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Biomedical subjects

S N Ho

Publications and source records attributed to S N Ho.

At least 19 recordsLinked to original sources
PubMed2000

The NFAT-related protein NFATL1 (TonEBP/NFAT5) is induced upon T cell activation in a calcineurin-dependent manner.

NFAT DNA binding complexes regulate programs of cellular activation and differentiation by translating receptor-dependent signaling events into specific transcriptional responses. NFAT proteins, originally defined as calcium/calcineurin-dependent regulators of cytokine gene transcription in T lymphocytes, are expressed in many different cell types and represent critical signaling intermediates that mediate an increasingly wide spectrum of biologic responses. Recent studies have identified a novel protein containing a region of similarity to the NFAT DNA binding domain. Here we demonstrate that this protein, designated NFATL1 (also known as tonicity enhancer binding protein and NFAT5) is expressed at high levels in the thymus but is undetectable in mature lymphocytes. However, NFATL1 can be induced in both primary quiescent T lymphocytes and differentiated Th1 and Th2 cell populations upon mitogen- or Ag receptor-dependent activation. The induction of NFATL1 protein, as well as NFATL1-dependent transcription, is inhibited by cyclosporin A and FK506, and expression of constitutively active calcineurin induces NFATL1-dependent transcription. Overexpression of NFATc1 and inhibition of NFATc activity through the use of a dominant negative NFATc1 protein have no affect on NFATL1-dependent transcription, indicating that NFATc proteins do not play a role in the calcineurin-dependent induction of NFATL1. Interestingly, induction of NFATL1 by a hyperosmotic stimulus is not blocked by the inhibition of calcineurin. Moreover, osmotic stress response genes such as aldose reductase are not induced upon T cell activation. Thus inducible expression of NFATL1 represents a mechanism by which receptor-dependent signals as well as osmotic stress signals are translated into transcriptional responses that regulate cell function.

Aldehyde Reductase↗
PubMed1997

Redundant expression but selective utilization of nuclear factor of activated T cells family members.

Nuclear factor of activated T cells (NF-AT) complexes regulate the induction of many early T cell activation molecules. Four related proteins can function as the cytoplasmic subunit of NF-AT, and their overlapping expression patterns and the mild phenotype of the NF-ATp null mice suggest that they may be functionally redundant. We characterized the distribution and activation of cytoplasmic NF-AT proteins in mature lymphocytes and found that NF-ATc, NF-ATp, and NF-AT4/x/c3 are co-expressed and co-regulated in mature T and B cells. Each protein forms independent DNA binding complexes, and at physiologic concentrations, NF-ATc and NF-ATp complexes out-compete NF-AT4/x/c3 for occupancy of NF-AT sites from the IL-2, IL-3/granulocyte-macrophage CSF, IL-4, and CD40 ligand genes. This predicts heavily redundant immune regulatory functions of NF-ATp and NF-ATc, but distinct activities for NF-AT4/x/c3. Additionally, Ab interaction with NF-ATp induces high affinity NF-kappaB site interaction, suggesting that nuclear partners may dramatically vary the specificity of the NF-AT family.

Animals↗
PubMed1997

Transcriptional control in keratinocytes and fibroblasts using synthetic ligands.

The skin is an attractive tissue for regulated target gene expression by virtue of its accessibility to topical regulating stimuli. We have used synthetic ligand-driven intracellular oligomerization to accomplish specific target gene regulation in human skin keratinocytes and fibroblasts. GAL4 DNA binding domains and VP16 transactivation domains, each linked to the FK506 binding protein, were expressed in normal human skin keratinocytes and fibroblasts. These hybrid proteins underwent heterodimerization via the novel intracellular dimerizing agent FK1012 to generate a heterodimeric activator of target gene expression in vitro. Dimeric FK1012, but not monomeric FK506M induced target gene expression in a dose-dependent fashion. FK1012 exerted no detectable nonspecific effects on expression of cutaneous genes and did not alter cellular proliferation kinetics. Controlled oligomerization of hybrid transcription activators offers a potential approach to target gene regulation in cells of normal human skin.

Cells, Cultured↗
PubMed1997

Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction.

The NF-AT family of transcription factors participates in the regulation of early immune response genes such as IL-2, IL-4, CD40 ligand, and Fas ligand in response to Ca2+/calcineurin signals initiated at the antigen receptor. Calcineurin activation leads to the rapid translocation of NF-AT family members from cytoplasm to nucleus, an event that is blocked by the immunosuppressive drugs cyclosporin A and FK506. We show that translocation requires two redundant nuclear localization sequences and that one sequence is in an intramolecular association with phosphorserines in a conserved motif located at the amino terminus of each NF-AT protein. Mutation of serines in this motif in NF-ATc both disrupts this intramolecular interaction and leads to nuclear localization, suggesting a model of NF-AT nuclear import in which dephosphorylation by calcineurin causes exposure of two nuclear localization sequences.

Amino Acid Sequence↗
PubMed1997

Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc.

Transcription factors of the NFAT family regulate the production of effector proteins that coordinate the immune response. The immunosuppressive drugs FK506 and cyclosporin A (CsA) act by blocking a Ca2+-mediated signalling pathway leading to NFAT. Although FK506 and CsA have enabled human organs to be transplanted routinely, the toxic side-effects of these drugs limit their usage. This toxicity might be absent in antagonists that target NFAT directly. As a first step in the structure-based search for NFAT antagonists, we now report the identification and solution structure of a 20K domain of NFATc (NFATc-DBD) that is both necessary and sufficient to bind DNA and activate transcription cooperatively. Although the overall fold of the NFATc DNA-binding domain is related to that of NF-kappaB p50 (refs 2, 3), the two proteins use significantly different strategies for DNA recognition. On the basis of these results, we present a model for the cooperative complex formed between NFAT and the mitogenic transcription factor AP-1 on the interleukin-2 enhancer.

Amino Acid Sequence↗
PubMed1996

Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression.

Cells need to distinguish between transient Ca2+ signals that induce events such as muscle contraction, secretion, adhesion and synaptic transmission, and sustained Ca2+ signals that are involved in cell proliferation and differentiation. The latter class of events is blocked in lymphocytes by the immunosuppressive drugs cyclosporin A and FK506, which inhibit calcineurin, a Ca2+-activated serine/threonine phosphatase necessary for the nuclear import of NF-AT transcription factors. Here we report that sustained high concentrations of Ca2+, but not transient pulses, are required to maintain NF-AT transcription factors in the nucleus, where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation. Furthermore, overexpression and constitutive nuclear localization of NF-AT, but not Jun, Fos, NF-kappaB, Oct or Ets family members, renders the interleukin-2 enhancer in Jurkat T lymphocytes resistant to FK506 and cyclosporin A. Thus a primary effect of these immunosuppressive reagents is to control the subcellular localization of the NF-AT family of transcription factors.

Animals↗
PubMed1996

Dimeric ligands define a role for transcriptional activation domains in reinitiation.

Eukaryotic transcriptional activators mediate transcriptional induction through stabilization of the preinitiation complex, probably through direct interactions with basal transcription factors. In vitro studies on the role of an activator in the maintenance of on-going transcription (reinitiation) have been contradictory, suggesting that, after formation of a preinitiation complex, an activator may or may not be necessary for transcription to be maintained. We have developed a means of regulating transcription in living cells through the use of both homodimeric and heterodimerizing synthetic ligands that allow the ligand-dependent association and disassociation of a transcriptional activation domain with a promoter. Here we report that maintaining the transcription of endogenous genes in vivo, in both yeast and human cells, requires the continuous presence of the activation domain. The use of synthetic ligands as a transcriptional on-off switch represents a powerful means of controlling the transcription in vitro and in vivo for both experimental and therapeutic purposes.

Alkaline Phosphatase↗
PubMed1996

Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization.

BACKGROUND: Genetic abnormalities in the Fas receptor or its trimeric ligand, FasL, result in massive T-cell proliferation and a lupus-like autoimmune syndrome, which was initially attributed to excessive lymphoproliferation but is now ascribed to the absence of Fas-mediated cell death. Although Fas is normally expressed on most thymocytes, negative selection seems to be unperturbed in Fas-deficient (lpr) mice. This suggests that Fas has an important function in peripheral, but not thymic, T cells. RESULTS: To explore the Fas-mediated cell death pathway both in vitro and in vivo, we used conditional alleles of the Fas receptor that can be triggered by an intracellularly active chemical inducer of dimerization known as FK1012. We found that membrane attachment is important for Fas function and, unlike previous results with anti-Fas monoclonal antibodies, we show that dimerization is sufficient to trigger apoptosis. Finally, the administration of FK1012 in vivo to transgenic animals expressing the conditional FAS receptor in thymocytes demonstrates that sensitivity to FAS-mediated apoptosis is restricted to CD4+CD8+ thymocytes. CONCLUSIONS: Here, we describe the first in vivo application of non-toxic, cell-permeable synthetic ligands to regulate signal transduction in transgenic mice expressing a conditional receptor. Using this system, we show that the Fas pathway is restricted to double-positive thymocytes in vivo, consistent with recent in vitro findings with thymocytes. This method promises to be more useful not only for developmental studies involving cell ablation, but also for studies involving the regulation of a wide variety of signaling molecules.

Animals↗
PubMed1996

Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins.

Extracellular growth and differentiation factors induce changes in gene expression in the nucleus by initiating a series of protein associations that alter the subcellular localization of intracellular signaling proteins. Initial events involve receptor homo- or heterodimerization and subsequent recruitment of cytosolic signaling proteins to the inner leaflet of the plasma membrane. Intermediate events involve the translocation of proteins into the nucleus. Late events involve the recruitment of transcriptional activators to the vicinity of specific genes in the nucleus, resulting in increased gene transcription. The ability to induce signals at each of these three phases of signaling pathways is illustrated by the use of a heterodimeric chemical inducer of dimerization that causes a proximal relationship between two different target proteins.

Animals↗
PubMed1995

NFATc3, a lymphoid-specific NFATc family member that is calcium-regulated and exhibits distinct DNA binding specificity.

Signals transduced by the T cell antigen receptor (TCR) regulate developmental transitions in the thymus and also mediate the immunologic activation of mature, peripheral T cells. In both cases TCR stimulation leads to the assembly of the NFAT transcription complex as a result of the calcium-dependent nuclear translocation of cytosolic subunits, NFATc, and the Ras/protein kinase C-dependent induction of a nuclear subunit, NFATn. To further understand the diverse roles of antigen receptor signaling throughout T cell development, we have identified a new NFATc family member, NFATc3, that is expressed at highest levels in the thymus. NFATc3 is the product of a gene on murine chromosome 8 that is not linked to the other NFATc genes. NFATc3, like other NFATc family members, contains a conserved rel similarity domain, and also defines a region conserved among NFATc family members, the SP repeat region, characterized by the repeated motif SPxxSPxxSPrxsxx (D/E)(D/E)swl. NFATc3 activates NFAT site-dependent transcription when overexpressed, yet exhibits a pattern of DNA site specificity distinct from other NFATc proteins. Additionally, thymic NFATc3 undergoes modifications in response to agents that mimic T cell receptor signaling, including a decrease in apparent molecular mass upon elevation of intracellular calcium that is inhibited by the immunosuppressant FK506. Given the preferential expression of NFATc3 in the thymus, NFATc family members may regulate distinct subsets of genes during T cell development.

Amino Acid Sequence↗
PubMed1995

Cloning and chromosomal localization of the human and murine genes for the T-cell transcription factors NFATc and NFATp.

The nuclear factor of activated T cells (NFAT) is a transcription factor complex involved in the activation of cytokines and cell surface molecules associated with coordinating the actions of different cells required for an immune response. Two different genes have recently been cloned that encode proteins capable of functioning as the pre-existing (p) and cytosolic (c) component of the NFAT transcription complex, NFATc of human and NFATp of murine origin (Northrop et al., 1994; McCaffrey et al., 1993b). We report here the partial cDNA cloning of the murine homolog of NFATc and the human homolog of NFATp, and the chromosomal localization of both genes in both species to conserved syntenic regions. Through the use of mapping panels of human x Chinese hamster and mouse x rodent cells hybrids, the NFATc genes were mapped to human and mouse chromosomes 18. By analyzing a chromosome 18 radiation hybrid panel, the human NFATc gene was localized to the q terminus, closely linked to STS marker D18S497. The murine Nfatc gene was sublocalized to chromosome band 18E4 by FISH. The NFATp genes were mapped by somatic cell hybrid analysis to human chromosome 20 and mouse chromosome 2. Human NFATp was assigned to chromosome region 20q13.2-->q13.3 by FISH. Based on the conserved syntenic region on human chromosome 20 and mouse chromosome 2, murine Nfatp is predicted to reside in the vicinity of a mutant locus wasted. Homozygous wst/wst mice display a phenotype reminiscent of severe combined immune deficiency or ataxia telangiectasia, disorders that could therefore be considered candidates for NFATp mutations.

Amino Acid Sequence↗
PubMed1994

Three-dimensional structure in solution of griseoviridin, a group A antibiotic.

The solution conformation of griseoviridin, a broad spectrum antibiotic, has been determined by 1H-NMR in deuterated dimethylsulfoxide. The structural determination is based on experimental data of NOE constraints Five structures were obtained from restrained molecular dynamics calculations, by imposing (the condition for) a minimum violation of distance constraints. These structures satisfy well the experimental restraints, with small values of NOE violation and total energies. On comparison with its crystal structure, a good agreement is noted with a backbone root-mean-square deviation value of 0.084 nm. However, a small variation between the structures is observed at the aminodecanoic acid part of the molecule.

Anti-Bacterial Agents↗
PubMed1994

Specific inhibition of formation of transcription complexes by a calicheamicin oligosaccharide: a paradigm for the development of transcriptional antagonists.

Sequence-specific DNA ligands that antagonize DNA-protein interactions represent a potentially powerful means of modulating gene expression. Calicheamicin gamma 1I, a member of the DNA-cleaving enediyne class of anticancer antibiotics, binds to specific DNA sequences through an aryltetrasaccharide domain. To take advantage of this unique sequence-specific recognition capability, the methyl glycoside of the aryltetrasaccharide of calicheamicin gamma 1I (CLM-MG) was used to investigate the ability of glycoconjugate DNA ligands to inhibit DNA-protein interactions. CLM-MG inhibits the formation of DNA-protein complexes at micromolar concentrations in a sequence-specific manner and rapidly dissociates preformed complexes. CLM-MG also inhibits transcription in vivo with similar sequence specificity. These results suggest a strategy for the development of a class of novel biological probes and therapeutic agents.

Aminoglycosides↗
PubMed1994

NF-AT components define a family of transcription factors targeted in T-cell activation.

The NF-AT transcription complex is required for the expression of a group of proteins that collectively coordinate the immune response. Here we purify two proteins encoded by separate genes that represent the pre-existing (p) and cytosolic (c) components of NF-AT. Expression of the full-length complementary DNA encoding NF-ATc activates the interleukin (IL-2) promoter in non-T lymphocytes, whereas a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon T-cell activation. The other protein, NF-ATp, is highly homologous to NF-ATc over a limited domain which shows similarity to the Dorsal/Rel family, but has a wider tissue distribution. Agents that increase intracellular Ca2+ or activate protein kinase C independently modify NF-ATc, indicating that distinct signalling pathways converge on NF-ATc to regulate its function.

Amino Acid Sequence↗
PubMed1994

FL-120A-D', new products related to kinamycin from Streptomyces chattanoogensis subsp. taitungensis subsp. nov. II. Isolation and structure determination.

Six new kinamycin antibiotics have been isolated from the culture filtrate of Streptomyces chattanoogensis. The structures of six related components were determined employing 1D and 2D NMR spectroscopy and mass spectrometry. These structures represent the first reported epoxide kinamycin (2, 3) and propionyl derivative of kinamycin (5), and new isobutyryl derivatives of kinamycin (1, 4, 6).

Anti-Bacterial Agents↗
PubMed1994

Solution conformation of enopeptin A, a depsipeptide antibiotic, using 2D NMR and restrained molecular dynamics studies.

Studies on the solution conformation of the cyclic depsipeptide antibiotic enopeptin A have been carried out using 2D NMR and molecular modelling techniques. The proton resonances of the antibiotic in DMSO-d6 have been assigned by the use of TOCSY and ROESY experiments. The interproton distance information obtained from the ROESY experiments have been used as the basis for elucidating the probable structures in solution. The restrained molecular dynamics technique was applied to calculate the structures in solution, and six resultant structures with fewer distance constraint violations were obtained that satisfy the experimental restraints very well. The conformation of the cyclic moiety of the molecules is well defined whereas the aliphatic chain segment is disordered.

Amino Acid Sequence↗
PubMed1990

Regulatory effects of transforming growth factor-beta on IL-2- and IL-4-dependent T cell-cycle progression.

Previous studies suggested that the potent immunosuppressive activities of transforming growth factor-beta (TGF-beta) were mediated in part through the inhibition of IL-2-dependent S-phase progression and mitosis of activated T cells. To further investigate the mechanism of T cell growth inhibition by TGF-beta, two constitutively activated murine T cell clones were employed as defined model systems for the growth factor-dependent phase of T cell proliferation. The Th cell line, HT-2, proliferated in response to either IL-2 or IL-4, whereas the cytotoxic T cell line, CT6, exhibited strict dependence on IL-2 for growth stimulation. In both cell lines, picomolar concentrations of TGF-beta inhibited S-phase progression stimulated by IL-2 or IL-4. TGF-beta pretreatment decreased the expression of high affinity IL-2R on HT-2 cells, but not on CT6 cells. In contrast, IL-2-stimulated transferrin receptor expression was markedly inhibited by TGF-beta in both T cell lines. Analyses of growth factor-dependent specific mRNA accumulation revealed that TGF-beta exerted selective inhibitory effects on gene expression in HT-2 and CT6 cells. TGF-beta significantly reduced early (1 to 2 h) increases in c-myc mRNA levels stimulated by IL-2 or IL-4 in both cell lines. In HT-2 cells, TGF-beta pretreatment also inhibited the early increase in granulocyte-macrophage CSF mRNA stimulated by IL-2 or IL-4. The inhibition of c-myc and granulocyte-macrophage cyte-macrophage CSF gene expression by TGF-beta was explained, at least in part, by suppression of the growth factor-dependent transcriptional activation of these genes. These studies suggest that inhibition of c-myc gene transcription may play a fundamental role in the antiproliferative effect of TGF-beta on IL-2- or IL-4-stimulated T cells.

Blotting, Northern↗