Biomedical subjects

E Y Choi

Publications and source records attributed to E Y Choi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Reorganization of myosin and focal adhesion proteins in Swiss 3T3 fibroblasts induced by transforming growth factor beta.

Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF-beta). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell-adhesion molecules were redistributed in TGF-beta-treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western-blot analysis. Changes in cell morphology became apparent within 12 h of the addition of TGF-beta and new RNA and protein synthesis was necessitated by the changes. While TGF-beta induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF-beta treatment, with an increased amount. In addition, the cell-shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell-shape change, and acto-myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF-beta in Swiss 3T3 fibroblasts.

3T3 Cells↗

PubMed1999

Involvement of protein kinase C and rho GTPase in the nuclear signalling pathway by transforming growth factor-beta1 in rat-2 fibroblast cells.

The transforming growth factor (TGF)-beta signal-transduction cascade from the cell membrane to the nuclear target is poorly characterised. Here we report that treatment with TGF-beta1 induces the levels of endogenous c-fos mRNA in Rat-2 fibroblast cells. In addition, by transient transfection analysis, TGF-beta1 was shown to stimulate c-fos serum response element (SRE)-driven reporter gene activity in a dose- and time-dependent manner, suggesting that SRE is one of the nuclear targets of TGF-beta1. To understand the signalling cascade by which TGF-beta1 mediates the transactivation of c-fos SRE, cells were either pre-treated with various inhibitors or co-transfected with expression plasmids encoding inhibitory proteins for Rho GTPase together with the SRE-luciferase reporter gene. Our results showed that an inhibition of protein kinase C (PKC) or RhoA selectively repressed the stimulation of c-fos SRE by TGF-beta1, implying the possible roles of PKC and RhoA GTPase in TGF-beta1-induced signalling to c-fos SRE.

Animals↗

PubMed1999

Production and characterization of isotype-specific monoclonal antibodies to bovine brain Rab GDI.

Small GTPases of the Rab family play a key role in controlling vesicular transport, and the Rab GDP-dissociation inhibitor (GDI) is a regulatory protein for the Rab proteins. Here we report the production and characterization of isotype-specific monoclonal antibodies (MAbs) to Rab GDI. Rab GDI was purified from bovine brain in several steps of column chromatography and was injected into BALB/c mice intraperitoneally. The resulting MAbs specifically recognized a single protein band of 55 kDa, which comigrates with purified bovine Rab GDI. To localize Rab GDI, we processed cells from different sources for indirect immunofluorescence microscopy. Interestingly, the MAb stained cytosol and vesicular structures in brain cells, whereas it predominantly stained cytosol in nonbrain cells. Next, we investigated the cross-reactivities of brain Rab GDI from some mammals. The immunoreactive bands on Western blots appeared to be the same in molecular mass, 55 kDa, in all mammalian species tested including human. In summary, we produced a panel of MAbs that are GDI-alpha/1 form-specific and we believe that the MAbs will be valuable tools in elucidating the function of Rab GDI isoforms.

Animals↗

PubMed1999

A monoclonal antibody against hamster tracheal mucin, which recognizes N-acetyl-galactosamine containing carbohydrate chains as an epitope.

Airway mucin that is present in airway secretion, plays an important role in host-defense by trapping airborne particles and removing them by mucociliary transport system. For the study of mucin, it is crucially important to have antibodies specific against mucin because other commonly used methods such as histologic stain for the detection of mucin usually suffer from varying levels of nonspecificity. In this study, we produced a monoclonal antibody (MAb) against hamster airway mucin, which is one of the most commonly used animal species for the study of mucin in vitro, and characterized its immunological properties along with the determination of the epitope it recognizes. The MAb, which was named MAb HTA, was IgM isotype and specific against mucin from both in vitro cell culture and in vivo airway secretion. In Western blot, MAb HTA specifically recognized high molecular weight airway mucin, which was also confirmed by the appearance of peak profile of immunological signal only on void volume fraction in Sepharose CL-4B gel filtration chromatography. It also immunoprecipitated high molecular weight hamster airway mucin with the aid of antimouse IgM agarose. In immunohistochemical stain of hamster trachea, it showed strong signal on airway epithelium and also on the mucin secreting goblet cell granules. The immunological signal was greatly increased by the treatment of endotoxin, which has been reported to cause airway secretory cell metaplasia. The MAb HTA recognized carbohydrate chains containing N-acetyl-galactosamine, one of the linking sugars of airway mucin, as an epitope. Treatment of mucin with N-acetyl-galactosaminidase caused great reduction of immunological signal. To the best of our knowledge, this is the first to report a MAb that recognizes N-acetylgalactosamine, a linking sugar of airway mucin. The specificity of MAb HTA against airway mucin and the clear demonstration of the epitope it recognizes should greatly aid the pharmacological and biochemical study of mucin in various physiological and pathological situations.

Acetylgalactosamine↗

PubMed1999

Percutaneous transmyocardial revascularization induces angiogenesis: a histologic and 3-dimensional micro computed tomography study.

The purpose of this study was to visualize the spatial patterns and connection of channels created after percutaneous transmyocardial revascularization (PTMR) in normal porcine hearts, and to estimate the relative contributions of transmyocardial and coronary perfusion. Six pigs underwent PTMR creating channels using radiofrequency ablative energy. Three-dimensional computed tomography imaging of channels 1 hr after PTMR showed the direct connection of PTMR channels to the myocardial capillary network and to epicardial coronary vessels. In the heart, examined 28 day after PTMR, there was a fine, extensive, network of microvessels originating from the site of the original PTMR channel, also connecting the left ventricular cavity to myocardial capillaries. Histopathologic examination of the 1-hr specimens showed numerous regions of myocardial hemorrhage and associated inflammatory cell infiltration. In the 28-day specimens, newly developed new vascular network suggested neovascularization within the core of these channel remnants. The immunoreactivity for basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were intense within myocardium and neovascular structure surrounding PTMR channel remnants. The vascular connections occur by direct communication with existing myocardial vasculature acutely, and angiogenesis in these channel remnant chronically.

Animals↗

PubMed1999

Apolipoprotein E polymorphism in non-diabetic patients with acute coronary syndrome.

Since a decade ago, apolipoprotein (apo) E polymorphism has been focussed as a risk factor for cardiovascular disease. ApoE plays a central role as a receptor ligand for the uptake of lipoproteins from the circulation. There was an agreement on apoE polymorphism being one of the major risk factors for coronary artery disease (CAD) by its effects on lipid profiles. However, the effects of apoE have not been noted in all populations and conflicting results in the risk of CAD have been noted. Recently, in situ expression of apoE on the atherosclerotic plaque has been studied. We, therefore, investigated the effects of apoE genotype on patients with acute coronary syndrome, including unstable angina and acute myocardial infarction, in non-diabetic patients. While we could not find significant risk effects of apoE on coronary artery disease and lipid profiles on simple comparison with the normal control group, we could find significantly decreased frequencies of apo epsilon 3 allele in patients with acute coronary syndrome compared with stable angina patients (77.8% vs 88.8%). We suggest that the apoE genotype could be associated with acute coronary events in CAD and further study with in situ biochemical methods will be needed on the effects of apoE polymorphism on plaque stability.

Apolipoproteins E↗

PubMed1998

Generation of cells with Hodgkin's and Reed-Sternberg phenotype through downregulation of CD99 (Mic2).

Despite the fact that Hodgkin's and Reed-Sternberg (H-RS) cells are morphological hallmarks of Hodgkin's disease (HD), the nature of H-RS cells still remains to be resolved. Here we report that downregulation of CD99 (Mic2) leads to the generation of cells with an H-RS phenotype. IM9 and BJAB B-cell lines that were transfected with an antisense CD99 expression construct showed the morphological and immunological characteristics of H-RS cells such as multinuclearity, expression of CD15, decreased expression of major histocompatibility complex (MHC) class I and CD45RB, and deregulated secretion of cytokines. The reduced expression of CD99 was also confirmed in H-RS cells of patient's lymph nodes and three HD-derived cell lines, L428, KM-H2, and HDLM-2. Moreover, features characteristic of H-RS cells were completely abolished by forced expression of CD99 and by a constitutively active form of Rac, which functions downstream of CD99. We suggest that CD99 molecules play a crucial role in regulating functions and morphology of cells through a Rac-Rho signaling pathway and that the loss of CD99 expression is a significant molecular event to generate H-RS cells.

12E7 Antigen↗

PubMed1998

Engagement of CD99 induces up-regulation of TCR and MHC class I and II molecules on the surface of human thymocytes.

CD99 is a cell surface molecule involved in the aggregation of lymphocytes and apoptosis of immature cortical thymocytes. Despite its high level expression on immature cortical thymocytes, the functional roles of this molecule during thymic selection are only now being elucidated. Examination of the effects of CD99 engagement on the expression kinetics of the TCR and MHC class I and II molecules, which are involved primarily in thymic positive selection, revealed a marked up-regulation of these proteins on the surface of immature thymocytes. This increase was the result of accelerated mobilization of molecules stored in cytosolic compartments to the plasma membrane, rather than increased RNA and protein synthesis. Confocal microscopic analysis revealed the changes in subcellular distribution of these molecules. When CD99 was engaged, TCR and MHC class I and II molecules were concentrated at the plasma membrane, particularly at cell-cell contact sites. The TCRlow subpopulation of immature double positive thymocytes was much more responsive to CD99-mediated up-regulation than was the TCRhigh population. These findings suggest that CD99-dependent up-regulation may have possible implication in positive selection during thymocyte ontogeny.

12E7 Antigen↗

PubMed1998

Development of a new anti-CD4 monoclonal antibody (YG23) which inhibits the formation of colonies of human bone marrow progenitor cells.

We have previously reported CD4 expression in CD34+ hematopoietic progenitor cells and suggested a role of CD4 in normal hematopoiesis and its possible relationship with the pathogenesis of acquired immunodeficiency syndrome (AIDS). To investigate whether CD4 expression in bone marrow progenitor cells can explain bone marrow suppression in AIDS, monoclonal antibodies (mAbs) against human CD4 were developed by immunizing Balb/c mice with human thymocytes. Three mAbs completely blocked the binding of Leu3a antibody, a well-known anti-CD4 mAb, to thymocytes, which indicates overlap between the epitopes recognized by these and Leu3a antibodies. Interestingly, one of these mAbs, YG23, significantly inhibited colony formation of human bone marrow progenitor cells treated with GM-CSF. This is the first demonstration that ligation of CD4 by an anti-CD4 mAb suppresses GM-CSF mediated proliferation and differentiation of human hematopoietic progenitor cells by modifying the intracellular signaling pathway through CD4 molecules. Based on these findings, we propose that alteration of CD4 signaling by either cross-linked gp120 or antibodies directed against a certain epitope shared with the YG23 binding site of the CD4 molecule may play a role in bone marrow dysfunction in AIDS patients.

Animals↗

PubMed1998

ILK (beta1-integrin-linked protein kinase): a novel immunohistochemical marker for Ewing's sarcoma and primitive neuroectodermal tumour.

ILK (beta1-integrin-linked protein kinase) is a recently identified 59-kDa serine/threonine protein kinase that interacts with the cytoplasmic domain of the beta1-integrin containing four ankyrin-like repeats. We have developed a polyclonal antibody against ILK and explored the ILK immunoreactivity in normal human cells and tissues. ILK was mainly expressed in cardiac muscle and skeletal muscles. Surprisingly, ILK expression was observed in Ewing's sarcoma (ES; 100%), primitive neuroectodermal tumour (PNET; 100%), medulloblastoma (100%), and neuroblastoma (33.3%), whereas other small round cell sarcomas were not stained by the anti-ILK antibody. These results suggest that ILK could be a novel marker for tumours with primitive neural differentiation. Our findings support the notion that ES is a tumour that is closely related to PNET and that both originate from the neuroectoderm. ILK may be a sensitive and specific immunohistochemical marker and useful for the positive identification of ES and PNET in formalin-fixed, paraffin-embedded tissue sections.

Adolescent↗

PubMed1998

Engagement of CD99 induces apoptosis through a calcineurin-independent pathway in Ewing's sarcoma cells.

Programmed cell death (PCD) is a prominent feature of the development of the immune and nervous systems. In both systems, widespread PCD occurs in primitive progenitor cells during development. In this study, we demonstrated that Ewing's sarcoma (ES) cells, undifferentiated neural precursors, underwent apoptosis upon engagement of CD99 with anti-CD99 monoclonal antibody. Apoptosis via CD99 occurred only in the undifferentiated state of ES cells, but not in differentiated ES cells. CD99-induced apoptosis in ES cells appeared to require de novo synthesis of RNA and protein as well as caspase activation. Cyclosporin A, known to be a potent inhibitor of both calcineurin activation and mitochondrial permeability transition pore opening, inhibited CD99-mediated apoptosis, whereas FK-506, a specific calcineurin inhibitor, did not, indicating the induction of CD99-mediated apoptosis through a calcineurin-independent pathway. Furthermore, the dying cells displayed the reduction of mitochondrial transmembrane potential (delta psi m). These results suggest that CD99 engagement induce CsA-inhibitable mitochondrial permeability transition pore opening, followed by a reduction of delta psi m and caspase activation, thereby leading to apoptosis. Based on these results, we suggest the possible involvement of CD99 in the apoptotic processes that occur during nervous system development and also its application in immunotherapeutic trials for ES cases.

12E7 Antigen↗

PubMed1998

A cell surface molecule, JL1; a specific target for diagnosis and treatment of leukemias.

We previously reported a novel differentiation antigen, which is specifically expressed in stage II double positive (CD4+CD8+) human cortical thymocytes (Park et al, J Exp Med 1993; 178: 1447-1451). This study was designed to investigate the expression pattern of JL1 in various types of leukemic cells from patients and normal hematopoietic cells to evaluate the possibility as a tool for diagnosis and treatment of leukemia. The expression of JL1 antigen was observed in 75.6% of leukemic cases (117 out of 154 leukemic patients tested) on flow cytometric analysis. The percentage of JL1-positive cases of T lineage acute lymphoblastic leukemia (T-ALL) (92.6%) was higher than that of other types of leukemias (75%). The presence of JL1 antigen was also confirmed by immunoblotting and immunoprecipitation. Since the JL1 antigen is selectively expressed on the surface of human leukemic cells but not on the mature human peripheral blood cells, normal bone marrow cells and various types of normal tissues, JL1 could be an excellent candidate for an immunodiagnostic and immunotherapeutic tool for hematopoietic malignancies such as leukemia.

Antibodies, Monoclonal↗

PubMed1998

Production and characterization of monoclonal antibodies against rat tracheal mucins.

The objective of this study was to generate and characterize monoclonal antibodies against rat airway mucins. Therefore, it should serve as a useful tool in studying the regulation of airway mucins using various in vivo rat models that are currently available. As an antigen, we used a high molecular mass mucin preparation purified from the spent media of rat tracheal surface epithelial cells in primary culture. Seven monoclonal hybridomas were obtained, among which mAbRT03 showed the highest immunoreactivity against the mucin. All of the antibodies secreted by these hybridomas recognized carbohydrate epitopes but not sialic acid residues, since their immunoreactivity was completely abolished by treatment of the mucin with 20 mM periodate but not with neuraminidase. Further characterization of mAbRT03 showed that (1) it belongs to the IgM type, (2) it binds to high molecular mass mucins based on Western blot, (3) it could indirectly immunoprecipitate rat airway mucin--and, as we know, this is the first demonstration of immunoprecipitation of airway mucin with anti-mucin antibodies--(4) it binds to the luminal side of tracheal epithelium as well as some goblet cells based on immunohistochemistry, and (5) it also recognizes in vivo airway mucins from rats, but not from human nor hamsters, which have been purified from the airway lavage fluids. This is the first monoclonal antibody against rat airway mucin, which has been developed against purified rat airway mucins. Therefore, mAbRT03 should be able to serve as an invaluable tool in studying the regulation of airway mucins using various intact rat models that are currently available.

Animals↗

PubMed1997

CD99 (MIC2) regulates the LFA-1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative regulators of cellular adhesion.

Despite the fact that integrin-mediated lymphocyte adhesion is a crucial event for an appropriate immune response, little is known about the mechanisms that control the adhesion and deadhesion processes generated by the engagement of CD99 between various types of immune cells. Here we report that the CD99 gene encodes two distinct proteins with opposite functions in the LFA-1/intercellular adhesion molecule 1 (ICAM-1)-mediated cell adhesion process. The two forms of the CD99 protein are produced by alternative splicing of the CD99 gene transcript. The major form induced homotypic adhesion of the human B lymphoblastoid cell line IM-9, whereas the minor, truncated form inhibited the adhesion process. Activation of the major form of CD99 with anti-CD99 monoclonal antibodies induced rapid aggregation of IM-9 cells, which was blocked by the addition of mAbs to LFA-1 or intracellular adhesion molecule 1. Overexpression of the minor truncated form of CD99 markedly down-regulated the expression of LFA-1. The two forms of CD99 are differentially expressed in most human cells tested and are highly conserved in monkey. Taken together, these observations suggest that the two forms of CD99 function in vivo in both positive and negative regulation of LFA-1-mediated adhesion of lymphocytes during an immune response.

12E7 Antigen↗

PubMed1997

Thymocytes positively select thymocytes in human system.

We previously demonstrated the expression of MHC class II molecules in a significant percentage of human fetal and postnatal thymocytes. These results, at that time, raised the question as to whether the MHC class II molecules on immature thymocytes could actively be involved in the selection of immature T cells. We have developed a human reaggregate culture system to address this issue. Surprisingly, despite the fact that thymic epithelial cells (TECs) have been shown to be a major selecting cell type of positive selection, we were clearly able to see the involvement of MHC class II+ thymocytes during selection process through T-T interaction. In addition, maturation to single positive (SP) cells occurred only in the presence of MHC class II molecules and immature thymocytes were found to be arrested at the double positive (DP) stage of differentiation by blocking of TCR recognition of MHC class II molecules. All these results strongly suggest that human MHC class II+ thymocytes actively participate in the selection of the TCR repertoire, for which TCR recognition of peptide/MHC class II may be an initial determining step.

Cell Aggregation↗

PubMed1997

Distribution of succinic semialdehyde reductase in rat brain.

Succinic semialdehyde reductase (SSR) that catalyzes the reduction of succinic semialdehyde (SSA) to gamma-hydroxybutyrate (GHB) has been identified as one of the NADPH-dependent aldehyde reductases. Reduction of SSA to GHB strongly supports the proposal that GHB biosynthesis may be an important step in the GABA shunt. It is pharmacologically significant in anesthesia, evoking the state of sleep, and an increase in brain dopamine level. Monoclonal antibodies against bovine brain succinic semialdehyde reductase were produced. Using the anti-succinic semialdehyde reductase antibodies, we investigated the distribution of brain succinic semialdehyde reductase in rat brain. The brain tissues were sectioned with a basis on the rat brain atlas of Paxinos and were stained by the immunoperoxidase staining method using monoclonal antibodies. In the section of the frontal lobe, immunoreactive cells were observed in the lateral septal area, the ventral pallidum, which belongs to the substantia innominata. We could observe immunoreactive cells in the reticular thalamic nucleus, which is closely related with 'sleeping', the basal nuclei of Meynert, which is associated with Alzheimer's disease, and hypothalamic nuclei. Immunoreactive cells were also shown in raphe nuclei or the reticular formation of the midbrain, cerebellum, and inferior olivary nuclei of the medulla oblongata. Succinic semialdehyde reductase-immunoreactive cells were distributed extensively in rat brain, especially immunoreactive cells were strongly observed in the areas associated with the limbic system and reticular formation.

Animals↗

PubMed1997

Molecular cloning and chromosomal localization of a human gene homologous to the murine R-PTP-kappa, a receptor-type protein tyrosine phosphatase.

Tyrosine phosphorylation of proteins plays an important role in cellular signaling and many cellular activities. The levels of cellular phosphorylation are reversibly controlled by protein tyrosine kinases and protein tyrosine phosphatases. The murine R-PTP-kappa, a receptor-type protein tyrosine phosphatase, has recently been cloned (Jiang et al. (1993) Mol. Cell. Biol. 13, 2942-2951). In order to identify the protein tyrosine phosphatases critical to the cellular signal transduction in human keratinocytes, a polymerase chain reaction (PCR)-based strategy was employed, and we have cloned a human homologue of the murine R-PTP-kappa. Here, we report the isolation of a complementary DNA encoding a human R-PTP-kappa. Of the several overlapping cDNA clones, one clone, which we originally termed p55-7, was found to encode a transmembrane protein of 1440 amino acids and was highly conserved with murine R-PTP-kappa with 98% identity at the amino-acid levels. The human R-PTP-kappa gene was localized to chromosome 6 by southern hybridization of DNA from a rodent/human somatic cell mapping panel. Northern blot analysis of RNA from several human tissues revealed, like the murine R-PTP-kappa, the presence of a major mRNA of approx. 7.0 kb and a minor mRNA of approx. 5.3 kb. In contrast to the expression of murine R-PTP-kappa which was highly expressed in liver and kidney, the human R-PTP-kappa was predominantly expressed in spleen, prostate, and ovary. However, the transcripts were detectable at various levels in all examined tissues (thymus, testis, small intestine, and colon) except for PBL (peripheral blood leukocytes). In addition, human R-PTP-kappa displayed a restricted pattern of expression among a series of cell lines, and was apparently expressed in an epidermal cells and cell lines (human normal keratinocytes, HaCaT, and A431), but was not detectable in other cell lines tested after longer exposure.

Amino Acid Sequence↗

PubMed1997

In vitro and in vivo properties of murine monoclonal antibody for a novel immature thymocyte-differentiated antigen, JL1.

JL1 is a novel thymocyte-differentiated antigen strictly confined to stage II immature cortical thymocytes. It is expressed in several types of leukemias and lymphomas. Murine anti-JL1 monoclonal antibody labeled with 131I and 99mTc showed 60-70% of immunoreactivity and 1.4-1.9 x 10(9) L/mol of affinity constant. The incubation of the radiolabeled antibody with Molt-4 cells showed no evidence of modulation or shedding. Localization indices increased from day 3 to day 5 in SCID mice bearing Molt-4 cells.

Animals↗