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T A Hamilton

Publications and source records attributed to T A Hamilton.

At least 181 records · Page 10Linked to original sources
PubMed1984

Fc-receptor mediated protein phosphorylation in murine peritoneal macrophages.

The effect of Fc receptor engagement on protein phosphorylation in murine peritoneal macrophages has been investigated. Treatment of macrophage cultures with insoluble immune complexes resulted in enhanced phosphorylation of six proteins at 73, 66, 53, 37, 31 and 25 kD. Comparison of the protein phosphorylation patterns induced by immune complexes with those induced by agents which mimic the actions of well known intracellular second messengers (i.e., A23187, dibutyryl cAMP, or phorbol myristate acetate) revealed substantial similarity between Fc receptor induced events and those induced in response to phorbol diesters. There were, however, two phosphorylated proteins which were only seen following stimulation with immune complexes. Thus, more than one kind of protein kinase activity appears to be involved in Fc receptor mediated stimulation of macrophage function.

Animals↗
PubMed1984

Murine monocytes express transferrin receptors: evidence for similarity to inflammatory macrophages.

Nonionic detergent extracts of murine monocytes contain a specific, high-affinity binding site for the serum glycoprotein transferrin. The binding activity was saturable and specific for 125I-labeled transferrin. The transferrin-receptor content of monocytes was compared with that of resident peritoneal macrophages and thioglycollate-elicited macrophages. Whereas resident cells showed no detectable activity, inflammatory macrophages and monocytes both bound transferrin to a similar degree. The calculated dissociation constant (2.3 X 10(-10)) and the number of sites per monocyte (11,400) compared favorably with those reported for transferrin receptors on inflammatory macrophages. Thus, based on the expression of the transferrin receptor, murine monocytes resemble murine inflammatory peritoneal macrophages rather than resident tissue macrophages.

Animals↗
PubMed1984

Quiescent lymphocytes express intracellular transferrin receptors.

Both quiescent and concanavalin A stimulated murine splenic lymphocytes were examined for the expression of surface and intracellular binding sites for the serum glycoprotein transferrin. Transferrin binding activity was observed on the surface of mitogen stimulated cells only. When soluble detergent extracts of both populations were studied, quiescent lymphocytes were shown to contain a significant pool of non-surface exposed, intracellular receptors which was approximately 20% of the total receptor complement of proliferating cells. Because the ratio of surface to intracellular binding sites was dramatically increased following mitogen stimulation, the regulation of transferrin receptor expression during this process may involve a substantial alteration in its subcellular distribution in addition to the well documented increase in number of binding sites.

Animals↗
PubMed1984

Expression of the transferrin receptor on murine peritoneal macrophages is modulated by in vitro treatment with interferon gamma.

The expression of transferrin receptors on murine peritoneal macrophages has been shown to be down regulated during functional activation in vivo. This observation suggested that the level of transferrin receptor expression varies in response to discrete extracellular signals known to induce macrophage activation. We have tested this concept directly and have shown that decreased transferrin receptor expression can be reproduced in vitro by treatment of inflammatory macrophages with preparations of interferon gamma derived from a T cell hybridoma supernatant. The ability of this agent to down regulate the expression of the transferrin receptor exhibited dose and time dependencies similar to those required for development of other macrophage functions in vitro. The addition of LPS produced no further decrease in receptor expression. Furthermore, murine gamma interferon, produced by recombinant DNA technology also caused a downshift in transferrin receptor expression at doses similar to those which have been shown previously to induce activation. The changes in receptor activity were the result of altered numbers of binding sites and the receptor:ligand affinity remained unaffected. These results indicate that altered expression of the transferrin receptor is one element of the pleiotypic change which macrophages undergo in response to IFN gamma. This system may, therefore, provide a useful model in which to study the biochemical basis of IFN gamma action in mononuclear phagocytes.

Animals↗
PubMed1984

Expression of the transferrin receptor in murine peritoneal macrophages is modulated in the different stages of activation.

A specific high affinity binding site for the serum glycoprotein transferrin was identified on murine peritoneal macrophages. The binding reached equilibrium within 60 min and was reversible, saturable, and specific for transferrin. Although the presence of this receptor was detected on the cell surface by studies carried out using intact cells, the majority (70 to 90%) of the binding activity was detectable only in detergent extracts of such cells. This suggests that a substantial portion of the binding activity is localized within the macrophage. The association constant (Ka) for binding to intact cells (6 to 9 X 10(8) M-1) was comparable to values reported for transferrin receptors described on other cell types. The expression of transferrin binding activity was examined in macrophages exhibiting qualitatively and quantitatively different degrees of functional activation. Resident peritoneal macrophages, exudate macrophages primed by elicitation with pyran copolymer, and activated macrophages induced by chronic infection of mice with bacillus Calmette-Guerin (BCG) or elicitation with heat killed Propionibacterium acnes (P. acnes) had low numbers of binding sites (1000 to 5000 total sites/cell). Macrophages elicited by sterile inflammatory agents (thioglycollate broth, fetal bovine serum, or casein) all exhibited a greater number of transferrin receptors (15,000 to 20,000 total sites/cell). This modulation did not appear to result from differential shifts between surface and internal loci. Our results suggest that the expression of the transferrin receptor may be a useful marker of the responsive stage of macrophage functional activation and the membrane changes that accompany activation.

Animals↗
PubMed1983

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase.

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.

Animals↗
PubMed1983

Receptor-mediated endocytosis and exocytosis of transferrin in Concanavalin A-stimulated rat lymphoblasts.

Iron-loaded transferrin has been shown to be necessary for the support of cell proliferation in culture. This function depends upon interaction of transferrin with a specific high-affinity cell surface receptor. The present report is directed toward determining the consequences of the interaction of transferrin with this receptor on Concanavalin A-stimulated rat lymphocytes. Three specific questions have been posed: a) Is transferrin endocytosed following binding to its specific receptor in a temperature-dependent fashion? b) Following endocytosis, is the carrier protein released from the cell in a structurally and functionally intact form? and c) Is the cell surface transferrin receptor also endocytosed following ligand binding? The results provide affirmative answers to all questions. Using two independent probes of the cell surface versus intracellular location of transferrin we observed that cell-bound transferrin moved from the cell surface to the inside of the cell and subsequently back to the medium. This process occurred in a temperature-dependent fashion. When cells containing only intracellular transferrin were further incubated at 37 degrees C approximately 80% of cell-bound transferrin was released to the medium. Nearly all of this material retained reactivity with antibody to transferrin. In addition, endocytosed transferrin exhibited qualitatively and quantitatively equivalent binding reactivity with the transferrin receptor and showed identical electrophoretic mobility on SDS gel electrophoresis. Finally, using similar methodology to that employed with transferrin itself, we provide evidence that the specific receptor is also endocytosed.

Animals↗
PubMed1982

Regulation of transferrin receptor expression in concanavalin A stimulated and Gross virus transformed rat lymphoblasts.

Expression of the cell surface receptor for the serum glycoprotein transferrin has been correlated with cellular proliferation in normal lymphocytes undergoing mitogen or antigen induced proliferative responses. In the present study, the expression of transferrin receptor in Concanavalin A stimulated rat lymphocytes or Gross virus transformed lymphoma cells has been examined with respect to the following questions: (1) is expression of receptor activity related to blastogenesis or to the subsequent IL-2 dependent DNA synthetic activity, and (2) is transferrin receptor expression regulated in similar fashion in both normal and malignant lymphoblasts? Scatchard analysis of saturation binding data illustrated that binding site number increased and subsequently decreased during the response while the receptor affinity for transferrin remained constant. These findings were confirmed by SDS-polyacrylamide gel electrophoretic analysis of radiolabeled cell surface proteins which specifically interact with transferrin. Examination of nonproliferating normal lymphoblasts (96 hr post Con A stimulation) compared with the same population of cells stimulated to reinitiate DNA Synthesis with a partially purified preparation of Interleukin 2 (IL-2) showed that transferrin receptor expression was tightly linked to the IL-2 dependent stimulation of DNA replication. This coordinate regulation of receptor expression was markedly less stringent in retrovirus transformed thymic lymphoma cells.

AKR murine leukemia virus↗
PubMed1982

Identification and molecular characterization of macrophage-associated antigens of the rat.

Rabbit antiserum to rat peritoneal exudate (PE) macrophage (M phi) antigens was prepared and its reactivity with cell surface proteins of M phi, granulocytes, and lymphocytes was studied by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). A total of 14 membrane antigens were identified of which three were found to be expressed only by M phi and granulocytes. By one-dimensional analysis, a protein with an approximate m.w. of 105,000 was present on splenic and PE M phi and on splenic lymphocytes. Two-dimensional analysis revealed that this band was heterogeneous and contained at least three species, one of which was restricted to expression on M phi and granulocytes. A second protein of 150,000 daltons was resolved into two species by two-dimensional analysis. Both of these species were present on M phi and granulocytes but not on lymphocytes. Both the 105,000- and 150,000-dalton proteins were glycosylated. Because the 105,000- and 150-000-dalton proteins expressed by M phi were also expressed by granulocytes, is is likely that these are differentiation antigens whose expression is a characteristic property of cells within both monocytoid and myeloid lineages. All three 105,000-dalton species and one of the two 150,000-dalton species were detected on mouse M phi, indicating their expression is not unique to the rat.

Absorption↗
PubMed1981

The effect of bromodeoxyuridine and dibutyryl cyclic AMP on the induction of placental alkaline phosphate in human choriocarcinoma cells.

The effect of 5-bromo-2'-deoxyuridine (BrdUrd) and dibutyryl cyclic AMP (Bt2cAMP) on the expression of the placental isoenzyme of human alkaline phosphatase was examined in BeWo choriocarcinoma cells. By using a combination of specific immunoprecipitation and polyacrylamide-gel electrophoresis of cells labelled either metabolically with [35S]methionine or cell-surface-labelled with 125I, both BrdUrd (5 micrograms/ml) and 1 mM-Bt2cAMP were shown to result in the enhanced accumulation of a specific protein. This protein has immunochemical identity and co-electrophoreses with placental alkaline phosphatase in two-dimensional gels. These results clearly demonstrate that the induction of placental alkaline phosphatase activity in choriocarcinoma cells treated with these agents is a consequence of the accumulation of specific enzyme protein rather than of altered catalytic activity.

Alkaline Phosphatase↗
PubMed1981

Expression in Escherichia coli of biologically active enzyme by a DNA sequence coding for the human plasminogen activator urokinase.

We have isolated clones of Escherichia coli strain K-12 that contain a hybrid pBR322 plasmid having a 4.2-kilobase insert of a DNA transcript of the mRNA of human plasminogen activator, urokinase. The bacterially produced enzyme has properties similar to those of urokinase from human fetal kidney cells. Both enzymes occur in discrete forms ranging from 32,000 to 150,000 daltons in size. They react with antibody to purified urokinase from human kidney cells, bind to a benzamidine-Sepharose column, and induce plasminogen-dependent lysis of a fibrin clot.

Base Sequence↗
PubMed1981

Characterization of the recognition of target cells sensitive or resistant to cytolysis by activated rat peritoneal macrophages.

The interaction between tumoricidal rat peritoneal exudate macrophages and normal or neoplastic target cells has been examined both with respect to the sensitivity of the target cells to macrophage-mediated cytolysis and the ability of such targets to compete with one another for the lytic activity of macrophages. The results demonstrate that, in confirmation of earlier work from other laboratories, target cells are either sensitive or largely resistant to lysis, and such sensitivity correlates well with the neoplastic phenotype of the target cell. However, in target cell competition experiments all cultured cells as well as Con A-stimulated splenocytes and freshly isolated thymocytes exhibit the capacity to block cytolysis of sensitive indicator targets. In contrast, freshly prepared non-mitogen-stimulated splenic or lymph node lymphocytes are completely devoid of competitive activity. These results show that macrophages can recognize and interact with both normal and tumorigenic cells and that such target recognition can be distinguished from target cytolysis. Furthermore, the evidence suggests that at least 1 target cell feature that correlates with recognition is cell cycle traverse.

Animals↗
PubMed1980

Expression of human placental cell surface antigens on peripheral blood lymphocytes and lymphoblastoid cell lines.

The expression of human placental cell surface antigens was examined in cells of lymphoid origin, including peripheral blood lymphocytes and cultured lymphoblastoid cells of bone marrow or thymus derivation. A select group of this defined set of surface antigens was detected on all three cell preparations. The most remarkable observation was the conspicuous absence of three subunits previously demonstrated to be present on all human cell surfaces examined to date. Antiserum directed against several placental components prevents adhesion and spreading of cells which grow attached to surfaces. These results suggest a role for these three glycoproteins in mediating cellular adhesion.

Antigens, Surface↗
PubMed1980

Independent expression of alpha-HCG and placental alkaline phosphatase in clonal human cell lines.

The expression of the placental isoenzyme of alkaline phosphatase and the alpha subunit of chorionic gonadotropin was examined in a series of cloned lines of Chang liver cells. both placental gene products show large quantitative variation in the amounts made (over 30-fold) among the various clones. This variation occurs independently for both proteins. These results suggest that these placental-specific genes are discordantly regulated. Because karyologic analysis of several clones shows significant variation in the chromosome content, we cannot determine whether such expression results from true independent mechanisms controlling expression or from variation in gene dosage.

Acid Phosphatase↗