RESUMO
Multidecadal droughts that occurred during the Medieval Climate Anomaly represent an important target for validating the ability of climate models to adequately characterize drought risk over the near-term future. A prominent hypothesis is that these megadroughts were driven by a centuries-long radiatively forced shift in the mean state of the tropical Pacific Ocean. Here we use a novel combination of spatiotemporal tree-ring reconstructions of Northern Hemisphere hydroclimate to infer the atmosphere-ocean dynamics that coincide with megadroughts over the American West, and find that these features are consistently associated with ten-to-thirty year periods of frequent cold El Niño Southern Oscillation conditions and not a centuries-long shift in the mean of the tropical Pacific Ocean. These results suggest an important role for internal variability in driving past megadroughts. State-of-the art climate models from the Coupled Model Intercomparison Project phase 5, however, do not simulate a consistent association between megadroughts and internal variability of the tropical Pacific Ocean, with implications for our confidence in megadrought risk projections.
RESUMO
Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1alpha activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/metabolismo , Inflamação/genética , Músculo Liso Vascular/imunologia , Receptor fas/metabolismo , Animais , Artérias Carótidas/imunologia , Artérias Carótidas/patologia , Caspases/metabolismo , Quimiocina CCL2/biossíntese , Proteína de Domínio de Morte Associada a Fas , Regulação da Expressão Gênica , Humanos , Interleucina-1/metabolismo , Interleucina-8/biossíntese , Ratos , Ratos Endogâmicos F344 , Regulação para CimaRESUMO
S100 beta produced in Escherichia coli from a synthetic gene (Van Eldik, L. J., J. L. Staecker, and F. Winningham-Major. 1988. J. Biol. Chem. 263:7830-7837) stimulates neurite outgrowth and enhances cell maintenance in cultures of embryonic chick cerebral cortex neurons. In control experiments, the neurite extension activity is reduced by preincubation with antibodies made against bovine brain S100 beta. When either of the two cysteines in S100 beta are altered by site-directed mutagenesis, the resultant proteins maintain the overall biochemical properties of S100 beta, but lose both the neurite extension and neuronal survival activities. However, another S100 beta mutant, in which the relative position of one of the two cysteines was changed, had neurotrophic activity similar to that of the unmodified protein. These and other results indicate that (a) specific neurite extension activity and neuronal survival activity are two related activities inherent to the S100 beta molecule; (b) a disulfide-linked form of S100 beta is required for full biological activity, and (c) the relative position of the cysteines can be modified. These data suggest potential in vivo roles for S100 beta in the development and maintenance of neuronal function in the central nervous system, and demonstrate the feasibility of the longer term development of selective pharmacological agents based on the S100 beta structure.
Assuntos
Axônios/ultraestrutura , Cisteína , Neurônios/citologia , Proteínas S100/farmacologia , Sequência de Aminoácidos , Animais , Axônios/efeitos dos fármacos , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Galinha , Cinética , Dados de Sequência Molecular , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas S100/genéticaRESUMO
Cells deprived of serum mitogens will either undergo immediate cell cycle arrest or complete mitosis and arrest in the next cell cycle. The transition from mitogen dependence to mitogen independence occurs in the mid-to late G1 phase of the cell cycle and is called the restriction point. Murine Balb/c-3T3 fibroblasts deprived of serum mitogens accumulated the cyclin-dependent kinase (CDK) inhibitor p27Kip1. This was correlated with inactivation of essential G1 cyclin-CDK complexes and with cell cycle arrest in G1. The ability of specific mitogens to allow transit through the restriction point paralleled their ability to down-regulate p27, and antisense inhibition of p27 expression prevented cell cycle arrest in response to mitogen depletion. Therefore, p27 is an essential component of the pathway that connects mitogenic signals to the cell cycle at the restriction point.
Assuntos
Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Fase G1 , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Supressoras de Tumor , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Becaplermina , Meios de Cultura , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Regulação para Baixo , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Mitógenos/farmacologia , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sisRESUMO
It has been hypothesized that the neutrophil chemoattractant interleukin-8 (IL-8) forms a gradient in the oral cavity, with the highest concentration of IL-8 produced closest to the bacterial biofilm. In periodontitis, this gradient is disrupted, impairing neutrophil chemotaxis to diseased sites. Treponema denticola is prominently associated with periodontal disease, yet little is known about its ability to modulate the production of inflammatory mediators by epithelial cells. Others have shown that dentilisin, the major outer membrane protease of T. denticola, degrades IL-8 in vitro. We now provide evidence that T. denticola also fails to induce IL-8 production from primary gingival epithelial cells (PGEC). The lack of IL-8 production is not explained by IL-8 degradation, because a protease mutant that does not degrade IL-8 does not induce IL-8 production with these stimuli either. The lack of innate immune mediator production may be a more global phenomenon because T. denticola fails to induce IL-6 or intercellular adhesion molecule 1 production from PGEC. T. denticola also fails to induce transcription of IL-8 and human beta-defensin-2 messenger RNA. The lack of immune mediator production is not explained by the failure of T. denticola to interact with Toll-like receptor 2 (TLR-2), as T. denticola stimulates nuclear factor-kappaB nuclear translocation in TLR-2-transfected HEK293 cells. Not only can T. denticola degrade the IL-8 present in the periodontal lesion, but this organism also fails to induce IL-8 production by PGEC. The lack of an epithelial cell response to T. denticola may contribute to the pathogenesis of periodontitis by failing to trigger chemotaxis of neutrophils into the periodontal pocket.
Assuntos
Células Epiteliais/metabolismo , Gengiva/metabolismo , Interleucina-8/biossíntese , Treponema denticola/imunologia , Infecções por Treponema/imunologia , Células Cultivadas , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Gengiva/imunologia , Gengiva/microbiologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-6/biossíntese , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismoRESUMO
BACKGROUND: The ability of cyclin-dependent kinases (CDKs) to promote cell proliferation is opposed by cyclin-dependent kinase inhibitors (CKIs), proteins that bind tightly to cyclin-CDK complexes and block the phosphorylation of exogenous substrates. Mice with targeted CKI gene deletions have only subtle proliferative abnormalities, however, and cells prepared from these mice seem remarkably normal when grown in vitro. One explanation may be the operation of compensatory pathways that control CDK activity and cell proliferation when normal pathways are inactivated. We have used mice lacking the CKIs p21(Cip1) and p27(Kip1) to investigate this issue, specifically with respect to CDK regulation by mitogens. RESULTS: We show that p27 is the major inhibitor of Cdk2 activity in mitogen-starved wild-type murine embryonic fibroblasts (MEFs). Nevertheless, inactivation of the cyclin E-Cdk2 complex in response to mitogen starvation occurs normally in MEFs that have a homozygous deletion of the p27 gene. Moreover, CDK regulation by mitogens is also not affected by the absence of both p27 and p21. A titratable Cdk2 inhibitor compensates for the absence of both CKIs, and we identify this inhibitor as p130, a protein related to the retinoblastoma gene product Rb. Thus, cyclin E-Cdk2 kinase activity cannot be inhibited by mitogen starvation of MEFs that lack both p27 and p130. In addition, cell types that naturally express low amounts of p130, such as T lymphocytes, are completely dependent on p27 for regulation of the cyclin E-Cdk2 complex by mitogens. CONCLUSIONS: Inhibition of Cdk2 activity in mitogen-starved fibroblasts is usually performed by the CKI p27, and to a minor extent by p21. Remarkably p130, a protein in the Rb family that is not related to either p21 or p27, will directly substitute for the CKIs and restore normal CDK regulation by mitogens in cells lacking both p27 and p21. This compensatory pathway may be important in settings in which CKIs are not expressed at standard levels, as is the case in many human tumors.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor , Animais , Células Cultivadas , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/metabolismo , Embrião de Mamíferos , Inibidores Enzimáticos/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Baço/imunologia , Linfócitos T/citologia , Linfócitos T/fisiologia , TransfecçãoRESUMO
The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.
Assuntos
Genes Fúngicos , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/citologia , Sequência de Aminoácidos , Diferenciação Celular , Clonagem Molecular , DNA Fúngico/genética , Dados de Sequência Molecular , Mutagênese Insercional , Nitrogênio/metabolismo , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Proteína Fosfatase 2 , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de SequênciaRESUMO
A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27(Kip1) and p21(Cip1). The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G1/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G1, demonstrating that cyclin E2 may be rate limiting for G1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Ciclo Celular , Linhagem Celular , Linhagem Celular Transformada , Clonagem Molecular , Quinase 2 Dependente de Ciclina , Ciclinas/genética , DNA Complementar , Fase G1 , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The ras proto-oncogene has been implicated during the formation of tumors in vivo as well as the transformation of cell lines in culture. Conditional expression of an activated ras mutant in Balb/c-3T3 fibroblasts failed to stimulate S phase entry in the absence of plasma-derived progression factors, but did shorten the G1 interval from 12 to 6 h and abrogate the normal proliferative requirement for platelet-derived growth factor. Ras-dependent alteration of the 3T3 cell cycle was accompanied by a dramatic increase in the expression of the G1 regulatory protein, cyclin D1, while expression of cyclin E and cyclin A proteins were only weakly induced. Cyclin/cdk complexes assembled in response to ectopic ras expression in the absence of growth factor stimulation bound the cdk inhibitory factor, Kip1, and were inactive. However, plasma-stimulated regulatory pathways functioned co-operatively with the oncogenic ras molecule to decrease Kip1 levels, induce the kinase activities associated with cyclins D, E and A, and trigger the initiation of DNA replication. Our results suggest that a ras-activated signal transduction pathway may link environmental mitogenic stimuli to the cell cycle machinery via modulation of G1 cyclin expression.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular , Genes ras , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Células 3T3 , Animais , Ciclina D1 , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , DNA/biossíntese , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Oncogênicas/genética , Plasma/fisiologia , RNA Mensageiro/análiseRESUMO
A screen for protein tyrosine phosphatases (PTPs) expressed in the chick inner ear yielded a high proportion of clones encoding an avian ortholog of protein tyrosine phosphatase receptor Q (Ptprq), a receptor-like PTP. Ptprq was first identified as a transcript upregulated in rat kidney in response to glomerular nephritis and has recently been shown to be active against inositol phospholipids. An antibody to the intracellular domain of Ptprq, anti-Ptprq, stains hair bundles in mice and chicks. In the chick ear, the distribution of Ptprq is almost identical to that of the 275 kDa hair-cell antigen (HCA), a component of hair-bundle shaft connectors recognized by a monoclonal antibody (mAb) that stains inner-ear hair bundles and kidney glomeruli. Furthermore, anti-Ptprq immunoblots a 275 kDa polypeptide immunoprecipitated by the anti-HCA mAb from the avian inner ear, indicating that the HCA and Ptprq are likely to be the same molecule. In two transgenic mouse strains with different mutations in Ptprq, anti-Ptprq immunoreactivity cannot be detected in the ear. Shaft connectors are absent from mutant vestibular hair bundles, but the stereocilia forming the hair bundle are not splayed, indicating that shaft connectors are not necessary to hold the stereocilia together; however, the mice show rapid postnatal deterioration in cochlear hair-bundle structure, associated with smaller than normal transducer currents with otherwise normal adaptation properties, a progressive loss of basal-coil cochlear hair cells, and deafness. These results reveal that Ptprq is required for formation of the shaft connectors of the hair bundle, the normal maturation of cochlear hair bundles, and the long-term survival of high-frequency auditory hair cells.
Assuntos
Cóclea/enzimologia , Células Ciliadas Auditivas/enzimologia , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Antígenos de Superfície/metabolismo , Separação Celular , Sobrevivência Celular/genética , Galinhas , Cílios/enzimologia , Cílios/ultraestrutura , Cóclea/citologia , Cóclea/embriologia , Surdez/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/ultraestrutura , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Faloidina , Monoéster Fosfórico Hidrolases/genética , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a ReceptoresRESUMO
Synthetic transcripts of satellite-2 (sat2) DNA from the newt undergo self-catalyzed, site-specific cleavage in vitro using the conserved hammerhead (HH) motif found in a number of infectious plant RNAs. We have analyzed sat2 transcripts from a variety of tissues to obtain evidence for the occurrence of self-cleavage in vivo. We found two distinct types of sat2 transcript populations. Monomeric transcripts in the tests and in somatic tissues such as the liver have ends that map to the in vitro self-cleavage site, and have end groups (5'-hydroxyls and phosphate blocked 3' ends) similar to those produced by self-cleavage. This is the first indication of HH-mediated self-cleavage occurring in animal cells. The monomeric sat2 transcripts found in the newt ovary have a different permutation of the basic DNA repeat sequence. These transcripts begin and end 47 nucleotides upstream from the in vitro self-cleavage site. Despite their apparent lack of utilization of the self-cleavage site, they have end groups which indicate that processing is involved in their formation.
Assuntos
DNA Satélite/genética , Processamento Pós-Transcricional do RNA/genética , RNA Catalítico/genética , Salamandridae/genética , Animais , Northern Blotting , Feminino , Fígado/metabolismo , Masculino , Conformação de Ácido Nucleico , Ovário/metabolismo , RNA Catalítico/fisiologia , Sequências Repetitivas de Ácido Nucleico/genética , Testículo/metabolismoRESUMO
Covalent attachment of lipids appears to be an important mechanism by which many proteins interact with membranes. As we learn more about how lipids and adjacent amino acids participate in addressing proteins to specific membranes within the cell, it should be possible to design more elegant and precise membrane targeting systems that can be used to guide proteins to functionally relevant destinations.
Assuntos
Ácidos Mirísticos/metabolismo , Ácidos Palmíticos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Proteínas de Ligação ao GTP/metabolismo , Vetores Genéticos , Mamíferos , Dados de Sequência Molecular , Mutagênese Insercional , Ácido Mirístico , Oligodesoxirribonucleotídeos , Ácido Palmítico , Reação em Cadeia da Polimerase/métodos , Prenilação de Proteína , Sinais Direcionadores de Proteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Mapeamento por Restrição , Proteínas ras/biossínteseRESUMO
Aspects of people's identification with groups may be understood by borrowing theoretical ideas and measurement strategies from research on attachment in close relationships. People have mental models of the self as a group member and of groups as sources of identity and esteem. These models affect thoughts, emotions, and behaviors related to group membership. Three studies show that two dimensions of attachment to groups, attachment anxiety and avoidance, can be assessed with good reliability, validity, and over-time stability. These factors are distinct from relationship attachment and from other measures of group identification. Group attachment predicts several important outcomes, including emotions concerning the group, time and activities shared with a group, social support, collective self-esteem, and ways of resolving conflict. This conceptualization provides new insights into the nature of people's psychological ties to groups.
Assuntos
Apego ao Objeto , Grupo Associado , Teoria Psicológica , Adulto , Conflito Psicológico , Humanos , Relações Interpessoais , Valor Preditivo dos Testes , Testes Psicológicos , Reprodutibilidade dos Testes , Autoimagem , Identificação Social , Inquéritos e QuestionáriosAssuntos
Negro ou Afro-Americano/psicologia , Moral , Pobreza , Idoso , Família , Feminino , Humanos , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Qualidade de VidaRESUMO
TGF-beta1 exerts diverse functions in tooth development and tissue repair, but its role in microbial defenses of the tooth is not well-understood. Odontoblasts extending their cellular processes into the dentin are the first cells to recognize signals from TGF-beta1 and bacteria in carious dentin. This study aimed to determine the role of TGF-beta1 in modulating odontoblast responses to oral bacteria. We show that these responses depend upon the expression levels of microbial recognition receptors TLR2 and TLR4 on the cell surface. Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum activated both TLRs, but TLR4 played a greater role. Lack of cell-surface TLR2 was associated with poor response to Streptococcus mutans, Enterococcus faecalis, and Lactobacillus casei. TGF-beta1 inhibited TLR2 and TLR4 expression and attenuated odontoblast responses. Our findings suggest that the balance between TLR-mediated inflammation and TGF-beta1 anti-inflammatory activity plays an important role in pulpal inflammation.
Assuntos
Cárie Dentária/imunologia , Odontoblastos/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células Clonais , Cárie Dentária/etiologia , Cárie Dentária/microbiologia , Infecções por Bactérias Gram-Negativas/complicações , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Positivas/complicações , Infecções por Bactérias Gram-Positivas/imunologia , Humanos , Odontoblastos/citologia , Odontoblastos/metabolismo , Pulpite/etiologia , Pulpite/imunologia , Pulpite/microbiologia , Interferência de RNA , RNA Interferente Pequeno/fisiologia , Análise de Regressão , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologiaRESUMO
The biologic activity of substance P has been demonstrated to be limited both in in vivo and in vitro by a membrane-bound protease, neutral endopeptidase (EC 3.4.24.11). The interaction of substance P with its receptor on guinea pig lung tissues was studied in the presence of an inhibitor of neutral endopeptidase under conditions that protect the peptide from degradation. Uptake of 0.1 nM [125I]-BH-substance P in lung membrane preparations was rapid at 4 degrees C, reaching equilibrium in 30 to 40 min, and binding was stable for at least 30 min thereafter. Binding was reversible and saturable. Scatchard analyses of saturation binding data are consistent with a single class of receptor molecules in both lung parenchymal and airway membranes, with a Kd of 2 to 3 nM and a receptor density of 4,000 to 5,000 fmol/g wet wt of tissue. In competitive binding experiments, neurokinin A and substance P methyl ester were equipotent and required approximately 100-fold higher concentrations to effect equivalent displacement than unlabeled substance P. Eledoisin also competed for [125I]-BH-substance P binding, but was less effective than the other analogs. The spasmogenically inactive derivative, substance P 1-9, did not compete for substance P binding at concentrations as high as 1 microM. Binding of [125I]-BH-substance P was rapidly and completely reversed by addition of 0.1 mM GTP, suggesting that association with a GTP binding protein is required for high affinity binding of substance P to its receptor in lung. The substance P receptor molecule was further characterized by covalently crosslinking [125I]-BH-substance P to membrane preparations followed by SDS-PAGE of the solubilized material.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Pulmão/metabolismo , Receptores de Neurotransmissores/metabolismo , Substância P/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas , Cobaias , Cinética , Masculino , Ensaio Radioligante , Receptores da Neurocinina-1 , Succinimidas/metabolismoRESUMO
Cellular responses to the AA isoform of platelet-derived growth factor (PDGF-AA) are mediated via the PDGF alpha receptor. Several studies suggest this receptor may signal pathways distinct from those activated by the PDGF beta receptor. Because alpha receptors are less well characterized than are beta receptors, and because the quantity of cell surface PDGF receptors governs the extent and perhaps type of PDGF-stimulated response, we examined the synthesis and degradation of alpha receptors in BALB/c-3T3 cells. Our data show that the ligand-independent half-life of alpha receptors is 3 h and that optimal turnover of alpha receptors requires protein synthesis. In the presence of ligand, the half-life of alpha receptors markedly decreases and is independent of protein synthesis. Although PDGF-AA accelerated the rate of alpha receptor turnover, pretreatment of cells with PDGF-AA and essentially complete down-regulation of alpha receptors did not correspondingly increase the level of alpha receptor synthesis. These findings indicate that the number of cell surface PDGF alpha receptors is regulated by the rate of internalization of these receptors. Lastly, we report that the recovery of PDGF-AA binding following down-regulation of alpha receptors is not affected by inhibition of RNA synthesis. Thus, repopulation of cell surface PDGF alpha receptors may not necessitate an increase in the level of PDGF alpha receptor mRNA.
Assuntos
Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Células 3T3 , Animais , Regulação para Baixo/fisiologia , Meia-Vida , Ligantes , Camundongos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/biossínteseRESUMO
The transcriptional promoter of satellite 2 from the eastern newt, Notophthalmus viridescens, was analyzed by assaying the activity of deleted or mutated satellite 2 clones in Xenopus laevis oocytes. Two elements in the promoter were found to be important for transcription. These elements have sequences that are similar to the sequences of the octamer and the proximal sequence element of vertebrate snRNA genes transcribed by RNA polymerase II. Furthermore, the organization of these elements and their respective roles in transcription are the same as their organization and roles in the snRNA genes. To further investigate the relationship between the satellite 2 and snRNA gene promoters, the ability of the satellite 2 promoter to drive transcription of a true snRNA gene was tested. The satellite 2 promoter initiated transcription of the Xenopus U1b2 snRNA gene as efficiently as the native U1b2 promoter, and the 3' ends of the resulting U1b2 transcripts were accurately formed. This latter result confirms that the satellite 2 promoter is a functional analog of the snRNA promoter, since 3'-end formation of snRNA genes transcribed by RNA polymerase II requires that transcription be initiated from a compatible promoter. The structural and functional similarities between the satellite 2 and the snRNA gene promoters suggest that these elements are evolutionarily related. These findings were used to extend a previously proposed model concerning the nature and derivation of satellite 2.
Assuntos
DNA Satélite/genética , Notophthalmus viridescens/genética , Regiões Promotoras Genéticas/genética , RNA Nuclear Pequeno/genética , Transcrição Gênica/genética , Animais , Sequência de Bases , Evolução Biológica , Dados de Sequência Molecular , Mutação/fisiologia , Oócitos , RNA Mensageiro/biossíntese , Xenopus laevisRESUMO
Platelet-derived growth factor (PDGF) stimulates the expression of a number of genes associated with entry of quiescent Balb/c-3T3 fibroblasts into the cell cycle. We determined that two of these genes, c-myc and c-fos, are induced equivalently in medium supplemented with platelet-poor plasma (PPP) and either PDGF-BB or PDGF-AA. The rate at which fibroblasts entered S phase was also similar in PDGF-BB- and AA-treated cells as was the expression of the late G1 gene, thymidine kinase (TK). However, PDGF-AA must be present for a period of 16 h to stimulate the proliferation of 90% of the cells, whereas PDGF-BB was required for only 4 h. Exposure of cells to PDGF-AA for 4 h, a time during which maximum expression of c-fos and c-myc occurred, only induced 20% of the cells in a quiescent population to enter the cell cycle. Therefore, PDGF-AA-mediated expression of the immediate early genes c-fos and c-myc may be necessary but is not sufficient to rapidly stimulate density-arrested Balb/c-3T3 fibroblasts into the competent state. Thus, these data suggest that PDGF-AA and PDGF-BB initiate traverse of the cell cycle by distinct mechanisms.
Assuntos
Divisão Celular/fisiologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Células 3T3 , Animais , Northern Blotting , Divisão Celular/genética , Expressão Gênica , Genes fos , Genes myc , Cinética , Camundongos , Testes de Precipitina , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Timidina Quinase/metabolismoRESUMO
Platelet-derived growth factor (PDGF) stimulates the proliferation of Balb/c-3T3 fibroblasts through binding and subsequent activation of PDGF receptors. Activation of the PDGF receptors has been proposed to involve receptor dimerization. PDGF-AB has been shown to bind PDGF alpha and beta receptor subunits to form PDGF alpha beta and alpha alpha receptor dimers. In this paper we demonstrate that, following the down-regulation of PDGF alpha receptors, the binding of PDGF-AB to beta receptors occurred at 37 degrees C but not at 4 degrees C. PDGF-AB stimulated the phosphorylation of PDGF beta receptor monomers in cells depleted of PDGF alpha receptors by prior exposure to PDGF-AA.