RESUMO
Heme oxygenase (HO)-1 degrades heme and protects against oxidative stress, but it has not been pharmacologically induced in humans. In this randomized study of 10 healthy volunteers, hemin (3 mg/kg intravenously in 25% albumin) was shown to increase plasma HO-1 protein concentration four- to fivefold and HO-1 activity ~15-fold relative to baseline at 24 and 48 h (placebo -56.41 +/- 6.31 (baseline), 69.79 +/- 13.00 (24 h), 77.44 +/- 10.62 (48 h) vs. hemin -71.70 +/- 9.20 (baseline), 1,126.20 +/- 293.30 (24 h), 1,192.20 +/- 333.30 (48 h)) in four of five subjects as compared with albumin alone (P = 0.03). This represents the overcoming of a fundamental hurdle to HO-1 research in humans.
Assuntos
Heme Oxigenase-1/efeitos dos fármacos , Hemina/farmacologia , Albumina Sérica/química , Adolescente , Adulto , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Feminino , Heme Oxigenase-1/sangue , Heme Oxigenase-1/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2.
Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/genética , Sistema y+ de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos Básicos , Transportador 1 de Aminoácidos Catiônicos/química , Transportador 2 de Aminoácidos Catiônicos/química , Bases de Dados de Ácidos Nucleicos , Éxons , Genes , Humanos , ÍntronsAssuntos
Proteínas de Transporte/metabolismo , Insulina/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Fator de Iniciação 4E em Eucariotos , Humanos , Proteínas Substratos do Receptor de Insulina , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Fatores de Iniciação de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Serina-Treonina Quinases TORRESUMO
The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.
Assuntos
Anticorpos/imunologia , Proteínas de Transporte , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Proteínas Quinases , Sirolimo/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Aminoácidos/farmacologia , Especificidade de Anticorpos , Proteínas de Ciclo Celular , Células Cultivadas , Humanos , Insulina/farmacologia , Dados de Sequência Molecular , Fosfoproteínas/imunologia , Fosforilação , Serina-Treonina Quinases TOR , Proteína 1A de Ligação a Tacrolimo/farmacologiaRESUMO
Results obtained with PHAS-I proteins having Ser to Ala mutations in the five known phosphorylation sites indicate that mTOR preferentially phosphorylates Thr36 and Thr45. The effects of phosphorylating these sites on eIF4E binding were assessed in a far-Western analysis with a labeled eIF4E probe. Phosphorylation of Thr36 only slightly attenuated binding of PHAS-I to eIF4E, while phosphorylation of Thr45 markedly inhibited binding. Phosphorylation of neither site affected the electrophoretic mobility of the protein, indicating that results of studies that rely solely on a gel-shift assay to assess changes in PHAS-I phosphorylation must be interpreted with caution.
Assuntos
Proteínas de Transporte , Fatores de Iniciação de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Análise Mutacional de DNA , Fator de Iniciação 4E em Eucariotos , Fatores de Iniciação de Peptídeos/genética , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Especificidade por Substrato , Serina-Treonina Quinases TOR , Treonina/metabolismoRESUMO
The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.
Assuntos
Proteínas de Transporte , Insulina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal , Androstadienos/farmacologia , Animais , Proteínas de Ciclo Celular , Ativação Enzimática , Fatores de Iniciação em Eucariotos , Antagonistas da Insulina/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Polienos/farmacologia , Proteínas Proto-Oncogênicas c-akt , Sirolimo , Serina-Treonina Quinases TOR , WortmaninaRESUMO
The influence of pulsed high-frequency electromagnetic fields emitted from a circularly polarized antenna on the neuroendocrine system in healthy humans was investigated (900 MHz electromagnetic field, pulsed with 217 Hz, average power density 0.02 mW/cm2). Nocturnal hormone profiles of growth hormone (GH), cortisol, luteinizing hormone (LH) and melatonin were determined under polysomnographic control. An alteration in the hypothalamo-pituitary-adrenal axis activity was found with a slight, transient elevation in the cortisol serum level immediately after onset of field exposure which persisted for 1 h. For GH, LH and melatonin, no significant effects were found under exposure to the field compared to the placebo condition, regarding both total hormone production during the entire night and dynamic characteristics of the secretion pattern. Also the evaluation of the sleep EEG data revealed no significant alterations under field exposure, although there was a trend to an REM suppressive effect. The results indicate that weak high-frequency electromagnetic fields have no effects on nocturnal hormone secretion except for a slight elevation in cortisol production which is transient, pointing to an adaptation of the organism to the stimulus.
Assuntos
Campos Eletromagnéticos , Hormônio do Crescimento Humano/sangue , Hidrocortisona/sangue , Hormônio Luteinizante/sangue , Melatonina/sangue , Adolescente , Glândulas Suprarrenais/fisiologia , Adulto , Eletroencefalografia , Humanos , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , Placebos , SonoRESUMO
The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.
Assuntos
Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polienos/farmacologia , Proteínas Quinases , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Androstadienos/farmacologia , Animais , Proteínas de Transporte/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular , Proteínas de Ligação a DNA/farmacologia , Fator de Iniciação 4E em Eucariotos , Fase G1 , Proteínas de Choque Térmico/farmacologia , Humanos , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Iniciação de Peptídeos/metabolismo , Fosfoproteínas/genética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Sirolimo , Serina-Treonina Quinases TOR , Proteínas de Ligação a Tacrolimo , Transfecção , Células Tumorais Cultivadas , WortmaninaRESUMO
Effects of endogenous nitric oxide (NO) on the release of mediators of the lipoxygenase and cyclo-oxygenase pathway from rat alveolar macrophages were studied. Alveolar macrophages, freshly isolated or after 18-h culture, were incubated in (amino acid-free) Krebs medium and labelled with [3H]arachidonic acid. The release of [3H]leukotriene B4 and [3H]prostanoids (separated by high performance liquid chromatography) was determined. A 23187 was used as stimulus, as rising intracellular Ca2+ activates directly the phospholipase A2 and lipoxygenase pathway. A 23187 (10 microM) enhanced [3H]leukotriene B4 release from freshly prepared alveolar macrophages about 65-fold, but only 5- to 6-fold from cultured alveolar macrophages. Evoked [3H]leukotriene B4 release and spontaneous [3H]prostanoid release were inhibited when L-arginine (300 microM) was added to the Krebs incubation medium of alveolar macrophages, in which marked NO synthase had been induced by culture with lipopolysaccharides (10 microg/ml). Inhibitory effects of L-arginine were prevented by N(G)-monomethyl-L-arginine (L-NMMA, 100 microM). Inhibition of NO synthase during the culture period by L-NMMA (culture medium, in contrast to Krebs medium, already contains the substrate of NO synthase, L-arginine), resulted in attenuation of the 'culture-dependent' decline of the evoked release of [3H]leukotriene B4 and allowed lipopolysaccharides to cause an increase in spontaneous [3H]prostanoid release (i.e., to induce cyclo-oxygenase activity). In conclusion, in rat alveolar macrophages, endogenous NO appears to inhibit the release of mediators of the cyclo-oxygenase and lipoxygenase pathway through multiple sites of action.
Assuntos
Leucotrieno B4/metabolismo , Macrófagos Alveolares/metabolismo , Óxido Nítrico/fisiologia , Animais , Ácidos Araquidônicos/metabolismo , Calcimicina/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Feminino , Lipoxigenase/metabolismo , Macrófagos Alveolares/química , Macrófagos Alveolares/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Sprague-Dawley , ômega-N-Metilarginina/farmacologiaRESUMO
The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [gamma-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.
Assuntos
Anticorpos/metabolismo , Proteínas de Transporte , Fosfoproteínas/metabolismo , Polienos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosfoproteínas/química , Fosforilação , Prolina/metabolismo , Ratos , Serina/metabolismo , Sirolimo , Especificidade por Substrato , Treonina/metabolismoRESUMO
The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.
Assuntos
Androstadienos/farmacologia , Proteínas de Transporte , Cromonas/farmacologia , Morfolinas/farmacologia , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Proteínas Quinases , Proteínas Adaptadoras de Transdução de Sinal , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Química Encefálica , Proteínas de Ciclo Celular , Linhagem Celular , Ditiotreitol/farmacologia , Inibidores Enzimáticos/farmacologia , Fatores de Iniciação em Eucariotos , Imunossupressores/farmacologia , Interleucina-2/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Ativação Linfocitária , Linfoma de Células T , Camundongos , Fosfatidilinositol 3-Quinases , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Polienos/farmacologia , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo , Linfócitos T/imunologia , Linfócitos T Citotóxicos , Serina-Treonina Quinases TOR , Células Tumorais Cultivadas , WortmaninaRESUMO
1. The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid-derived mediators from rat isolate tracheae. 2. Tracheae were preincubated with [3H]-arachidonic acid and the outflow of 3H-compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor-preferring antagonist rho-fluoro-hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine. 3. High performance liquid chromatography analysis (methanol gradient) of the released 3H-compounds showed that one peak, co-eluting with [14C]-prostaglandin E2([14C]-PGE2) and [3H]-PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]-arachidonic acid remained unaffected. 4. Using an acetonitril gradient separation it was shown that [3H]-PGE2, [3H]-PGD2 and [3H]-PGF2alpha are released spontaneously, but [3H]-PGE2 represented the major fraction of 3H-prostaglandins. Acetylcholine enhanced the release of all three 3H-prostaglandins, but the effect on PGE2 was most pronounced and most consistent. 5. After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H-prostaglandins ([3H]-PGE2/PGD2 peak) was abolished. 6. Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa-dependent manner. 7. After inhibition of cyclo-oxygenase by 3 microM indomethacin, the outflow of 3H-prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release [3H]-arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 microM) also blocked the effect of acetylcholine on the outflow of 3H-prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]-arachidonic acid. 8. In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa.
Assuntos
Ácido Araquidônico/farmacologia , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Prostaglandinas/metabolismo , Traqueia/efeitos dos fármacos , Acetonitrilas/química , Acetilcolina/farmacologia , Animais , Ácido Araquidônico/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Técnicas In Vitro , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Traqueia/metabolismoRESUMO
The proliferation of activated T lymphocytes is critically dependent on the binding of the T-cell growth factors, interleukin (IL)-2 and IL-4, to distinct but evolutionarily related cell surface receptors. Previous results suggest that the IL-2 receptor (IL-2R) and IL-4R are coupled to both overlapping and distinct intracellular signaling pathways in T lymphocytes. In this study, we demonstrate that activation of Janus tyrosine kinases (JAKs) and STAT transcription factors is rapidly induced by exposure of factor-dependent murine T-cell lines to IL-2 or IL-4. Both IL-2 and IL-4 stimulated the rapid activation of JAK1 and JAK3, whereas JAK2 activity was unaffected by either cytokine. These responses were accompanied by the appearance in cell nuclei of 3 DNA binding activities that recognized a high-affinity binding site for STAT factors. In transient transfection assays, this STAT factor target sequence conferred IL-2 and IL-4 inducibility on a synthetic luciferase reporter gene. Antibody supershifting experiments indicated that IL-2 induces the formation of STAT dimers containing STAT3 and STAT1 alpha. Although IL-4 also activated STAT1 alpha, the major IL4-induced STAT factor is not STAT3 and remains undefined. Pretreatment of the T-cells with the protein-tyrosine kinase inhibitor herbimycin A blocked both the nuclear translocation of STAT factors and STAT-dependent reporter gene transcription. Immunoblot analyses confirmed that cytoplasmic STAT3 was heavily phosphorylated on tyrosine in IL-2-stimulated cells, and that phosphorylated STAT3 appeared in the nuclei of these cells. These results indicate that identical JAKs and partially overlapping sets of STATs are activated by IL-2 and IL-4 in T lymphocytes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Humanos , Janus Quinase 1 , Janus Quinase 2 , Janus Quinase 3 , Luciferases/biossíntese , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3 , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transativadores/metabolismo , TransfecçãoRESUMO
Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores do Crescimento/farmacologia , Purinas/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Cinetina , Ativação Linfocitária/efeitos dos fármacos , Mitose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The immunosuppressant drug, rapamycin (RAP), is a potent inhibitor of IL-2-dependent T-cell proliferation. The antiproliferative effect of RAP is mediated through the formation of an active complex with its cytosolic receptor protein, FKBP12. The molecular target of the FKBP12.RAP complex is a putative lipid kinase termed the mammalian Target Of Rapamycin (mTOR). This review will discuss recent findings suggesting that mTOR is a novel regulator of G1- to S-phase progression in eukaryotic cells.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Eucarióticas/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Imunossupressores/farmacologia , Fosfatidilinositol 3-Quinases , Polienos/farmacologia , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular , Clonagem Molecular , Ciclinas/metabolismo , Ciclosporina/farmacologia , DNA Complementar/genética , Células Eucarióticas/fisiologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Fase G1/fisiologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/farmacologia , Interleucina-2/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Linfoma de Células T/patologia , Mamíferos/metabolismo , Modelos Imunológicos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Sirolimo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo , Células Tumorais CultivadasAssuntos
Ácido Araquidônico/metabolismo , Macrófagos Alveolares/fisiologia , Traqueia/fisiologia , Animais , Animais Recém-Nascidos , Ácido Araquidônico/farmacologia , Líquido da Lavagem Broncoalveolar , Calcimicina/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Inflamação , Macrófagos Alveolares/efeitos dos fármacos , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Traqueia/efeitos dos fármacosRESUMO
The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Polienos/metabolismo , Proteínas Quinases , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular , Primers do DNA , DNA Complementar , Proteínas Fúngicas/metabolismo , Humanos , Linfoma de Células T/metabolismo , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Sirolimo , Serina-Treonina Quinases TOR , Proteínas de Ligação a Tacrolimo , Células Tumorais CultivadasRESUMO
1. Rat or guinea pig isolated tracheae were labelled with [3H]-choline to measure evoked tritium outflow, which reflects neuronal release of [3H]-acetylcholine. Tritium outflow was evoked either by electrical stimulation of the extrinsic vagal nerve (rat tracheae) or by 27 mM potassium (guinea pig tracheae). 2. In rat tracheae isoprenaline (0.01, 0.1 microM) inhibited evoked [3H]-acetylcholine release, whereas beta 2-adrenoceptor-selective agonists (fenoterol, formoterol, salbutamol) were ineffective. 3. The inhibitory effect of isoprenaline was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 1-selective antagonist CGP 20712 A (0.1 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 4. In rat isolated tracheae prelabelled with [3H]-arachidonic acid, isoprenaline (0.1 microM) but not formoterol (0.01 microM) enhanced the outflow of [3H]-prostaglandins (PGD2, PGE2). This effect was blocked by 0.1 microM CGP 20712 A. 5. In guinea pig tracheae electrical stimulation of the extrinsic vagal nerve did not cause a constant release of [3H]-acetylcholine, but 27 mM potassium elicited a reproducible release of [3H]-acetylcholine. In this species both isoprenaline (0.1 microM) and formoterol (0.01 microM) inhibited evoked [3H]-acetylcholine release. Inhibition was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 2-selective antagonist ICI 118551 (0.3 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 6. In conclusion, the present experiments have demonstrated that activation of beta-adrenoceptors localized in the mucosa mediates inhibition of [3H]-acetylcholine release from the neuroeffector junctions of the pulmonary, parasympathetic nerves most probably by the liberation of inhibitory prostaglandins from the airway mucosa. The adrenoceptor subtype involved differs in rat (beta 1 subtype) and guinea pig (beta 2 subtype) airways.