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J R Gnarra

Publications and source records attributed to J R Gnarra.

49 records · Page 3Linked to original sources
PubMed1990

Structure, function, and regulation of the interleukin-2 receptor and identification of a novel immune activation gene.

This chapter is divided into two sections, the first dealing with a novel immune activation gene, denoted Act-2. This gene encodes a secreted protein that may represent a new cytokine. The Act-2 protein shares significant homology with proteins in two related families of small secreted proteins. Act-2 is rapidly synthesized by activated T cells, B cells and monocytes. The second section deals with interleukin-2 receptors. These receptors are now known to be comprised of three distinct classes of receptors, formed by various combinations of two IL-2 binding proteins, the alpha and beta chains. The low-affinity receptors contain alpha, but not beta chains; the intermediate-affinity receptors contain beta, but not alpha chains, and the high-affinity receptors contain both alpha and beta chains. The beta chain appears to be tyrosine phosphorylated. We discuss evidence for the existence of another protein of relative molecular mass 100,000, which appears to be a subunit of at least the high-affinity receptor.

Amino Acid Sequence↗
PubMed1990

A 100-kilodalton protein is associated with the murine interleukin 2 receptor: biochemical evidence that p100 is distinct from the alpha and beta chains.

Two proteins that specifically bind the T-cell growth factor interleukin 2 (IL-2) have been identified previously on the surface of T cells; these proteins have been designated IL-2R alpha and IL-2R beta for the alpha and beta chains of the IL-2 receptor (IL-2R). The association of these independent binding proteins with each other on the surface of activated T cells correlates with the generation of high-affinity binding sites. These high-affinity sites transduce the major mitogenic signal of IL-2, yet the mechanisms of association of the alpha and beta chains with each other as well as signal transduction in response to IL-2 are unknown. Cotransfection experiments of cDNAs encoding the alpha and beta chains in T cells and fibroblasts have suggested functional requirements for other T cell-specific factor(s). We now provide biochemical evidence for a distinct 100-kDa protein that interacts with the alpha or beta chains, or both, on the surface of the IL-2-dependent cell line CTLL-2 as well as activated murine splenocytes. This same 100-kDa protein is capable of being chemically cross-linked to 125I-labeled IL-2.

Animals↗
PubMed1990

Human interleukin 2 receptor beta-chain gene: chromosomal localization and identification of 5' regulatory sequences.

Interleukin 2 (IL-2) binds to and stimulates activated T cells through high-affinity IL-2 receptors (IL-2Rs). Such receptors represent a complex consisting of at least two proteins, the 55-kDa IL-2R alpha chain and the 70-kDa IL-2R beta chain. The low-affinity, IL-2R alpha chain cannot by itself transduce a mitogenic signal, whereas IL-2 stimulates resting lymphocytes through the intermediate-affinity, IL-2R beta receptor. We report here identification of the genomic locus for IL-2R beta. The exons are contained on four EcoRI fragments of 1.1, 9.2, 7.2, and 13.7 kilobases. The 1.1-kilobase EcoRI fragment lies at the 5'-most end of the genomic locus and contains promoter sequences. The promoter contains no TATA box-like elements but does contain the d(GT)n class of middle repetitive elements, which may play an interesting regulatory role. The IL-2R beta gene is localized to chromosome 22q11.2-q12, a region that is the locus for several lymphoid neoplasias.

Base Sequence↗
PubMed1989

The beta-chain of the IL-2 receptor (p70) is tyrosine-phosphorylated on YT and HUT-102B2 cells.

IL-2 has previously been shown to rapidly induce activity of a tyrosine kinase. High-affinity IL-2 receptors that mediate the major mitogenic signals of IL-2 contain both p70 and p55 chains. p55 has no potential tyrosine phosphorylation sites and lacks consensus sequences found in protein tyrosine kinases. Inasmuch as the phosphorylation of hormone receptors is generally an important mechanism for regulating receptor function, we have now investigated the phosphorylation status of p70. By using anti-phosphotyrosine antibodies to immunoprecipitate affinity-labeled IL-2 receptors and to probe Western blots, we provide data suggesting that p70, but not p55, is constitutively tyrosine-phosphorylated on the leukemic cell lines studied.

Cell Line, Transformed↗
PubMed1988

Possible association between IL-2 receptors and class I HLA molecules on T cells.

In the process of evaluating murine hybridomas for an antibody to the beta-subunit of the IL-2R (p70) we identified an antibody that immunoprecipitated a 55- to 57-kDa complex from cross-linked lysates. We demonstrate that this complex is composed of IL-2 (15.5 kDa) cross-linked to the H chain of HLA class I (40 to 42 kDa), suggesting a molecular interaction between HLA class I molecules and IL-2R. Although the exact role of this association remains to be determined, the specific cross-linking of IL-2 to HLA class I Ag is intriguing in view of published claims for a role of HLA class I in OKT3-induced lymphocyte proliferation and in NK cell lytic activity.

Animals↗
PubMed1988

Spatial and temporal characteristics of the increase in intracellular Ca2+ induced in cytotoxic T lymphocytes by cellular antigen.

The increase in intracellular Ca2+ concentration [( Ca2+]i) in cytolytic T lymphocytes in response to target cell binding was investigated by ratio image fluorescence microscopy. Ca2+ mobilization from intracellular stores occurred at a site distal to target cell contact and was transient. Extracellular Ca2+ influx resulted in an increase in [Ca2+]i that was prolonged and distributed more proximal to the target cell. Oscillations in [Ca2+]i were observed after target cell contact, although the periodicity was dependent on the presence of extracellular Ca2+. The Ag-specific reorientation of cytoplasmic granules occurred well after [Ca2+]i had begun to decline to a resting level, but was dependent on extracellular Ca2+. These studies indicate that the Ag-stimulated increase in [Ca2+]i exhibits considerable spatial and temporal variation, and that these characteristics are altered by the availability of extracellular Ca2+. The results also suggest that these changes in [Ca2+]i may play a role in the cytoplasmic events that accompany T cell-mediated cytolysis.

Animals↗
PubMed1988

Early events in target-cell lysis by cytotoxic T cells.

Using ratio-imaging fluorescence microscopy, we have investigated the changes in intracellular Ca2+ [( Ca2+]i) that occurred in cytotoxic T lymphocytes (CTL) upon target-cell binding. This process resulted in a rapid increase in [Ca2+]i, which was localized in the region of the CTL in contact with the target cell. This increase was mediated both by influx from the external medium as well as by release from intracellular stores. Although the magnitude of the initial increase in [Ca2+]i was not dependent upon the presence of extracellular Ca2+, influx was necessary for sustained elevation of [Ca2+]i. Inasmuch as target-cell lysis by the CTL clone used is dependent on extracellular Ca2+, this suggests that a prolonged elevation of [Ca2+]i is necessary for lytic function. It was also shown that the increase in [Ca2+]i and its subsequent decay show several pulsations. The mechanism by which these variations are generated and their possible function is not known. Finally, a role for K+ efflux in the control of the antigen-induced increase in [Ca2+]i was demonstrated. Thus it is becoming clear that signal transduction in CTL is remarkably complex, involving several ionic species and second messengers.

Animals↗
PubMed1987

The role of K+ in the regulation of the increase in intracellular Ca2+ mediated by the T lymphocyte antigen receptor.

The regulation of the increase in intracellular calcium ([Ca2+]i) occurring in cytolytic T lymphocytes (CTLs) upon their interaction with antigen was examined. This [Ca2+]i increase and lytic function were insensitive to verapamil, a Ca channel blocker. An antigen-independent increase in [Ca2+]i was not induced by depolarization of CTLs with excess extracellular K+, suggesting that Ca2+ influx is not mediated by the ubiquitous voltage-gated Ca channel. The antigen-induced [Ca2+]i increase was inhibited by prior membrane hyperpolarization with valinomycin. Hyperpolarization occurred under normal circumstances in CTLs exposed to antigen-receptor-specific antibodies. This potential change was Ca2+-dependent and inhibited by K channel blockade. Conversely, K channel blockade augmented the antigen-specific [Ca2+]i increase while markedly decreasing the K+ efflux associated with CTL lytic function. Therefore, either membrane potential or intracellular K+ regulates the antigen-specific [Ca2+]i increase in CTLs.

Animals↗
PubMed1987

Demonstration of a calcium influx in cytolytic T lymphocytes in response to target cell binding.

By using the Ca2+-sensitive dye indo-1, an antigen-specific increase in intracellular Ca2+ in cloned cytolytic T lymphocytes (CTL) was measured under conditions that were permissive for T cell-mediated cytolysis. To synchronize lethal hit delivery in a suspension of effector and target cells, a modification of the cation pulse method in which Ca2+ is added to preformed conjugates of CTL and target cells was used. Conjugate formation was unaffected by the absence of extracellular Ca2+ under these conditions. Lytic activity of these cloned CTL was markedly reduced in the absence of extracellular Ca2+ and was restored upon Ca2+ repletion. When indo-1-loaded CTL were preincubated with target cells in the absence of extracellular Ca2+, a marked antigen-specific increase in indo-1 fluorescence, indicative of an increase in intracellular Ca2+, was observed after repletion of extracellular Ca2+. This increase in intracellular Ca2+ was shown to be due solely to changes in the CTL and not the target cell within the time course of the experiment, and results from the influx of extracellular Ca2+. Antibody to the T cell receptor for antigen also evokes a similar increase in intracellular Ca2+ in CTL under these conditions. This method provides a means for the direct examination of the response of CTL to cellular antigen as well as soluble antibody and is a versatile and valuable tool for the study of CTL function.

Animals↗
PubMed1986

Derivation of cytotoxic T cell lines that express T cell receptor but exhibit reversible loss of stable antigen binding.

Stable cell lines lacking cytotoxic activity against specific target cells were derived from highly active murine CTL clones by the omission of antigen from the culture for several weeks. Several independent CTL clones cultured in the absence of antigen showed a gradual decline in cytotoxic activity, resulting in complete loss by 5 to 10 wk. Such noncytotoxic (NC) cells lacked the ability to form stable conjugates with specific target cells, but were able to kill all target cells tested in the presence of Con A. It was shown by subcloning at limiting dilution that all cells in the starting population were cytolytically active, and that all cells in the NC population derived from such a clone were cytolytically inactive against target cells bearing an appropriate antigen under normal assay conditions. By using the monoclonal antibody F23.1, which reacted with the antigen receptors of two of the CTL clones, it was shown that the NC cells derived from these clones continued to express the receptor at normal levels. Levels of expression of Thy-1.2, Lyt-2.2, and LFA-1 were also similar in all cytotoxic cell lines and their noncytotoxic derivatives. The F23.1 antibody induced an increase in cytoplasmic free Ca2+ in both CTL and NC cells, and NC cells lysed F23.1 hybridoma cells in the absence of Con A. When cells expressing appropriate target cell antigen were added back to cultures of NC cells, cytotoxic activity of appropriate specificity was fully recovered in 2 wk. These results indicate that expression of an apparently functional antigen receptor alone is insufficient for stable binding of CTL to specific target cells, and that other factors dependent upon antigen stimulation may be involved in the recognition process. A difference in affinity for antigen between CTL and NC cells is suggested as a possible explanation for these observations.

Animals↗
PubMed1982

Effectors of three beta-glucosidases from human liver.

1. A third beta-glucosidase from human liver has been isolated using a mild (0.02-0.10%) Triton X-100 extraction of the exhaustively washed high speed (200,000 X g, 30 min) particulate fraction, QAE-Sephadex and concanavalin A-Sepharose chromatography. This new beta-glucosidase, referred to as TX beta-glucosidase, possesses a distinctive set of chemical properties such that it is similar to both, glucocerebrosidase and cytoplasmic beta-glucosidase, but it is not identical to either enzyme. 2. The TX beta-glucosidase hydrolyzes glucocerebroside as well as the beta-D-glucose, beta-D-galactose, beta-D-fucose, beta-D-xylose and alpha-L-arabinose derivatives of 4-methylumbelliferone. Like the cytoplasmic beta-glucosidase, the TX beta-glucosidase is inhibited by bile salts, and unaffected by conduritol B epoxide and heat stable activator protein. 3. All three beta-glucosidases were inhibited by N-hexylpsychosine, and all showed the same, mixed type inhibition kinetics, indicating a common hydrophobic binding site in all three enzymes. 4. The TX beta-glucosidase, which constitutes only a few percent of the total beta-glucosidase activity of human liver, is absent from liver from two cases of neurologic Gaucher disease and present in reduced amounts in a third case with CNS disease. Liver from a case of type 1 Gaucher disease contained normal amounts of the TX beta-glucosidase.

Bile Acids and Salts↗