E-cadherin expression can alter the specificity of gap junction formation

Specificity of gap junction formation produces communication compartments, groups of cells joined to each other by gap junctions (homologous communication) but more rarely to cells in adjacent compartments (heterologous communication). Specificity of junction formation can be studied in mixed cultures of different cell types. In these model systems, compartmentation is often associated with sorting out, a process that produces separate domains of the different cells. The borders of the physically distinct domains correlate with the functional boundaries of the communication compartments. Compartments have also been observed in vivo where they are believed to play a role in separating groups of cells following different differentiation pathways. Two classes of cell surface molecule, connexins and cell adhesion molecules, are candidates for a role in the control of specificity. A representative of each class appears to be necessary for gap junction formation and both are expressed in a tissue specific manner. We have shown that mixed cultures of rat epithelial (BRL) cells and rat (BICR) fibroblasts show specificity, form communication compartments and sort out. Both cell types express the same connexin (connexin 43) but different cell adhesion molecules (BRL, P-cadherin and 125-kDa N-cadherin; BICR, 140-kDa N-cadherin). Transfection of both cell types with E-cadherin results in a 10-fold increase in heterologous communication. These data suggest that E-cadherin plays a role in the control of specificity of gap junction formation.     

Dual specificity of Src homology 2 domains for phosphotyrosine peptide ligands

SH2 domains mediate protein-protein interactions and are involved in a wide range of intracellular signaling events. SH2 domains are 100-amino acid stretches of protein that bind to other proteins containing phosphotyrosine residues. A current major research goal is formulation of the structural principles which govern peptide-binding specificity in SH2 domains. Several structures (both X-ray and NMR) of SH2 domains have now been determined. Short peptide fragments on the carboxyl-terminal side of the phosphotyrosine residue carry the sequence specific information for SH2 recognition. The bound peptides are held in an extended conformation. However, for the GRB2 SH2 domain, the peptide adopts a beta-turn as the motif for recognition [Rahuel, J., et al. (1996) Nat. Struct. Biol. 3, 586-589]. Our SAR data and molecular modeling studies suggest that many SH2 domains, such as the SH2 domains of Lck, Src, and p85, can interact with high affinity with short peptide sequences at least in two ways which are sequence-dependent. The peptide forms either an extended chain across the D-strand of SH2 domains with anchors at pY and pY+3 or, as in the case of GRB2 SH2, a beta-turn with anchors at pY and pY+2. Due to a bulky tryptophan in its EF1 loop, GRB2 SH2 cannot bind peptide conformations such as the extended chain and thus has a unique specificity.     

Concerted formation of macromolecular Suppressor-mutator transposition complexes

Transposition of the maize Suppressor-mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA-DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein-protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range.     

Two-domain MHC class II molecules form stable complexes with myelin basic protein 69-89 peptide that detect and inhibit rat encephalitogenic T cells and treat experimental autoimmune encephalomyelitis

We designed and expressed in bacteria a single-chain two-domain MHC class II molecule capable of binding and forming stable complexes with antigenic peptide. The prototype "beta1alpha1" molecule included the beta1 domain of the rat RT1.B class II molecule covalently linked to the amino terminus of the alpha1 domain. In association with the encephalitogenic myelin basic protein (MBP) 69-89 peptide recognized by Lewis rat T cells, the beta1alpha1/MBP-69-89 complex specifically labeled and inhibited activation of MBP-69-89 reactive T cells in an IL-2-reversible manner. Moreover, this complex both suppressed and treated clinical signs of experimental autoimmune encephalomyelitis and inhibited delayed-type hypersensitivity reactions and lymphocyte proliferation in an Ag-specific manner. These data indicate that the beta1alpha1/MBP-69-89 complex functions as a simplified natural TCR ligand with potent inhibitory activity that does not require additional signaling from the beta2 and alpha2 domains. This new class of small soluble polypeptide may provide a template for designing human homologues useful in detecting and regulating potentially autopathogenic T cells.     

Studies on the formation and stability of aminoacyl-tRNA synthetase complexes from Ehrlich ascites cells

Nine aminoacyl-tRNA synthetases from Ehrlich ascites cells were examined with respect to their ability to be isolated as high molecular weight complexes, soluble enzymes, and ribosome-bound enzymes. Several different methods were employed for cell homogenization and enzyme isolation, with particular attention paid to the effects of hypotonic, isotonic, and hypertonic buffers on enzyme isolation. The binding of all synthetases to ribosomes was eliminated if the low ionic strength of the isolation buffer was raised to isotonic levels. In contrast, neither the ionic strength or composition of the buffers, nor the procedures used for cell homogenization or enzyme isolation had any significant effect on the isolation of the high molecular weight synthetase complex. Certain enzymes (lysyl-, methionyl- and isoleucyl-tRNA synthetases) formed very stable complexes and high molecular weight species were the predominant forms of these enzymes under all conditions of cell homogenization and enzyme isolation. Other enzymes (glycyl-, tyrosinyl- and threonyl-tRNA synthetases) formed complexes very weakly, if at all, and always appeared predominately in the soluble enzyme fraction. Isolated soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases did not associate to form significant amounts of complex upon re-isolATION, SUGGESTING THAT A COMPONENT NECESSARY FOR COMPLEX FORMATION WAS MISSING FROM THE SOLUBLE ENZYME FRACTION. However, the soluble forms of these enzymes, but not the glycyl-, tyrosinyl- and threonyl-tRNA synthetases, did for complexes when mixed with ribosomal RNA or polyuridylic acid. Preliminary experiments showed no significant differences between the complexed and soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases with respect to Km values or ability to charge different isoaccepting tRNAs.     

Binding constants in the formation of mammalian protein synthesis initiation complexes and the role of mRNA

Acute idiopathic thrombocytopaenic purpura is the most common of the thrombocytopaenias of childhood. Clinically it is associated with petechiae, mucocutaneous bleeding and occasionally haemorrhage into tissues. The oral mucosa is frequently involved. This paper describes a case presenting in general dental practice.     

On the formation of cytochrome P-450 product complexes during the metabolism of phenylalkylamines

Differences have been found, which are usually 1% or less, between the left ventricular ejection time measured from the external carotid pulse tracing, and from the rate of change of thoracic impedance (dZ/dt) waveform, using either the second heart sound or the X-point of the dZ/dt tracing as the end-point. The Heather Index obtained from the ECG and dZ/dt tracings has been correlated with other indices of cardiac performance. The changes observed in the physiological variables during head-up and head-down tilting were in the expected directions.     

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.     

Topoisomerase II.etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.     

Dynamics of serum IgM autoreactive repertoires following immunization: strain specificity, inheritance and association with autoimmune disease susceptibility

Immunization of Lewis rats with myelin basic protein (MBP) in complete Freud's adjuvant (CFA) provokes experimental allergic encephalomyelitis (EAE). Here we compare, irrespective of antigen specificity, the structure and dynamics of serum IgM autoreactive repertoires following immunization with MBP/CFA in EAE-susceptible Lewis and relatively resistant Fischer rats. Prior to the appearance of clinical symptoms, Lewis rats developed a specific modification of serum IgM autoreactivities that, scored on other determinants than MBP itself, showed a prognostic association with EAE symptoms. Although comparable in their production of MBP-specific serum IgM and IgG antibodies, Fischer rats did not share these MBP/CFA-induced IgM autoreactivities of Lewis rats when immunized in the same manner. Moreover, while the Lewis-type repertoire reaction was specific for MBP/CFA alone, the respective Fischer reaction was not qualitatively different from that observed in this strain upon non-pathogenic immunization with self-related or -unrelated antigens. In general, the repertoire reactions differed qualitatively between the strains, consisting of components with typical behavior and strain preferences. The EAE-associated, as well as the other components of both Lewis- and Fischer-type repertoire reactions were usually co-dominantly inherited in F1 animals. These results indicate that a global antibody repertoire analysis may serve as a tool to describe prototypical response structures, possibly involved in immune regulation and susceptibility to pathogenic autoimmunity.     

Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue

Activation of autoreactive T cells can lead to autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). The initiation and maintenance of IDDM by dendritic cells (DC), the most potent professional antigen-presenting cells, were investigated in transgenic mice expressing the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) under the control of the rat insulin promoter (RIP-GP mice). We show that after adoptive transfer of DC constitutively expressing the immunodominant cytotoxic T lymphocyte (CTL) epitope of the LCMV-GP, RIP-GP mice developed autoimmune diabetes. Kinetic and functional studies of DC-activated CTL revealed that development of IDDM was dependent on dose and timing of antigenic stimulation. Strikingly, repeated CTL activation by DC led to severe destructive mononuclear infiltration of the pancreatic islets but also to de novo formation of islet-associated organized lymphoid structures in the pancreatic parenchyma. In addition, repetitive DC immunization induced IDDM with lymphoid neogenesis also in perforin-deficient RIP-GP mice, illustrating that CD8(+) T cell-dependent inflammatory mechanisms independent of perforin could induce IDDM. Thus, DC presenting self-antigens not only are potent inducers of autoreactive T cells, but also help to maintain a peripheral immune response locally; therefore, the induction of autoimmunity against previously ignored autoantigens represents a potential hazard, particularly in DC-based antitumor therapies.     

Tissue layer and organ specificity of trichome formation are regulated by GLABRA1 and TRIPTYCHON in Arabidopsis

In animal development, cellular diversity is generated within tissues which in turn are derived from germ layers. Similar to the germ layers in animals, plants establish three distinct tissue layers early in development which each give rise to a distinct set of cell types. To investigate the role of tissue-layer-specific cues in generating plant cellular diversity we studied the spatial regulation of an epidermal cell type, trichomes (hairs), by the two genes, GLABRA1 (GL1) and TRIPTYCHON (TRY). Ubiquitous expression of the positive regulator GL1 in the absence of the negative regulator TRY leads to ectopic trichome formation not only on additional organs but also in subepidermal tissue layers. Trichomes in inner tissue layers can differentiate the same morphology and show a spacing pattern comparable to trichomes in the epidermis. This clearly shows that cell type specification takes place downstream of tissue-specific cues. We propose a model of how the tissue and organ specificity of trichome induction is regulated in normal development.