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J L Pace

Publications and source records attributed to J L Pace.

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

Molecular, biochemical, and functional characteristics of tumor necrosis factor-alpha produced by human placental cytotrophoblastic cells.

Previous studies have shown that the TNF-alpha gene is transcribed and translated in fully differentiated human placental syncytiotrophoblast. In this study, TNF transcripts were identified by in situ hybridization in cytotrophoblastic cells, a progenitor subpopulation that proliferates rapidly in early gestation tissues. To establish molecular and biochemical characteristics of cytotrophoblastic TNF and to evaluate potential utilization, experiments were conducted on two cytotrophoblastic cell lines, Jar and JEG-3. Northern blot hybridization and immunocytochemical tests showed that Jar and JEG-3 cells contained TNF mRNA and specific protein. Enzyme immunoassays demonstrated production of TNF, and immunoprecipitation experiments showed that Jar cell TNF protein was the same molecular mass as macrophage TNF. DNA synthesis in both lines was promoted by rTNF, and experiments employing 17-mer TNF antisense and sense oligonucleotides showed specific inhibition of DNA synthesis by antisense sequences. Both p60 and p80 TNF-R mRNA were present in the choriocarcinoma cell lines, and DNA synthesis was inhibited by antibody to the p60 TNF-R. Although the two lines were similar in many respects, Jar cells produced more TNF and demonstrated a greater reliance on TNF for their growth. Collectively, the results indicate that: 1) the TNF gene is expressed in both normal and malignant cytotrophoblast; 2) certain molecular, immunologic, and biochemical characteristics of trophoblast-derived TNF are similar to macrophage TNF; and 3) the p60 TNF-R facilitates utilization of TNF as an autocrine growth factor by choriocarcinoma cells. Although TNF apparently serves important functions in cytotrophoblast during the course of placental development that might include promotion of proliferation and invasion, constitutive expression of this gene in neoplastic cells could account in part for the remarkable ability of trophoblastic tumors to overcome host defenses.

Base Sequence↗
PubMed1993

The mouse macrophage activation-associated marker protein, p71/73, is an inducible prostaglandin endoperoxide synthase (cyclooxygenase).

The inducible protein p71/73 marks the response of mouse macrophages to one of several stimuli (e.g., bacterial lipopolysaccharide or poly I:C) that trigger the expression of cytolytic activity when these cells have previously been primed for tumor cell killing by interferon-gamma (IFN-gamma). The results reported here identify this marker protein as the inducible prostaglandin endoperoxide synthase (PES), TIS10/PES-2. Identification was based on four findings: (1) p71/73, like the TIS10/PES-2 protein, was associated with cellular membranes; (2) the sequence of amino acids in the NH2 terminus of both p71 and p73 was 96% identical to the predicted NH2-terminal sequence of the TIS10/PES-2 protein; (3) a polyclonal antiserum raised against the COOH-terminal region of the TIS10/PES-2 gene product recognized p71/73 in immunoblots; and (4) dexamethasone, which blocks induction of TIS10/PES-2 expression, inhibited the induction of both p71/73 synthesis and tumoricidal activity in macrophage. Several regulatory roles for this protein in the activation process are possible.

Amino Acid Sequence↗
PubMed1993

Expression of tumor necrosis factor-alpha in mouse spermatogenic cells.

Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNF alpha) in vitro by bioassay and for the presence of TNF alpha mRNA by Northern blot analysis. Small quantities of bioactive TNF alpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNF alpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids appeared uninduced by LPS treatment. In situ hybridization for the TNF alpha message by using digoxigenin label antisense TNF alpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNF alpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNF alpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.

Animals↗
PubMed1992

The N-terminus and C-terminus of IFN-gamma are binding domains for cloned soluble IFN-gamma receptor.

The mechanism of binding of murine IFN-gamma to its receptor has not been determined. We have studied this mechanism by examining the binding of overlapping synthetic peptides of IFN-gamma to cloned soluble murine IFN-gamma R. IFN-gamma (1-39) and IFN-gamma (95-133) were able to compete with [125I]IFN-gamma for binding to cloned soluble receptor. Peptides corresponding to the inner region of IFN-gamma--IFN-gamma (36-60), IFN-gamma (54-91), and IFN-gamma (78-107)--showed a markedly reduced ability to compete with [125I]IFN-gamma for receptor binding relative to the N-terminal and C-terminal peptides. In direct binding studies, the binding of [125I]-IFN-gamma (1-39) to soluble receptor could only be competed by IFN-gamma (1-39) and IFN-gamma and not by any of the other peptides including IFN-gamma (95-133). This suggests that the N- and C-termini of IFN-gamma bind to different regions of the receptor. These data in conjunction with previous structure/function studies and x-ray crystallographic data have allowed us to formulate a "velcro-key" model of IFN-gamma binding to receptor that involves both the N- and C-terminal domains. The N-terminus binds in the classical "lock-and-key" manner characterized by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct from the N-terminus likely through the polycationic region, which is conserved across species barriers. Binding of this type would exhibit high affinity and low specificity similar to a piece of velcro. This interaction becomes specific when the C-terminus is in the context of the whole IFN-gamma molecule and may act to increase the affinity of receptor binding and/or facilitate signal transduction.

Amino Acid Sequence↗
PubMed1992

Characterization and use of monoclonal and polyclonal antibodies against the mouse interferon-gamma receptor.

To facilitate investigation of its physical and functional properties, 11 monoclonal antibodies (mAbs) and a goat polyclonal IgG specific for the mouse interferon- (IFN-gamma) receptor were characterized and their potential uses studied. Eight of the mAbs interacted with epitopes on the extracellular domain of the receptor, two interacted with epitopes on the intracellular domain, and one interacted with an epitope that could not be localized definitively to either region. Of the 11 mAbs, the majority (8) were IgGs, 2 were IgMs, and 1 was an IgA. Relative avidities of the seven that could be determined ranged from 333 to 0.002 microM-1. Both the polyclonal goat IgG and mAb GR-20 (the latter specific for an epitope in the binding site for IFN-gamma) blocked binding of the ligand and, as expected, prevented induction by IFN-gamma of priming of macrophages for tumor cell killing. None of the other mAbs had an effect despite the fact that GR-22 partially (greater than 50%) blocked binding of IFN-gamma. Neither the polyclonal IgG nor any of the mAbs had an agonist effect. The relative usefulness of the antibodies for immunoprecipitation, immunoblotting, immunoassay, and cell staining with and without prior fixation is described. The results of immunocytochemical staining directly confirmed that the majority of immunologically reactive receptor protein expressed by cells is intracellular. To facilitate use by other investigators, the hybridomas that produce these mAbs will be offered to the American Type Culture Collection.

Animals↗
PubMed1991

Stable expression of a secreted form of the mouse IFN-gamma receptor by rat cells.

The biologic effects of IFN-gamma are mediated through a receptor that is expressed in relatively low abundance on normal mammalian cells. As a consequence, investigations of the physicochemical and ligand-binding properties of the purified receptor have been limited. The work reported here characterizes a secreted form of the receptor for mouse IFN-gamma, made by deletion of the nucleotides that code for the anchoring domain from a cDNA that encodes the receptor binding protein and its related signal peptide. When transfected into rat XC cells, this construct produced up to approximately 1 mg/liter of a secreted protein that had the characteristics of the native receptor. Both the secreted protein and its mRNA were of sizes that were consistent with loss of the transmembrane region. The protein was detectable by a mAb that is specific for an epitope that is found in the ligand binding site of the receptor for mouse IFN-gamma, as well as by a goat polyclonal IgG that is monospecific for the mouse IFN-gamma R. Supernates that contained the secreted protein blocked binding of IFN-gamma to mouse IFN-gamma R and inhibited in a dose-dependent manner the IFN-gamma-mediated priming of mouse bone marrow culture-derived macrophages for tumor cell killing. Availability of relatively large amounts of a secreted protein that retains ligand-binding activity should facilitate purification and basic studies of the receptor binding protein and could provide new approaches to the treatment/prevention of diseases that arise due to inappropriate response of cells to IFN-gamma. In addition, because this secreted receptor, unlike others, consists of both the extracellular and intracellular domains, it is likely that it will be useful in determining how the cytoplasmic portion of the receptor is involved in receptor function.

Amino Acid Sequence↗
PubMed1991

Activation-associated marker proteins: peptide mapping and their expression in macrophage cell lines.

The activation-associated markers, p47b and p71/73, have been recognized as minor proteins in peritoneal and bone marrow culture-derived macrophages activated for tumor cell killing. Proteins with identical characteristics of migration on 2-dimensional gels and comparable Cleveland peptide maps are described here in macrophage cell lines that could be activated for tumor cell killing (J774A.1, RAW 264, UNC-2). Macrophage cell lines that could not be activated (P388D1 and PU5-1.8) did not express the markers. The expression of these markers in activatable macrophage cell lines strengthens their association with the activation process and provides a bulk source of the proteins for purification studies.

Animals↗
PubMed1991

Purification and partial characterization of an avian thymic hormone. Avian thymic hormone.

An avian thymic hormone, originally designated the T1-antigen, was purified from chicken thymus by Sephadex G-75-40 chromatography and affinity chromatography, following enrichment by heat and pH treatments. It was characterized as an acidic polypeptide rich in phenylalanine, alanine and serine, lacking in histidine, tryptophan, methionine and cysteine, and having a blocked N-terminal amino acid. The hormone also was rich in hydrophobic amino acid residues, which gave it a propensity to form aggregates. Its molecular weight was estimated by gel electrophoresis and low speed sedimentation equilibrium to be 12-13 Kd, and by molecular sieving chromatography to be 15-16 Kd. The hormone was lacking in carbohydrates and amino sugars.

Amino Acids↗
PubMed1990

Induction of macrophage-mediated tumor cytotoxicity by a hamster monoclonal antibody with specificity for lipopolysaccharide receptor.

Experiments have been carried out to assess the immunostimulatory activity of a hamster IgM mAb (mAb5D3) with specificity for an 80-kDa LPS-binding protein expressed on murine macrophages and monocytes. The addition of mAb5D3 to cultures of murine bone marrow-derived macrophages activated these cells to become tumoricidal for mastocytoma cells in vitro. The activity of mAb5D3 was enhanced in the presence of IFN-gamma. Neither mAb5D3 nor LPS were able to activate macrophages from the LPS-hyporesponsive C3H/HeJ mouse, although these cells responded normally to heat-killed Listeria monocytogenes. The results of several experiments establish that the observed LPS-like activity of mAb5D3 was not due to contaminating endotoxin: 1) the activity of mAb5D3 but not LPS was heat labile at 100 degrees C; 2) the activity of LPS but not mAb5D3, was inhibited by addition of polymyxin B; and 3) quantitative estimates of endotoxin contamination by Limulus amoebocyte lysate reactivity. These experiments thus demonstrate that mAb5D3 can serve as an agonist for LPS-dependent macrophage responses and, when considered with those of our companion paper showing specificity of mAb5D3 for the 80-kDa LPS-binding protein, provide strong support for the concept that the 80-kDa LPS-binding protein previously identified serves as a functional receptor for LPS on murine macrophages.

Animals↗
PubMed1990

Differential responses of rat trophoblast cells and embryonic fibroblasts to cytokines that regulate proliferation and class I MHC antigen expression.

TNF-alpha and type I IFN (IFN-alpha/beta) are present in the uteroplacental unit during the course of normal gestation. IFN-gamma is likely to be present during infections. To identify potential effects on two types of blastocyst-derived cells, TNF-alpha, IFN-alpha/beta, and IFN-gamma were tested for the ability to modulate proliferation and the expression of class I MHC Ag by rat trophoblast cells and embryonic fibroblasts. The three cytokines had opposite influences on cellular proliferation by the two types of cells. Growth of the trophoblast cells was inhibited by TNF-alpha, IFN-alpha/beta, and IFN-gamma, whereas both TNF-alpha and IFN-alpha/beta enhanced fibroblast proliferation. The two endogenous cytokines had different effects on class I Ag expression by trophoblast cells and fibroblasts: TNF-alpha failed to induce trophoblast cell class I Ag and IFN-alpha/beta was a poor inducer whereas fibroblast Ag were induced by both cytokines. Moreover, combinations of TNF-alpha and IFN did not increase trophoblast cell class I Ag whereas the same combinations synergized to induce class I Ag expression by fibroblasts. In contrast, IFN-gamma was a highly efficient inducer on both types of cells. The results suggest that 1) cytokines in the uteroplacental unit may orchestrate some of the events associated with placental and embryonic development by exerting differential effects on two embryologically distinct types of cells and that 2) infections may disrupt normal events.

Animals↗
PubMed1989

Macrophage activation-associated proteins. Characterization of stimuli and conditions needed for expression of proteins 47b, 71/73, and 120.

The expression of cellular proteins was analyzed by two-dimensional gel electrophoresis during and after exposure of mouse macrophages to either mouse rIFN-gamma or natural MuIFN-beta sufficient to prime macrophages for tumor cell killing. The reversible inhibitor of protein synthesis, cycloheximide (CY), was included in some experiments during exposure to IFN. While it was present, CY suppressed protein synthesis by greater than 90%, but did not affect priming for tumor cell killing that was induced by either kind of IFN, as measured in cytotoxicity assays. Further analysis showed that, after CY and IFN were removed, protein synthesis recovered fully within 1 h. p47b, a protein that has been associated closely with the induction of the primed state in mouse macrophages, was then substantially expressed despite no new stimulation by IFN. Thus, macrophages in which protein synthesis had been reversibly inhibited delayed full processing of a signal delivered by IFN, until after protein synthesis had resumed. Such a delay in processing may explain how macrophages subsequently became activated, despite treatment with CY. The expression of the protein doublet, p71/73, was induced, regardless of which of three dissimilar agents (LPS, heat killed Listeria monocytogenes, poly I:C) was used to trigger the expression of cytolytic activity by primed macrophages. Therefore, the likelihood was increased that p71/73, expressed with p47b, is a valid phenotypic marker for fully activated, cytolytic macrophages. By contrast, p120, another protein that has been proposed as a marker of full activation in peritoneal macrophages, was expressed by bone marrow culture-derived macrophages regardless of whether or not they were cytolytic for tumor cells. It cannot be regarded as a reliable marker of macrophage activation in all circumstances, therefore.

Animals↗
PubMed1989

A monoclonal antibody against the ligand binding site of the receptor for mouse interferon-gamma.

A rat monoclonal IgG2a antibody, GR-20, has been produced against the receptor for mouse interferon-gamma (MuIFN-gamma). Comparison of competitive binding studies performed with either 125I-labeled GR-20 or recombinant (r) MuIFN-gamma, as well as a variety of other studies, suggested that the epitope recognized by the monoclonal antibody (mAb) is in the domain of the receptor that binds ligand. The binding of GR-20 to cells of the monomyelocytic line WEHI-3 was of high affinity (1-2 nM). Approximately 20,000 binding sites were found per cell, a value that is in close agreement with the number of MuIFN-gamma receptors quantified on cells of the same type by ligand binding studies. The mAb also bound to a variety of other mouse cells, suggesting that the same epitope is shared by receptors for MuIFN-gamma, regardless of cell type. The epitope was not detected on two human cell types that were tested, while cells of a rat cell line shown to be minimally responsive to rMuIFN-gamma gave equivocal binding results when they were interacted with GR-20. Binding of the mAb to the receptor did not mimic the effects of ligand. In fact, the opposite was true: binding blocked the induction of three biological effects of MuIFN-gamma, including priming of macrophages for tumor cell killing, upregulation of the expression of class II major histocompatibility antigens (Ia) on the same cell type, and induction of antiviral activity in L cells. Following intravenous injection, initial removal of GR-20 was precipitous, followed after 1 h by a phase which was more gradual, resulting in 5-10% of biologically active mAb remaining in the circulation after 24 h. Such retention should make this mAb useful in a variety of studies in vivo.

Animals↗
PubMed1988

Synergistic interactions between IFN-gamma and IFN-beta in priming murine macrophages for tumor cell killing.

The ability of MuIFN-beta and MuIFN-gamma to potentiate the development of tumoricidal activity in proteose peptone-elicited murine peritoneal macrophages was investigated. Macrophages were stimulated with increasing concentrations of either MuIFN-beta or MuIFN-gamma, alone and in combination, in the presence of 1 ng/ml lipopolysaccharide (LPS). The priming activities attributable to the interferons (IFNs) were quantified using the dose-response curves obtained for these samples. The priming activity observed for mixtures of MuIFN-beta and MuIFN-gamma was greater than that expected if MuIFN-beta and MuIFN-gamma had acted in an additive manner. Isobologram analysis of data obtained when macrophages were stimulated with combinations of IFNs demonstrated that MuIFN-beta and MuIFN-gamma acted synergistically to prime macrophages for tumor cell killing. The greatest degree of synergy was observed when macrophages were stimulated with suboptimal and nearly equivalent concentrations of each class of IFN. Further studies demonstrated that macrophages stimulated with combinations of MuIFN-beta and MuIFN-gamma were more sensitive to the trigger signal provided by LPS than were cells primed with either IFN alone. Thus, the synergistic effects observed were quantitative in nature in that macrophages perceived combinations of MuIFN-beta and MuIFN-gamma as having higher priming activities than expected.

Animals↗
PubMed1988

Purification and partial characterization of a receptor protein for mouse interferon gamma.

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.

Animals↗