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R Hyman

Publications and source records attributed to R Hyman.

At least 37 records · Page 2Linked to original sources
PubMed1993

Antibody-induced activation of the hyaluronan receptor function of CD44 requires multivalent binding by antibody.

CD44 can function as a receptor for hyaluronan (HA). However, many cell lines and normal hematopoietic cells that express CD44 do not constitutively bind HA. A monoclonal antibody (mAb) specific for CD44 (IRAWB 14) has been described previously which induces CD44-mediated binding of HA rapidly (seconds to minutes) in some cell lines and in normal murine T cells. Of 16 CD44-specific mAb tested in the present study, only 3 exhibited this activity. Monovalent Fab fragments were prepared from two IgG2a antibodies that induce HA binding (IRAWB 14 and IRAWB 26) and used to determine whether multivalent binding was required for induction of HA receptor function. Fab from both antibodies had a tendency to form multivalent aggregates. After addition of iodoacetamide to prevent further aggregation, multimeric and monovalent forms were separated by gel filtration. This made it possible to compare the inducing activity of monovalent and multivalent antibody fragments of identical composition in the absence of Fc determinants. Multimeric forms were very active at inducing binding of fluorescein-conjugated HA (Fl-HA). Monovalent Fab fragments of both antibodies had 20- to 50-fold lower binding activity than intact antibody or multimer. IRAWB 26 Fab monomers were completely inactive in the induction of HA-binding. The observed weak inducing activity of IRAWB 14 Fab monomer could be attributed to very low levels of contaminating multimer. Induction of HA binding could also be achieved by using anti-immunoglobulin to cross-link Fab monomers of IRAWB 26. Thus, multivalent binding was required for the activation of HA binding by CD44-specific antibody, suggesting that the distribution of CD44 molecules on the cell surface is important for HA receptor function. In kinetic studies, induction of HA receptor function occurred simultaneously with antibody binding at 0 degrees C (ice water bath). Furthermore, antibody could induce HA binding in paraformaldehyde-fixed cells, which were permeable to propidium iodide and trypan blue, suggesting that intracellular signaling mechanisms were not involved in induction of receptor function. We conclude, therefore, that these CD44-specific antibodies are inducing HA binding by directly influencing the distribution of CD44 on the cell surface. The possibility of a concurrent change in CD44 conformation is not ruled out. We discuss possible mechanisms by which CD44 might be activated to bind HA in vivo.

Animals↗
PubMed1993

CD44 and its interaction with extracellular matrix.

It is now generally accepted that CD44 is a cell adhesion receptor and that hyaluronan is one of its ligands. Like many cell adhesion receptors, CD44 is broadly distributed, and its ligand, hyaluronan, is a common component of extracellular matrices and extracellular fluids. Yet a great variety of responses has been reported to result from CD44 ligation. These include cell adhesion, cell migration, induction (or at least support) of hematopoietic differentiation, effects on other cell adhesion mechanisms, and interaction with cell activation signals. This diversity of responses indicates that downstream events following ligand binding by CD44 may vary depending on the cell type expressing CD44 and on the environment of that cell. CD44 is expressed on cells in the early stages of hematopoiesis and has been shown to participate in at least some aspects of the hematopoietic process. In mature lymphocytes, CD44 is upregulated in response to antigenic stimuli and may participate in the effector stage of immunological responses. Along with other adhesion receptors that show alterations in expression after activation, CD44 probably contributes to differences in the recirculation patterns of different lymphocyte subpopulations. CD44 ligand-binding function on lymphocytes is strictly regulated, such that most CD44-expressing cells do not constitutively bind ligand. Ligand-binding function may be activated as a result of differentiation, inside-out signaling, and/or extracellular stimuli. This regulation, which in some situations can be rapid and transient, potentially provides exquisite specificity to what would otherwise be a common interaction. CD44 is not a single molecule, but a diverse family of molecules generated by alternate splicing of multiple exons of a single gene and by different posttranslational modifications in different cell types. It is not yet clear how these modifications influence ligand-binding function. The significance of the multiple isoforms of CD44 is not understood, but association of some isoforms with malignancies has been observed. And in at least some experimental systems, a contribution of CD44 isoforms to metastatic behavior has been demonstrated.

Animals↗
PubMed1993

Differential effects of expression of the CD45 tyrosine protein phosphatase on the tyrosine phosphorylation of the lck, fyn, and c-src tyrosine protein kinases.

Expression of the CD45 tyrosine protein phosphatase is required for the response of functional lymphocytes to stimulation through the antigen receptor. One or more of its substrates may therefore be essential for signal transduction during lymphocyte activation. We have studied the phosphorylation of the closely related lck, fyn, and c-src tyrosine protein kinases in leukemic murine T-cell lines that have lost the expression of CD45. The phosphorylation of the lck kinase at an inhibitory site of tyrosine phosphorylation, Tyr-505, was increased by two-, six-, and eightfold in three different cell lines. Phosphorylation of the fyn kinase at the homologous site, Tyr-531, was unaltered in one of these cell lines, but increased by 2.5-fold in the two others. The phosphorylation of p60c-src at the homologous tyrosine was essentially unchanged in the one CD45-negative cell line in which it was examined. The expression of CD45 therefore regulates the phosphorylation and potentially the activity of the lck and fyn tyrosine protein kinases, but the effect on the lck kinase is much greater than on the fyn kinase. This finding and the observation that CD45 had no effect on the phosphorylation of p60c-src suggest that CD45 exhibits polypeptide substrate specificity in vivo. Additionally, these findings are consistent with the hypothesis that the unresponsiveness of CD45-negative lymphoid cells to antigenic stimulation is due largely to hyperphosphorylation of the lck kinase.

Animals↗
PubMed1993

Influence of the apoA-II gene locus on HDL levels and fatty streak development in mice.

Previous studies have shown that distal mouse chromosome 1 contains the apolipoprotein AII (apoAII) gene, encoding the second most abundant apolipoprotein in high density lipoproteins (HDLs), as well as a gene termed Ath-1 that controls aortic fatty streak development and HDL cholesterol levels in response to a high-fat, high-cholesterol diet. We report genetic studies confirming that the genes are distinct. Using molecular markers for mouse chromosome 1, we have further mapped the two genes, and our results indicate that they are separated by a minimum of 2 cM. We also report evidence that in mice on a low-fat chow diet, the apoAII gene locus influences HDL cholesterol levels. Thus, statistical analysis of two sets of recombinant inbred strains revealed concordant segregation patterns of HDL cholesterol levels and the apoAII gene locus. The effect of apoAII expression on HDL cholesterol levels was further tested by using a congenic strain that exhibits increased apoAII synthesis in comparison to the background strain. The results support the concept that increased synthesis of apoAII results in increased HDL cholesterol levels. Unexpectedly, increased expression of apoAII appeared to promote rather than retard aortic fatty streak development.

Animals↗
PubMed1992

Contribution of de-novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo.

The relative contribution of de-novo and salvage synthesis to tissue pyrimidine nucleotide pools is an important parameter in the rational design of anti-pyrimidine therapies, but has not been measured in vivo. We have measured the contribution of de-novo synthesis to the total acid-soluble uracil nucleotide pool in mouse tissues by analysis of the incorporation of label after intra-peritoneal infusion of L-[15N]alanine. The contribution of salvage synthesis was measured by the incorporation of radiolabel after intravenous infusion of [14C]uridine. The results show that de-novo synthesis makes the larger contribution to the intestine uracil nucleotide pool, salvage synthesis makes the larger contribution to the kidney pool, and de-novo and salvage synthesis make roughly equal contributions to the liver pool. In tumors studied (L1210, P388, B16, Nettesheim), the contribution of de-novo synthesis was at least five times the contribution of salvage synthesis. The measurements were repeated 24 hours after a 400-mg/kg dose of N-phosphonacetyl-L-aspartic acid. De-novo synthesis was substantially inhibited in all tissues and tumors after this treatment, although significant residual activity was observed in the intestine and L1210 cells. Nettesheim carcinoma was the only tumor or tissue to show a significant increase in salvage synthesis after N-phosphonacetyl-L-aspartic acid treatment.

Animals↗
PubMed1992

Tcl-30, a new T cell-specific gene expressed in immature glucocorticoid-sensitive thymocytes.

Glucocorticoid treatment induces programmed cell death (apoptosis) in susceptible T lymphocytes. We have previously isolated and characterized 13 genes induced by agents that cause apoptosis in the murine thymoma cell line, WEHI-7TG. One of these genes, now designated Tcl-30, encodes a 2.4-kb mRNA that is specifically expressed in thymus. Sequence analysis of a full-length cDNA for Tcl-30 reveals a potential open reading frame of 454 amino acids that shares sequence identity to a human placental-specific protein of unknown function, PP11. The putative protein encoded by Tcl-30 also contains a cysteine-rich somatomedin B-like domain found in vitronectin, PP11, and the plasma cell membrane glycoprotein, PC-1. Subpopulations of murine thymocytes sorted on the basis of their expression of the CD4, CD8, and surface heat-stable Ag (HSA) were analyzed by an RNase protection assay to determine the expression of Tcl-30 as a function of T cell development. Tcl-30 was expressed exclusively in the HSA+ T cell populations (CD4-CD8-HSA+; CD4+CD8-HSA+; CD4-CD8+HSA+; and CD4+CD8+HSA+) and not in the HSA- single positive T cell populations (CD4+CD8- or CD4-CD8+) of the thymus or spleen. Therefore, we conclude that Tcl-30 expression is lost during T cell maturation and is absent at the most mature stages of T cell development. The function of Tcl-30 is unknown; however, the CD4+CD8+ double-positive subpopulation expressing Tcl-30 represents thymocytes destined to undergo massive intrathymic cell death. The possibility that Tcl-30 expression may define a population of T lymphocytes that is sensitive to glucocorticoid-mediated apoptosis is discussed.

Amino Acid Sequence↗
PubMed1992

Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody.

The CD44-negative T lymphoma AKR1 (CD44.2 genotype) was transfected with a CD44.1 cDNA. The intact cDNA conferred on the transfected cells the ability to bind hyaluronic acid (HA) both from solution and immobilized on culture plates. It also conferred a CD44-dependent and hyaluronidase-sensitive increase in adhesion to a lymph node endothelial cell line. A mutant cDNA which codes for a CD44 molecule lacking most of the cytoplasmic domain of CD44 was also transfected into AKR1, and cell sorting was used to select transfectants expressing levels of cell surface CD44 expression comparable with the line transfected with the wild-type CD44 cDNA. The cells transfected with the mutant construct bound fluoresceinated HA from solution very poorly, but did adhere to immobilized HA, though less well than cells transfected with the wild-type construct. This result indicates that the cytoplasmic domain of CD44 is necessary for binding of HA from solution but is not required for binding to immobilized HA, although it may contribute to adhesion following ligand recognition. A monoclonal antibody (mAb), IRAWB 14, which reacts with CD44 on all CD44+ cells dramatically induced HA binding by some CD44+ cell lines that did not constitutively bind HA. The transfectant expressing a CD44 molecule with a truncated cytoplasmic domain could be induced by this antibody to bind fluoresceinated-HA from solution. Splenic T cells did not bind fluoresceinated HA constitutively. In the presence of the IRAWB 14 mAb, virtually all CD44+ splenic T cells bound HA. Induction was immediate and occurred equally well at room temperature and at 4 degrees C, indicating that the new HA-binding activity was due to preexistent CD44 molecules. These results are compatible with an antibody-induced activation of CD44 by either a conformational change in the CD44 molecule or a change in the distribution of CD44 molecules on the cell surface.

Animals↗
PubMed1992

CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells.

The hyaluronic acid (HA)-binding function of CD44 expressed on the cell surface of normal hematopoietic cells has been studied by assaying binding of fluoresceinated hyaluronic acid (F1-HA) and adhesion to immobilized HA. As has been observed previously, normal hematopoietic cells from bone marrow and spleen do not constitutively bind HA. A CD44-specific monoclonal antibody, IRAWB 14, which has been shown to rapidly induce HA binding in some CD44+ cell lines, was used to activate the HA-binding function of CD44 in these normal cells. Only splenic T cells were activated by the IRAWB 14 antibody to bind F1-HA. Upon activation, F1-HA binding correlated with the level of CD44 expression. Activation of HA binding allowed splenic T cells to adhere to HA immobilized on plastic and to an endothelial cell line in an HA-dependent manner. BALB/c and AKR/J splenic T cells differ in their level of CD44 expression, and this correlated with differences in their ability to bind HA upon antibody activation. The minor subpopulation of MEL-14- T cells were among the brightest F1-HA-staining cells. We propose, on the basis of these and other results, that there are three states of CD44 function with respect to HA binding: (a) a non-activatable, resting state, which cannot be rapidly activated to bind HA, as seen in most hematopoietic cells; (b) an activatable state, which can be rapidly converted to HA-binding function, in this case by the IRAWB 14 antibody, illustrated by T cells as shown here; and (c) a constitutively active state, which can bind HA without antibody activation, seen in some cell lines.

Animals↗
PubMed1992

Activation of Lyt-2 associated with distant upstream insertion of an SL3-3 provirus.

Two Lyt-2+ mutants of the T-cell lymphoma SL12.4.10 were selected by fluorescence activated cell sorting. Both mutants expressed Lyt-2 (CD8 alpha-chain) but not Lyt-3 (CD8 beta-chain). Derivatives of one Lyt-2+ mutant that expressed Lyt-3 could be isolated by sorting for Lyt-3+ cells. Southern blotting analysis indicated that both mutants had structural rearrangements within or immediately 3' of the Lyt-3 gene, accompanied by demethylation of at least one Hpa II site within the Lyt-2 gene. Gene cloning analysis of one mutant demonstrated that the structural rearrangement was due to insertion of an SL3-3 provirus 35 kb 5' to the Lyt-2 gene. It is likely that Lyt-2 gene activation is a direct or indirect consequence of proviral insertion at this site.

Animals↗
PubMed1992

Coordinate change in phenotype in a mouse cell line selected for CD8 expression.

A CD4+, CD8+ derivative of the CD4+, CD8- cell line SAKRTLS 12.1 was isolated by fluorescence activated cell sorting for CD8+ cells. This derivative showed a co-ordinate change in a number of independent characters: The parental cell line was CD4+, CD8-, CD3+, CD5hi, HSA+, DEXR, CD44hi, while the derivative was CD4+, CD8+, CD3-, CD5(10), HSA+, DEXS, CD44(10). The derivative expressed the Thy-1.1, Ly-2.1, and Ly-3.1 surface antigens, consistent with origin from the SAKRTLS 12.1 parental cell line, and showed a drug resistance profile identical to that of the parent. It was not possible to isolate revertants with a phenotype identical to that of the parental cell line. Activation of the structural gene coding for CD8 alpha chain was correlated with demethylation at several sites. We interpret these results to mean that this CD8+ derivative of SAKRTLS 12.1 arose as a result of an alteration of a gene that coordinately regulates multiple genes whose expression changes during thymocyte differentiation. Gene methylation may contribute, directly or indirectly, to some or all of the changes in gene expression observed.

Animals↗
PubMed1992

Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition.

We previously found that the CD44 glycoprotein on some lymphocytes can mediate adhesion to hyaluronate (HA) bearing cells. However, many questions remain about the molecular heterogeneity of CD44 and mechanisms which control its recognition of this ligand. In vitro mutagenesis and DNA sequencing have now been used to investigate the importance of the membrane proximal region of murine CD44 for recognition of soluble or cell surface HA. CD44 with an 83 amino acid deletion in this region mediated binding to soluble ligand and the apparent avidity increased markedly in the presence of a particular antibody to CD44, IRAWB14. The shortened CD44 was however inefficient in mediating adhesion of transfected cells to HA immobilized on cell surfaces. Four new murine isoforms of CD44 were isolated from a carcinoma line by use of the polymerase chain reaction. Only two of them correspond to ones recently discovered in rat and human cells. The longest variant nearly doubled the length of the extracellular portion of the molecule and introduced an additional 20 potential sites for glycosylation. When expressed on T lymphoma cells, all four of the new murine CD44 isoforms were capable of mediating adhesion to HA bearing cells. This result contrasts with a report that a related human CD44 isoform lacks this ability when expressed on B lineage lymphoma cells. The new murine isoforms also conferred the ability to recognize soluble HA and were very responsive to the IRAWB14 antibody. A brief survey of normal murine cell lines and tissues revealed that the hemopoietic isoform was the most abundant species. These findings indicate that the NH2-terminal portion of CD44 is sufficient for HA recognition and that this function is not necessarily abrogated by variations which occur in the membrane proximal domain. They add to the known molecular diversity of CD44 and provide another experimental model in which isoform specific functions can be investigated.

Amino Acid Sequence↗
PubMed1991

Vascular smooth muscle expresses a truncated Na+, K(+)-ATPase alpha-1 subunit isoform.

By regulating transmembrane Na+ and K+ concentrations and membrane potential, the Na+,K(+)-ATPase plays an important role in regulating cardiac, skeletal, and smooth muscle function. A high degree of amino acid sequence and structural identity characterizes the three Mr 100,000 Na+,K(+)-ATPase alpha subunit isoforms expressed in cardiac and skeletal muscle. Strikingly, vascular smooth muscle utilizes alternative RNA processing of the alpha-1 gene to express a structurally distinct Mr approximately 65,000 isoform, alpha 1-T (truncated). Analysis of both its mRNA and protein structure reveals that alpha-1-T represents a major, evolutionarily conserved, truncated Na+,K(+)-ATPase isoform expressed in vascular smooth muscle. This demonstrates an unexpected complexity in the regulation of vascular smooth muscle Na+,K(+)-ATPase gene expression and suggests that a structurally novel, truncated alpha subunit may play a role in vascular smooth muscle active ion transport.

Amino Acid Sequence↗
PubMed1991

Identification of defects in glycosylphosphatidylinositol anchor biosynthesis in the Thy-1 expression mutants.

A number of eukaryotic proteins are anchored to the membrane by glycosylphosphatidylinositol (GPI), of which the core structure is conserved from protozoan to mammalian cells. Here, we used a panel of thymoma mutants, which synthesize Thy-1 but cannot express it on the cell surface, to study the GPI biosynthetic pathway in mammalian cells. These mutants have been assigned into six complementation classes (A, B, C, E, F, H) by the technique of somatic cell hybridization. Using a combination of metabolic labeling and chemical/enzymatic tests, the biosynthetic defects were mapped to four different steps. Class A, C, and H mutants cannot transfer N-acetylglucosamine (GlcNAc) to a phosphatidylinositol acceptor, suggesting that the first step of GPI synthesis is regulated by at least three genes. The Class E mutant does not synthesize dolichol-phosphate-mannose, the donor for the first mannose residue transferred to the GPI core, and thus cannot form any mannose-containing GPI precursors. Class B and F mutants are defective in the addition of the third mannose residue or ethanolamine phosphate, respectively, to the elongating GPI core. Our findings have implications for the biosynthesis and attachment of the mammalian GPI anchor.

Autoradiography↗
PubMed1991

Aldosterone-mediated regulation of Na+, K(+)-ATPase gene expression in adult and neonatal rat cardiocytes.

By altering the Na+/K+ electrochemical gradient, Na+,K(+)-ATPase activity profoundly influences cardiac cell excitability and contractility. The recent finding of mineralocorticoid hormone receptors in the heart implies that Na+,K(+)-ATPase gene expression, and hence cardiac function, is regulated by aldosterone, a corticosteroid hormone associated with certain forms of hypertension and classically involved in regulating Na+,K(+)-ATPase gene expression and transepithelial Na+ transport in tissues such as the kidney. The regulation by aldosterone of the major cardiac Na+,K(+)-ATPase isoform genes, alpha-1 and beta-1, were studied in adult and neonatal rat ventricular cardiocytes grown in defined serum-free media. In both cell types, aldosterone-induced a rapid and sustained 3-fold induction in alpha-1 mRNA accumulation within 6 h. beta-1 mRNA was similarly induced. alpha-1 mRNA induction occurred over the physiological range with an EC50 of 1-2 nM, consistent with binding of aldosterone to the high affinity mineralocorticoid hormone receptor. In adult cardiocytes, this was associated with a 36% increase in alpha subunit protein accumulation and an increase in Na(+)-K(+)-ATPase transport activity. Aldosterone did not alter the 3-h half-life of alpha-1 mRNA, indicating an induction of alpha-1 mRNA synthesis. Aldosterone-dependent alpha-1 mRNA accumulation was not blocked by the protein synthesis inhibitor cycloheximide, whereas amiloride inhibited both an aldosterone-dependent increase in intracellular Na+ [Na+]i) and alpha-1 mRNA accumulation. This demonstrates that aldosterone directly stimulates Na+,K(+)-ATPase alpha-1 subunit mRNA synthesis and protein accumulation in cardiac cells throughout development and suggests that the heart is a mineralocorticoid-responsive organ. An early increase in [Na+]i may be a proximal event in the mediation of the hormone effect.

Aging↗
PubMed1990

Natural autoantibodies to thymocytes: origin, VH genes, fine specificities, and the role of Thy-1 glycoprotein.

15 SM/J mouse hybridoma antibodies that show antithymocyte autoantibody (ATA) activity by immunofluorescence staining were studied. Half of these antibodies react with determinants whose expression is associated with Thy-1, as shown by blocking experiments with anti-Thy-1 and loss of reactivity with Thy-1- mutant cell lines. The Thy-1 dependence of three of these ATA is further confirmed by their reexpression on a Thy-1 gene transfectant. However, the remaining antibodies exhibited binding that showed little or no dependence on Thy-1. Furthermore, we find that most ATA derives from the Ly-1 B subpopulation, as demonstrated by lipopolysaccharide-induced ATA secretion in vitro and by comparison of ATA hybridoma frequencies. VH region gene sequence data of 14 monoclonal ATA from Ly-1 B cell-derived hybridomas reveal the utilization of nine VH genes belonging to four different VH families (J558, 3609, Q52, and Vgam3.8). While we find that two of these hybridomas arose from a clonal expansion, we also find four examples of a 3609 family VH gene utilized in clonally independent lines showing similar specificity. Yet another example of identical VH gene usage by clonally unrelated cells is found in two J558 ATA of a distinct fine specificity. These data suggest that the enrichment of ATA B cells in the Ly-1 B subset is primarily due to repeated independent recruitment of B cells by antigen resulting in the expression of a restricted set of VH genes.

Animals↗