Selection of estrogen receptor beta- and thyroid hormone receptor beta-specific coactivator-mimetic peptides using recombinant peptide libraries.

Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.

Antimalarial, antiproliferative, and antitumor activities of artemisinin-derived, chemically robust, trioxane dimers.

Nine C-10 non-acetal derivatives of the natural trioxane artemisinin (1) were prepared as dimers using some novel chemistry. As designed, each dimer was stable chemically. C-10 Olefinic dimers 7 and C-10 saturated dimers 8-13 all showed good to excellent antimalarial and antiproliferative activities in vitro. Dimers 8, 10, and 12 were especially potent and selective at inhibiting growth of some human cancer cell lines in the NCI in vitro 60-cell line assay.

Expression of cre recombinase as a reporter of signal transduction in mammalian cells.

BACKGROUND: Cell-based reporter assays, which rely on a reporter gene under the control of a regulated promoter, are widely used to screen chemical libraries for novel receptor ligands. Here, we describe a reporter system that is based on ligand-induced DNA recombination to express the reporter gene. This system converts a transient activation of a signal transduction pathway into an amplified, constitutive and heritable expression of the reporter gene. RESULTS: We constructed gene fusions of Cre recombinase and mammalian promoters regulated by calcium, nuclear receptors or cyclic AMP. Reporter systems, comprising a Cre gene fusion and a loxP/reporter gene, were used to study the kinetics and dose responses to compounds that activate or inhibit the corresponding signal transduction pathway. We compared these reporters with conventional reporter systems in which the reporter gene is under the direct control of the responsive promoter. Reporter gene expression of the Cre reporters was greater than that of conventional reporters and could be measured more than a week after adding the stimulus. For all pathways studied here, the dose responses of the Cre reporters are nearly identical to those of conventional reporter systems. CONCLUSIONS: We have shown that Cre recombinase can be regulated by a variety of signal transduction pathways. It should therefore be possible to use receptor ligands to induce phenotypic conversion of mammalian cells for use in a variety of applications. One such application is high-throughput screening, and we developed loxP/luciferase reporter genes that provide an amplified and sustained luminescent response.

Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.

Orally active, hydrolytically stable, semisynthetic, antimalarial trioxanes in the artemisinin family.

In only three chemical operations, natural trioxane lactone artemisinin (1) was converted into a series of C-10 carbon-substituted 10-deoxoartemisinin compounds 4-9. The three steps involved lactone reduction, replacement of the anomeric lactol OH by F using diethylaminosulfur trifluoride, and finally boron trifluoride-promoted substitution of F by aryl, heteroaryl, and acetylide nucleophiles. All of these C-10 nonacetal, chemically robust, enantiomerically pure compounds 4-9 have high antimalarial potencies in vitro against Plasmodium falciparum malaria parasites, and furans 5a and 5b and pyrrole 7a are antimalarially potent also in vivo even when administered to rodents orally.

Feasibility of a healthcare proxy counseling program for patients with Alzheimer's disease.

BACKGROUND: Although significant progress has been made in the implementation of advance directive counseling programs for cognitively intact patients, there is a paucity of information on the outcome of these programs with patients with Alzheimer's disease. This study investigated the prevalence of completed healthcare proxies in a sample of Alzheimer's disease outpatients, and the feasibility of a systematic proxy counseling program for this population. METHODS: The setting was a geriatric psychiatry clinic. Ninety-four patients with Alzheimer's disease were surveyed for their previous completion of a healthcare proxy. All patients with capacity and without a proxy were approached to complete the advance directive with a lay counselor. RESULTS: Thirty-two percent (n = 30) of patients had completed a proxy prior to the initiation of a counseling program. Of patients without proxies (n = 64), 89% had capacity to complete one. Seventy-nine percent subsequently completed a proxy through the counseling program. Hispanics were least likely to have had a proxy prior to initiation of the program, yet were very willing to complete the document. CONCLUSIONS: The majority of patients with Alzheimer's disease in an outpatient setting did not have healthcare proxies, yet had the capacity and motivation to complete this advance directive. With physician input regarding the presence of decisional capacity, a lay counselor successfully implemented the counseling process. These results support the initiation of similar counseling programs for Alzheimer's outpatients.

Working hours. Minute stakes.

The working time directive has major implications for NHS employers. Many NHS employers believe current systems for recording hours worked will not meet the directive's requirements. The introduction of leave entitlement for bank and agency nurses is likely to cost the NHS an extra 40 m Pounds or more a year. The directive does not currently apply to junior doctors. But if, as expected, they are brought within it, the implications for the NHS are huge.

Human resources. Happy families or snap?

Family-friendly initiatives are popular with staff, but little research has been done to determine their effect. Employers should ask staff what sort of schemes would be most useful to avoid wasting managers' time. Under half the employers surveyed offered creches, nurseries or holiday play schemes. Where they are offered, they are very popular with staff. Employers should specify what they offer in advertisements for staff.

TNF alpha-mediated inhibition and reversal of adipocyte differentiation is accompanied by suppressed expression of PPARgamma without effects on Pref-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNF alpha inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARgamma and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARgamma occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNF alpha-mediated inhibition of adipocyte differentiation, PPARgamma messenger RNA (mRNA) expression stays at low levels. In contrast, TNF alpha treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors [such as TNF alpha, basic fibroblast growth factor, transforming growth factor beta, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARgamma mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNF alpha to fully differentiated adipocytes results in the rapid disappearance of PPARgamma protein expression and the rapid loss of PPARgamma DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNF alpha-generated signals, whereas PPARgamma expression remains sensitive to TNF alpha at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.

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.

The mechanism of action of cyclosporin A and FK506.

The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin suppress the immune response by inhibiting evolutionary conserved signal transduction pathways. CsA, FK506, and rapamycin bind to their intracellular receptors, immunophilins, creating composite surfaces that block the activity of specific targets. For CsA/cyclophilin and FK506/FKBP the target is calcineurin. Because of the large surface area of interaction of the drug-immunophilin complex with calcineurin, FK506 and CsA have a specificity for their biologic targets that is equivalent to growth factor-receptor interactions. To date, all the therapeutic as well as toxic effects of these drugs have been shown to be due to inhibition of calcineurin. Inhibition of the action of calcineurin results in a complete block in the translocation of the cytosolic component of the nuclear factor of activated T cells (NF-AT), resulting in a failure to activate the genes regulated by the NF-AT transcription factor. These genes include those required for B-cell help such as interleukin (IL-4) and CD40 ligand as well as those necessary for T-cell proliferation such as IL-2. The purpose of this article is to illustrate the means by which these drugs produce immunosuppression.

Characterization of the roles of SH2 domain-containing proteins in T-lymphocyte activation by using dominant negative SH2 domains.

Activation of the T-cell antigen receptor initiates a complex signaling cascade leading to changes in cytokine gene transcription. Several proteins containing Src homology 2 (SH2) domains, capable of interacting with phosphotyrosine-containing sequences within other proteins, are involved in the activation of signaling intermediates such as p2l(ras) and phospholipase Cgamma1. In this study, we used dominant negative SH2 domains to determine the importance of SH2 domain-containing proteins for T-cell activation. We show that tandem SH2 domains of either Zap70 or Syk tyrosine kinase are potent inhibitors of signaling initiated by the T-cell receptor zeta chain in vivo while individual SH2 domains are ineffective. Thus, while only the C-terminal SH2 domains appear to have significant affinity for immunoreceptor tyrosine-based activation motifs in vitro, the N-terminal SH2 domains are necessary in vivo. We find the spacing between the tandem SH2 domains of Zap70 to be critical for in vivo interactions. The SH2 domain of the adapter protein Grb2 is an effective inhibitor in our dominant negative assay, although it has little affinity for immunoreceptor tyrosine-based activation motifs. A single point mutation that abolishes phosphotyrosine binding renders the Grb2 SH2 domain incapable of this inhibition. In contrast, the SH2 domain of Shc does not inhibit this signaling cascade. We conclude that Grb2, but not Shc, is involved in T-cell receptor signaling.

BMP-4 regulates the dorsal-ventral differences in FGF/MAPKK-mediated mesoderm induction in Xenopus.

Recent studies on Xenopus development have revealed an increasingly complex array of inductive, prepatterning, and competence signals that are necessary for proper mesoderm formation. In this study, we establish that fibroblast growth factor (FGF) signals through mitogen-activated protein kinase kinase (MAPKK) to induce mesodermal gene expression. We demonstrate that a partially activated form of MAPKK restores expression of the mesodermal genes Xcad-3 and Xbra, eliminated by the dominant-negative FGF receptor (delta FGFR). Similar to the results reported earlier with delta FGFR, expression of a dominant-negative form of MAPKK (MAPKKD) preferentially eliminates the dorsal expression of Xcad-3 and Xbra. We tested whether the regional localization of bone morphogenetic protein-4 (BMP-4) could explain why both MAPKKD and delta FGFR eliminate the dorsal and not the ventral expression of Xcad-3 and Xbra. We show that ectopic expression of BMP-4 is sufficient to maintain the dorsal expression of Xcad-3 and Xbra in embryos containing delta FGFR and that expression of a dominant-negative BMP receptor reduces the dorsal-ventral differences in delta FGFR embryos. These results indicate that regional localization of BMP-4 is responsible for the dorsal-ventral asymmetry in FGF/MAPKK-mediated mesoderm induction.

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.

Fibroblast growth factor, but not activin, is a potent activator of mitogen-activated protein kinase in Xenopus explants.

Isolated explants from the animal hemisphere of Xenopus embryos were incubated with Xenopus basic fibroblast growth factor (XbFGF) or human activin A. XbFGF incubation resulted in the rapid activation of mitogen-activated protein kinase (MAPK) and ribosomal S6 protein kinase (pp90rsk) in a dose-dependent manner with the highest levels of activation occurring at 50 ng/ml. Maximal activation occurred within 6-10 min after the addition of growth factor, and the activity of both kinases declined to unstimulated levels after 30 min. Activin was unable to activate either MAPK or pp90rsk in the Xenopus explants to a substantial level, although it induced dorsal mesoderm better than XbFGF under the same experimental conditions. The regulatory protein Xwnt-8 did not activate MAPK, nor did it enhance the activation of MAPK by XbFGF. XbFGF was able to activate MAPK through at least the midgastrula stage, suggesting that this family of growth factors may have a role in gastrula-stage events.

Dorsal-ventral differences in Xcad-3 expression in response to FGF-mediated induction in Xenopus.

We have identified a dorsal-ventral difference in the specification of mesoderm in vivo by examining the effect of the dominant-negative FGF receptor on a new member of the Xenopus caudal gene family, Xcad-3. Xcad-3 is expressed throughout the marginal zone during the gastrula stages and serves as a useful marker for events occurring within the mesoderm. Disruption of the FGF signaling pathway by the dominant-negative FGF receptor, disrupts the Xcad-3 expression pattern, eliminating expression preferentially from the dorsal regions of the embryo. We also find that the expression of the Xenopus brachyury homolog, Xbra, is more readily eliminated from the dorsal than the ventral region of the embryo by the dominant-negative FGF receptor, indicating that the observed dorsal-ventral differences are not unique to Xcad-3. These results demonstrate the importance of regional effects on FGF-mediated induction in vivo and suggest that FGF-dependent expression of mesodermal genes depends upon the localization of other factors which establish dorsal-ventral differences within the embryo.

Cloning and characterization of NF-ATc and NF-ATp: the cytoplasmic components of NF-AT.

Present evidence indicates a pathway of signal transmission in T cells that is outlined in figure 1. The elevation in intracellular calcium that is induced by interactions at the antigen receptor leads to the activation of the calcium-dependent phosphatase calcineurin. This in turn leads to the nuclear association of the cytosolic component of NF-ATc. The activation of calcineurin and the nuclear import of NF-ATc can both be blocked by cyclosporin A or FK506 in complex with their respective immunophilins. Once in the nucleus, NF-ATc interacts with NF-ATn to form an active transcriptional complex. NF-ATn is a ubiquitous protein, can be synthesized in response to PMA, and has many similarities to AP-1. The mechanism by which NF-ATc enters the nucleus is unknown, and although it appears to require calcineurin, NF-ATc has not yet been shown to be an in vivo substrate of calcineurin. Alternative mechanisms include the possibility that NF-ATc operates on some cytoplasmic anchor or that other proteins that are controlled by calcineurin carry out the nuclear import of NF-ATc. Although NF-ATp copurifies with NF-ATc, there is as yet no understanding of how NF-ATp is functioning in vivo. Now that these proteins are purified and cloned, the major goals will be to understand their role and the roles of other family members in thymic development.