2 by somatic cell hybrid mapping, fluorescent in situ hybridization (FISH) and radiation hybrid mapping.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart.

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific four and a half LIM-only protein 2 (FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12-q13 by fluorescent in-situ hybridization (FISH).

Amino Acid Sequence↗