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Young-Tae Chang

Publications and source records attributed to Young-Tae Chang.

At least 19 recordsLinked to original sources
PubMed2006

Forward chemical genetic approach identifies new role for GAPDH in insulin signaling.

Insulin and insulin-like growth factor have an essential role in growth, development and the maintenance of metabolic homeostasis, including glucose uptake from the bloodstream. Researchers have identified mutations in insulin receptors that cause severe insulin resistance, and a temperature-sensitive daf-2 (a gene encoding an insulin receptor-like protein) mutant in Caenorhabditis elegans has served as an insulin resistance model. Here we report a forward chemical genetic approach with a tagged library that we used to identify a small molecule, GAPDH segregator (GAPDS), that suppresses the dauer formation induced by the daf-2 mutant. Like insulin, GAPDS increased both glucose uptake and the concentration of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in mammalian preadipocytes. Using affinity matrices and RNA interference, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a GAPDS target. We discovered that GAPDH stimulates phosphatase activity against not only PtdIns(3,4,5)P(3) but also PtdIns(4,5)P(2). These results suggest that GAPDH is both an active regulator in the phosphoinositide-mediated signaling pathway and a potential new target for insulin resistance treatment.

Adipocytes↗
PubMed2006

A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's peptide.

Aggregation of the Alzheimer's peptide Abeta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of Abeta aggregation from large libraries of inactive candidates. The screen uses a fusion of Abeta42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of Abeta42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Abeta42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic Abeta42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of Abeta42 aggregation and/or amyloidogenesis.

Alzheimer Disease↗
PubMed2006

Visual artificial tongue for quantitative metal-cation analysis by an off-the-shelf dye array.

A chemical-probe array composed of 47 off-the-shelf dyes was prepared in solution format (New York Tongue 1: NYT-1) and was tested in the identification and quantitation of 47 cation analytes, including 44 metal ions, in addition to H(+), NH(4) (+), and tetrabutylammonium (TBA). The cation solutions were tested in a series of concentrations and the fold-change in effective absorbance was analyzed by principal-component analysis (PCA), hierarchical-cluster analysis (HCA), and nearest-neighbor decision to determine both identity and quantity of the analytes. Apart from alkali-metal ions (Na(+), K(+), Li(+), Cs(+), and Rb(+)), which behave very similarly to each other due mainly to their low response, most of the cations were clearly distinguishable at 10 mM concentration. The practical detection limit of each analyte was also determined by a sequential dilution and the nearest-neighbor decision method. In the finalized working analyte concentration range (approximately 10 mM down to 0.33 microM), by considering alkali metals as one analyte group, most of the analytes were correctly identified (99.4 %). Furthermore, the success rate at which the concentration of each analyte was correctly determined was also high (96.8 %).

Cations↗
PubMed2006

Inhibition of interleukin-4 production in activated T cells via the downregulation of AP-1/NF-AT activation by N-lauroyl-D-erythro-sphingosine and N-lauroyl-D-erythro-C20-sphingosine.

Allergic diseases are hypersensitivity disorders that are associated with the generation of specific immunoglobulin E (IgE) in response to environmental allergens. Interleukin (IL)-4, which is primarily produced by the CD4(+) T cells, is an important stimulus for the switching of the antibody isotype to IgE in both mice and humans. In a previous study, we demonstrated that ceramide derivatives coupled with a lauroyl group exerted strong inhibitory effects on IL-4 production in T cells. In this study, we attempted to characterize the mechanisms underlying the inhibition of IL-4 production in T cells. Two ceramide derivatives, N-lauroyl-D-erythro-sphingosine and N-lauroyl-D-erythro-C(20)-sphingosine (hereafter abbreviated as LES and LECS, respectively), were shown to significantly inhibit the production of IL-4 in both primary CD4(+) T cells and EL4 T thymoma cells in a dose-dependent manner. LES and LECS also inhibited the activity of the IL-4 gene promoter in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells that had been transfected with an IL-4 promoter construct deleted of a P4 site harboring the AP-1 and NF-AT binding sites. Furthermore, LES and LECS inhibited the DNA binding activities of both AP-1 and NF-AT transcription factors. In addition, LES and LECS were demonstrated to suppress PMA-stimulated PKC activity, although they exerted no significant effects on the protein levels of the conventional PKCs. These results indicate that the ceramides, LES and LECS, may inhibit the production of IL-4 in the activated T cells, via the downregulation of AP-1/NF-AT activation and PKC activity.

Animals↗
PubMed2006

RNA-selective, live cell imaging probes for studying nuclear structure and function.

The higher-order structural organization of the cell nucleus reflects the underlying genome-wide transcriptional activity and macromolecular transport processes. To study the microscopic organization of RNA distribution within the nucleus, a combinatorial library of fluorescent styryl molecules was synthesized and screened for an in vitro RNA response and live cell nuclear imaging. Four different cell lines (HeLa, A549, 3T3, and 3T3-L1) were analyzed in terms of higher-order nuclear organization. We identified RNA-selective dyes with better imaging properties relative to commercially available SYTORNASelect dye; the selected dyes were also cell permeant, photostable, and well tolerated by the cells. Our dyes also had very good counterstain compatibility with Hoechst and DAPI, which could help to image the DNA distribution in relation to RNA distribution in live cells and therefore reveal different patterns of RNA-DNA colocalization.

Animals↗
PubMed2006

Myoseverin is a potential angiogenesis inhibitor by inhibiting endothelial cell function and endothelial progenitor cell differentiation.

Myoseverin, a new microtubule-binding molecule, acts reversibly on myoblast proliferation without the cytotoxic effects displayed by nonpurine-based microtubule-disrupting molecules, like taxol, vinblastine, nocodazole, and the colchicines. In this study, we examined the effects of myoseverin on in vitro function of endothelial cells and endothelial progenitor cell differentiation in order to explore the possibility for the application of myoseverin as a reversible antiangiogenic agent. Myoseverin potently inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner with an IC50 of approximately 8 microM. When myoseverin was removed after treatment for 3 days, all the cells pretreated at a concentration range of 2.5-80 microM resumed the cell growth. It also inhibited VEGF-induced HUVEC migration dose dependently. When mononuclear cells (MNCs) isolated from human cord blood were cultured on fibronectin-coated plates for 7 days, myoseverin decreased the number of adherent cells in a dose-dependent manner with IC50 of approximately 9 microM. It also suppressed the development of ac-LDL uptake ability as well as the expression of endothelial lineage markers, KDR, CD31, and vWF. Finally, it inhibited formation of HUVECs or ex vivo cultivated EPCs into capillary-like structure on Matri-gel and in vivo angiogenesis on the chick chorioallantoic membrane. Therefore, these results suggest that myoseverin can be effectively used for the inhibition of new vessel growth by inhibiting endothelial cell function and differentiation of progenitor cells.

Angiogenesis Inhibitors↗
PubMed2005

Identification of a new class of prostaglandin transporter inhibitors and characterization of their biological effects on prostaglandin E2 transport.

Prostaglandins (PGs) are involved in several major signaling pathways. Their effects are terminated when they are transported across cell membranes and oxidized intracellularly. The transport step of PG metabolism is carried out by the prostaglandin transporter (PGT). Inhibition of PGT would therefore be expected to change local or circulating concentrations of prostaglandins, and thus their biological effects. To develop PGT-specific inhibitors with high affinity, we designed a library of triazine compounds and screened 1842 small molecules by using Madin-Darby canine kidney cells stably expressing rat PGT. We found several effective PGT inhibitors. Among them, the most potent inhibitor had a Ki of 3.7 +/- 0.2 microM. These inhibitors allowed us to isolate the efflux process of PGE2 and to demonstrate that PGT does not transport PGE2 outwardly under physiological conditions.

3T3 Cells↗
PubMed2005

Identification of 12Cysbeta on tubulin as the binding site of tubulyzine.

We have undertaken quantitative binding site studies in order to identify the binding site of the known microtubule destabilizing agents, the tubulyzines, in the tubulin dimer. Two different approaches were employed that utilized the tubulyzines and their derivatives. The first approach was based on a chemical affinity labeling method using tubulyzine affinity derivatives, and the second approach employed the mass spectrometric measurement of the differential reactivity of cysteines using the tubulyzines and monobromobimane. Based on overlapping data from these two approaches, we propose that the tubulyzines bind at the guanosine-5'-triphosphate binding site of beta-tubulin. Interestingly, we also show that the tubulyzines' binding to tubulin induces a conformational change in tubulin that prevents further interaction of the 239Cysbeta with other reagents.

Amino Acid Sequence↗
PubMed2005

Fcepsilon RI control of Ras via inositol (1,4,5) trisphosphate 3-kinase and inositol tetrakisphosphate.

The inositol (1,4,5) trisphosphate 3-kinase (ITP3K) phosphorylates Ins (1,4,5) P3 to produce Ins (1,3,4,5) P4. The ITP3K substrate, InsP3, and its product, InsP4, both have the potential to regulate mast cell function. Here, we explore the effects of dominant inhibition of ITP3K upon secretory responses and Ras GTPase activation following antigenic cross-linking of the mast cell immunoreceptor, FcvarepsilonRI. Inhibition of ITP3K potentiates both calcium release from intracellular stores and calcium-dependent secretory responses in mast cells. Moreover, mast cells with dominantly inhibited ITP3K display constitutive activation of Ras and certain Ras effector pathways. We propose three mechanisms by which ITP3K inhibition could influence Ras activation. The protection of InsP3 that results from ITP3K inhibition may lead to enhanced activation of calcium-sensitive Ras-GAPs or -GRFs. Similarly, the deficit in InsP4 may change the behavior of the InsP4 receptor, the GAP1(IP4BP). Our data are inconsistent with calcium-sensitive Ras-GAP activation being the primary consequence of ITP3K inhibition in mast cells. Rather, we observe potentiation of Ras responses in mast cells transfected with dominant negative GAP1(IP4BP). Moreover, shRNA-mediated knockdown of GAP1(IP4BP) potentiates FcvarepsilonRI-mediated Ras activation, indicating that this InsP4-binding GAP protein may be used by the FcvarepsilonRI immunoreceptor to regulate Ras.

Animals↗
PubMed2005

Phytic acid synthesis and vacuolar accumulation in suspension-cultured cells of Catharanthus roseus induced by high concentration of inorganic phosphate and cations.

We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP(6)) synthesis in suspension-cultured cells of Catharanthus. InsP(6) and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP(6) was less than 50 nM, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP(6) was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K(+), Ca(2+), or Zn(2+). However, there was a strong tendency for InsP(6) to accumulate in the vacuole in the presence of Ca(2+) and in nonvacuolar compartments when supplied with Zn(2+), possibly due to precipitation of InsP(6) with Zn(2+) in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP(6) accumulation. The amounts of both Ins(3)P(1) myo-inositol monophosphate synthase, a key enzyme for InsP(6) synthesis, and Ins(1,4,5)P(3) kinase were unrelated to the level of accumulation of InsP(6). The mechanisms for InsP(6) synthesis and localization into vacuoles in plant cells are discussed.

Calcium↗
PubMed2005

Inhibitory activity of a ceramide library on interleukin-4 production from activated T cells.

Allergic diseases are hypersensitivity disorders associated with the production of specific immunoglobulin E (IgE) to environmental allergens. Interleukin (IL)-4, produced primarily by CD4(+) T cells, is an important stimulus for the switch of the antibody isotype to IgE in both mice and humans. In this study we investigated the inhibitory activity of IL-4 production in activated T cells by screening ceramide derivatives prepared by solid phase combinatorial chemistry. Many ceramide derivatives significantly inhibited IL-4 production in T cells. In particular, ceramide derivatives with a lauroyl group showed strong inhibitory activities on IL-4 production in both phorbol 12-myristate 13-acetate (PMA)-activated EL4 T cells and antigen-primed cells, suggesting that they can be used as compounds for the development of anti-allergic agents.

Animals↗
PubMed2005

Structural and functional modeling of human lysozyme reveals a unique nonapeptide, HL9, with anti-HIV activity.

We previously reported that lysozyme accounts for anti-HIV activity associated with the beta-core fraction of human chorionic gonadotropin [Lee-Huang, S., Huang, P. L., Sun, Y., Kung, H. F., Blithe, D. L. & Chen, H. C. (1999) Proc Natl Acad Sci U S A 96, 2678-81]. To define the structural and sequence requirements for anti-HIV activity, we carried out peptide fragmentation and activity mapping of human lysozyme. We identified two peptides that consist of 18 and 9 amino acids of human lysozyme (HL18 and HL9), corresponding to residues 98-115 and 107-115. HL18 and HL9 are potent inhibitors of HIV-1 infection and replication with EC(50)s of 50 to 55 nM, comparable to intact lysozyme. Scrambling the sequence or substitution of key arginine or tryptophan residues results in loss of antiviral activity. HL9, with the sequence RAWVAWRNR, is the smallest peptide we identified with full anti-HIV activity. It forms a pocket with its basic residues on the surface of the molecule. HL9 exists as an alpha-helix in native human lysozyme, in a region of the protein distinct from the muramidase catalytic site. Monte Carlo peptide folding energy minimizing simulation modeling and CD studies indicate that helical propensity does not correlate with antiviral activity. HL9 blocks HIV-1 viral entrance and replication, and modulates gene expression of HIV-infected cells, affecting pathways involved in survival, stress, TGFbeta, p53, NFkappaB, protein kinase C and hedgehog signaling.

Amino Acid Sequence↗
PubMed2005

Identification of the F1F0 mitochondrial ATPase as a target for modulating skin pigmentation by screening a tagged triazine library in zebrafish.

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells.

Amino Acid Sequence↗
PubMed2005

Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of alpha-galactosylceramides.

A form of alpha-galactosylceramide, KRN7000, activates CD1d-restricted Valpha14-invariant (Valpha14i) natural killer (NK) T cells and initiates multiple downstream immune reactions. We report that substituting the C26:0 N-acyl chain of KRN7000 with shorter, unsaturated fatty acids modifies the outcome of Valpha14i NKT cell activation. One analogue containing a diunsaturated C20 fatty acid (C20:2) potently induced a T helper type 2-biased cytokine response, with diminished IFN-gamma production and reduced Valpha14i NKT cell expansion. C20:2 also exhibited less stringent requirements for loading onto CD1d than KRN7000, suggesting a mechanism for the immunomodulatory properties of this lipid. The differential cellular response elicited by this class of Valpha14i NKT cell agonists may prove to be useful in immunotherapeutic applications.

Animals↗
PubMed2005

Small molecule microarrays: recent advances and applications.

Directed or exploratory drug development programs constantly seek robust screening platforms for the high fidelity identification and validation of potential targets. Small-molecule microarrays (SMMs) have risen to this call by elegantly forging the capability of combinatorial chemistry in producing myriad compounds with the powerful throughput afforded by microarrays. This synergism offers scientists a versatile tool for rapid compound analysis and discovery. Microarrays of small molecules have already been successfully applied in important areas ranging from protein profiling to the discovery of therapeutic leads. Recent interesting developments towards improved immobilization strategies and library creation methods, together with novel advances herein described, have set the stage for SMMs to take on wider and more routine applications in academia and industry. As a rapidly maturing technology, SMMs pave the way forward in high-throughput exploration, both in the identification of biologically significant natural and synthetic small molecules and in harnessing their vast potential towards medicinal and diagnostic applications.

Combinatorial Chemistry Techniques↗