Isolation of monotreme T-cell receptor α and β chains

Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the mammalian immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR -chain cDNAs (TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR -chain cDNA (TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.     

Isolation and sequence analysis of cDNA for the dog T-cell receptor Tcrα and Tcrβ chains

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M97510 and M97511.     

The helper T-cell repertoire of mice expressing class II major histocompatibility complex β chains in the absence of α chains

 Mutant mice generated by disrupting the H2-Aa ^b major histocompatibility complex (Mhc) gene are demonstrated here to express Aβ^b chains in the absence of α chains. These mice possess a CD4⁺ helper T cell (Th) repertoire that uses predominantly the Vβ7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the α-free Aβ molecule. As an alloantigen, the Aα-free Aβ molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal α/β heterodimers. Indeed, molecular modeling suggests a β/β homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vβ usage, toward a series of foreign α/β class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal α/β heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature. Received: 30 September 1996 / Revised: 18 October 1996     

Sequence analysis of sheep T-cell receptor β chains

The nucleotide sequences reported in this paper have been submitted to the GenBank database and have been assigned the accession numbers M94181-M94183.     

Haptoglobin-hemoglobin binding involves the heavy chain of haptoglobin and the α and β chains of hemoglobin

Previous studies from this laboratory have demonstrated unambiguously that the isolated β chain of human adult hemoglobin binds human haptoglobin (Hp). In the present work, the ability of the isolated subunits of haptoglobin and hemoglobin to form complexes is investigated. In quantitative radiometric adsorbent titrations, the H chain of haptoglobin bound to hemoglobin whereas the L chain had no binding activity. Also, the H chain of haptoglobin bound to the isolated α and β subunits of hemoglobin, but its binding to the α or β chain was less than the binding it exhibits to hemoglobin. The isolated L chain was able to reassociate with the H chain to form a complex that binds to hemoglobin or its subunits. Although the L chains had no binding activity, its association with the H chain increased the binding of the latter to Hb or its isolated α and β subunits suggesting a more indirect role for the L chain in haptoglobin-hemoglobin interactions.     

Structure and diversity of the T-cell receptor α chain in the Mexican axolotl

 Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) α chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRα-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRα-V segments were all provided by sequences belonging to the human TCRα-V1 family and the mouse TCRα-V3 and TCRα-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 α chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 α chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Cα domain had the typical structure of mammalian and avian Cα domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. Received: 12 June 1996 / Revised: 11 September 1996     

Active peptides from the carboxyl-terminal globular domain of laminin α2 and Drosophila α chains

The laminin α1 chain carboxyl-terminal globular domain (G domain) contains multiple biological activities. Recently, we identified five cell binding sequences from the G domain by screening with overlapping 12-mer peptides encompassing the entire domain. The structures of these five sequences in the α1 chain are conserved in the corresponding regions of the different laminin α chains. Here we characterize the adhesion activities of the corresponding peptide segments from both the mouse laminin α2 chain and Drosophila laminin α chain using peptide-coated plastic plates and peptide-conjugated Sepharose beads. Using several cell lines, the laminin α2 chain peptides showed cell attachment and/or spreading activities with cell type specificities. Cell spreading on MG-10 was inhibited by integrin antibodies. Four of the Drosophila laminin peptides showed cell attachment activities. These results suggest that biologically active regions in the G domain are conserved in the laminin α1 and α2 chains, and that these regions in laminin play an important role in cell surface receptor interactions.     

Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with heme

Kinetics of the reconstitution of hemoglobin from semihemoglobins and with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins and are occupied by heme in the and chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin , the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin . Correspondence to: Y. Kawamura-Konishi     

DQ α and β RFLP reveals the composition of the DQ molecule recognized by T-cell clones

Pst I RFLP, revealed with DQ and DQ probes, was compared with Taq I RFLP using a panel of DR-homozygous cell lines and HLA-typed family members. Taq I patterns, characteristic for each DR-associated DQ and allelic forms, were recognized in the homozygous state and then proven to segregate in the heterozygous members of informative families. The presence of both specific and chains was found to be necessary to form the type of DQ molecule specifically recognized by two alloreactive T-cell clones. Particular and associations also seem to be responsible for some Dw splits of the DRw6-positive cells. Taq I RFLP analysis may be more complex than the Pst I analysis, but is certainly more informative and complete, considering the type of information we were seeking by performing these types of experiments.Abbreviations used in this paper BSA bovine serum albumin - GLO glyoxalase - kb kilobase(s) - LCL lymphoblastoid cell line - MHC major histocompatibility complex - PBL peripheral blood lymphocyte - PLT primed lymphocyte test - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - SSC standard sodium citrate - SSCP sodium, sodium citrate, sodium phosphate - TBE Tris-borate, boric acid, ethylenediaminetetraacetate (EDTA) - TCGF T-cell growth factor     

Exploration of the activation pathway of Δα-Chymotrypsin with molecular dynamics simulations and correlation with kinetic experiments

Correlating the experimentally observed kinetics of protein conformational changes with theoretical predictions is a formidable and challenging task, due to the multitude of degrees of freedom (>5,000) in a protein and the huge gap between the timescale of the kinetic event of interest (ms) and the typical timescale of computer simulations (ns). In this study we show that using the targeted molecular dynamics (TMD) method it is possible to simulate conformational changes of the ms time range and to correlate multiple simulations of single pathways with ensemble experiments on both the structural and energetic basis. As a model system we chose to study the conformational change of rat-Δα-chymotrypsin from its inactive to its active conformation. This activation process has been analyzed previously by experimental and theoretical methods, i.e. fluorescence stopped-flow spectroscopy (FSF), molecular dynamics (MD) and TMD. Inspired by the results of these studies on the wild type (WT) enzyme, several mutants were constructed to alter the conformational pathway and studied by FSF measurements. In the present work WT and mutant N18G were subjected to multiple MD and subsequent TMD simulations. We report the existence of two main activation pathways, a feature of chymotrypsin activation that has been hitherto unknown. A method to correlate the energetics of the different pathways calculated by TMD and the kinetic parameters observed by experimental methods such as FSF is presented. Our work is relevant for experimental single molecule studies of enzymes in general. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induction of prophage λ by daunorubicin and derivatives correlation with antineoplastic activity

The antineoplastic drug daunorubicin and 15 other anthracyclines were tested for their ability to induce prophage λ in Escherichia coli K12. Prophage λ induction by daunorubicin was obtained in excision-repair deficient uvr⁻ bacteria at doses about 3-fold lower than in excision-repair proficient uvr⁺ cells; this suggests that some of the lesions produced in DNA by daunorubicin are subject to excision repair and may be adducts. Daunorubicin seems to be converted to active species capable of causing prophage inducing lesions in DNA by bacterial enzymes. The antineoplastic and prophage inducing potencies of the anthracyclines were compared in a blind test. These two parameters were correlated for two thirds of the compounds. Such a correlation supports the idea that the antineoplastic activity of the anthracyclines is a consequence of their capacity to damage DNA.     

NH2-terminal sequence of the α and β chains of human DC-1 antigen isolated from the JY cell line. Homology with murine I-A molecules

The DC-1 antigen has been isolated from the JY cell line (DR4, w6). The amino terminal sequences of its α and β chains are both reported and are homologous to the murine I-A antigens. The JY and previously reported LB DC-1 α chain sequences appear to be variants of the.DC α chains reported by other authors. The JY DC-1 β chain sequence appears to be identical with that deduced from a β chain cDNA clone and thus identifies this clone. The JY and LB DC-1 β-chains are clearly different since the latter has a blocked amino terminus.     

Apoptotic cell-mediated suppression of streptococcal cell wall-induced arthritis is associated with alteration of macrophage function and local regulatory T-cell increase: a potential cell-based therapy?

Introduction  

Experimental streptococcal cell wall (SCW)-induced arthritis is characterized by two successive phases of the disease. The acute phase occurs early and is associated with an inflammatory process and neutrophil infiltration into the synovium. The second chronic phase is related to effector T-cell activation and the dysregulation of macrophage function. Creation of an immunomodulatory environment has been attributed to apoptotic cells themselves, apoptotic cell uptake by phagocytes as well as a less sensibility of phagocytes capturing apoptotic bodies to activation. Therefore we evaluated the potential of apoptotic cell injection to influence the course of inflammation in SCW-induced arthritis in rats.