Inhibition of T-cell receptor beta-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck.

The variable region genes of the T cell receptor (TCR) alpha and beta chains are assembled by somatic recombination of separate germline elements. During thymocyte development, gene rearrangements display both an ordered progression, with beta chain formation preceding alpha chain, and allelic exclusion, with each cell containing a single functional beta chain rearrangement. Although considerable evidence supports the view that the individual loci are regulated independently, signaling molecules that may participate in controlling TCR gene recombination remain unidentified. Here we report that the lymphocyte-specific protein tyrosine kinase p56lck, when overexpressed in developing thymocytes, provokes a reduction in V beta--D beta rearrangement while permitting normal juxtaposition of other TCR gene segments. Our data support a model in which p56lck activity impinges upon a signaling process that ordinarily permits allelic exclusion at the beta-chain locus.     

Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.Abbreviations C-GAG, collagen type I-glycosaminoglycans - C-C6S, collagen type I-chondroitin-6-sulfate - C-H, collagen type I-heparin     

Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn)

Engagement of the clonotypic antigen receptor (TCR) on T lymphocytes provokes an activation response leading to cell proliferation and lymphokine secretion. To examine the molecular basis of T cell signaling, we generated transgenic animals in which a lymphocyte-specific nonreceptor protein-tyrosine kinase p59fyn(T) is 20-fold overexpressed in developing T lineage cells. Thymocytes from these mice, analyzed using both cellular and biochemical assays, were remarkably hyperstimulable. Moreover, the responsiveness of normal thymocytes to TCR-derived signals correlated well with the extent to which p59fyn was expressed in these cells. Overexpression of a catalytically inactive form of p59fyn substantially inhibited TCR-mediated activation in otherwise normal thymocytes. These effects are unique to p59fyn; overexpression of a closely related T cell-specific tyrosine kinase, p56lck, elicits dramatically different phenotypes. Our results suggest that p59fyn is a critically important component of the TCR signal transduction apparatus.     

Dirofilaria immitis: surface properties of third- and fourth-stage larvae

The objective of this study was to analyze surface properties of larval Dirofilaria immitis with potential relevance to protective immunity. Comparisons were made between third (L3)- and fourth-stage larvae (L4) based on their net surface charge, surface carbohydrate and antigen composition, ability to nonspecifically absorb host proteins, complement activation, and nonspecific cellular adherence. It was determined that L3 had a net negative surface charge, whereas L4 had either a neutral or weakly positive surface charge. The lectin Con A, but not any of the other lectins tested, bound only to the surface of L4, and not to that of L3. Monoclonal antibodies were prepared which reacted with the surface of L3 or with the surface of L4, but never both. L4 were found to nonspecifically adsorb host protein to their surfaces, whereas L3 did not. Both L3 and L4 were found to activate complement through the alternate pathway. Finally, nonspecific cellular adherence was found on L3 both in vitro and in vivo but not on L4. The surfaces of L3 and L4 were thus shown to be significantly different and, potentially, in ways which would have great impact in the generation and effectiveness of a protective immune response.     

Forms and intracellular distribution of alpha-D-mannosidases in murine liver and spleen

1. The intracellular distribution of alpha-D-mannosidase in homogenates of murine liver and spleen was investigated by differential and gradient density centrifugation. 2. In both tissues an enzyme with a neutral pH optimum was found in the cytosol together with an alpha-D-mannosidase with optimal activity between pH 5.5 and 6.0 which was also partially membrane-bound. 3. In liver the acidic alpha-D-mannosidase was obtained almost entirely in a particulate form distributed equally between a heterogeneous low density region and heavy density lysosomes. 4. The lysosomal form of the liver enzyme was purified to electrophoretic homogeneity and shown to be a glycoprotein composed of four identical subunits of molecular weight 65 kDa. 5. Antibody raised against the purified liver alpha-D-mannosidase immunoprecipitated a polypeptide from spleen which had the same molecular size. This acidic enzyme was the predominant type of alpha-D-mannosidase in spleen, but in contrast to liver, it was obtained mainly in a cytosoluble form, the remaining activity being present in the heterogeneous light density compartment. 6. Although both tissues contain the same molecular form of the acidic alpha-D-mannosidase, in murine spleen this enzyme does not appear to be associated with stable heavy density lysosomes.     

The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing

The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.