Cloning of a lymphocyte homing receptor reveals a lectin domain

Lymphocytes express cell surface molecules, termed homing receptors, that mediate their selective attachment to specialized high endothelial venules found within secondary lymphoid organs. Previous work has demonstrated that the adhesive interaction between lymphocytes and the endothelium of peripheral lymph nodes appears to involve a lectin-like activity. Moreover, MEL-14, a monoclonal antibody that blocks lymphocyte-peripheral lymph node binding and presumably recognizes the homing receptor mediating this adhesive interaction, appeared to detect the lectin-like receptor. In this paper we describe the cloning of a murine cDNA that encodes the antigen recognized by the MEL-14 antibody. Characterization of the cDNA encoding the putative mouse peripheral lymph node-specific homing receptor shows that it contains a lectin domain that appears to be involved in the binding of lymphocytes to peripheral lymph node endothelium, thus defining a new type of cellular adhesion molecule. This result supports a novel mechanism for the distribution of lymphocyte populations to various lymphoid organs.     

Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.     

Bioethics and academic freedom

The author describes the events surrounding his attempts to lecture on the subject of euthanasia in West Germany in June 1989. Singer, who defends the view that active euthanasia for some newborns with handicaps may be ethically permissible, had been invited to speak to professional and academic groups. Strong public protests against Singer and his topic led to the cancellation of some of his engagements, disruptions during others, and harrassment of the German academics who had invited him to speak. These incidents and the subject of euthanasia became matters of intense national debate in West Germany, but there was little public or academic support for Singer's right to be heard. Singer argues that bioethics and bioethicists must have the freedom to challenge conventional moral beliefs, and that the events in West Germany illustrate the grave danger to that freedom from religious and political intolerance.     

Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line

Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.     

Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos

We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.     

Comparative study of the tyrosine phosphorylation of proteins in Swiss 3T3 fibroblasts stimulated by a variety of mitogenic agents

We have carried out a comparative study of the protein tyrosine phosphorylation induced by a wide range of mitogenic stimuli on a single cell type, Swiss 3T3 mouse fibroblasts. For this purpose we have used high-affinity antibodies directed to phosphotyrosine residues on proteins (Wang: Mol. Cell. Biol. 5:3640-3643, 1985) in immunoblotting and immunofluorescence microscopy experiments. Immunoblotting experiments showed that all of the mitogens tested, including epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, fetal calf serum, trypsin, and 12-O-tetradecanoylphorbol-13-acetate, increased the phosphorylation on tyrosine of a number of proteins. Most of the increase in tyrosine phosphorylation induced by each factor involved a small set of proteins with apparent molecular weights (Mr) above 50,000. Following stimulation with epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor, increased phosphotyrosine modification of proteins with molecular weights corresponding to those of the respective receptors was observed. A protein band of apparent Mr 160,000 contained substantially increased levels of phosphotyrosine following insulin treatment, but tyrosine phosphorylation of the insulin receptor was apparently below the level of detectability. The phosphotyrosine content of proteins with apparent Mr of 220,000, 120,000, and 70,000 was increased by all the agents tested. Phosphorylation on tyrosine of most of the proteins increased within a few minutes of the mitogenic stimulation, reached a peak, and returned more slowly to basal levels. Immunofluorescence labeling with the antibodies specific for phosphotyrosine showed a substantial increase in the amount of phosphotyrosine containing proteins only in the presence of platelet-derived growth factor and fetal calf serum. This finding suggests that most of the proteins phosphorylated on tyrosine in Swiss 3T3 fibroblasts are not concentrated in specific subcellular structures, but rather are diffusely distributed throughout the cell and are therefore not detectable by immunofluorescence microscopy.     

Induction of anti-CD8 resistant cytotoxic T lymphocytes by anti-CD8 antibodies. Functional evidence for T cell signaling induced by multi-valent cross-linking of CD8 on precursor cells

The effector function of most MHC class I allospecific CTL is inhibited by anti-CD8 mAb. In the present study, we report the surprising observation that multi-valent cross-linking of CD8 molecules on precursor cells by specific antibody actively induces the generation of CD8+ class I allospecific CTL whose lytic function is resistant to anti-CD8 antibody inhibition, and actively induces down-modulation of cell surface CD8 expression on these cells. In marked contrast, bi-valent cross-linking of CD8 inhibits the generation of CD8+ CTL from precursor cells and fails to induce down-modulation of cell surface CD8 expression. These results demonstrate that CD8 can transduce net positive signals, but only when the molecule is extensively cross-linked.     

Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

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.