RESUMO
UNLABELLED: Newly synthesized erythromycin derivatives were screened for synergy with oxacillin and other ß-lactam antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). MRSA ATCC43300 and some clinically isolated MRSA were used. Several erythromycin derivatives were found to possess high synergism with oxacillin against MRSA. The newly synthesized erythromycin derivatives were also tested for their inhibitory effects against MRSA, either separately or in combination with oxacillin, using serial broth dilution, disc diffusion, Etest strips, growth curves and time-kill curves. A representative derivative, SIPI-8294, could potentiate almost all ß-lactam antibiotics tested against the model strain MRSA ATCC43300 from 4 to 128 times and had synergism with oxacillin against 12 of 16 clinical isolates of MRSA under one-fourth of the minimum inhibitory concentration (MIC) of the compounds. This is the first report on the synergistic activity of these new erythromycin derivatives. These findings provide a new choice for the treatment of infection caused by MRSA and lead us to further study the synergistic mechanism. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the synergy of anti-MRSA between new erythromycin derivatives and ß-lactam antibiotics in vitro. The results show that although the erythromycin derivatives have poor anti-MRSA effects alone, they possess high synergism with oxacillin against MRSA ATCC43300 and clinically isolated MRSA. These novel compounds can significantly reduce the dosage of ß-lactam antibiotics against MRSA, while this synergistic effect is different from the combination of ß-lactams and ß-lactamase inhibitors. The research may provide a new choice for the treatment of infection caused by MRSA and be useful to the research and development of new combination of medicines.
Assuntos
Antibacterianos/farmacologia , Eritromicina/análogos & derivados , Eritromicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Farmacorresistência Bacteriana Múltipla , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade MicrobianaRESUMO
The membrane redistribution and phosphorylation of focal adhesion kinase (FAK) have been reported to be important for cell migration. We previously showed that Lysophosphatidic acid (LPA) induced FAK membrane redistribution and autophosphorylation in ovarian cancer SK-OV3 cells and the signaling pathway consisting of Gi-Ras-MEKK1 mediated LPA-induced FAK membrane redistribution but not FAK autophosphorylation. We also showed that the disruption of the Gi-Ras-MEKK1 pathway led to a significant reduction in LPA-stimulated cell migration. These findings raised the question of whether LPA-induced FAK autophosphorylation was required for LPA-stimulated cell migration and what signaling mechanism was involved in LPA-induced FAK autophosphorylation. In this study, we expressed the membrane anchored wild-type FAK (CD2-FAK) in SK-OV3 cells and found that the expression of CD2-FAK greatly rescued LPA-stimulated cell migration in Gi or Ras-inhibited cells. However, Gi inhibitor pertussis toxin or dominant-negative H-Ras still significantly inhibited LPA-stimulated cell migration in cells expressing the membrane anchored FAK containing a mutation in the autophosphorylation site [CD2-FAK(Y397A)]. These results suggest that FAK autophosphorylation plays a role in LPA-stimulated cell migration. With the aid of p115RhoGEF-RGS, G12 and G13 minigenes to inhibit G12/13, we found that the G12/13 pathway was required for LPA-induced FAK autophosphorylation and efficient cell migration. Moreover, LPA activated RhoA and Rho kinase (ROCK) in a G12/13-dependent manner and their activities were required for LPA-induced FAK autophosphorylation. However, Rho or ROCK inhibitors displayed no effect on LPA-induced FAK membrane redistribution although they abolished LPA-induced cytoskeleton reorganization. Our studies show that the G12/13-RhoA-ROCK signaling pathway mediates LPA-induced FAK autophosphorylation and contributes to LPA-stimulated cell migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Lisofosfolipídeos/farmacologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/genética , Adesões Focais/enzimologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Genes ras/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas , Proteínas ras/fisiologia , Quinases Associadas a rhoRESUMO
Bradykinin (BK), a pluripotent nonameric peptide, is known for its proinflammatory functions in both tissue injury and allergic inflammation of the airway mucosa and submucosa. To understand the mechanisms by which BK serves as an inflammatory mediator, the human lung fibroblast cell line WI-38 was stimulated with BK and the expression of IL-1beta gene was examined. BK at nanomolar concentrations induced a marked increase in immunoreactive IL-1beta, detectable within 2 h in both secreted and cell-associated forms. BK-induced IL-1beta synthesis was inhibited by a B2-type BK receptor antagonist and by treatment of the cells with pertussis toxin, indicating the involvement of a BK receptor that couples to the G(i)/G(o) class of heterotrimeric G proteins. Whereas cycloheximide and actinomycin D both inhibited BK-induced IL-1beta synthesis, results from Northern blot and nuclear run-on assays suggested that BK acted primarily at the transcription level which led to the accumulation of IL-1beta message in stimulated cells. Gel mobility shift assays were used with nuclear extracts from stimulated WI-38 cells to examine the transcription mechanism for BK-induced IL-1beta expression. A DNA binding activity specific for the decameric kappaB enhancer was detected and was found to contain the p50 and p65 subunits of the NF-kappaB/rel protein family. BK-induced NF-kappaB activation correlated with IL-1beta message upregulation with respect to agonist concentration, time course, sensitivity to bacterial toxins, and blockade by the B2 receptor antagonist. After BK stimulation, a significant increase in the activity of chloramphenicol acetyltransferase was observed in WI-38 cells transfected with a reporter plasmid bearing the kappaB enhancers from the IL-1beta gene. Deletion of the kappaB enhancer sequence significantly reduced BK-induced chloramphenicol acetyltransferase activity. These findings suggests a novel function of BK in the activation of NF-kappaB and the induction of cytokine gene expression.
Assuntos
Bradicinina/fisiologia , Interleucina-1/fisiologia , NF-kappa B/fisiologia , Receptores da Bradicinina/fisiologia , Células Cultivadas , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Toxina Pertussis , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-kappaB-dependent pathway. We now provide evidence that protein kinase C-delta (PKC-delta) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-delta and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-delta inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-delta. Inhibition of PKC-delta or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-kappaB activity and resultant ICAM-1 expression. The effects of PKC-delta inhibition were secondary to inhibition of IKKbeta activation and of subsequent NF-kappaB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IkappaBalpha degradation without affecting the DNA binding function of nuclear NF-kappaB. Thus, PKC-delta signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKbeta, which mediates NF-kappaB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-kappaB p65 (RelA).
Assuntos
Endotélio Vascular/fisiologia , Molécula 1 de Adesão Intercelular/genética , Isoenzimas/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteína Quinase C/fisiologia , Trombina/fisiologia , Células Cultivadas , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , NF-kappa B/fisiologia , Proteína Quinase C-delta , Transdução de Sinais/efeitos dos fármacos , Trombina/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
A cDNA designated as AZ3B has been isolated from a differentiated HL-6 0 cell cDNA library with a probe derived from the N-formyl peptide receptor gene. The 1.97-kb cDNA encodes a novel G protein-coupled receptor (GPCR) with 482 amino acids. In addition to the predicted 7 transmembrane domains common to all GPCRs, the protein encoded by AZ3B contains a large extracellular loop of approximately 172 amino acids between the fourth and the fifth transmembrane domains, a feature unique among the hundreds of GPCRs identified to date. High sequence homology exists between the AZ3B protein and a number of chemoattractant receptors in the amino-terminal 170 residues and the carboxyl-terminal 150 residues. Northern and flow cytometric analyses suggested that the AZ3B message and protein are widely expressed in several differentiated hematopoietic cell lines, in the lung, placenta, heart, and endothelial cells. We postulate that the AZ3B protein defines a distinct group of receptors within the GPCR superfamily.
Assuntos
DNA Complementar/genética , Proteínas de Ligação ao GTP/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/química , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Estrutura Secundária de Proteína , Receptores de Formil Peptídeo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
N-formyl peptides, such as fMet-Leu-Phe, are one of the most potent chemoattractants for phagocytic leukocytes. The interaction of N-formyl peptides with their specific cell surface receptors has been studied extensively and used as a model system for the characterization of G-protein-coupled signal transduction in phagocytes. The cloning of the N-formyl peptide receptor cDNA from several species and the identification of homologous genes have allowed detailed studies of structural and functional aspects of the receptor. Recent findings that the receptor is expressed in nonhematopoietic cells and that nonformylated peptides can activate the receptor suggest potentially novel functions and the existence of additional ligands for this receptor.
Assuntos
Receptores Imunológicos , Receptores de Peptídeos , Sequência de Aminoácidos , Animais , Fatores Quimiotáticos/metabolismo , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/fisiologia , Receptores de Formil Peptídeo , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/fisiologia , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Accumulating evidence indicates that G-protein-coupled receptors (GPCRs) play an active role in transcriptional regulation. In leukocytes, activation of receptors for several chemokines and classic chemoattractants has been associated with enhanced expression of proinflammatory cytokines and chemokines. GPCRs in endothelial and epithelial cells also regulate transcription and contribute to the expression of cytokines, adhesion molecules, and growth factors that are essential for extravasation of leukocytes and tissue repair. Nuclear factor (NF) kappaB is one of the most important transcription factors responsible for the expression of these proinflammatory genes. Recent studies have shown that GPCRs utilize several different pathways to activate NF-kappaB. These pathways differ from the ones induced by classic cytokines in that they are initiated by heterotrimeric G-proteins, but they converge to IkappaB phosphorylation and nuclear translocation/modification of the NF-kappaB proteins. GPCR-induced NF-kappaB activation provides an effective means for local expression of cytokine and growth factor genes due to the wide distribution of these receptors. Chemokine-induced, GPCR-mediated production of chemokines constitutes an autocrine regulatory mechanism for the growth of certain malignant tumors and enhances the recruitment of leukocytes to sites of inflammation.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Leucócitos/fisiologia , NF-kappa B/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Animais , Comunicação Autócrina , Humanos , Ativação TranscricionalRESUMO
Fractalkine is an endothelial cell-derived CX3C chemokine that is chemotactic mainly to mononuclear cells. Fractalkine was induced in rat aortic endothelial cells (RAEC) by interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) transcriptionally and translationally. This induction correlated with increased NF-kappaB DNA binding activity as determined by gel mobility shift assay. Supershift assays revealed that the NF-kappaB subunits p50 and p65 were responsible for kappaB binding. Accordingly, we examined the role of NF-kappaB in fractalkine induction in RAEC through the use of an adenovirus-mediated mutant IkappaB as a specific inhibitor. Delivery of a dominant-negative form of IkappaBalpha in RAEC dramatically reduced the induction of fractalkine by these stimuli, suggesting a role for NF-kappaB activation in fractalkine induction. The inhibition of fractalkine expression by two potent NF-kappaB inhibitors, sulfasalazine and sanguinarine, further supported the central role of NF-kappaB in fractalkine transcription regulation and suggested a novel therapeutic target aimed at modulating leukocyte endothelial cell interaction.
Assuntos
Quimiocinas CX3C , Quimiocinas CXC/biossíntese , Endotélio Vascular/metabolismo , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/biossíntese , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Aorta , Células Cultivadas , Quimiocina CX3CL1 , Ratos , Regulação para Cima/efeitos dos fármacosRESUMO
A recombinant human neutrophil N-formyl peptide receptor (rFPR) expressed in transfected mouse fibroblasts (TX2 cells) was analyzed for its ability to couple physically with the heterotrimeric G protein, Gi. Immunoprecipitation of photoaffinity-labeled rFPR and endogenous neutrophil formyl peptide receptor (nFPR) with an anti-FPR peptide antibody demonstrated that the receptors were identical in both size and extent of glycosylation. Coupling of rFPR with endogenous TX2 Gi was demonstrated by coimmunoprecipitation of the two proteins with an anti-Gi antibody. Moreover, rFPR was able to form a physical complex with purified Gi in a soluble reconstitution system. We observed similar affinities of rFPR and nFPR for Gi. This report provides the first direct evidence that rFPR associates physically with Gi and provides a foundation for analysis of the G protein coupling capacity of mutant rFPRs.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Centrifugação com Gradiente de Concentração , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Imunoensaio , Cinética , Camundongos , Peso Molecular , Receptores de Formil Peptídeo , Receptores Imunológicos/genética , Receptores Imunológicos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.
Assuntos
Proteínas de Membrana , Receptores de Complemento/química , Receptores de Complemento/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Complemento C3a/química , Complemento C3a/metabolismo , Citometria de Fluxo , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We investigated the function of proteinase-activated receptor-1 (PAR-1) in the regulation of pulmonary microvascular permeability in response to thrombin challenge using PAR-1 knockout mice (-/-). Lungs were isolated and perfused with albumin (5 g/100 ml)-Krebs solution at constant flow (2 ml/min). Lung wet weight and pulmonary artery pressure (P(pa)) were continuously monitored. We determined the capillary filtration coefficient (K(fc)) and (125)I-labeled albumin (BSA) permeability-surface area product (PS) to assess changes in pulmonary microvessel permeability to liquid and albumin, respectively. Normal and PAR-1-null lung preparations received in the perfusate: 1) thrombin or 2) selective PAR-1 agonist peptide (TFLLRNPNDK-NH(2)). In control PAR-1 (+/+) mouse lungs, (125)I-albumin PS and K(fc) were significantly increased over baseline (by approximately 7- and 1.5-fold, respectively) within 20 min of alpha-thrombin (100 nM) challenge. PAR-1 agonist peptide (5 microM) gave similar results, whereas control peptide (5 microM; FTLLRNPNDK-NH(2)) was ineffective. At relatively high concentrations, thrombin (500 nM) or PAR-1 agonist peptide (10 microM) also induced increases in P(pa) and lung wet weight. All effects of thrombin (100 or 500 nM) or PAR-1 agonist peptide (5 or 10 microM) were prevented in PAR-1-null lung preparations. Baseline measures of microvessel permeability and P(pa) in the PAR-1-null preparations were indistinguishable from those in normal lungs. Moreover, PAR-1-null preparations gave normal vasoconstrictor response to thromboxane analog, U-46619 (100 nM). The results indicate that the PAR-1 receptor is critical in mediating the permeability-increasing and vasoconstrictor effects of thrombin in pulmonary microvessels.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Circulação Pulmonar/efeitos dos fármacos , Receptores de Trombina/genética , Trombina/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Relação Dose-Resposta a Droga , Genótipo , Técnicas In Vitro , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , Cadeias Leves de Miosina/metabolismo , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , RNA/genética , RNA/metabolismo , Receptor PAR-1 , Receptores de Trombina/fisiologia , Vasoconstritores/farmacologiaRESUMO
This study delineates the isolation and characterization of a novel chemokine receptor gene, the putative chicken CXC receptor 1 (cCXCR1). Using a human CXCR1 probe, we isolated several positive clones from a chicken genomic library. One of the clones contained a fragment of approximately 5000bp that hybridized strongly with the hCXCR1 probe. This fragment was sequenced and subjected to a variety of computer analyses. The open reading frame for this gene predicts a seven transmembrane domain protein with all the characteristics of a chemokine receptor and with 67% sequence homology to hCXCR1, 65% to hCXCR2 and also with considerable sequence homology to other human chemokine receptors such as hCXCR4 (50%), hCCR2 (49%) and hCCR1 (49%). However, the homology to a previously isolated potential G-protein-coupled receptor for chickens (AvCRL1) is only 47%. Using 5' RACE, two transcription initiation sites were identified suggesting the potential for the expression of two protein isoforms (I and II) in vivo. The promoter for the putative cCXCR1 contains a variety of consensus transcription factor binding elements that can potentially be involved in the expression of this chicken receptor upon stimulation by stress-inducing agents. RT-PCR analysis was used to determine the pattern of expression of the larger isoform (I) of this receptor in a variety of tissues. This form of the receptor is expressed primarily in the organs of the gastrointestinal tract, tissues that are frequently exposed to stress-inducing agents, but not in the central nervous system, tissues that are protected from insult by the blood barrier. Using the same RT-PCR approach we show that stress-inducing agents, such as 'first-hand' and 'second-hand' cigarette smoke components, tumor promoters and thrombin, differentially stimulate the expression of the isoform I in primary fibroblasts. Thrombin is an enzyme that plays many important roles in thrombosis, angiogenesis and wound healing and exposure to both cigarette smokes and/or to tumor promoters can lead to tumorigenesis. Therefore, upregulation of chemokines and their receptors by stress-inducing agents can confer highly regulated modulation of cellular responses to traumatic and pathological situations.
Assuntos
Genes/genética , Receptores de Quimiocinas/genética , Receptores de Interleucina-8A/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Galinhas , Clonagem Molecular , DNA/química , DNA/genética , DNA/isolamento & purificação , Regulação da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Ésteres de Forbol/farmacologia , Isoformas de Proteínas/genética , RNA/genética , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Trombina/farmacologia , Distribuição Tecidual , Transcrição GênicaRESUMO
The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.
Assuntos
Hemodinâmica/fisiologia , Receptores de Trombina/deficiência , Animais , Regulação da Expressão Gênica , Camundongos , Camundongos Mutantes , Fenótipo , Receptores de Trombina/genéticaRESUMO
Receptors for leukocyte chemoattractants, including chemokines, are traditionally considered to be responsible for the activation of special leukocyte functions such as chemotaxis, degranulation, and the release of superoxide anions. Recently, these G-protein-coupled serpentine receptors have been found to transduce signals leading to gene transcription and translation in leukocytes. Transcription factors, such as NF kappa B and AP-1, are activated upon stimulation of the cells with several chemoattractants at physiologically relevant concentrations. Activation of transcription factors through these receptors involves G-protein coupling and the activation of protein kinases. The underlying signaling pathways appear to be different from those utilized by TNF-alpha, a better characterized cytokine that induces the transcription of immediate-early genes. Chemoattractants stimulate the expression of several inflammatory cytokines and chemokines, which in turn may activate their respective receptors and initiate an autocrine regulatory mechanism for persistent cytokine and chemokine gene expression.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Citocinas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Células CHO , Cricetinae , DNA/genética , DNA/metabolismo , Expressão Gênica , Humanos , Técnicas In Vitro , Interleucina-8/biossíntese , Dados de Sequência Molecular , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
PAF stimulation of NF-kappa B activation and transcription of immediate-early genes have been investigated in peripheral blood mononuclear cells and in transfected Chinese hamster ovary cells expressing the cloned PAF receptor. These studies identified a G protein-coupled pathway for PAF induction of gene expression and transcription factor activation, which differs from the mechanisms employed by other immediate-early gene inducers. Potential significance of PAF induced NF-kappa B activation is discussed.
Assuntos
Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Animais , Células CHO , Cricetinae , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , NF-kappa B/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , TransfecçãoAssuntos
Quimiotaxia de Leucócito/fisiologia , Leucócitos/fisiologia , Receptores de Superfície Celular/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Quimiocinas/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Humanos , Inflamação/fisiopatologia , Inflamação/terapia , Dados de Sequência Molecular , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/imunologia , Homologia de Sequência de AminoácidosRESUMO
The chemotactic peptide f-Met-Leu-Phe (fMLP) stimulates leukocyte functions through binding and activation of a specific G-protein-coupled formyl peptide receptor (FPR). Recent studies have shown that stimulation of neutrophils with fMLP induces the activation of two members of the mitogen-activated protein kinase (MAP kinase) family, ERK1 and ERK2, through mechanisms that are not completely understood but may involve the phosphorylation of the adapter protein SHC by the Src-related kinase Lyn. In this study, transfected fibroblasts expressing the rabbit FPR were used to investigate further the role of Lyn and SHC phosphorylation in fMLP-stimulated MAP kinase activation. Stimulation of transfected cells with fMLP resulted in the time- and dose-dependent increase in tyrosine phosphorylation and activation of ERK1 and ERK2 and the activation of MEK, the MAP kinase/ERK kinase. The activation of both ERKs and MEK was inhibited by preincubation of the cells with pertussis toxin, indicating that activation was dependent upon a Gi/Go-like protein that couples to the receptor. Our data also show that, unlike neutrophils, FPR-transfected fibroblasts do not express the Src-related kinase Lyn. In the absence of Lyn, fMLP stimulation did not result in an increased tyrosine phosphorylation of the adapter protein SHC, whereas it was still able to induce MAP kinase activation. These data suggest that Lyn and SHC are not the only upstream signals for activation of the MAP kinase/ERK pathway by fMLP and demonstrate the potential application of the FPR-transfected cells for the delineation of additional signaling mechanisms stimulated by fMLP.
Assuntos
N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Adulto , Animais , Linhagem Celular , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Toxina Pertussis , Fosforilação , Coelhos , Receptores de Formil Peptídeo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins.
Assuntos
Glicoproteínas/biossíntese , Sinais Direcionadores de Proteínas/metabolismo , Animais , Linhagem Celular , Glicoproteínas/genética , Cinética , Manose/metabolismo , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Coelhos , Reticulócitos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Trítio , Tunicamicina/farmacologiaRESUMO
Two nearly full-length cDNAs for placental plasminogen activator inhibitor (PAI) have been isolated from a human placenta lambda gt11 cDNA library. One positive (lambda PAI-75.1) expressed a protein that could adsorb and purify anti-PAI antibodies. The expressed protein inhibited the activity of human urokinase in a fibrin autography assay, and formed a 79-kDa (reduced) covalent complex with 125I-urokinase that could be immunoprecipitated with anti-PAI. The cDNA insert of the longer isolate (lambda PAI-75.15) consisted of 1909 base pairs, including a 5'-noncoding region of 55 base pairs, an open reading frame of 1245 base pairs, a stop codon, a 3'-noncoding region of 581 base pairs, and a poly(A) tail. The size of the mRNA was estimated to be 2.0 kilobases by Northern blot analysis. The translated amino acid sequence consisted of 415 amino acids, corresponding to a 46.6-kDa protein. The sequence was related to members of the serpin gene family, particularly ovalbumin and the chicken gene Y protein. Like these avian proteins, placental PAI appears to lack a cleavable NH2-terminal signal peptide. Residues 347-376 were identical to the partial sequence reported recently for a PAI isolated from the human monocytic U-937 cell line. Placental PAI mRNA was apparently expressed at low levels in human umbilical vein endothelial cells, but was not detectable in HepG2 hepatoma cells. It was present in U-937 cells and was inducible at least 10-fold by phorbol 12-myristate 13-acetate. Thus placental PAI is a unique member of the serpin gene family, distinct from endothelial-type PAI. It is probably identical to monocyte-macrophage PAI.
Assuntos
Clonagem Molecular , DNA/metabolismo , Escherichia coli/genética , Genes , Glicoproteínas/genética , Placenta/metabolismo , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Sequência de Aminoácidos , Animais , Sequência de Bases , Enzimas de Restrição do DNA , Feminino , Humanos , Gravidez , Biossíntese de Proteínas , Homologia de Sequência do Ácido NucleicoRESUMO
The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.