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I S Trowbridge

Publications and source records attributed to I S Trowbridge.

At least 73 records · Page 4Linked to original sources
PubMed1986

Identification of lymphocyte integral membrane proteins as substrates for protein kinase C. Phosphorylation of the interleukin-2 receptor, class I HLA antigens, and T200 glycoprotein.

The interleukin-2 (IL-2) receptor, the leukocyte-specific membrane glycoprotein, T200, and the class I major histocompatibility antigens (HLA) have been identified as substrates for protein kinase C in vitro. IL-2 receptors on normal human T lymphocytes and the leukemic cell line, HUT102B2, are rapidly phosphorylated in vivo in response to the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Tryptic peptide analysis showed that the in vitro and in vivo 32P-labeled IL-2 receptors were phosphorylated on the same sites. A synthetic peptide corresponding to the carboxyl-terminal cytoplasmic tail of the IL-2 receptor was shown to be phosphorylated in vitro by protein kinase C. Tryptic digestion of the peptide generated the same 32P-labeled species as those found for the IL-2 receptor. From these studies, it was concluded that Ser-247 is the major site of phosphorylation in the IL-2 receptor and that Thr-250 is a minor site. These results also provide direct evidence that the in vivo phosphorylation of the IL-2 receptor stimulated by TPA is catalyzed by protein kinase C. The sites phosphorylated in the HLA antigens in vitro by protein kinase C or in vivo after TPA stimulation were also localized to the carboxyl-terminal cytoplasmic domain of the heavy chain by limited proteolysis.

Amino Acid Sequence↗
PubMed1986

Different classes of T lymphocytes have different mRNAs for the leukocyte-common antigen, T200.

The leukocyte common antigen, T200, is expressed on all white blood cells but not on other differentiated cells. Within the hematopoietic lineage, specific cell types display characteristic structural forms of the molecule on their surface. We show that murine cytotoxic T lymphocyte clones and helper T cell clones contain different size mRNA for this molecule, and that the early precursors of T200 glycoprotein made in the helper and cytotoxic T cells differ in Mr. Thus, in addition to differences in posttranslational modifications, it is highly likely that a difference in protein structure contributes to the distinct forms of T200 glycoprotein found on these functional T cell subsets.

Animals↗
PubMed1986

Identification and characterization of the human Pgp-1 glycoprotein.

Two monoclonal antibodies have been raised against human Pgp-1 by the immunization of mice with human fibroblasts. The human molecule, like the previously identified mouse counterpart, is an abundant membrane protein (Mr approximately 95 000) with a broad tissue distribution. Pgp-1 is phosphorylated, and phosphoamino acid analysis demonstrates that this occurs exclusively on serine residues. A major difference between the mouse and the human is that 50-60% of human thymocytes are Pgp-1+ compared to 5-10% of mouse thymocytes at an equivalent stage in development. Immunofluorescence studies of cryostat sections showed that the majority of human medullary thymocytes are strongly stained with Pgp-1-specific antibody, whereas the expression of Pgp-1 on cortical thymocytes is much more heterogeneous.

Age Factors↗
PubMed1986

Structure and function of transferrin receptors and their relationship to cell growth.

The transferrin receptor binds the major serum iron-transport protein, transferrin, and mediates cellular iron uptake. The receptor is a major immunodominant cell surface glycoprotein of cultured cells and its expression on the cell surface is co-ordinately regulated with cell growth. Recent structural and functional studies of the transferrin receptor are reviewed. The properties of monoclonal antibodies against the transferrin receptor that inhibit transferrin-mediated iron uptake are described. Studies with these antibodies establish that the transferrin receptor plays an important role in cell growth and suggest that monoclonal antibodies that interfere with the function of growth-related receptors may be useful in regulating tumour cell growth.

Animals↗
PubMed1985

Antibodies against the transferrin receptor block the induction of cytotoxic T lymphocytes. A new method for antigen-specific negative selection in vitro.

The induction of cytotoxic T lymphocytes (CTL) specific for alloantigen in a primary mixed lymphocyte culture (MLC) was used as a model system to study the potential usefulness of monoclonal antibodies directed against the murine transferrin receptor in eliminating antigen-activated T cells. Two monoclonal antibodies were used which are able to block the transferrin-mediated uptake of iron by growing cells. One of these antibodies is cytotoxic in the presence of complement (C). Treatment of MLC cells from control, uninhibited cultures with the cytotoxic anti-transferrin receptor antibody plus C completely knocked out all CTL effector activity showing that all of the effector cells expressed the receptor. However, surviving cells from such antibody-plus C-treated populations were not depleted of precursors since they were able to mount a strong CTL response to the original alloantigen upon restimulation. Blocking antibody against the transferrin receptor, in the absence of C, when present during the course of a primary MLC, completely inhibited the generation of effector CTL. Conditions were optimized such that antibody present during the course of the MLC could completely and specifically eliminate all antigen-activated T cells including precursors as assessed by secondary restimulation with the original, plus third-party antigens, in the absence of blocking antibody. This method for producing specifically unreactive T cell populations appears to be superior to previously described techniques.

Animals↗
PubMed1985

Monoclonal antibody to a membrane glycoprotein inhibits the acrosome reaction and associated Ca2+ and H+ fluxes of sea urchin sperm.

Monoclonal antibodies were prepared using sea urchin (Strongylocentrotus purpuratus) sperm or isolated sperm plasma membrane vesicles as immunogen. Two monoclonal antibodies, J4/4 and J10/14, react with different epitopes on external domains of the same Mr = 210,000 integral-membrane glycoprotein. At Fab fragment concentrations of equivalent cell surface binding, J10/14 inhibits the egg jelly-induced exocytotic acrosome reaction and associated Ca2+ influx and H+ efflux, whereas J4/4 does not. Immunofluorescent localization shows that both monoclonal antibodies react with a narrow collar of plasma membrane over the acrosomal complex and also with the entire flagellum.

Acrosome↗
PubMed1985

Cross-linking of human T cell receptor proteins: association between the T cell idiotype beta subunit and the T3 glycoprotein heavy subunit.

Bifunctional cross-linking reagents DSP, DSS, and BSOCOES were used to cross-link 125I-surface-labeled viable T lymphocytes. The cross-linked cells were solubilized in Nonidet-P40, immunoprecipitated with anti-Ti (monoclonal antibody T40/25) or anti-T3 (monoclonal antibodies UCHT-1 or OKT3), and analyzed by SDS-PAGE. With all three cross-linkers, the intact cross-linked products obtained with monoclonal antibody T40/25 from HPB-ALL cells were 20-30 kd heavier than the Ti dimer (Mr 80,000). When the DSP cross-linked product was isolated using either anti-Ti or anti-T3 monoclonal antibodies and then cleaved, bands having molecular weights identical with both the Ti and T3 subunits were obtained. The two-dimensional SDS-PAGE analysis (nonreducing followed by reducing conditions) of the DSS and BSOCOES cross-linked products revealed the specifically cross-linked bands to have Mr 40,000 and Mr 28,000. These data indicate that the Ti molecule and the T3 molecule are spatially associated on the cell surface and suggest the predominant association is between the Ti beta subunit (Mr 40,000) and the T3 heavy subunit (Mr 28,000).

Antibodies, Monoclonal↗
PubMed1985

The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis.

The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis.

Animals↗
PubMed1985

Immune phenotype heterogeneity in AML.

Blood cells from 46 patients with acute myeloid leukaemia were studied for expression of various surface markers, defined by a panel of 12 monoclonal antibodies and the expression of Fc gamma receptors. Corresponding studies were done on normal bone marrow cells. Antibodies which bound to leukaemic cells in high frequencies were those which most frequently also bound to normal bone marrow cells. Immunophenotypic analysis revealed a marked antigenic heterogeneity in AML, also evident within single FAB subclasses. However, leukaemic cells of FAB subclass M1 significantly more often expressed HLA class I antigen than those of FAB subclass M5a, whereas Fc gamma receptors which were expressed only on a few cells in M5a, were increasingly frequent on leukaemic cells of M1-M2, M4, and M5b leukaemias. The frequency of cells reacting with the monoclonal antibody T50/12,11,2 was related to the complete remission rate of the patients. Patients with high frequencies of cells reacting with this antibody had a better complete remission rate than patients with fewer cells binding to this antibody. The immunophenotypic heterogeneity an AML may reflect a great biological variability of this disease. This variability may be of importance for the classification and treatment of AML.

Adult↗
PubMed1984

Induction of expression and phosphorylation of the human interleukin 2 receptor by a phorbol diester.

The receptor for interleukin 2 (IL 2) is induced on normal mature T cells activated by antigen or mitogen. Receptor-negative human T leukemic cell lines were induced to express the IL 2 receptor after incubation with 12-O-tetradecanoyl phorbol 13-acetate. The Mr of the IL 2 receptor expressed on the surface of four different T cell lines and normal peripheral blood lymphocytes varied. The source of the heterogeneity appeared to be due to differences in post-translational processing of the receptor. The precursor of the IL 2 receptor isolated from tunicamycin-treated HUT102B2 or 12-O-tetradecanoyl phorbol 13-acetate-induced CCRF-CEM cells had the same Mr and isoelectric point. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate also induced the rapid phosphorylation of serine and threonine residues on the IL 2 receptor from HUT102B2 cells and activated peripheral blood lymphocytes. This phosphorylation may be mediated by the Ca2+ and phospholipid-dependent protein kinase C.

Cell Line↗
PubMed1984

Identification of shared antigenic determinants of the putative human T lymphocyte antigen receptor.

A mouse antiserum, anti-gp40,49 was obtained by immunizing BALB/c mice with the putative T cell antigen receptor isolated from HPB-MLT cells. This antiserum reacted with peripheral blood lymphocyte (PBL) and a panel of immunocompetent T cell lines and clones in each case precipitating from lysates of cells labeled by surface iodination, a disulfide-linked dimer consisting of an alpha subunit Mr (46,000-49,000) and a beta subunit Mr (40,000-45,000). Variability in Mr of the two subunits, particularly of the beta (light) subunit, was observed when the receptors of immunocompetent T cell lines with different antigen specificities were compared. Anti-gp40,49 serum reacted selectively with the alpha subunit after reduction and alkylation of the protein complex. These results confirm the relationship between the gp40,49 protein complex of HPB-MLT cells and the putative T cell antigen receptor on normal immunocompetent T cells and indicate that the alpha subunit of the human receptor expressed shared determinant(s) that are immunogenic in the mouse. Some features of the T cell antigen receptor appear to be unusual in that even with a xenoantiserum against the purified molecule, only antibodies against clonotypic determinants could be detected at the cell surface by quantitative immunofluorescence analysis.

Animals↗
PubMed1984

Effect of an anti-murine transferrin receptor-ricin A conjugate on bone marrow stem and progenitor cells treated in vitro.

A monoclonal antibody with specificity for murine transferrin receptor was conjugated with the toxic A subunit of ricin. The dose range, specificity, and kinetics of inhibition of protein synthesis of the conjugate were determined on the murine T-lymphoma cell line, BW5147. When toxin was present throughout the period of culture, in vitro myeloid (CFUc) and erythroid (CFUe and BFUe) bone marrow colonies were inhibited by doses of conjugate comparable to those that inhibit protein synthesis in murine cell lines (IC50 of 5 X 10(-11)M). Bone marrow exposed briefly (30 min to 6 h) to anti-transferrin receptor antibody-ricin A conjugate was assayed for myeloid (CFUc) and erythroid (CFUe and BFUe) progenitors in vitro and for in vivo spleen colony formation (CFUs). Only CFUe were depleted by this pulse exposure, consistent with the higher frequency of proliferating cells and transferrin receptor expression in the CFUe population relative to other progenitors.

Animals↗
PubMed1983

A monoclonal antibody useful for the differential diagnosis between malignant lymphoma and nonhematopoietic neoplasms.

A monoclonal antibody designated as T29/33, which recognizes a major cell surface glycoprotein of hematopoietic cells, was tested as a possible tool to distinguish lymphoma from nonhematopoietic tumors having a similar morphologic appearance by light microscopy. Each of the 40 lymphomas studied by immunofluorescence and/or immunoperoxidase on frozen sections revealed the presence of the corresponding antigen, whereas 110 control tumors of various nonhematopoietic origins were negative. It was concluded that the T29/33 antibody may be of value in resolving the diagnostic problems presented by tumors in which the differential diagnosis is between non-Hodgkin's lymphoma and nonlymphoid tumors.

Aged↗
PubMed1983

Enhancement of toxicity of antitransferrin receptor antibody-Pseudomonas exotoxin conjugates by adenovirus.

Cytotoxic conjugates were constructed by chemically coupling Pseudomonas exotoxin to antitransferrin receptor antibodies. Toxicity of these conjugates, due to entry via the transferrin receptor, was enhanced 100- to 300-fold in the presence of adenovirus. By electron microscopy and immunofluorescence it was determined that antitransferrin receptor antibodies and the conjugates derived from them entered cells from coated pits into receptosomes. We believe that the enhanced toxicity resulted when adenovirus and the toxin conjugates were internalized into the same receptosomes. In the process of infection, adenovirus enters cells and brings about a virus-mediated disruption of receptosomes; and this disruption can liberate many more toxin molecules into the cytosol than is possible in the absence of virus.

ADP Ribose Transferases↗
PubMed1983

Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

Biological Transport, Active↗