Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

In vitro inhibition, by loratadine and descarboxyethoxyloratadine, of histamine release from human basophils, and of histamine release and intracellular calcium fluxes in rat basophilic leukemia cells (RBL-2H3).

The effect of the H1-antihistamine drug loratadine and its active metabolite descarboxyethoxyloratadine upon histamine release was examined on anti-immunoglobulin E (IgE) triggered human basophils and 2,4-dinitrophenyl (DNP) triggered rat basophilic leukemia (RBL-2H3) cells. In both experimental systems, dose-dependent inhibition of histamine release was observed at descarboxyethoxyloratadine and loratadine doses above 2 and 7 microM, respectively. In the RBL-2H3 experimental system, inhibition by loratadine increased when the concentration of extracellular Ca2+ was reduced from 1.8 to 0.45 mM. We further investigated the effect of loratadine and descarboxyethoxyloratadine on the increase in cytosolic calcium concentration (Ca2+)i, an early step in biochemical events leading to exocytosis. The effect of these two drugs upon (Ca2+)i changes was measured using the fluorescent probe fura-2 loaded into RBL-2H3 cells passively sensitized with DNP-specific IgE. Both drugs inhibited, in a dose-dependent manner (2.5-25 microM), the (Ca2+)i rise induced by DNP-BSA challenge in sensitized RBL cells, a process observed in both the presence and absence of extracellular Ca2+. Loratadine also inhibited the Mn2+ influx into these cells, thus reflecting the Ca2+ influx. These results suggest that loratadine and descarboxyethoxyloratadine impair the increase in (Ca2+)i following cell activation by decreasing both the influx of extracellular Ca2+ and the release of Ca2+ from intracellular stores.

Expression of the alpha chain of human Fc epsilon RI in transfected rat basophilic leukemia cells: functional activation after sensitization with human mite-specific IgE.

The actual dilemma in studying the binding and triggering capacity of IgE from allergic patients is the lack of cultured basophils or mast cell analogs of human origin. Human IgE binds with exquisite species specificity to the high affinity IgE receptor (Fc epsilon RI) expressed on the surface of these cells. In rodents this receptor has been characterized as a tetrameric plasma membrane protein composed of an IgE-binding alpha chain, a beta chain and two disulfide-linked gamma chains. In order to establish a cell line expressing the alpha chain of human Fc epsilon RI which can be triggered with IgE from human patients and specific allergen, we transfected the cDNA coding for the human alpha subunit into rat basophilic leukemia cells. The resulting transfectants express the human alpha chain on the cell surface in the form of a hybrid complex associated with endogenous rat gamma chains. After sensitization with human IgE from mite-specific patients, the transfectant produces a calcium response upon incubation with allergen. The established cell line can be used as a model system to study the mechanism of mast cell triggering through IgE from allergic patients.

Inhibition of DNA synthesis and DNA fragmentation in stimulated splenocytes by the concerted action of topoisomerase I and II poisons.

Stimulated splenocytes were used as a model system to investigate the effects of topoisomerase inhibitors on normal, non-transformed, non-tumoral proliferating cells. The concerted action of camptothecin (a poison of topoisomerase I) and etoposide (a poison of topoisomerase II) lead to nearly complete inhibition of DNA synthesis in concanavalin A-stimulated splenocytes. Analysis of replicated cellular DNA after a short treatment with both drugs revealed a DNA cleavage to medium size fragments. This effect was additive, suggesting that cleavable complexes were formed independently by both topoisomerases on their respective DNA sites. In contrast, prolonged contact with both drugs was followed by degradation of the bulk cellular DNA to nucleosome size fragments, indicating that apoptosis took place in these cells. Combination of camptothecin and etoposide enhanced this phenomenon, consistent with the fact that degradation was the result of secondary events which may amplify the signal. Thus, aphidicolin, an inhibitor of eukaryotic replicases which blocks replication, also triggered DNA degradation in proliferating splenocytes.

Topoisomerase inhibitors induce irreversible fragmentation of replicated DNA in concanavalin A stimulated splenocytes.

Etoposide, a nonintercalative antitumor drug, is known to inhibit topoisomerase II. Its effects have been tested in concanavalin A stimulated splenocytes, a system of cell proliferation in which topoisomerase II is induced. The primary effect of etoposide was a strong inhibition of DNA synthesis and the production of reversible DNA breaks, presumably associated with topoisomerase II. However, prolonged (20 h) contact with the drug resulted in a secondary fragmentation by irreversible double-strand breaks that yielded unusually small DNA fragments. Surprisingly, the same effect was obtained with novobiocin, which does not produce topoisomerase II associated DNA breaks. Moreover, long-term treatment with camptothecin, a specific inhibitor of topoisomerase I which is known to induce single-strand breaks in vitro and in vivo, also produced double-strand breaks and DNA fragmentation into small pieces. These findings suggest that prolonged treatment of proliferating splenocytes by etoposide and other topoisomerase inhibitors induced DNA fragmentation by a mechanism that does not directly involve topoisomerases.

DNA topoisomerase activities in concanavalin A-stimulated lymphocytes.

Topoisomerase activities have been measured in nuclear extracts of concanavalin A-stimulated lymphocytes. In parallel with the wave of DNA synthesis, type II topoisomerase activity was considerably increased. After 72 h treatment, this activity was stimulated approx. 20-fold over the activity in untreated cells. In contrast, type I topoisomerase was poorly stimulated after 24 h treatment, and 4-5-fold after 72 h. These findings, together with our previous results on regenerating rat liver, suggest a major role of topoisomerase II in DNA replication.

Threonine starvation of concanavalin A treated lymphocytes impairs DNA polymerase alpha activity.

When cultured in a threonine-deficient medium, concanavalin A treated guinea-pig lymphocytes do not incorporate tritiated thymidine. DNA polymerase activity is strongly affected. The addition of the missing amino acid is followed by an early increase in protein and RNA synthesis and a delayed rise in DNA polymerase alpha activity associated with the onset of DNA synthesis.

Influence of amino acid deficiency and tRNA aminoacylation on DNA synthesis and DNA polymerase activity during the secondary immune response in vitro.

DNA synthesis and DNA polymerase activity are increased when KLH-primed guinea-pig lymphocytes are restimulated in vitro with the homologous antigen. This response can be modulated by glutamine deficiency and by an inhibitor of the histidyl-tRNA synthetase.

Influence on certain amino acids on steps leading to DNA synthesis in concanavalin A-treated guinea-pig lymphocytes.

When guinea-pig lymph node cells were exposed to ConA in a culture medium lacking glutamine or cysteine, no DNA synthesis occurred. The addition of the missing acid to ConA-treated lymphocytes submitted to glutamine or cysteine starvation for 40 h allowed the synthesis of DNA to take place after a period of only 10-12 h. The synthesis of DNA is preceded by a rapid increase of 3H-uridine incorporation into RNA and of 3H-leucine incorporation into protein which occurred a few hours after addition of the missing amino acid. When cycloheximide was added to lymphocytes exposed to ConA in a glutamine or cysteine deprived medium, a relative enhancement of uridine incorporation was observed. No such effect was provoked by puromycin. These results suggest the possibility of a control system in lymphocytes similar to those described in microbial cells for amino acid control of RNA synthesis.