Association of SWAP-70 with the B cell antigen receptor complex.

SWAP-70 is a component of an enzyme complex that recombines Ig switch regions in vitro. We report here the cloning of the human cDNA and its B lymphocyte-specific expression. Although its sequence contains three nuclear localization signals, in small resting B cells, SWAP-70 is mainly found in the cytoplasm. On stimulation, SWAP-70 translocates to the nucleus. In activated, class-switching B cell cultures, it is associated with membrane IgG, but not IgM. The membrane Ig association requires a functional pleckstrin homology domain and is controlled by the C terminus. We suggest that SWAP-70 is involved not only in nuclear events but also in signaling in B cell activation.

Mapping of the SWAP70 gene to mouse chromosome 7 and human chromosome 11p15.

The protein SWAP-70 was isolated as part of a DNA recombination complex in B lymphocytes, where it is predominantly expressed. In resting B cells, SWAP-70 is found in the cytoplasm; upon B-cell activation, it is transported both into the nucleus and to the cell membrane, where it is associated with the B-cell receptor complex and may play a role in signal transduction. In the nucleus, its involvement in heavy-chain class switch recombination has been suggested. In this report, using restriction fragment length polymorphism, simple sequence length polymorphism, and fluorescence in situ hybridization, we map the chromosomal localization of the mouse and the human genes to syntenic regions of mouse mid Chromosome (Chr) 7 and human Chr 11p15.

Cellular, intracellular, and developmental expression patterns of murine SWAP-70.

SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.

Analysis of the expression of intercellular adhesion molecule-1 and MUC18 on benign and malignant melanocytic lesions using monoclonal antibodies directed against distinct epitopes and recognizing denatured, non-glycosylated antigen.

Intercellular adhesion molecule (ICAM)-1 and mucin isotype MUC18 were originally identified as melanoma progression antigens by monoclonal antibodies (MAb) generated in a search for molecules expressed by melanomas but not detectable on benign naevi. As MAb detect single epitopes whose accessibility may be modulated, a new panel of antibodies directed against distinct epitopes and reacting with denatured nonglycosylated antigen as well as native antigen were used to examine expression of these molecules on melanocytic lesions. The antibodies were analysed in a binding inhibition assay and divided into groups defining independent epitopes. Three anti-ICAM-1 and four anti-MUC18 antibodies representing these groups were then tested on frozen sections of 10 benign naevi and 10 melanoma lymph-node metastases. The anti-ICAM-1 antibodies demonstrated concordant reactivities on both the malignant and benign lesions and reacted with all samples suggesting that antibodies that detect differences in ICAM-1 expression between these two lesions detect altered epitopes. Three of the four antibodies directed to MUC18 showed concordant reactivities and indicated that this molecule was expressed in nine melanomas and three naevi. However, one antibody (MUC18BA.3) reacted strongly with all lesions indicating either crossreactivity with another melanocyte molecule or the expression of a different form of MUC18 on naevi.

Inducible expression of the beta 1 subunit of the sodium pump.

The Na,K-ATPase, or sodium pump, a ubiquitous transmembrane enzyme in higher eukaryotes, consists of an alpha and a beta subunit. Here we investigate the expression pattern of the two beta isotypes in mouse B cell lines. Neither primary cells nor cell lines express beta 2. Abelson virus-transformed pre-B cells express beta 1, while B lymphomas and plasmacytomas do not. Thus, beta 1 expression in transformed cells follows that of their untransformed counterparts. Some subclones of pre-B cell line 70Z/3 express beta 1, and others do not, but lipopolysaccharide induces the beta 1-negative cells to become beta 1-positive.

Loss of the beta 1 subunit of the sodium pump during lymphocyte differentiation.

The Na,K-ATPase, or sodium pump, is responsible for maintaining cellular volume and is involved in receptor-mediated endocytosis; it is a ubiquitous transmembrane enzyme in higher eukaryotes and consists of an alpha and a beta subunit. In the mouse, two isotypes of beta with no known function have been identified: beta 1 and beta 2. We have studied the expression of beta 1 and beta 2 in lymphocytes from bone marrow, spleen, peripheral blood, and thymus. The beta 2 subunit is not expressed in any of the lymphocytes tested. Pre-B lymphocytes and the majority of mature, resting B cells in the bone marrow express the beta 1 subunit, as do all pre-T cells and mature thymocytes. In the spleen and in blood, beta 1 expression defines subsets of T and B lymphocytes. Mitogen-stimulated T and B cells lose beta 1 expression and do not express beta 2. While there is no indication that there is a change in alpha subunit isoform expression as a result of lymphocyte activation or that it is expressed in smaller amounts, there is a switch in the expression of the beta isoform.

A quasi-monoclonal mouse.

As a model for studying the generation of antibody diversity, a gene-targeted mouse was produced that is hemizygous for a rearranged V(D)J segment at the immunoglobulin (Ig) heavy chain locus, the other allele being nonfunctional. The mouse also has no functional kappa light chain allele. The heavy chain, when paired with any lambda light chain, is specific for the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). The primary repertoire of this quasi-monoclonal mouse is monospecific, but somatic hypermutation and secondary rearrangements change the specificity of 20 percent of the antigen receptors on B cells. The serum concentrations of the Ig isotypes are similar to those in nontransgenic littermates, but less than half of the serum IgM binds to NP, and none of the other isotypes do. Thus, neither network interactions nor random activation of a small fraction of the B cell population can account for serum Ig concentrations.

A simpler sort of antibody.

The monoclonal antibody NEMO is directed against a molecule expressed by human cells of the melanocytic lineage. Although obtained by conventional immunization and fusion procedures, NEMO consists solely of kappa light chain. SDS/PAGE analysis indicates that the kappa chains are present as both monomers and dimers. When these two forms were separated by gel filtration, only the monomeric form bound antigen. As kappa light chains from the myeloma MOPC-41 and the hybridoma MORK do not bind to the melanocytic cells, we conclude that the binding specificity of NEMO resides in the variable region.

High resolution of multiple forms of red blood cell enzymes using a Toyopearl DEAE 650 S.

We have investigated a new anion exchange chromatographic support (Toyopearl DEAE 650 S) which simultaneously allows easily to remove hemoglobin from hemolysates and to obtain a very high resolution of enzymes present in multiple forms. The results obtained are better than those obtainable using an anion-exchange HPLC column. The data obtained at analytical level suggest a wider use of this new matrix also for preparative purposes without significant changes in the resolution.

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.

Analysis of amino acids as DABS-derivatives with a sensitivity to the femtomole level using RP-HPLC narrow-bore columns.

In this paper we report the complete separation of amino acids as DABS-derivatives using a 3µm Supelcosil LC-18 (25 cm × 2.1 mm I.D.) narrowbore column. The system described makes it possible to perform the analysis of DABS-amino acids with a sensitivity to the femtomole level. We have also studied the conditions necessary for using the narrow-bore columns for routine analysis, paying particular attention to the problem of providing adequate protection for the analytical column. We have found it very suitable to use a (2 cm × 2.1 mm I.D.) guard column filled with a 40µm Pelliguard LC-18, pellicular packing resin, without affecting the complete resolution of the DABS-amino acids. Comparing the results obtained using conventional HPLC columns (3-5µm Supelcosil LC-18) of different lengths (15 and 25 cm × 4.6 mm I.D.) with those obtainable with the narrow-bore columns used in this work, it is possible to achieve a much greater sensitivity using the narrow-bore columns. In short, using the appropriate guard column and the "standard" HPLC apparatus used, the narrow-bore columns are very useful for routine analyses of DABS-amino acids with a sensitivity at the femtomole level.

Free radicals promote "in vitro" a different intracellular decay of rabbit reticulocyte and erythrocyte glycolytic enzymes.

Rabbit red blood cells (RBC) were exposed in vitro to an oxygen-radical-generating system represented by iron and ascorbic acid. Under these experimental conditions we have investigated the effect of this system on some intracellular rabbit reticulocyte and erythrocyte enzymes. The results obtained have shown a pronounced decay of hexokinase activity both in the erythrocytes and reticulocytes when exposed to these radical species. We have found that the amount of hexokinase inactivated is at least three times higher in a blood sample with a percentage of reticulocytes of 50-60%. This different behaviour of the hexokinase decay in the erythrocytes and reticulocytes could be due to its different intracellular distribution related to the two distinct cells. In addition we have evaluated some important intracellular compounds involved in maintaining the redox and the energetic state of the cell such as the reduced glutathione and the adenine nucleotides and their degradation products, in order to understand if there is any correlation between the hexokinase decay and a change concerning the metabolic conditions of the rabbit reticulocytes and erythrocytes exposed to free radicals.