RESUMEN
The development of more efficient, ethical, and effective means of assessing the effects of chemicals on human health and the environment was a lifetime goal of Gilman Veith. His work has provided the foundation for the use of chemical structure for informing toxicological assessment by regulatory agencies the world over. Veith's scientific work influenced the early development of the SAR models in use today at the US Environmental Protection Agency. He was the driving force behind the Organisation for Economic Co-operation and Development QSAR Toolbox. Veith was one of a few early pioneers whose vision led to the linkage of chemical structure and biological activity as a means of predicting adverse apical outcomes (known as a mode of action, or an adverse outcome pathway approach), and he understood at an early stage the power that could be harnessed when combining computational and mechanistic biological approaches as a means of avoiding animal testing. Through the International QSAR Foundation he organized like-minded experts to develop non-animal methods and frameworks for the assessment of chemical hazard and risk for the benefit of public and environmental health. Avoiding animal testing was Gil's passion, and his work helped to initiate the paradigm shift in toxicology that is now rendering this feasible.
Asunto(s)
Alternativas a las Pruebas en Animales , Relación Estructura-Actividad , Pruebas de Toxicidad/métodos , Simulación por Computador , Modelos Químicos , Relación Estructura-Actividad Cuantitativa , Medición de RiesgoRESUMEN
The role of the anti-phospholipid antibodies (APLA) and anti-endothelial cell antibodies (AECA) in the pathogenesis of anti-phospholipid syndrome (APS) is unclear. Differences in the reported involvement of APLA may be due, in part, to the polyclonal nature of these antibodies and the use of serum and serum fractions for analysis. To circumvent this issue, we generated monoclonal antibodies (MAB) from three patients with APS and two healthy controls. We then compared the antigen binding patterns and the heavy chain variable region (VH) DNA sequences of the MAB derived from patients with APS to those from healthy controls. The results of this study indicate that APLA and AECA comprise a highly heterogeneous population of antibodies with respect to the antigens they recognize, as well as VH gene usage. MAB derived from patients with APS do not differ from those derived from normal individuals based on either antigen recognition or VH gene usage. These results suggest the importance of additional predisposing factors in the pathogenesis of APS.
Asunto(s)
Anticuerpos Antifosfolípidos/genética , Anticuerpos Monoclonales/genética , Endotelio Vascular/inmunología , Secuencia de Aminoácidos , Anticuerpos Anticardiolipina/sangre , Anticuerpos Anticardiolipina/genética , Anticuerpos Anticardiolipina/inmunología , Anticuerpos Antifosfolípidos/sangre , Anticuerpos Antifosfolípidos/inmunología , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/genética , Síndrome Antifosfolípido/inmunología , Secuencia de Bases , ADN/análisis , ADN/genética , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Glicoproteínas/sangre , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Datos de Secuencia Molecular , Fosfolípidos/metabolismo , Reacción en Cadena de la Polimerasa , beta 2 Glicoproteína IRESUMEN
The objective of this study was to determine whether cultured human trophoblasts migrate in response to changes in oxygen tension or temperature. Human trophoblastic cells distributed homogenously within individual wells of standard culture plates were subjected to oxygen and thermal gradients. The redistribution of cells was determined 90 min to 18 h after these gradients had been established. Trophoblastic cells did not migrate in response to gradients of oxygen or carbon dioxide applied in this manner. In contrast, the cells migrated in response to thermal gradients of less than 1 degree C in the direction of the warmer temperature. The response began within minutes, was reversed by a change in the direction of the thermal gradient, and was inhibited at high cell concentrations. Migration was independent of proliferation or protein synthesis, but required microfilament assembly. The capacity of trophoblasts to migrate in response to small difference of temperature within the physiologic range may contribute to the initiation of placental development before contact with the maternal circulation has been established.
Asunto(s)
Movimiento Celular , Calor , Trofoblastos/citología , Arterias/citología , Dióxido de Carbono/sangre , Femenino , Humanos , Oxígeno/sangre , Embarazo , Útero/irrigación sanguíneaRESUMEN
Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI-1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell-bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.
Asunto(s)
Proteínas de la Membrana/inmunología , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetinae , Epítopos/química , Epítopos/inmunología , Glicosilfosfatidilinositoles/deficiencia , Glicosilfosfatidilinositoles/metabolismo , Proteínas de la Membrana/química , Unión Proteica , Conformación Proteica , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/metabolismo , Transducción de Señal , Solubilidad , alfa-Macroglobulinas/metabolismoRESUMEN
We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2(425aa), or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A. Furnari, E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S. Huang, J. Virol. 67:4981-4991, 1993; C. Hagemeier, S. Walker, R. Caswell, T. Kouzarides, and J. Sinclair, J. Virol. 66:4452-4456, 1992; R. Jupp, S. Hoffman, R. M. Stenberg, J. A. Nelson, and P. Ghazal, J. Virol. 67:7539-7546, 1993) but also with its ability to interact with the transcription factors which bind to the upstream element of promoters it activated (e.g., SP1 and Tef-1 but not Oct-1). This ability to have multiple interactions with the promoter complex may be crucial for transcriptional activation, since the IE proteins cannot activate promoters having only a TATA element or only an upstream transcription factor binding site. In addition, we show that proteins which bind IEP86 also bind to IEP55. Thus, the negative effect on transcription noted with IEP55 may be the result of competition with IEP86 for interaction with the promoter complex. The synergy caused by IEP72 appears to be mediated by a more indirect mechanism. This is suggested by our observation that IEP72 could not bind to any of the proteins tested (TBP, Tef-1, or Oct-1) or to IEP86.
Asunto(s)
Citomegalovirus/genética , Proteínas Inmediatas-Precoces/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Antígenos Transformadores de Poliomavirus/metabolismo , Secuencia de Bases , Citomegalovirus/metabolismo , ADN Viral , Humanos , Datos de Secuencia Molecular , Factores de Transcripción/metabolismoRESUMEN
The early proteins of simian virus 40 (SV40) large T and small t antigen (T/t antigen) can each cause the transcriptional activation of a variety of cellular and viral promoters. We showed previously that simian cellular DNA-binding factors (the Band A factors) bind to sequences within the SV40 late promoter which are important for transcriptional activation in the presence of the SV40 early proteins. Band A factors isolated from simian cells which produce T/t antigen (COS cells or SV40-infected CV-1 cells) have altered binding properties in comparison with the factors from normal simian cells (CV-1). This suggests that the transcriptional activation mediated by T/t antigen may be due to either modification of existing factors or induction of new members of a family of factors. We have purified the Band A factors from both COS and CV-1 cells and have determined the binding site by methylation interference and DNase protection footprinting. The COS cell factors have altered chromatographic properties on ion-exchange columns and have higher-molecular-weight forms than the CV-1 cell factors. Major forms of the CV-1 factors migrate between 20 and 24 kilodaltons, while the COS factors migrate between 20 and 28 kilodaltons. The binding sites for the factors from CV-1 and COS cells are similar, covering a rather broad region within the 72-base-pair repeat comprising the AP-1 site and the two-octamer binding protein (OBP100/Oct 1) sites, OBP I and OBP II. Specific binding competition analyses indicate that the two general regions within the binding site (the AP-1-OBP II site and the OBP I site) each retain partial binding ability; however, the factors bind best when the two regions are adjacent in a relatively specific spatial arrangement. The binding site for the Band A factors corresponds very well to sequences necessary for the activation of the late promoter as defined by deletion and base substitution mutagenesis studies (J. M. Keller and J. C. Alwine, Mol. Cell. Biol. 5:1859-1869, 1985; E. May, F. Omilli, M. Emoult-Lange, M. Zenke, and P. Chambon, Nucleic Acids Res. 15:2445-2461, 1987). These data, in combination with the data showing that the Band A factors are modified or induced in the presence of T/t antigen, strongly suggest that T/t antigen mediates its transcriptional activation function, at least in part, through the Band A factors.