RESUMEN
Pulmonary hypertension (PH) leads to an increased right ventricular workload, cardiac failure and death. In idiopathic pulmonary arterial hypertension (PAH) the vasodilating vasoactive intestinal peptide (aviptadil) is deficient. The aim of the present study was to test the acute effects on haemodynamics and blood gases, and the safety, of a single dose of inhaled aviptadil in chronic PH. A total of 20 patients with PH (PAH in nine, PH in lung disease in eight and chronic thromboembolic PH in three) inhaled a single 100-microg dose of aviptadil during right-heart catheterisation. Haemodynamics and blood gases were measured. Aviptadil aerosol caused a small and temporary but significant selective pulmonary vasodilation, an improved stroke volume and mixed venous oxygen saturation. Overall, six patients experienced a pulmonary vascular resistance reduction of >20%. In patients with significant lung disease, aviptadil tended to improve oxygenation. The pulmonary vasodilating effect of aviptadil aerosol was modest and short-lived, did not cause any side-effects and led to a reduced workload of the right ventricle without affecting systemic blood pressure. Aviptadil inhalation tended to improve oxygenation in patients with significant lung disease. Further studies are needed to evaluate the full therapeutic potential of aviptadil aerosol, including higher doses and chronic treatment.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/fisiopatología , Fentolamina/administración & dosificación , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/metabolismo , Adulto , Aerosoles , Anciano , Presión Sanguínea , Combinación de Medicamentos , Femenino , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/patología , Humanos , Enfermedades Pulmonares/patología , Masculino , Persona de Mediana Edad , Oxígeno/metabolismoRESUMEN
Helicobacter pylori has a major aetiological role in human gastric carcinogenesis but the cellular and molecular pathways by which infection promotes transformation remain to be resolved. This study demonstrates that H. pylori exposure to MKN-1, ST42, and MKN-28 gastric epithelial tumour cells results in the activation of HB-EGF gene expression and EGFR tyrosine phosphorylation. These cell responses are induced by both cagPAI positive and cagPAI negative H. pylori strains and are dependent on cell surface expression of the HB-EGF precursor. The induction of HB-EGF gene transcription by H. pylori requires metalloprotease-, EGFR-, and Mek1-activities, indicating the involvement of the "triple membrane passing signal" (TMPS) for EGFR transactivation. Moreover, the release of the inflammatory cytokine IL-8 by cells exposed to H. pylori is significantly impaired by inhibitors of TMPS pathway elements. Our findings support a model in which H. pylori triggers constitutive EGFR signal activation, which enhances IL-8 production, and initiates neoplastic transformation of gastric epithelial cells.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Helicobacter pylori/metabolismo , Metaloendopeptidasas/metabolismo , Activación Transcripcional , Northern Blotting , Western Blotting , Línea Celular , Membrana Celular/enzimología , Células Cultivadas , Técnicas de Cocultivo , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Epitelio/patología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Transducción de Señal , Estómago/patología , Neoplasias Gástricas/enzimología , Factores de Tiempo , Tirosina/metabolismoRESUMEN
Recent data suggest that mast cells (MCs) in patients with systemic mastocytosis or mast cell leukemia express a CD2-reactive antigen. To explore the biochemical nature and function of this antigen, primary MCs as well as the MC line HMC-1 derived from a patient with mast cell leukemia were examined. Northern blot experiments revealed expression of CD2 messenger RNA in HMC-1, whereas primary nonneoplastic MCs did not express transcripts for CD2. In cell surface staining experiments, bone marrow (BM) MCs in systemic mastocytosis (n = 12) as well as HMC-1 cells (30%-80%) were found to express the T11-1 and T11-2 (but not T11-3) epitopes of CD2. By contrast, BM MCs in myelodysplastic syndromes and nonhematologic disorders (bronchiogenic carcinoma, foreskin phimosis, uterine myeomata ) were consistently CD2(-). All MC species analyzed including HMC-1 were found to express LFA-3 (CD58), the natural ligand of CD2. To study the functional role of CD2 on neoplastic MCs, CD2(+) and CD2(-) HMC-1 cells were separated by cell sorting. CD2(+) HMC-1 cells were found to form spontaneous aggregates and rosettes with sheep erythrocytes in excess over CD2(-) cells, and a T11-1 antibody inhibited both the aggregation and rosette formation. Moreover, exposure of CD2(+) HMC-1 cells to T11-1 or T11-2 antibody was followed by expression of T11-3. In addition, stimulation of neoplastic MCs through T11-3 and a second CD2 epitope resulted in histamine release. These data show that neoplastic MCs express functionally active CD2. It is hypothesized that expression of CD2 is associated with pathologic accumulation and function of MCs in systemic mastocytosis.
Asunto(s)
Antígenos CD2/fisiología , Epítopos/análisis , Expresión Génica , Leucemia de Mastocitos/inmunología , Mastocitos/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Northern Blotting , Células de la Médula Ósea/inmunología , Antígenos CD2/análisis , Antígenos CD2/genética , Línea Celular , Células Cultivadas , Eritrocitos/inmunología , Femenino , Sangre Fetal/citología , Genitales Masculinos , Liberación de Histamina , Humanos , Pulmón/inmunología , Masculino , Síndromes Mielodisplásicos/patología , ARN Mensajero/análisis , Formación de Roseta , Ovinos , Piel/inmunología , Células Madre , Útero/inmunologíaRESUMEN
Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 5 [corrected].
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 4 , ADN Complementario/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Algoritmos , Animales , Northern Blotting , Encéfalo/metabolismo , Clonación Molecular , Etiquetas de Secuencia Expresada , Glutatión Transferasa/metabolismo , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Riñón/metabolismo , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Miocardio/metabolismo , Fosforilación , Proteínas Tirosina Quinasas , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Distribución TisularRESUMEN
Dendritic cells (DCs), nature's adjuvant, must mature to sensitize T cells. However, although the maturation process is essential, it is not yet fully understood at the molecular level. In this study, we investigated the course of expression of the unique hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A), which is part of a particular RNA nuclear export pathway, during in vitro generation of human DCs. We show that eIF-5A expression is significantly upregulated during DC maturation. Furthermore, an inhibitor of the hypusine modification, GC7 (N(1)-guanyl-1, 7-diaminoheptane), prevents CD83 surface expression by apparently interfering with nucleocytoplasmic translocation of the CD83 mRNA and, importantly, significantly inhibits DC-mediated T lymphocyte activation. The data presented suggest that CD83 mRNA is transported from the nucleus to the cytoplasm via a specific nuclear export pathway and that hypusine formation appears to be essential for the maturation of functional DCs. Therefore, pharmacological interference with hypusine formation may provide a new possibility to modulate DC function.
Asunto(s)
Núcleo Celular/metabolismo , Células Dendríticas/citología , Inmunoglobulinas/biosíntesis , Lisina/análogos & derivados , Glicoproteínas de Membrana/biosíntesis , Factores de Iniciación de Péptidos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Presentación de Antígeno/efectos de los fármacos , Antígenos CD , Transporte Biológico/efectos de los fármacos , Compartimento Celular/efectos de los fármacos , Diferenciación Celular , Células Dendríticas/inmunología , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Inmunoglobulinas/genética , Activación de Linfocitos , Lisina/metabolismo , Glicoproteínas de Membrana/genética , Procesamiento Proteico-Postraduccional , Linfocitos T/inmunología , Regulación hacia Arriba , Factor 5A Eucariótico de Iniciación de Traducción , Antígeno CD83RESUMEN
Ribosomal protein L5 is part of the 60 S ribosomal subunit and localizes in both the cytoplasm and the nucleus of eukaryotic cells, accumulating particularly in the nucleoli. L5 is known to bind specifically to 5 S rRNA and is involved in nucleocytoplasmic transport of this rRNA. Here, we report a detailed analysis of the domain organization of the human ribosomal protein L5. We show that a signal that mediates nuclear import and nucleolar localization maps to amino acids 21-37 within the 297-amino acid L5 protein. Furthermore, carboxyl-terminal residues at positions 255-297 serve as an additional nuclear/nucleolar targeting signal. Domains involved in 5 S rRNA binding are located at both the amino terminus and the carboxyl terminus of L5. Microinjection studies in somatic cells demonstrate that a nuclear export signal (NES) that maps to amino acids 101-111 resides in the central region of L5. This NES is characterized by a pronounced clustering of critical leucine residues, which creates a peptide motif not previously observed in other leucine-rich NESs. Finally, we present a refined model of the multidomain structure of human ribosomal protein L5.
Asunto(s)
Señales de Localización Nuclear , Proteínas Ribosómicas/química , Secuencia de Aminoácidos , Nucléolo Celular/metabolismo , Clonación Molecular , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Indicadores y Reactivos , Proteínas Luminiscentes , Datos de Secuencia Molecular , ARN Ribosómico 5S/metabolismoRESUMEN
Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans-activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Productos del Gen rev/metabolismo , Carioferinas , Leucina , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN , Receptores Citoplasmáticos y Nucleares , Secuencia de Aminoácidos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Clonación Molecular , Citoplasma/metabolismo , Glutatión Transferasa/metabolismo , VIH-1/fisiología , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Microinyecciones , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Factor 5A Eucariótico de Iniciación de Traducción , Proteína Exportina 1RESUMEN
Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.
Asunto(s)
Fibrinólisis , Mastocitos/enzimología , Activador de Tejido Plasminógeno/biosíntesis , Línea Celular , Células Cultivadas , Endotelio Vascular/química , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Humanos , Inmunohistoquímica , Pulmón/química , Pulmón/citología , Pulmón/enzimología , Mastocitos/química , Mastocitos/metabolismo , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Inhibidor 1 de Activador Plasminogénico/análisis , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/biosíntesis , Activador de Tejido Plasminógeno/análisis , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/fisiología , Venas UmbilicalesRESUMEN
The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.
Asunto(s)
Productos del Gen rev/metabolismo , VIH-1/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Productos del Gen rev/química , VIH-1/fisiología , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Activación Transcripcional , Replicación Viral , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.
Asunto(s)
Productos del Gen rev/metabolismo , VIH-1/genética , Factores de Iniciación de Péptidos/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Núcleo Celular/metabolismo , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/química , Saccharomyces cerevisiae , Alineación de Secuencia , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Recent data suggest that mast cells (MC) are involved in the regulation of leukocyte accumulation in inflammatory reactions. In this study, expression of leukocyte-chemotactic peptides (chemokines) in purified human lung MC (n = 16) and a human mast cell line, HMC-1, was analyzed. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) showed baseline expression of monocyte chemoattractant protein (MCP)-1 mRNA in unstimulated MC. Exposure of MC to recombinant stem cell factor (rhSCF, 100 ng/mL) or anti-IgE (10 microgram/mL) was followed by a substantial increase in expression of MCP-1 mRNA. Neither unstimulated nor stem cell factor (SCF )-stimulated lung MC expressed transcripts for interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, or RANTES by Northern blotting. The mast cell line HMC-1, which contains a mutated and intrinsically activated SCF-receptor, was found to express high levels of MCP-1 mRNA in a constitutive manner. Exposure of HMC-1 cells to rhSCF resulted in upregulation of MCP-1 mRNA expression, and de novo expression of MIP-1beta mRNA. The SCF-induced upregulation of MCP-1 mRNA in lung MC and HMC-1 was accompanied by an increase in immunologically detectable MCP-1 in cell supernatants (sup) (lung MC [<98%], control medium, 1 hour: 159 +/- 27 v SCF, 100 ng/mL, 1 hour: 398 +/- 46 pg/mL/10(6) cells; HMC-1: control, 1 hour: 894 +/- 116 v SCF, 1 hour: 1,536 +/- 265 pg/mL/10(6)). IgE-dependent activation was also followed by MCP-1 release from MC. MC-sup and HMC-1-sup induced chemotaxis in blood monocytes (Mo) (control: 100% +/- 12% v 2-hour-MC-sup: 463% +/- 38% v HMC-1-sup: 532% +/- 12%), and a monoclonal antibody (MoAb) to MCP-1 (but not MoAb to IL-8) inhibited Mo-chemotaxis induced by MC-sup or HMC-1-sup (39% to 55% inhibition, P < .05). In summary, our study identifies MCP-1 as the predominant CC-chemokine produced and released in human lung MC. MCP-1 may be a crucial mediator in inflammatory reactions associated with MC activation and accumulation of MCP-1-responsive leukocytes.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Quimiocina CCL2/biosíntesis , Pulmón/metabolismo , Mastocitos/metabolismo , Factor de Células Madre/farmacología , Quimiotaxis/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Pulmón/citología , ARN Mensajero/metabolismoRESUMEN
Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine, a modification that appears to be required for cell proliferation. This hypusine-modified protein stimulates synthesis of methionyl-puromycin in an in vitro assay which mimics the formation of the first peptide bond during protein synthesis, although the exact role of eIF-5A in vivo is still unknown. The unexpected finding that eIF-5A is a cellular cofactor of the HIV-1 Rev trans-activator protein may, however, provide a novel opportunity to reveal precisely what function eIF-5A performs in eukaryotic cells. In this review article, we first present a brief description of HIV-1 Rev function, followed by an overview of the data that identified eIF-5A as a Rev cofactor and, finally, discuss novel findings with respect to cellular eIF-5A activities.
Asunto(s)
Productos del Gen rev/fisiología , VIH-1/fisiología , Factores de Iniciación de Péptidos/fisiología , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Transducción de Señal , Replicación Viral , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
SDZ NIM 811 is a cyclosporin A (CsA) analogue that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. Binding to cyclophilin A, the intracellular receptor for cyclosporins, is a prerequisite for HIV-1 inhibition by cyclosporins. Cyclophilin A was demonstrated to bind to HIV-1 p24gag and this cyclophilin-Gag interaction leads to the incorporation of cyclophilin A into HIV-1 virions. SDZ NIM 811 inhibits this protein interaction, and this is likely to be the molecular basis for its antiviral activity. Here, we show that in activated primary T cells SDZ NIM 811 interferes with two stages of the virus replication cycle: (i) translocation of pre-integration complexes into the nucleus and (ii) production of infectious virus particles. SDZ NIM 811 not only inhibits translocation of HIV-1 pre-integration complexes in primary T cells, but also in a growth-arrested T cell line. In vivo, most T lymphocytes are quiescent, but serve nevertheless as a major and inducible HIV-1 reservoir in infected individuals. Significant amounts of cyclophilin A were found to be associated with virus particles propagated in primary T cells. SDZ NIM 811 caused a strong reduction in the amount of incorporated cyclophilin A, thereby reducing infectivity. Thus, cyclophilin A seems to be necessary for HIV-1 replication in primary T cells.
Asunto(s)
Fármacos Anti-VIH/farmacología , Ciclosporina/farmacología , Infecciones por VIH/virología , VIH-1/fisiología , Linfocitos T/virología , Replicación Viral/efectos de los fármacos , División Celular , Ciclosporina/metabolismo , Interacciones Farmacológicas , VIH-1/efectos de los fármacos , Humanos , Linfocitos T/patología , Virión/efectos de los fármacosRESUMEN
The urokinase receptor system is involved in several biological processes including extracellular proteolysis, cell invasion, and chemotaxis. Mast cells are multifunctional perivascular cells that play an important role in the regulation of microenvironmental events. We report that primary human mast cells and the human mast cell line HMC-1 express the receptor for urokinase. As assessed by Northern blotting and reverse transcription polymerase chain reaction technique, purified human lung mast cells and HMC-1 cells expressed urokinase receptor mRNA in a constitutive manner. Using a toluidine blue/immunofluorescence double staining technique and monoclonal antibodies, surface expression of urokinase receptor was demonstrable in lung, skin, uterus, heart, and tonsil mast cells, whereas the low density lipoprotein receptor-related protein was not detectable. Binding of monoclonal antibody VIM5 (recognizing the urokinase binding domain of urokinase receptor) to HMC-1 could be blocked by high molecular weight but not low molecular weight urokinase. Binding analyses performed with 123I-urokinase revealed expression of 271,000 +/- 55,000 high affinity urokinase binding sites per HMC-1 cell, with a calculated dissociation constant of 1. 29 +/- 0.3 nM. Purified urokinase induced dose-dependent migration of primary mast cells and HMC-1 in a chemotaxis assay without inducing release of histamine. The mast cell agonist stem cell factor also induced migration of HMC-1 and caused up-regulation of expression of urokinase receptor mRNA. Together, our data show that human mast cells express functional receptors for urokinase. Expression of urokinase receptors on mast cells may have implications for mast cell-dependent microvascular processes associated with fibrinolysis, migration, or local tissue repair.
Asunto(s)
Mastocitos/metabolismo , Receptores de Superficie Celular/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Quimiotaxis de Leucocito , Liberación de Histamina , Humanos , Hibridación in Situ , Ensayo de Unión Radioligante , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo UroquinasaAsunto(s)
Proteínas Portadoras/genética , Cromosomas Humanos Par 2 , Productos del Gen rev/metabolismo , Productos del Gen rex/genética , VIH-1/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/genética , Proteínas de Unión al ARN , Mapeo Cromosómico , Humanos , Hibridación Fluorescente in Situ , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Previously, we described two mutants of the cellular Rev co-factor, eukaryotic initiation factor 5A (eIF-5A M13 and M14), which suppress human immunodeficiency virus type 1 (HIV-1) SF2 replication in clonal T cell lines. This study introduced the notion that it is possible to develop gene therapies against infectious agents on the basis of mutant host factors required for viral replication. In this report, we provide further evidence to support this new paradigm and describe murine leukemia virus (MLV)-based retroviral vectors expressing three different eIF-5A mutants from the viral long terminal repeat (LTR). HIV-1 replication (SF2, HXB-3) was reduced up to 2 orders of magnitude in transduced, polyclonal T cell populations. All eIF-5A mutants also showed antiviral activity (approximately seven-fold reduction) in a chronic HIV-1 infection model. Expression of eIF-5A mutant M13 delta in peripheral blood lymphocytes (PBLs) showed no difference in proliferation and metabolic activity as determined in a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)-assay, suggesting that expression of this type of mutant protein is not associated with cellular toxicity. In summary, these data suggest that gene therapy for HIV-1 infection can be developed on the basis of mutants of the Rev co-factor eIF-5A.
Asunto(s)
VIH-1/fisiología , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN , Replicación Viral/efectos de los fármacos , Northern Blotting , Southern Blotting , Estudios de Factibilidad , Terapia Genética/métodos , Vectores Genéticos , Humanos , Mutagénesis Sitio-Dirigida , Retroviridae , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Recent data suggest that local overexpression of the tissue-hormone c-kit ligand (stem cell factor [SCF]) is associated with accumulation of mast cells (MCs) and a decrease in expression of c-kit in the accumulated MCs [28]. In the present study, the effects of recombinant human (rh) SCF on expression of c-kit mRNA and c-kit protein in isolated human MCs and a human mast cell line, HMC-1, were analyzed. Incubation of isolated lung MC with rhSCF (100 ng/mL) for 120 minutes resulted in decreased expression of c-kit mRNA (optical density [OD], control: 100% vs. rhSCF: 37%). Almost identical results were obtained with HMC-1 cells (OD, control: 100% vs. rhSCF: 40 to 45%). As assessed by flow cytometry and monoclonal antibodies (mAbs) to c-kit, the SCF-induced decrease of c-kit mRNA in HMC-1 was associated with a substantial decrease in surface expression of c-kit (MFI, control: 100 +/- 21%, vs. MFI in cells incubated with rhSCF [100 ng/mL at 37 degrees C for 12 hours]: 8 +/- 2%, vs. MFI in cells incubated with rhSCF, 100 ng/mL, at 4 degrees C: 34 +/- 3%). The effects of rhSCF on c-kit expression in HMC-1 cells were dose- and time-dependent with maximum effects observed with 10-100 ng/mL of rhSCF after 4 to 12 hours. The SCF-dependent loss of c-kit was also accompanied by a decreased chemotactic response to rhSCF (control: 100%; rhSCF: 71 +/- 2%). This study shows that exposure of human lung MC and HMC-1 cells to recombinant SCF results in downregulation of c-kit mRNA and surface c-kit expression. These data may explain the partial loss of c-kit on MCs in areas of SCF overexpression.
Asunto(s)
Pulmón/citología , Mastocitos/citología , Proteínas Proto-Oncogénicas c-kit/fisiología , Factor de Células Madre/farmacología , Northern Blotting , Línea Celular , Quimiotaxis/efectos de los fármacos , Regulación hacia Abajo , Humanos , Técnicas para Inmunoenzimas , Mastocitos/química , Proteínas de la Membrana/biosíntesis , Sondas de Oligonucleótidos/análisis , Proteínas Proto-Oncogénicas c-kit/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.
Asunto(s)
Productos del Gen rev/metabolismo , VIH-1/fisiología , Factores de Iniciación de Péptidos/fisiología , Proteínas de Unión al ARN , Linfocitos T/virología , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Células Cultivadas , Genes env , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Linfocitos T/metabolismo , Activación Transcripcional , Replicación Viral , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Leukocyte adhesion and transmigration through the endothelial cell (EC) layer plays a crucial role in inflammation. IL-1 alpha and TNF alpha increase EC-adhesiveness for leukocytes by stimulating surface expression of ICAM-1 (intercellular adhesion molecule 1, CD54), VCAM-1 (vascular cell adhesion molecule 1, CD106) and E-selectin (CD62E). In this study, the effects of ibuprofen on IL-1 alpha and TNF alpha-induced expression of ICAM-1, VCAM-1 and E-selectin on cultured human umbilical vein EC (HUVEC) were analyzed. Exposure to IL-1 alpha or TNF alpha resulted in an increased expression of VCAM-1, ICAM-1, and E-selectin. Ibuprofen was identified as a potent inhibitor of IL-1 alpha and TNF alpha-induced surface expression of VCAM-1 and a less potent inhibitor of pyrogen-induced expression of ICAM-1, whereas no effect on E-selectin was found. The effects of ibuprofen on VCAM-1 expression were dose-dependent (IC50 [IL-1 alpha]: 0.5 mM; IC50 [TNF alpha]: 0.5 mM) and time-dependent with maximum responses observed after 18 h. Moreover, ibuprofen abrogated pyrogen-dependent adhesion of leukocytes to HUVEC. Ibuprofen also inhibited VCAM-1 mRNA expression in pyrogen activated EC. VCAM-1-downregulation on EC by ibuprofen may contribute to the anti-inflammatory actions of the drug.