RESUMEN

BACKGROUND: The incidence rates of osteoporosis and fractures are increased after cardiac transplantation. METHODS: We performed a cross-sectional analysis of cardiac transplant recipients in a late post-transplantation period (4.4 [2.5] years after cardiac transplantation, n = 27). We measured bone mineral density (BMD) by DXA at the hip and lumbar spine and by quantitative ultrasound (QUS) at the calcaneus. Vertebral fracture (vfx) prevalence was analysed by anterior-posterior and lateral radiographs of the thoracic and lumbar spine. RESULTS: Overall, vfx were present in 13 of 27 patients (48.2%, n = 51 vfx). Vfx were observed in 1 out of 5 patients with normal DXA results, 7 out of 14 osteopenic and 5 out of 8 osteoporotic cardiac transplant recipients. BMD at the femoral neck and more prominently at Ward's triangle were significantly lower in vfx patients compared to patients without vfx, with adjusted mean values (95% CI) of 0.804 [0.750-0.859] vs. 0.915 [0.860-0.969] g/cm2 and 0.573 [0.501-0.646] vs. 0.766 [0.697-0.836] g/cm2, respectively. CONCLUSIONS: These findings suggest an association between DXA measurements of the hip with vertebral fractures in a late post-transplantation period and thus extend knowledge from previous reports on cardiac transplant recipients studied earlier after CTX. In particular, our data pinpoint a potentially interesting role for BMD at Ward's triangle.

Asunto(s)

Densidad Ósea , Trasplante de Corazón , Osteoporosis/epidemiología , Complicaciones Posoperatorias/epidemiología , Fracturas de la Columna Vertebral/epidemiología , Absorciometría de Fotón , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/etiología , Osteoporosis/metabolismo , Complicaciones Posoperatorias/diagnóstico por imagen , Prevalencia , Fracturas de la Columna Vertebral/diagnóstico por imagen , Fracturas de la Columna Vertebral/etiología , Ultrasonografía

RESUMEN

In order to achieve neuron-restricted expression of antiapoptotic proteins, cellular promoters were investigated for their expression profiles in the context of adenoviral vectors. Both the synapsin 1 gene and the tubulin alpha1 gene promoters were strictly neuron specific in cocultures of primary neurons with their essential feeder cells. The neuron-specific enolase gene promoter exhibited only weak activity in cultured hippocampal neurons and was not neuron specific in preparations of cerebellar granule cells. By attaining virtually 100% transduction efficiency we were able to generate "quasi-transgenic" primary neuron cultures using both differentiated and completely undifferentiated hippocampal neurons. In a functional assay, we used the synapsin promoter to evaluate the effect of Bcl-X(L) overexpression on potassium-withdrawal-induced apoptosis of cerebellar granule neurons. We found nearly complete inhibition of caspase-9 and -3 activation and apoptosis, indicating a major role for mitochondrial pathways in this paradigm of neuronal cell death. The excellent suitability of the synapsin promoter as a strong panneuronal promoter was further demonstrated by its restricted neuronal activity in various brain regions of adult rats in vivo.

Asunto(s)

Adenoviridae/genética , Vectores Genéticos/genética , Neuronas/metabolismo , Regiones Promotoras Genéticas/genética , Sinapsinas/genética , Animales , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Caspasa 3 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Técnicas de Cocultivo , Citomegalovirus/genética , Expresión Génica , Vectores Genéticos/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Hipocampo/metabolismo , Mitocondrias/metabolismo , Neuroglía/citología , Neuronas/citología , Neuronas/efectos de los fármacos , Potasio/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Ratas , Ratas Sprague-Dawley , Transgenes , Tubulina (Proteína)/genética , Proteína bcl-X

RESUMEN

Tissue factor, the high-affinity receptor and cofactor for the plasma serine protease VII/VIIa, is the primary cellular initiator of the blood coagulation cascade. Inside the vasculature, expression of the tissue factor gene must be tightly controlled. Whereas the endothelium normally does not express tissue factor, on stimulation with inflammatory cytokines or endotoxin the gene is transcriptionally upregulated leading to a procoagulant state. We have now detected a repressive cis-acting element in the tissue factor promoter that downmodulates tissue factor transcription in endothelial cells. In reporter gene assays, deletion of this element leads to an increase of tissue factor transcription and insertion of a trimerized site reduces transcription. Specific protein/DNA complexes are formed on the element with nuclear extracts in electrophoretic mobility shift assays and cross-linking of the proteins followed by SDS-PAGE detects the presence of at least 2 subunits of approximately 40 and 60 kDa, respectively. After transfection of different cell types with the reporter genes, the suppressive effect of the element can only be revealed in endothelial cells. These data suggest that this element represents a novel transcription factor target sequence that functions to suppress expression of the tissue factor gene, preferentially in endothelial cells thereby supporting a noncoagulant state.

Asunto(s)

Endotelio Vascular/fisiología , Proteínas Represoras/fisiología , Tromboplastina/genética , Animales , Bovinos , Células Cultivadas , Secuencia Conservada , ADN/metabolismo , Humanos , Regiones Promotoras Genéticas , Porcinos , Transcripción Genética

RESUMEN

Vascular endothelial cell growth factor (VEGF) is a major regulator of angiogenesis. We report here that treatment of endothelial cells with VEGF leads to upregulation of tissue factor mRNA and protein expression on the cell surface. Reporter gene studies show that transcriptional activation of the tissue factor gene by VEGF is mediated by a GC-rich promoter element containing overlapping binding sites for Sp1 and EGR-1. As shown by immunofluorescence and electrophoretic mobility shift assays, upon VEGF treatment EGR-1 rapidly accumulates in the nucleus and binds to its respective recognition site in the tissue factor promoter. Sp1 occupies this element in unstimulated cells and seems to be partially displaced by increasing amounts of EGR-1. Transfection of endothelial cells with an EGR-1 expression plasmid mimics the upregulation of tissue factor transcription observed after VEGF treatment. In contrast, NFkappaB, the major transcription factor involved in tissue factor upregulation by inflammatory stimuli, is not activated by VEGF. These data show that VEGF induces a response in endothelial cells largely distinct from inflammatory stimuli, and suggest that EGR-1 is a major mediator of the activation of the tissue factor and possibly other VEGF-responsive genes.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/metabolismo , Linfocinas/farmacología , Tromboplastina/biosíntesis , Factores de Transcripción/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Inflamación , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

NKG2-C is a member of the recently discovered NKG2 family of genes and proteins, which are preferentially expressed on human natural killer (NK) cells. These potential NK cell receptors belong to a larger class of type II transmembrane proteins with a C-type lectin domain. We show here that NKG2-C is expressed as a 36-kDa glycoprotein by translation in vitro, recombinant expression and immunoprecipitation from a human NK cell clone. Further, a recombinant soluble NKG2-C-receptor binds specifically to K562 cells, which are target cells for NK cell killing, and to RPMI 8866 cells, which are feeder cells for NK cells; several other hematopoietic cell lines tested do not show any binding. The binding structures on the surface of K562 cells disappear, concomitant with a loss in susceptibility to killing when the cells are induced to differentiate with phorbol ester and Ca2+ ionophore. Our data suggest the presence of specific target molecules for NKG2-C on K562 cells, since overall glycosylation, Lewis X and Lewis Y structures, as well as the mucin-like CD43 molecule, do not change following induction of the cells. We propose that NKG2-C mediates a specific interaction of NK cells and their target cells with functional importance for NK cell killing.

Asunto(s)

Células Asesinas Naturales/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Inmunológicos/fisiología , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Citotoxicidad Inmunológica , ADN Complementario/genética , Perros , Glicosilación , Humanos , Ionomicina/farmacología , Células Asesinas Naturales/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Glicoproteínas de Membrana/genética , Subfamília C de Receptores Similares a Lectina de Células NK , Nucleopoliedrovirus/genética , Receptores Inmunológicos/genética , Receptores de Células Asesinas Naturales , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo

RESUMEN

Tissue factor is up-regulated on endothelial cells and monocytes in response to cytokines and endotoxin and is the main trigger of the extrinsic pathway of the coagulation cascade. We have isolated the porcine tissue factor gene and studied the regulation of the promoter, which has not been investigated previously in endothelial cells. Comparison of the promoter sequences with the respective human and murine genes reveals short stretches of homology, which encompass potential binding sites for AP-1, NF kappa B, and Sp1 transcription factors. Using DNase I footprinting, we detect binding of nuclear factors to these promoter elements. Transfection experiments demonstrate that a 300-base pair fragment containing the conserved elements can mediate induced transcription and that the NF kappa B-like element is essential. In accordance, electrophoretic mobility shift assays show a strong increase in the binding of factors to the NF kappa B-like site following induction. We further provide evidence that RelA (p65), c-Rel, and possibly novel polypeptides bind to the tissue factor NF kappa B element. In addition, we show constitutive binding of members of the Fos/Jun and Sp1 families to the AP-1 and Sp1 sites, respectively. We propose a concerted action of AP-1-, NF kappa B-, and Sp1-like factors in transcription from the tissue factor promoter in endothelial cells.

Asunto(s)

Endotelio Vascular/metabolismo , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Tromboplastina/genética , Factor de Transcripción AP-1/metabolismo , Animales , Secuencia de Bases , Coagulación Sanguínea , Células Cultivadas , Endotelio Vascular/citología , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-rel , Homología de Secuencia de Ácido Nucleico , Porcinos , Tromboplastina/metabolismo , Factor de Transcripción ReIA

RESUMEN

Genomic clones of ECI-6 (endothelial cell inducible), the porcine I kappa B alpha gene that encodes a cytoplasmic inhibitor of the transcription factor NF-kappa B, were isolated, spanning the entire transcribed region plus 2.1 and 0.35 kb of 5'- and 3'-flanking sequences, respectively. The gene contains five introns ranging in size from 0.6 to 0.1 kb. Four of the introns are located in the coding regions for four of the five ankyrin-like repeats in the central part of the I kappa B alpha protein at similar positions. The fifth intron is located in the C-terminal region. Southern blot analysis indicates the presence of a single copy of ECI-6/I kappa B alpha in the porcine genome.

Asunto(s)

Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Exones , Proteínas I-kappa B , Intrones , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , ADN Complementario , Genoma , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Porcinos

Asunto(s)

Fracturas Óseas/terapia , Traumatismos Vertebrales/terapia , Vértebras Torácicas/lesiones , Adolescente , Adulto , Anciano , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Pronóstico