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Biomedical subjects

H D Royer

Publications and source records attributed to H D Royer.

At least 37 records · Page 2Linked to original sources
PubMed1991

Delineation of human papillomavirus type 18 enhancer binding proteins: the intracellular distribution of a novel octamer binding protein p92 is cell cycle regulated.

The enhancer of human papillomavirus type 18 consists of two functionally redundant domains, one is partially conserved between HPV18 and HPV16, both mediate strong transcriptional enhancement. In contrast, short fragments of the enhancer mediate low transcriptional enhancement, suggesting that there is functional cooperation between HPV enhancer binding factors. Previously interactions of the enhancer with NF-1, AP1 and steroid receptors were shown by EMSA. Here we show by binding site blotting, that four novel sequence specific proteins p110, p92, p42 and p40 bind to the enhancer. Nuclear proteins p110 and p92 bind at repeated sites in the enhancer, proteins p42 and p40 only at one site. Recognition sequences for p110 and p92 were identified in a TTGCTTGCATAA sequence motif and consist of an overlapping p110 and p92 recognition site. The specific interaction of p110 with G residues of this 12 nucleotide long sequence was demonstrated by a mutant recognition site. Single recognition sites for p42 and p40 were localized in the enhancer by the use of overlapping oligonucleotides. In addition, electrophoretic mobility shift analysis identified Oct-1 and AP2 interactions with the enhancer. The AP2 binding site was mapped to a AGGCACATATT motif. The p92 protein binds to enhancer oligonucleotides, containing at least one copy of Oct-1 like recognition sequences, these oligonucleotides also bind synthetic Oct-1 protein. During serum starvation or at high saturation density, p92 moves from the nucleus into the cytoplasm. Immunoblots of cytoplasmic extracts with anti-Oct-1 antisera showed, that p92 is a novel octamer binding factor, which is not immunologically related to the Oct-1 protein. The intracellular p92 distribution is regulated at the G0/G1 boundary of the cell cycle, by nucleo-cytoplasmic translocation.

Base Sequence↗
PubMed1987

Multiple nuclear proteins bind upstream sequences in the promotor region of a T-cell receptor beta-chain variable-region gene: evidence for tissue specificity.

DNA-nuclear protein binding interactions were examined in the promoter region of a representative T-cell receptor Ti beta-chain variable-region gene by means of electrophoretic mobility-shift and DNase I-protection analysis. Within 175 bases upstream of the transcription initiation site, four protected regions ("footprints") were identified on the coding strand, at nucleotides -46 to -68 (I), -72 to -92 (II), -113 to -134 (III), and -136 to -175 (IV). Nuclear proteins (0.6 M NaCl fraction from a heparin-Sepharose column chromatography of nuclear extracts) of a variety of cell types produced footprints I, III, and IV and a fifth footprint (beyond nucleotide -200). In contrast, footprint II was produced only by T-cell extracts, although nuclear extracts of a transformed B-lymphoblastoid line produced a partial footprint in this region. Furthermore, footprint analysis of the noncoding strand showed that a continuous region of protection corresponding to the entire region of footprints I and II was generated, along with a DNase I-hypersensitive site, by nuclear proteins derived from T cells but not other cell types. Footprint I has the sequence structure of many well-defined protein-DNA binding sites. Nucleotide sequences in the region of footprint II bore no homology to known sequences, whereas those in the areas of footprints III and IV were similar to motifs within immunoglobulin and other enhancers. These findings may have implications for the tissue specificity of human Ti beta-chain gene transcription.

Base Sequence↗
PubMed1987

The human T cell receptor for antigen: structure, ontogeny and gene expression.

T cell receptor molecules are now well characterized as well as the genes encoding alpha and beta chains of this molecular complex. The genome organisation of alpha and beta chain genes is similar to the genomic organisation of immunoglobulin genes. In T cell differentiation immature precursor cells move into the human thymus, where they mature and subsequently are released into the periphery as immuno-competent T cells with a variety of different functions. The processes at work in thymic ontogeny are understood, only in part, and await further study. One aspect of T cell differentiation is the acquisition of immunocompetence by T cells in thymic ontogeny. This process is associated with T cell receptor gene rearrangements and T cell receptor gene expression. The mechanisms leading to gene expression have been studied in many systems and basic principles are now emerging. The enzyme RNA polymerase II, which synthesizes m-RNA requires additional factors for its activity, and these factors have, at least in part, been identified as proteins. Experiments aimed at identifying DNA-protein interactions at the V beta upstream regulatory region are discussed.

Gene Expression Regulation↗
PubMed1986

Expression of the NKTa clonotype in a series of human natural killer clones with identical cytotoxic specificity.

Over a period of 3 yr, a series of ten NK clones that express a unique clonotypic T cell receptor-like structure, termed NKTa, has been generated from a single individual. These clones were derived from either peripheral blood nonadherent cell fractions (JT9, JT10, JT11), NKH2-purified cells (CNK8, CNK9), or NKTa-purified cells (CNK11, CNK12, CNK13, CNK14, CNK15). Flow cytometric analysis of peripheral blood mononuclear cells from this individual showed that NKTa+ cells occur with a frequency of approximately 0.15%. The existence of NKTa+ cells in peripheral blood was confirmed by use of immunorosette enrichment techniques, flow cytometric purification, and subsequent clonal expansion of NKTa+ cells. Phenotypic analysis of NKTa+ clones showed that all expressed NKH1 as well as T3, T8, T11, T12, and Mo1 antigens. Only five of ten clones expressed NKH2 antigen. All NKTa+ clones had broad cytolytic activity against a series of seven different target cells that was similar to that of other NK clones. In addition, cytotoxicity of each clone could be inhibited by preincubation of effector cells with monoclonal anti-NKTa or by preincubation of target cells with monoclonal anti-TNKTAR. Although half of the NKTa+ clones appeared phenotypically different from the other half with regard to the expression of NKH2 antigen, analysis of T cell receptor gene rearrangements indicated that all NKTa+ clones contained identical gene rearrangements of C beta 2.

Antibodies, Anti-Idiotypic↗
PubMed1986

The ontogeny, structure and function of the human T lymphocyte receptor for antigen and major histocompatibility complex.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 kDa Ti heterodimer and the invariant 20 and 25 kDa T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunological competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous interleukin-2 production, release and subsequent binding to interleukin-2 receptors. The implications of these findings for understanding of human T cell growth and its regulation in disease states are discussed.

Amino Acid Sequence↗
PubMed1986

The structure and function of the T3-Ti molecular complex on human T lymphocytes.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex composed of a clonotypic 90-kDa Ti heterodimer and the invariant 20- and 25-kDa T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The Ti alpha and Ti beta subunits bear no precursor-to-product relationship and are encoded by separate germ line V, D, J, and C segments, which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous IL 2 production, release, and subsequent binding to IL 2 receptors.

Amino Acid Sequence↗
PubMed1985

Regional location of T cell receptor gene Ti alpha on human chromosome 14.

The chromosomal location of Ti alpha was determined by hybridization of a radiolabeled cDNA for the alpha chain of human T cell receptor with 12 human X mouse cell hybrid DNAs cleaved with BamHI. Seven hybrids contained human Ti alpha, while the remaining five lacked it. Only human chromosome 14 matched the distribution of human Ti alpha signal across the mapping panel. Hybrids segregating a chromosome 14 translocation were used to demonstrate that Ti alpha is in the region 14pter greater than 14q21. Thus, the alpha and beta chain genes that contribute structural components to the Ti moiety of the human T cell receptor lie on different chromosomes. In humans, the immunoglobulin heavy chain locus and Ti alpha are in different regions of chromosome 14, with Ti alpha more proximal and the immunoglobulin heavy chain locus more distal.

Animals↗
PubMed1985

Analysis of T-cell receptor gene rearrangement and expression in human natural killer clones.

A series of clones of human natural killer (NK) cells was characterized with respect to expression of the Ti alpha and Ti beta genes of the T-cell receptor. T11+T3+ NK clones contained Ti alpha and Ti beta RNA transcripts and expressed disulfide-linked heterodimers, demonstrating the presence of a functional T-cell receptor. In contrast, T11+T3- NK clones expressed only 1.0-kilobase truncated Ti beta transcripts, without a Ti alpha transcript and no detectable surface Ti protein. Since previous studies demonstrated that Ti beta gene activation precedes Ti alpha gene activation in thymic ontogeny, the T11+T3- NK cells appear to be derived from T-lineage precursors.

Clone Cells↗
PubMed1985

Molecular analysis of T cell receptor (Ti) variable region (V) gene expression. Evidence that a single Ti beta V gene family can be used in formation of V domains on phenotypically and functionally diverse T cell populations.

We examine the rules governing Ti beta variable (V) gene segment usage in the formation of T cell antigen-MHC receptors in diverse regulatory and effector T lymphoid subpopulations. To this end, a single Ti beta V gene family and its products were analyzed. A monoclonal antibody, termed anti-Ti3A, which was shown to be reactive with an epitope encoded by members of the REX cell line Ti beta V gene family, and which was expressed on 2% of human T lymphocytes was used in selection of clones from unprimed peripheral T lymphocytes. Both T4+, as well as T8+ T cell clones with inducer, suppressor, and/or cytotoxic function were defined. Southern analysis, isoelectric focusing and two-dimensional peptide mapping indicated that individual members of the REX V gene family were linked to different Ti beta diversity and/or joining and constant region segments. Moreover, the Ti alpha chains of such clones were distinct. These results imply that Ti beta V gene usage is not restricted to any functionally or phenotypically defined T cell subsets, and there is presumably little, if any, restriction on the mechanisms that generate combinational, junctional or chain association-mediated diversity.

Antibodies, Monoclonal↗
PubMed1985

The human T-cell receptor.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to the identification of the human T-cell antigen receptor as a surface complex comprised of a clonotypic 90-kD Ti heterodimer and the invariant 20- and 25-kD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate genes. Moreover, the presence of unique peptides following proteolysis of different Ti molecules isolated by non-cross-reactive anticlonotypic monoclonal antibodies supports the notion that variable regions exist within both the alpha and the beta subunits. N-Terminal amino acid sequencing and molecular cloning of the Ti beta subunit further show that it bears an homology to the first V-region framework of immunoglobulin light chains and represents the product of a gene that rearranges specifically in T lymphocytes. Triggering of the T3-Ti receptor complex gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors. The implications of these findings for understanding human T-cell growth and its regulation in disease states are discussed.

Amino Acid Sequence↗
PubMed1985

Genes encoding the T-cell receptor alpha and beta subunits are transcribed in an ordered manner during intrathymic ontogeny.

To further characterize sequential events involved in activation of genes encoding the T-cell receptor (a complex of T3 molecules and a disulfide-linked heterodimer designated Ti, T3-Ti) for antigen and the major histocompatibility complex during intrathymic ontogeny, cDNA probes specific for Ti alpha and Ti beta subunits were used for transcriptional analysis. Ti beta transcript levels were minimal in stage I thymocytes, maximal in stage II thymocytes, and intermediate in state III thymocytes. In contrast, Ti alpha transcriptional activity was virtually undetectable in stage I, was low in stage II, and achieved high levels only in the stage III compartment. Analysis of tumor populations derived from individual stages of thymic differentiation confirmed these observations and demonstrated that clonal stage I-II cells often express Ti beta RNA in the absence of Ti alpha RNA. The latter was not a consequence of functional Ti alpha-subunit isotypy because each of 13 interleukin 2-dependent T-cell clones, including inducer, suppressor, and cytotoxic T cells, contain transcripts that hybridize with the Ti alpha probe. From these data it is concluded that Ti beta gene activation precedes Ti alpha gene activation. Moreover, the high level of Ti beta mRNA preceding Ti alpha mRNA expression implies that Ti beta mRNA and/or its protein products may regulate Ti alpha gene transcription.

Aging↗
PubMed1985

The human T lymphocyte receptor complex for antigen and MHC.

Recent studies using cloned antigen specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 KD Ti heterodimer and the in-variant 20 and 25 KD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors. The implications of these findings for understanding of human T cell growth and its regulation in disease states are discussed.

Amino Acid Sequence↗
PubMed1984

Chromosomal location of human T-cell receptor gene Ti beta.

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.

Animals↗
PubMed1984

Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex.

Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) showed rearrangement of the TiC beta 1 gene on Southern blot analysis with or without deletion of the other TiC beta 1 allele. In contrast, TiC beta 2 always remained in germline configuration. Moreover, the finding that one additional suppressor clone deleted both TiC beta 1 alleles, maintained a germline TiC beta 2 configuration, and yet actively transcribed TiC beta 2 message suggested that TiC beta 2 is not a pseudogene. Rather, it appeared to be used less frequently than the TiC beta 1 gene and in the absence of detectable DNA rearrangements. Together, these results demonstrate that the functional repertoire (or isotype) of a given subclass of T cells is not encoded within the Ti beta genes.

Clone Cells↗
PubMed1984

Anti-idiotypic antibodies as probes of cell surface receptors.

Anti-idiotypic antibodies have proven to have unique applications as probes in both functional and biochemical studies of cell surface receptors. Anti-idiotypic receptor antibodies have been prepared to antibodies which bind to purified ligand, as in the case of insulin, retinol-binding protein, the mammalian reovirus receptor, and the neutrophil chemotactic receptor, and to natural ligand analogs, such as the beta-adrenergic antagonist alprenolol. These systems have documented the usefulness of anti-idiotypic antibodies in the quantitation and modulation of specific membrane receptors on a variety of cell types. Anti-idiotypic antibodies have also been utilized for the isolation of specific membrane receptors, e.g., reovirus and B-1H globulin receptors. Some anti-idiotypic receptor antibodies, e.g., insulin and reovirus systems, have been shown to mimic the physiological properties of ligand upon binding to cellular receptors. These antibodies enable a new dimension of both receptor based cellular studies and therapeutic regimens. This review focuses on the past use of anti-idiotypic antibodies as probes of cell surface receptors, and on the methodologies required for the successful application of anti-idiotypic antibodies for use in further membrane receptor studies, and of the genes which encode and regulate these receptors. We also discuss the use of anti-idiotypic antibodies in the understanding of and therapeutic approach to receptor related diseases.

Animals↗