RESUMEN
B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB-1 and SAB-2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic mu chain-positive pre-B cells or mature B cells to the BM stromal cell line. Injection of both SAB-1 and SAB-2 antibodies into pregnant mice reduced by 90% the number of B220+n B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU-preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB-1 is present on CS 1.3 stroma cells and on a pre-B cell line while SAB-2 is present on pro-B and pre-B cell lines, but not on CS 1.3 stromal cells. SAB-1 and SAB-2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB-1 and SAB-2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB-1 and SAB-2 probably recognize novel cell-cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/citología , Células Madre Hematopoyéticas/citología , Animales , Anticuerpos Monoclonales/química , Antígenos de Superficie/biosíntesis , Adhesión Celular/inmunología , Moléculas de Adhesión Celular/inmunología , Diferenciación Celular/inmunología , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Endopeptidasa K , Feto/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Hialuronoglucosaminidasa/farmacología , Hígado/citología , Hígado/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Neuraminidasa/farmacología , Placenta/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Serina Endopeptidasas/farmacología , Distribución Tisular , Tripsina/farmacología , Células Tumorales CultivadasRESUMEN
The soluble cAMP-dependent protein kinase activities of two estrogen receptor-containing (MCF-7 and ZR-75-1) and two estrogen receptor-lacking (BT-20 and MDA-MB-231) established human mammary tumor cell lines were analyzed by DEAE-cellulose chromatography and photoaffinity labeling with 8-azido-[32P]cAMP. Predominantly, type I isoenzyme was present in MDA-MB-231 cells, type II protein kinase was the main form in ZR-75-1 and BT-20 cells; whereas MCF-7 cytosols contained equal amounts of both protein kinase types. No correlations between estrogen receptor content and cAMP-dependent protein kinase holoenzyme ratios of isoenzymes were found. A distinctly greater heterogeneity of charge isomers of cAMP-binding proteins (regulatory subunits) was observed in estrogen receptor-containing cells.
Asunto(s)
Neoplasias de la Mama/análisis , Proteínas Portadoras/análisis , Proteína Receptora de AMP Cíclico , Proteínas Quinasas/análisis , Receptores de Estrógenos/análisis , Línea Celular , Cromatografía DEAE-Celulosa , Femenino , Humanos , Isoenzimas/análisis , Peso MolecularRESUMEN
Two isoenzymes of cAMP-dependent protein kinases were found in MCF-7 cytosol. The regulatory (cAMP-binding) subunit of protein kinase I (predominant form) has an apparent molecular weight (MW) of 49,000 and the two forms of regulatory subunits of protein kinase II have MWs of 52,000 and 54,000. Substantial amounts of the 49,000 protein cochromatographed on DEAE cellulose with protein kinase II. The quantities of protein kinase holoenzyme activity are strongly influenced by extraction procedures: the use of EDTA and of the protease inhibitor benzamidine can lead to extensive dissociation. On the other hand, high yields of cAMP-dependent protein kinase holoenzyme activity were consistently obtained with 150 mM KCl.
Asunto(s)
Neoplasias de la Mama/análisis , Proteínas Portadoras/análisis , Proteína Receptora de AMP Cíclico , Isoenzimas/análisis , Proteínas de Neoplasias/análisis , Proteínas Quinasas/análisis , Línea Celular , Cromatografía DEAE-Celulosa , Citosol/análisis , Femenino , Humanos , Peso MolecularRESUMEN
The cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins of dysplastic (control) and neoplastic human breast tissue cytosols were investigated after photoaffinity labeling with 8-azido-cyclic adenosine 3':5'-[32P]monophosphate (8-N3-cAMP) by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Four main binding proteins, all specific for cAMP, were identified, with molecular weights of 52,000, 49,000, 39,000, and 37,000. According to their molecular weights, elution on diethylaminoethyl cellulose, and in vitro phosphorylation, the Mr 49,000 and 52,000 species correspond to the regulatory subunits (R-I, R-II) of cAMP-dependent protein kinases types I and II. The smaller cAMP receptors (Mr 39,000 and 37,000) are proteolytic fragments of the intact R-proteins. Dissociation constants (Kd) with 8-N3-cAMP of 0.8 nM for R-I, and 0.12 microM for R-II were obtained; the proteolytic fragments exhibited Kd's similar to that of R-I. No difference in the 8-N3-cAMP affinities and labeling efficiencies was found between control and neoplastic tissues. Although the average incorporation of 8-N3-cAMP was 0.29 +/- 0.02 (S.E.) pmol/mg protein for control and 0.45 +/- 0.06 pmol/mg protein for neoplastic breast tissue cytosol, this difference does not reflect different cellular concentrations of cAMP receptors since the content of blood protein components is lower in tumor tissue. However, tumor cytosols exhibited an increased content of proteolytic R-fragments, and the ratio of intact cAMP receptors versus proteolyzed R-proteins was significantly (p less than 0.01) higher in control (8.3 +/- 0.9) than in tumor (3.0 +/- 0.5) tissue. The average R-I/R-II ratio was greater than 1 in each case, but no significant difference was observed between control and neoplastic tissue. Inverse relationships were obtained, especially between proteolyzed R-fragments and estrogen receptors, when the contents and ratios of cAMP-binding proteins were correlated with the contents of estrogen and progesterone receptors in tumor tissue by a Spearman rank correlation coefficient r = -0.55 (significance of difference from zero being p less than 0.01).
Asunto(s)
Azidas , Neoplasias de la Mama/análisis , Proteínas Portadoras/análisis , Proteína Receptora de AMP Cíclico , Enfermedad Fibroquística de la Mama/análisis , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Enfermedades de la Mama , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Citosol/análisis , Femenino , Humanos , Proteínas Quinasas/análisisRESUMEN
An ATP-dependent proton pump drives epinephrine transport in chromaffin granule ghosts. When ghosts are suspended in a medium free of permeant anions, ATP addition leads to an increase in membrane potential (interior positive) and epinephrine uptake but not to a change in intravesicular pH. Since ATP does not affect the pH gradient, the energy for transport must be drawn from the membrane potential (delta psi), and epinephrine uptake must result in a net efflux of positive charge. This can be achieved by an antiport (exchange diffusion) mechanism in which each catecholamine cation is taken up in exchange for more than one H+. Measurements indicate that the stoichiometry is close to 2 H+/epinephrine cation, so the equilibrium epinephrine gradient is theoretically [E]in/[E]out = ([H+]in/[H+]out)2eFdelta psi/(RT). In deenergized ghosts, the epinephrine concentration gradient equals the [H+] gradient. This is consistent with a situation in which the H+ concentration gradient is in equilibrium with the membrane potential as described by the Nernst equation. Then, in the equation above, the membrane potential term (eFdelta psi/(RT)) will exactly cancel one power of the [H+] gradient, leaving [E]in/[E]out equal to [H+]in/[H+]out.