No abstract

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Despite a well documented tubulin-stabilizing effect, many side effects of taxol therapy cannot be explained by cytoskeletal mechanisms. In the present study submicromolar concentrations of taxol, mimicking concentrations found in patients, induced cytosolic calcium (Ca 2؉ ) oscillations in a human neuronal cell line. These oscillations were independent of extracellular and mitochondrial Ca 2؉ but dependent on intact signaling via the phosphoinositide signaling pathway. We identified a taxol binding protein, neuronal Ca 2؉ sensor 1 (NCS-1), a Ca 2؉ binding protein that interacts with the inositol 1,4,5-trisphosphate receptor from a human brain cDNA phage display library. Taxol increased binding of NCS-1 to the inositol 1,4,5-trisphosphate receptor. Short hairpin RNA-mediated knockdown of NCS-1 in the same cell line abrogated the response to taxol but not to other agonists stimulating the phosphoinositide signaling pathway. These findings are important for studies involving taxol as a research tool in cell biology and may help to devise new strategies for the management of side effects induced by taxol therapy.calcium imaging ͉ calcium release ͉ display cloning ͉ drug-induced side effects ͉ hypersensitivity reactions

The natural product rapamycin has been used to provide temporal and quantitative control of gene expression in animals through its ability to interact with two proteins simultaneously. A shortcoming of this approach is that rapamycin is an inhibitor of cell proliferation, the result of binding to FKBP12-rapamycin-associated protein (FRAP). To overcome this limitation, nontoxic derivatives of rapamycin bearing bulky substituents at its C16-position were synthesized, each in a single step. The isosteric isopropoxy and methallyl substituents with the nonnatural C16-configuration abolish both binding to FRAP and inhibition of T cell proliferation. Binding proteins for these derivatives were identified from libraries of cDNAs encoding mutants of the FKBP12-rapamycin-binding (FRB) domain of FRAP by using a mammalian three-hybrid transcription assay. Targeting of the mutations was guided by the structure of the FKBP12-rapamycin-FRB ternary complex. Three compensatory mutations in the FRB domain, all along one face of an ␣-helix in a rapamycin-binding pocket, were identified that together restore binding of the rapamycin derivatives. Using this mutant FRB domain, one of the nontoxic rapamycin derivatives induced targeted gene expression in Jurkat T cells with an EC 50 below 10 nM. Another derivative was used to recruit a cytosolic protein to the plasma membrane, mimicking a process involved in many signaling pathways.

The transition metal catalyzed reaction of x-diazo ciirbonyl compounds has found numerous applications in organic synthesis, and its L I S~ in either heterocyclic or carbocyclic ring formation is well prccedcnted. Early work in this area made use of insoluble copper catalysts. Although these catalysts are still employed today. their use has decreased signifhntly with the advent o f homogeneous copper cxtalysts and catalysts based on other metals. The discovery that Rh" carboxylates facilitate nitrogen loss from diazo compounds rekindled significant interest in the field of diazo/ carbenoid chemistry. Since the realization that Rh" carboxylates are superior catalysts for the generation of transient electrophilic metal carbenoids from adiazo carbonyl compounds, intramolecular carbenoid addition and insertion reactions have assumed strategic importance in C-C bond-forming reactions in organic synthesis. In contrast to other catalysts that are suitable for carbenoid reactions of diazo compounds, those constructed with the dirhodium(r1) framework are most amenable to ligand modifications that, in turn, can influence reaction selectivity. This article will emphasize the chemical behavior of transition metal carbenoid complexes that are greatly affected by the nature of the ligand groups attached to the metal center. Much of the discussion will center on the ability of the dirhodium(i1) ligands to determine reaction preference toward different functional groups on the same molecule.

Kaiso, a p120 catenin-binding protein, is expressed in the cytoplasmic and nuclear compartments of cells; however, the biological consequences and clinical implications of a shift between these compartments have yet to be established. Herein, we report an enrichment of nuclear Kaiso expression in cells of primary and metastatic prostate tumors relative to the normal prostate epithelium. Nuclear expression of Kaiso correlates with Gleason score (P < 0.001) and tumor grade (P < 0.001). There is higher nuclear expression of Kaiso in primary tumor/normal matched samples and in primary tumors from African American men (P < 0.0001). We further found that epidermal growth factor (EGF) receptor up-regulates Kaiso at the RNA and protein levels in prostate cancer cell lines, but more interestingly causes a shift of cytoplasmic Kaiso to the nucleus that is reversed by the EGF receptor-specific kinase inhibitor, PD153035. In both DU-145 and PC-3 prostate cancer cell lines, Kaiso inhibition (short hairpin RNA-Kaiso) decreased cell migration and invasion even in the presence of EGF. Further, Kaiso directly binds to the E-cadherin promoter, and inhibition of Kaiso in PC-3 cells results in increased E-cadherin expression, as well as re-establishment of cell-cell contacts. In addition, Kaiso-depleted cells show more epithelial morphology and a reversal of the mesenchymal markers N-cadherin and fibronectin. Our findings establish a defined oncogenic role of Kaiso in promoting the progression of prostate cancer.

Autoimmunity leads to the activation of innate effector pathways, pro-inflammatory cytokine production, and end-organ injury. Macrophage migration inhibitory factor (MIF) is an upstream activator of the innate response that mediates the recruitment and retention of monocytes via CD74 and associated chemokine receptors, and it has a role in the maintenance of B lymphocytes. High-expression MIF alleles also are associated with end-organ damage in different autoimmune diseases. We assessed the therapeutic efficacy of ISO-1, an orally bioavailable, MIF antagonist, in two distinct models of systemic lupus erythematosus (SLE): the NZB/NZW F1 and the MRL/lpr mouse strains. ISO-1, like anti-MIF, inhibited the interaction between MIF and its receptor, CD74, and in each model of disease, it reduced functional and histological indices of glomerulonephritis, CD74+ and CXCR4+ leukocyte recruitment, and pro-inflammatory cytokine and chemokine expression. Neither autoantibody production nor T and B cell activation were significantly affected, pointing to the specificity of MIF antagonism in reducing excessive pro-inflammatory responses. These data highlight the feasibility of targeting the MIF–MIF receptor interaction by small molecule antagonism and support the therapeutic value of downregulating MIF-dependent pathways of tissue damage in SLE.

While Ras activation has been shown to play an important role in signal transduction by the T-lymphocyte antigen receptor, the mechanism of its activation in T cells is unclear. 9810The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

In eukaryotes, sequence-specific DNAbinding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo. The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.Each of the roughly 100,000 genes encoded in the human genome is subject to individual dosage control. The systems that regulate gene expression respond to a wide variety of developmental and environmental stimuli, thus allowing each cell type to express a unique and characteristic subset of its genes, and to adjust the dosage of particular gene products as needed. The importance of dosage control is underscored by the fact that targeted disruption of key regulatory molecules in mice often results in drastic phenotypic abnormalities (1), just as inherited or acquired defects in the function of genetic regulatory mechanisms contribute broadly to human disease. These findings have fueled efforts aimed at understanding fundamental mechanisms of gene regulation, with a eye toward discovering means of overriding endogenous regulatory controls or of creating new signaling circuitry in cells (2-5). Of particular interest in this regard are synthetic molecules designed to modulate gene transcription in living cells (2-6). To date, attention has been focused mostly on the discovery of organic molecules that interact sequence-specifically with DNA and thereby antagonize transcriptional stimulation by activator proteins (6). Here we describe a strategy for conditional activation of gene expression using organic molecules that simultaneously target the transcriptional machinery and a DNA-binding protein.The regulatory mechanisms controlling the transcription of protein-coding genes by RNA polymerase II have been extensively studied. In the current model, RNA polymerase II and its host of associated proteins are recruited to the core promoter through noncovalent contacts with sequencespecific DNA binding proteins (7,8). An especially prevalent and important subset of such proteins, known as transactivators, typically bind DNA at sites outside the core promoter and activate transcription via through-space contacts with components of the transcriptional machinery, including chromatin remodeling proteins (7-10). The DNA-binding and activation functions of transactivators generally reside on separate domains whose operation is portable to heterologous fusion proteins (11). Though activation domains must be physically associated with a DNA-binding domain to attain proper...