RESUMEN
We have reported the generation and characterization of four HIV-1 neutralizing human monoclonal antibodies. Three antibodies recognize a conformational epitope within the CD4-binding site of HIV-1 gp120 and one recognizes a linear epitope located within the hypervariable V3 domain of gp120. In the present study we report the nucleotide sequences of the cDNAs encoding the variable regions of the heavy and light chains of these antibodies. Molecular characteristics, closet germline genes, and the putative extent of somatic mutation are presented. Two of the four heavy chain variable (VH) regions are derived from the VH1 gene family, one from the VH3 gene family, and one from the VH5 gene family. In addition, the VH chain of a previously described human monoclonal antibody, directed against HIV-1 gp41, is derived from the VH3 gene family. The degree of nucleotide variation between these five antibodies and their closest germline counterparts ranges from 4 to 12%, mainly located in the complementarity-determining regions. Significant nucleotide sequence homology with previously described germline diversity (D) genes could be found for only two of five antibody D segments. Joining (JH) gene segments utilized are JH4 or JH6. Two light chain variable (VL) regions are derived from a VK1 gene segment, one from a V kappa 4, one from a V lambda 2, and one from a lambda 6 gene segment.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Anti-VIH/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Secuencia de Bases , ADN Complementario , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Seropositividad para VIH/inmunología , Humanos , Región de Unión de la Inmunoglobulina/genética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Rat basophilic leukemia cells exhibit 12-lipoxygenase activity only upon cell disruption. 12-Lipoxygenase may also possess 15-lipoxygenase activity, as is indicated by the formation of low amounts of 15(S)-HETE, in addition to the predominant product 12(S)-HETE, upon incubation of partially purified 12-lipoxygenase with arachidonic acid. With 5(S)-HPETE as substrate not only 5(S), 12(S)-diHETE and 5(S), 15(S)-diHETE are formed, but also LTA4, as was indicated by the presence of LTA4-derived LTB4-isomers. 12-Lipoxygenase from rat basophilic leukemia cells has many features in common with 12-lipoxygenase from bovine leukocytes. As was suggested for the latter enzyme, 12-lipoxygenase from rat basophilic leukemia cells may represent the remaining LTA4-synthase activity of 5-lipoxygenase, of which the 5-dioxygenase activity has disappeared upon cell disruption. Such a possible shift from 5-lipoxygenase activity to 12-lipoxygenase activity could not simply be induced by interaction of cytosolic 5-lipoxygenase with a membrane fraction after cell disruption, but may involve release of membrane-associated 5-lipoxygenase upon disruption of activated rat basophilic leukemia cells.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Leucemia Basofílica Aguda/enzimología , Animales , Araquidonato 12-Lipooxigenasa/aislamiento & purificación , Araquidonato 5-Lipooxigenasa/aislamiento & purificación , Fraccionamiento Celular , Cromatografía Líquida de Alta Presión , Citosol/enzimología , Ácidos Hidroxieicosatetraenoicos/aislamiento & purificación , Ácidos Hidroxieicosatetraenoicos/metabolismo , Cinética , Ratas , Células Tumorales CultivadasRESUMEN
Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively.
Asunto(s)
Asma/sangre , Basófilos/metabolismo , SRS-A/biosíntesis , Anticuerpos Antiidiotipos/fisiología , Asma/inmunología , Basófilos/efectos de los fármacos , Calcimicina/farmacología , Liberación de Histamina , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina E/fisiología , Proteínas Opsoninas , Prostaglandina D2/biosíntesis , SRS-A/sangre , ZimosanRESUMEN
12-Lipoxygenase from rat basophilic leukemia cells was purified about 300-fold by protein-HPLC in a single run. Maximal 12-lipoxygenase activity was observed at pH 7.5, while the enzyme became almost inactive at pH 6 and 9. Although Ca2+ was not essential for 12-lipoxygenase activity, the partially purified enzyme was stimulated approx. 2-fold in the presence of 0.1-5.0 mM Ca2+. Contrary to 5-lipoxygenase from RBL-1 cells, 12-lipoxygenase was not inactivated by preincubation with Ca2+ for 1-10 min, nor was it stimulated by 0.1-10 mM ATP.
Asunto(s)
Araquidonato 12-Lipooxigenasa/aislamiento & purificación , Leucemia Basofílica Aguda/enzimología , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Inhibidores de la Lipooxigenasa , Ratas , Células Tumorales Cultivadas/enzimologíaRESUMEN
12-Lipoxygenase and 5-lipoxygenase from rat basophilic leukemia cells were separated by protein-HPLC in a single step. Upon incubation in the presence of Ca2+, 12-lipoxygenase converted arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and linoleic acid into 13(S)-hydro(pero)xyoctadecadienoic acid. The reaction products were analyzed by reversed-phase and chiral straight-phase HPLC with ultraviolet-detection. Using the cytosolic fraction of rat basophilic leukemia cells, optimal 12-lipoxygenase activity was observed at 10 degrees C. At 37 degrees C 12-lipoxygenase was very rapidly inactivated by its own product, hydroperoxy fatty acid, at low concentrations (10-100 nM).
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Leucemia Experimental/enzimología , Animales , Araquidonato 12-Lipooxigenasa/aislamiento & purificación , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Ácidos Grasos/metabolismo , Ratas , Temperatura , Células Tumorales CultivadasRESUMEN
The presence of Fc receptors for IgE on epidermal Langerhans cells (LC) from patients with atopic dermatitis (AD) was demonstrated by three different types of experiments. Firstly, cell-bound IgE on LC was removed by acid elution and restored by highly purified human myeloma IgE (IgE kappa). Secondly, after pepsin digestion of cell-bound IgE the number of LC staining with anti-human light chain (kappa, lambda) antibodies significantly decreased in contrast to the number of LC staining with anti-human epsilon heavy chain antibody. Thirdly, LC formed rosettes with sheep red blood cells (SRBC) coated with IgE kappa. Epidermal LC from normal non-atopic controls, did not form rosettes with SRBC-IgE. The SRBC-IgE rosette formation could be inhibited by preincubation with IgE kappa and BB10 (MoAb directed against the Fc receptor for IgE on human eosinophils, platelets and macrophages), but also with human IgG, whereas the SRBC-IgG rosette formation could be inhibited neither by IgE kappa nor by BB10. Both the SRBC-IgE and the SRBC-IgG rosette formation could be inhibited by OKT6 (anti-CD1) antibody. The results of inhibition studies with OKT6 antibody on the reconstitution of IgE on epidermal LC after acid elution suggest an associated expression of the CD1 antigen and the Fc receptor for IgE.