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Biomedical subjects

A B Frey

Publications and source records attributed to A B Frey.

At least 37 records · Page 2Linked to original sources
PubMed1996

Analysis of the structure and function of the von Willebrand factor A1 domain using targeted deletions and alanine-scanning mutagenesis.

von Willebrand factor (vWF) mediates the primary adhesion of platelets to sites of vascular damage through interaction with glycoprotein Ib (GPIb) of the platelet GPIb/IX complex. To investigate the vWF/GPIb interaction we introduced both in-frame deletions and substitutions into the vWF A1 domain. The introduction of nine sequential 20-amino acid deletions within the Cys509-Cys695 loop of the A1 domain caused the defective secretion of vWF from mammalian cells, and resulted in multimeric vWF without platelet-binding activity. In other experiments we substituted alanine for charged amino acids (residues 524, 534, 549, 552, 569-573, and 642-645) in proposed functional domains within the Cys509-Cys695 loop. All six substitution mutants showed normal secretion from transfected mammalian cells and bound to fixed platelets in the presence of botrocetin. In contrast, only mutants vWF-R524A and vWF-K549A showed significant binding to platelets in the presence of ristocetin. Mutant vWF-K549A showed increased platelet-binding at suboptimal concentrations of both botrocetin and ristocetin. These results suggest that the substituted amino acids do not play a critical role in the activation of vWF by botrocetin or in the direct interaction of vWF with the GPIb/IX complex. However, the charged amino acids at positions 534, 552, 569-573, and 642-645 do play an important role in the ristocetin-induced binding of vWF to platelets. The interaction of vWF with heparin was significantly reduced by substitution of Lys residues 642-645, indicating that these residues may form part of a heparin-binding domain in the carboxy-terminal half of the Cys509-Cys695 loop.

Alanine↗
PubMed1996

Role of host antigen receptor-bearing and antigen receptor-negative cells in immune response to rat adenocarcinoma 13762.

Involvement of individual immune cell populations in the immune response to rat breast adenocarcinoma 13762 was studied by selective depletion treatments in vivo. Depletion of Ag nonspecific host defense cells from naive animals before tumor challenge resulted in statistically significant acceleration of tumor growth (p less than 0.01 or less), whereas depletion of either CD4+ T cells or CD8+ T cells had no effect on the incidence or kinetics of tumor development. In contrast to naive animals treated before tumor challenge, animals actively immunized by injection of irradiated tumor before depleting treatments were shown to require CD4+ T cells to reject tumorigenic challenge. Depletion of either macrophages or neutrophils from immune animals also increased tumor development, whereas NK cells were not involved. Depletion of CD8+ T cells from immune animals permitted transient growth of tumors that were subsequently rejected, implying a role in tumor rejection. Transfer of immune antiserum to naive animals at the time of tumor challenge was without effect on tumor development. Depletion of CD4+ T cells, neutrophils, or macrophages in the priming phase of antitumor immune response abrogated tumor immunity, but depletion of CD8+ T cells or NK cells was without effect on the ability to prime animals by immunization with irradiated cells. Collectively, these data suggest that host natural defense cells that do not express Ag receptor are primarily responsible for resistance of adenocarcinoma 13762 growth in naive animals. In contrast, tumor immunity induced by active immunization requires Ag receptor-bearing CD4+ T cells and involves participation of CD8+ T cells, neutrophils, or macrophages in elimination of tumor.

Adenocarcinoma↗
PubMed1995

Dominant-negative p53 can restore tumorigenicity of L-929 cells in immunocompetent mice.

To study the effect of a transforming allele of the tumor suppressor p53 upon the anti-tumor immune response, antigenic L-929 cells were transfected with the dominant-negative valine135 mutant of murine p53. Several p53val135-expressing transfectants formed non-regressing tumors in immunocompetent hosts. The growth rates of tumorigenic and non-tumorigenic clones were equivalent in vitro in sublethally irradiated C3H/HeN mice and in nude mice. Tumorigenic and non-tumorigenic p53val135-expressing L-929 clones expressed equivalent levels of cell surface class I major histocompatibility complex (MHC) glycoproteins. Immunization with a tumorigenic Lp53val135 clone protected mice from subsequent challenge and primed MHC class I-restricted cytotoxic T-lymphocytic precursors. Secretion of an immunosuppressive cytokine, transforming growth factor beta-1 and sensitivity to tumor necrosis factor-alpha were equivalent from tumorigenic and non-tumorigenic cell lines. These data suggest that expression of a transforming allele of p53 can allow L-929 cells to escape the host immune system.

Alleles↗
PubMed1995

Expression of activated H-rasval12 in nontumorigenic and non-cross-reactive syngeneic cells induces tumor antigens cross-reactive with rat mammary adenocarcinoma 13762.

Adenocarcinoma 13762 expresses tumor Ags that can induce protective immunity to tumorigenic challenge. Syngeneic fibroblast Rat1 cells transformed by expression of H-rasval12 (Rat1/ras) but not parental Rat1 cells, and p53-transformed Rat1 cells, or other syngeneic cells or tumors, can immunize rats against tumorigenic challenge of 13762 tumor. Coincident with induced resistance to 13762 tumors, immunization of rats with Rat1/ras tumor induces CD4+ T cells that cross-react with adenocarcinoma 13762 in vitro and transfer protective immunity to tumorigenic 13762 challenge in vivo. Cross-reactive tumor Ags expressed in Rat1/ras tumor are not derived from ras protein, because immunization with purified H-rasval12 protein induces protective immunity to challenge by Rat1/ras tumor but not to adenocarcinoma 13762. In addition, immunization with H-rasval12 protein induces anti-ras CD4+ T cells that are uniquely reactive with Rat1/ras tumor: anti-ras T cells are not reactive with 13762 tumor in vitro and do not confer protective immunity to challenge with 13762 tumor in vivo. Tumor Ags expressed in Ras-transformed Rat1 cells that elicit cross-protective immunity likely arise as a consequence of transformation mediated by activated ras oncogene but are not derived from the Ras protein sequence.

Adenocarcinoma↗
PubMed1995

Apparent split tolerance of CD8+ T cells from beta 2-microglobulin-deficient (beta 2m-/-) mice to syngeneic beta 2m+/+ cells.

beta 2-microglobulin-deficient (beta 2m-/-) mice express reduced levels of MHC class I molecules and, consequently, have impaired positive selection of CD8+ T lymphocytes in the thymus. However, small numbers of CD8+ CTLs can be found in beta 2m-/- mice after immunization with allogeneic as well as syngeneic beta 2m+/+ tumor or spleen cells. It has been proposed, therefore, that because of the low ligand density in beta 2m-/- mice, negative selection does not remove cells capable of recognizing syngeneic MHC class I expressed at normal levels. We report here that beta 2m-/- CD8+ T cells are partially tolerant to syngeneic beta 2m+/+ cells. Despite the ability of beta 2m-/- mice to raise CD8+ CTLs against syngeneic beta 2m+/+ cells, these CD8+ cells do not proliferate and do not secrete IFN-gamma or IL-3/granulocyte-macrophage-CSF upon in vitro stimulation with syngeneic beta 2m+/+ cells. In contrast, all of these cellular responses are displayed by the beta 2m-/- CD8+ cells upon recognition of the allogeneic MHC class I. These in vitro findings of partial responsiveness to syngeneic and of full responsiveness to allogeneic MHC class I correlate well with the ability of beta 2m-/- mice to reject allogeneic, but not syngeneic, tumors in vivo. It appears, thus, that the significantly reduced levels of MHC class I molecules found in beta 2m-/- mice, although not capable of inducing deletion of all reactive clones, can induce deletion of high affinity clones and, therefore, maintain tolerance to self-MHC class I, even when expressed at much higher (beta 2m+/+) levels.

Animals↗
PubMed1995

Mouse embryo fibroblasts transformed by activated ras or dominant-negative p53 express cross-reactive tumor rejection antigens.

To study the immune response against oncogene-transformed tumors, C3H/HcN mouse embryo fibroblasts (MEF) were transfected with an activated allele of the H-ras proto-oncogene VaII2 and a dominant-negative allele of the murine p53 tumor suppressor gene VaII35. Transformed cell lines were derived and found to be tumorigenic in syngeneic mice. Immunization with irradiated p53 + ras-transformed MEF, but not primary MEF or unrelated syngeneic cells, protected mice from subsequent challenge with live tumor cells. The role of different immune cell subsets in the effector phase of anti-tumor immunity induced by immunization with p53 + ras-transformed MEF was investigated by in vivo antibody depletion experiments. Immunized mice depleted of CD8+ T, NK or B cells were resistant, but depletion of CD4+ T cells rendered mice susceptible to tumorigenic challenge. In contrast to the tumor-specific immune responses mounted against most chemically or UV-induced tumors, a series of independently derived p53 + ras-transformed MEF were cross-reactive in tumor rejection assays. In addition, immunization with C3H-derived L-929 cell lines expressing single gene products H-ras or p53 did not protect mice against tumorigenic challenge with p53 + ras-transformed tumors. However, MEF transformed by expression of either H-ras or p53 were cross-protective in vivo. Our data suggest that the p53 + ras-transformed MEF share tumor rejection antigens which are also induced by single gene transformation of the parental primary cell but are not the products of oncogenic ras or p53 protein.

Animals↗
PubMed1995

Rat mammary adenocarcinoma 13762 expressing IFN-gamma elicits antitumor CD4+ MHC class II-restricted T cells that are cytolytic in vitro and tumoricidal in vivo.

Rat adenocarcinoma 13762 was modified by transfection to express IFN-gamma, and the tumor-forming potential of cytokine-producing cells was found to be dramatically impaired. Animals resistant to inocula of IFN-gamma-modified tumor were resistant to subsequent challenge with unmodified 13762 tumor. Induced immunity was tumor specific in that syngeneic but non-cross-reactive tumor grew with normal kinetics in animals injected with IFN-gamma-producing 13762 tumor. Antitumor T cells were derived from animals primed with IFN-gamma-producing 13762 tumor and expanded into a cell line by coculture in vitro with IFN-gamma-producing 13762 cells. Anti-13762-gamma T cells were cytotoxic in vitro toward IFN-gamma-producing 13762 tumor and were not reactive with other syngeneic tumors or spleen B cells. Anti-13762-gamma T cells were determined to be CD4+ by Ab staining and flow cytometric analysis, and recognition of 13762-gamma in vitro was inhibited by anti-MHC class II Ab. Anti-13762-gamma T cells were not reactive in vitro with wild-type 13762 tumor unless treated with exogenous rIFN-gamma, which induced expression of cell surface MHC class II. However, adoptively transferred anti-13762-gamma T cells could effect regression of wild-type 13762 tumor or dramatically inhibit progressive growth in animals carrying significant tumor burden, and the antitumor phenotype did not require CD8+ T cells in vivo. These experiments demonstrate that although antitumor T cells elicited against cytokine-modified tumor may fail to demonstrate reactivity with unmodified tumor in vitro, antitumor properties may be manifest in vivo.

Adenocarcinoma↗
PubMed1995

Protective resistance to experimental Borrelia burgdorferi infection of mice by adoptive transfer of a CD4+ T cell clone.

The anti-spirochete T cell immune response to immunization with Borrelia burgdorferi was investigated. The cellular immune response to vaccination of immunocompetent BALB/c mice was characterized initially in vitro by assay of the proliferative response of primary lymph node cells to B. burgdorferi sonicate. Subsequently, an anti-spirochete T cell line (RBN2.1) and clone (97.1) were derived from lymph node cells of BALB/c mice primed with B. burgdorferi antigen. Both the line and clone were CD4+ by flow cytometric analysis. Significantly, RBN2.1 and clone 97.1 were able to transfer resistance to infection to syngeneic naive recipients. Assay of antigen-specific interleukin-2, interleukin-4, tumor necrosis factor-alpha, and interferon-gamma production demonstrated that clone 97.1 was of the Th2 subclass. When B. burgdorferi sonicate was fractionated on SDS-PAGE and then electroeluted, clone 97.1 was reactive exclusively to a spirochete protein of approximately 21 kDa. These data suggest that T-cell-mediated protective immunity to infection by B. burgdorferi can be elicited by active immunization.

Animals↗
PubMed1995

Single exposure of mice to Borrelia burgdorferi elicits immunoglobulin G antibodies characteristic of secondary immune response without production of interleukin-4 by immune T cells.

Borrelia burgdorferi antigen can elicit immunoglobulins (Igs) characteristic of the primary and secondary immune responses without the contribution of an interleukin-4-producing helper T-cell population. Single exposure of mice to soluble B. burgdorferi antigen elicited both Th1-type and Th2-type antispirochete antibodies. Production of the Ig classes showed different patterns with increasing time postinjection (IgM levels decreased; IgG1, IgG2a, IgG2b, and IgG3 levels increased; IgE was not detected), and Ig patterns were similar to those produced in infected mice. Upon infectious challenge, immunized mice achieved maximal titers of all antispirochete IgG subclasses more quickly than unimmunized mice did. In contrast to the antibody responses which showed both Th1- and Th2-type patterns, T-cell immune response to either immunization or infection was characterized by interleukin-2 and gamma interferon production; interleukin-4 and interleukin-5 were undetectable. Injection with whole spirochetes induced a pattern of antibodies and cytokine production similar to those obtained by injection with soluble antigen. In addition, mouse strains of different major histocompatibility complex backgrounds produced similar patterns of Ig in response to immunization. None of the various parameters of immunization tested resulted in detectable interleukin-4 production by primary or secondary immune T cells. The production of both IgM and IgG1 at early times following a single exposure to spirochete antigen clearly differs from immune responses to haptens or model protein antigens. Production of similar Ig classes in infected and immune mice implies that antigen-specific antibody is responsible for passive immunizing activity found in immune sera.

Adjuvants, Immunologic↗
PubMed1993

Rat adenocarcinoma 13762 expresses tumor rejection antigens but tumor-bearing animals exhibit tumor-specific immunosuppression.

Rat adenocarcinoma 13762 was adapted to continuous growth in culture and used in a variety of experiments to investigate the immune response to inoculation of animals with replication-defective tumor cells. The results demonstrate that 13762 cells express tumor-specific tumor rejection antigens that elicit protective immunity to tumorigenic challenge. By several criteria there is no apparent humoral component of the anti-tumor immunity; however, anti-tumor immunity is characterized by nylon-wool nonadherent spleen T cells. Anti-tumor T cells demonstrate tumoricidal activity in local adoptive transfer assays and are not found in spleens of naive animals or animals immunized against either nontumorigenic Rat 1 cells or a syngeneic fibrosarcoma. Despite the expression of tumor rejection antigens 13762 tumor, and the demonstrable ability of injection of irradiated tumor to induce anti-tumor immunity, tumors elicited in unimmunized syngeneic animals grow progressively. The reasons for growth of antigenic tumor are unknown but are shown not to be due to defective antigen expression in 13762 tumor since, in addition to being able to elicit T cell immune response in immunized animals, 13762 tumor expresses MHC Class I molecules and can be a target for allogeneic T cell recognition in vitro. These data suggest that in tumor-bearing animals an effective anti-tumor immune response is either not initiated or down-regulated. Since animals bearing 13762 tumors can be immunized against an unrelated syngeneic sarcoma, can produce humoral responses to several protein antigens, and can produce delayed type hypersensitivity response against dinitrofluorobenzene, the immune response to 13762-induced tumors appears specifically suppressed. In support of this contention, 13762 cells express high levels of transforming growth factor beta 1 in vitro which is postulated to impact upon the nascent anti-tumor immune response.

Adenocarcinoma↗
PubMed1991

Differential expression of MAG isoforms during development.

The myelin-associated glycoproteins (MAG) mediate the cell interactions of oligodendrocytes and Schwann cells with axons that are myelinated. MAG exists in two developmentally regulated isoforms: large MAG (L-MAG) and small MAG (S-MAG). In this paper, we have studied the tissue-specific and developmentally regulated alternative splicing of these isoforms using monospecific antibodies that recognize epitopes common to both isoforms or that are present only on L-MAG. In the central nervous system (CNS), L-MAG is the major form synthesized early in development, and it persists as a significant proportion of the MAG present in the adult. In the peripheral nervous system (PNS), L-MAG is expressed at modest levels during development; it is virtually absent in the adult. Thus, the expression of L-MAG is not limited to the CNS, as was formerly believed, suggesting that it plays a common role during the early stages of myelin formation by both oligodendrocytes and Schwann cells. In both the CNS and PNS, S-MAG is the predominant isoform in the adult. A higher-molecular-weight form of MAG is present in the PNS at low abundance, that is developmentally regulated, and appears to be a glycosylation variant. An analysis of the carbohydrate residues on MAG demonstrates that it contains both N-linked and O-linked sugars that could be modulated during development. These results suggest a possible mechanism for the regulation of MAG function during myelinogenesis via the expression of alternative isoforms and carbohydrate modifications.

Animals↗
PubMed1990

Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction.

Protein zero (P0), an integral membrane glycoprotein synthesized by Schwann cells, is the major glycoprotein of peripheral nerve myelin. The predicted disposition of P0 with respect to the membrane bilayer postulates the existence of extracellular and intracellular domains, that mediate compaction of the myelin lamellae. We used in vitro translations programmed with sciatic nerve mRNA and cells transfected with a P0 cDNA construct to study the biosynthesis and topology of P0 in the bilayer. The behavior of P0 at the cell surface, when expressed under physiological conditions, was also examined. We have verified the topological predictions of an earlier model, derived from analysis of a P0 cDNA, and provide evidence that the extracellular domain of P0 mediates homotypically cell-cell interactions in the transfectants.

Amino Acid Sequence↗
PubMed1989

p53-plus-ras-transformed rat embryo fibroblasts express tumor-specific transplantation antigen activity which is shared by independently transformed cells.

p53-plus-ras-transformed rat cell lines express a tumor-specific transplantation antigen that is common to a number (85%) of independently derived p53-plus-ras-transformed cell lines. This has been shown by immunizing rats with irradiated p53-plus-ras-transformed cells and demonstrating protection of these animals by subsequent live-cell tumor challenge. Several c-myc-plus-ras-transformed cell lines (54% of the lines tested) and one adenovirus E1a-plus-ras-transformed cell line (9% of those tested) were shown to share a common tumor-specific transplantation antigen by their ability to immunize a rat against a p53-plus-ras cell line challenge. Several experimental approaches have been used to fractionate and identify the antigen common to these cell lines. The experimental results reported here make it clear that the p53 protein common to most of these transformed cell lines is not likely to be the tumor-specific transplantation antigen.

Animals↗
PubMed1988

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP.

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.

Adenosine Triphosphate↗
PubMed1987

Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies.

Fifteen peptides, ranging in length from 6 to 31 amino acids and corresponding in sequence to portions of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P-450 PB-4), were previously synthesized chemically and used to prepare site-specific rabbit antibodies (Frey, A. B., D.J. Waxman, and G. Kreibich, 1985, J. Biol. Chem., 260:15253-15265). The antipeptide antibodies were affinity purified using Sepharose resins derivatized with the respective peptides and 14 preparations were obtained that in an ELISA assay showed affinities to immobilized P-450 judged to be adequate for binding studies on intact rat liver microsomes. The binding of these antibodies to rough microsomes from the livers of phenobarbital treated rats was assessed using 125I-labeled IgG and by immunoelectron microscopy employing protein A-gold as a marker. It was found that many of the antibodies bound to the cytoplasmic surface of the membrane but none bound to the luminal face of ruptured or inverted microsomal vesicles or to contaminating membranes of other organelles present in the preparations. These observations eliminate previously proposed models for the transmembrane disposition of P-450 that postulate the existence of multiple transmembrane domains and the exposure of several polar segments of the polypeptide on the luminal side of the membrane. The fact that an antibody raised to the first 31 residues of P-450 bound well to the purified P-450 but very poorly to rough microsomes, whereas an antibody to a peptide comprising residues 24-38 showed relatively strong binding to intact microsomes, is consistent with the proposal that the amino terminal segment of P-450 extending approximately to residue 20 is embedded in the phospholipid bilayer and the immediately following segment is exposed on the cytoplasmic surface of the membrane. All these results favor a model in which the cytochrome P-450 molecule is largely exposed on the cytoplasmic surface of the endoplasmic reticulum membrane to which it is anchored by its short amino terminal hydrophobic segment.

Amino Acid Sequence↗
PubMed1987

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.

Amino Acid Sequence↗