技术资料

Retinoic acid derivatives (retinoids) exert their pleiotropic effects on cell development through specific nuclear receptors,the retinoic acid receptors and retinoid X receptors. Despite recent progress in understanding the cellular and molecular mechanisms of retinoid activity,it is unknown which of the retinoid receptor pathways are involved in the specific processes of sebocyte growth and development. In this study,we investigated the roles of specific retinoid receptors in sebocyte growth and differentiation,by testing the effects of selective retinoic acid receptor and retinoid X receptor ligands at concentrations between 10-10 M and 10-6 M in a primary rat preputial cell monolayer culture system. Cell growth was determined by number of cells and colonies,and cell differentiation by analysis of lipid-forming colonies. All-trans retinoic acid and selective retinoic acid receptor agonists (CD271 = adapalene,an RAR-beta,gamma agonist; CD2043 = retinoic acid receptor pan-agonist; and CD336 = Am580,an RAR-alpha agonist) caused significant decreases in numbers of cells,colonies,and lipid-forming colonies,but with an exception at high doses of all-trans retinoic acid (10-6 M),with which only a small number of colonies grew but they became twice as differentiated as controls (42.2 +/- 4.0% vs 22.6 +/- 2.7%,mean +/- SEM,lipid-forming colonies,p textless 0.01). Furthermore,the RAR-beta,gamma antagonist CD2665 antagonized the suppressive effects of all-trans retinoic acid,adapalene,and CD2043 on both cell growth and differentiation. In contrast,the retinoid X receptor agonist CD2809 increased cell growth slightly and lipid-forming colonies dramatically in a clear dose-related manner to a maximum of 73.7% +/- 6.7% at 10-6 M (p textless 0. 001). Our data suggest that retinoic acid receptors and retinoid X receptors differ in their roles in sebocyte growth and differentiation: (i) retinoic acid receptors,especially the beta and/or gamma subtypes,mediate both the antiproliferative and antidifferentiative effects of retinoids; (ii) retinoid X receptors mediate prominent differentiative and weak proliferative effects; (iii) the antiproliferative and antidifferentiative effects of all-trans retinoic acid are probably mediated by retinoic acid receptors,whereas its differentiative effect at high dose may be mediated by retinoid X receptors via all-trans retinoic acid metabolism to 9-cis retinoic acid,the natural ligand of retinoid X receptors. View Publication

We report here the cloning,expression,and characterization of human PDE11A1,a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits textless/=50% amino acid identity with the catalytic domains of all other PDEs,being most similar to PDE5,and has distinct biochemical properties. The human PDE11A1 cDNA isolated contains a complete open reading frame encoding a 490-amino acid enzyme with a predicted molecular mass of 55,786 Da. At the N terminus PDE11A1 has a single GAF domain homologous to that found in other signaling molecules,including PDE2,PDE5,PDE6,and PDE10,which constitutes a potential allosteric binding site for cGMP or another small ligand. Tissue distribution studies indicate that PDE11A mRNA occurs at highest levels in skeletal muscle,prostate,kidney,liver,pituitary,and salivary glands and testis. PDE11A is expressed as at least three major transcripts of approximately 10.5,approximately 8.5,and approximately 6.0 kb,thus suggesting the existence of multiple subtypes. This possibility is further supported by the detection of three distinct proteins of approximately 78,approximately 65,and approximately 56 kDa by Western blotting of human tissues for PDE11A isoforms. Recombinant human PDE11A1 hydrolyzes both cGMP and cAMP with K(m) values of 0.52 microM and 1.04 microM,respectively,and similar V(max) values. Therefore,PDE11A represents a dual-substrate PDE that may regulate both cGMP and cAMP under physiological conditions. PDE11A is sensitive to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) as well as zaprinast and dipyridamole,inhibitors that are generally considered relatively specific for the cGMP-selective PDEs,with IC(50) values of 49.8 microM,12.0 microM,and 0.37 microM,respectively. View Publication

Potent inhibition of Janus kinase 3 was found for a series of naphthyl(beta-aminoethyl)ketones (e.g. 7,pIC50 = 7.1+/-0.3). Further studies indicated that these compounds fragment in less than 1 h by retro-Michael reaction in the Jak3 in vitro ELISA assay procedure. The breakdown product of 7,2-naphthylvinyl ketone (22,pIC50 = 6.8+/-0.3) showed very similar inhibitory activity to 7. Compounds 7 (in neutral buffer) and 22 will be useful pharmacological tools for the investigation of the Janus tyrosine kinase Jak3. View Publication

Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system,we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast,repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal,and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses. View Publication

The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin. They include A2439,G2505,and G2553 for E. coli,and G2058,A2503,G2505,and G2553 for Hf. gibbonsii (using the E. coli numbering system). Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III,respectively,of E. coli 23S rRNA. In further experiments,puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P' site of E. coli ribosomes. Moreover,it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m2A2503/psi2504 at an adjacent site in E. coli 23S rRNA. These data strongly support the concept that puromycin,along with other peptidyl-transferase antibiotics,in particular the streptogramin B drugs,bind to an RNA structural motif that contains several conserved and accessible base moieties of the peptidyl transferase loop region. This streptogramin motif is also likely to provide binding sites for the 3' termini of the acceptor and donor tRNAs. In contrast,the effects at A508 and A1579,which are located at the exit site of the peptide channel,are likely to be caused by a structural effect transmitted along the peptide channel. View Publication

BACKGROUND: Compelling evidence indicates that paclitaxel kills cancer cells through the induction of apoptosis. Paclitaxel binds microtubules and causes kinetic suppression (stabilization) of microtubule dynamics. The consequent arrest of the cell cycle at mitotic phase has been considered to be the cause of paclitaxel-induced cytotoxicity. However,the biochemical events,downstream from paclitaxel's binding to microtubules,that lead to apoptosis are not well understood. METHODS: The authors examined recent scientific literature about the mechanisms by which paclitaxel exerts cytotoxicity. RESULTS: In addition to an arrest of the cell cycle at the mitotic phase in paclitaxel-treated cells,recent discoveries of activation of signaling molecules by paclitaxel and paclitaxel-induced transcriptional activation of various genes indicate that paclitaxel initiates apoptosis through multiple mechanisms. The checkpoint of mitotic spindle assembly,aberrant activation of cyclin-dependent kinases,and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) are shown to be involved in paclitaxel-induced apoptosis. Consistent with observations that microtubules of different status (e.g.,cytoskeletal microtubules vs. mitotic spindles) have different sensitivity to paclitaxel,the concentration of paclitaxel appears to be the major determinant of its apoptogenic mechanisms. CONCLUSIONS: Advances in research of the cell cycle and apoptosis have extended our understanding of the mechanisms of paclitaxel-induced cell death. Further elucidation of resistance and enhancement of paclitaxel-induced apoptosis should expedite the development of better paclitaxel-based regimens for cancer therapy. View Publication

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan,a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase,glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta,CDK1/cyclin B,and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs,the details of which were recently revealed by crystallographic analysis. GSK-3 beta,along with CDK5,is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75,thereby mimicking one of the effects of dopamine in the striatum. Finally,we show that many,but not all,reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described. View Publication

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Cytokines are important in the regulation of haematopoiesis and immune responses,and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells,which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells,and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15,and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow,in the proliferation of mature B-cell populations co-stimulated with anti-CD40,and in the proliferation of T cells co-stimulated with anti-CD3. View Publication

Neprilysin (NEP),a thermolysin-like zinc metalloendopeptidase,plays an important role in turning off peptide signalling events at the cell surface. It is involved in the metabolism of a number of regulatory peptides of the mammalian nervous,cardiovascular,inflammatory and immune systems. Examples include enkephalins,tachykinins,natriuretic and chemotactic peptides. NEP is an integral plasma membrane ectopeptidase of the M13 family of zinc peptidases. Other related mammalian NEP-like enzymes include the endothelin-converting enzymes (ECE-1 and ECE-2),KELL and PEX. A number of novel mammalian homologues of NEP have also recently been described. NEP family members are potential therapeutic targets,for example in cardiovascular and inflammatory disorders,and potent and selective inhibitors such as phosphoramidon have contributed to understanding enzyme function. Inhibitor design should be facilitated by the recent three-dimensional structural solution of the NEP-phosphoramidon complex. For several of the family members,however,a well-defined physiological function or substrate is lacking. Knowledge of the complete genomes of Caenorhabditis elegans and Drosophila melanogaster allows the full complement of NEP-like activities to be analysed in a single organism. These model organisms also provide convenient systems for examining cell-specific expression,developmental and functional roles of this peptidase family,and reveal the power of functional genomics. View Publication

The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-contained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule,truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the plasmids were shown to be optimal for gene therapy applications,as there was a rapid reduction in expression levels following removal of Dox. Co-transfection of cells with an autoregulatory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of using this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregulatory plasmids display the requirements for gene therapy applications in chronic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis,where novel protein therapeutics and combination therapies are needed. Gene Therapy (2000) 7,2061-2070. View Publication

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However,the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However,5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood,as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted,mediated by CD8(+) lymphocytes,and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959) View Publication