PubMed Health⌕ Search

Biomedical subjects

M J Robertson

Publications and source records attributed to M J Robertson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Phosphorylation of src family lck tyrosine kinase following interleukin-12 activation of human natural killer cells.

Interleukin-12 (IL-12) is a heterodimeric cytokine that augments the cytolytic activity of human NK cells and T cells but has little mitogenic activity on resting lymphocytes. The intracellular signaling pathways involved in NK cell activation by IL-12 have not been fully characterized. In the present studies we found that IL-12 induces tyrosine phosphorylation of a 56-kDa protein, identified in Western blot experiments as p56lck, in resting NK cells. IL-12 was active in the range of 0.1 to 1000 U/ml, with maximal activity between 10 and 100 U/ml (30 and 300 pM). The maximal effect was noted 5 min after stimulation and was almost completely inhibited by genistein. IL-12 induced similar effects in resting and IL-2 activated NK cells. Following IL-12 stimulation of resting NK cells, immunoprecipitated lck kinase exhibited increased in vitro autophosphorylation activity 5 and 10 min after activation. Several additional substrates were phosphorylated in vitro following IL-12 stimulation, including proteins of 70 and 110 kDa. These studies indicate that lck tyrosine kinase is involved in the IL-12 signaling pathway in human NK cells. As the primary functional effect of IL-12 on resting NK cells is the enhancement of cytolytic activity and secretory function rather than cell proliferation, these findings suggest that lck tyrosine kinase is involved in these signaling pathways in human NK cells.

Animals↗
PubMed1995

Interleukin 2 signaling involves the phosphorylation of Stat proteins.

One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat-related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function.

Blotting, Western↗
PubMed1995

Renal pharmacology of GR138950, a novel non-peptide angiotensin AT1 receptor antagonist.

This paper describes the renal pharmacology of the novel, specific, non-peptide angiotensin AT1 receptor antagonist, GR138950 (1-[[3-bromo-2-[2-[[(trifluoromethyl) sulphonyl] amino] phenyl]-5-benzofuranyl] methyl]-4-cyclopropyl-2-ethyl-1H-imidazole-5- carboxamide). When administered to anaesthetised salt-replete dogs, GR138950 caused renal vasodilatation and significant increases in sodium and urine excretion. No change in glomerular filtration rate was observed indicating that the natriuresis was a consequence of inhibition of tubular sodium reabsorption. Qualitatively similar but less marked changes in renal function were observed in response to the angiotensin converting enzyme inhibitor, captopril, although in contrast to GR138950, captopril also caused a small but significant fall in mean blood pressure. Intra-renal artery infusion of exogenous angiotensin II resulted in dose-related renal vasoconstriction and decreases in urine excretion, sodium excretion, fractional excretion of sodium and glomerular filtration rate. These renal effects of angiotensin II were all markedly antagonised by GR138950. We conclude that GR138950 is an effective antagonist of the renal haemodynamic and excretory actions of endogenous and exogenous angiotensin II.

Angiotensin I↗
PubMed1995

Inhibition of selective signaling events in natural killer cells recognizing major histocompatibility complex class I.

Many studies have characterized the transmembrane signaling events initiated after T-cell antigen receptor recognition of major histocompatibility complex (MHC)-bound peptides. Yet, little is known about signal transduction from a set of MHC class I recognizing receptors on natural killer (NK) cells whose ligation dramatically inhibits NK cell-mediated killing. In this study we evaluated the influence of MHC recognition on the proximal signaling events in NK cells binding tumor targets. We utilized two experimental models where NK cell-mediated cytotoxicity was fully inhibited by the recognition of specific MHC class I molecules. NK cell binding to either class I-deficient or class I-transfected target cells initiated rapid protein tyrosine kinase activation. In contrast, whereas NK cell binding to class I-deficient targets led to inositol phosphate release and increased intracellular free calcium ([Ca2+]i), NK recognition of class I-bearing targets did not induce the activation of these phospholipase C-dependent signaling events. The recognition of class I by NK cells clearly had a negative regulatory effect since blocking this interaction using anti-class I F(ab')2 fragments increased inositol 1,4,5-trisphosphate release and [Ca2+]i and increased the lysis of the targets. These results suggest that one of the mechanisms by which NK cell recognition of specific MHC class I molecules can block the development of cell-mediated cytotoxicity is by inhibiting specific critical signaling events.

Antibodies, Monoclonal↗
PubMed1995

Functional consequences of APO-1/Fas (CD95) antigen expression by normal and neoplastic hematopoietic cells.

Murine monoclonal antibody (mAb) 7C11 binds to the same cell surface epitope as anti-APO-1 and anti-Fas and reacts specifically with cells transfected with a cDNA encoding the human Fas antigen. Furthermore, incubation with 7C11 causes death of hematopoietic cell lines that express APO-1/Fas but not APO-1/Fas-negative cell lines. 7C11 therefore recognizes the human APO-1/Fas (CD95) antigen, a 40 to 50 kDa cell surface glycoprotein that can trigger apoptosis or programmed cell death. Expression of APO-1/Fas antigen by normal and neoplastic hematopoietic cells was determined by flow cytometry using 7C11. APO-1/Fas is expressed by approximately 30 to 40% of resting peripheral blood T cells, B cells, and monocytes and by approximately 5% of resting NK cells and thymocytes. It was not detected on granulocytes, erythrocytes, or platelets. Approximately 80 to 90% of activated T cells, B cells, and thymocytes express APO-1/Fas, as do the majority of activated NK cells. Perturbation of APO-1/Fas by 7C11 does not affect the viability of resting lymphocytes or monocytes. In contrast, activated T cells and NK cells undergo apoptosis within 3 hours of exposure to 7C11. Other mAb that stimulate T cells or NK cells do not cause rapid induction of programmed cell death. APO-1/Fas antigen is expressed by many cell lines of lymphoid and myeloid lineage. However, this antigen was detected on neoplastic cells from only one of 69 patients with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, or multiple myeloma. Only 3 out of 25 tumor samples from patients with non-Hodgkin's lymphoma were found to express APO-1/Fas. All three of these lymphomas harbored the bcl-2-Ig fusion gene associated with the chromosomal translocation t (14;18). Conversely, only 27% of lymphomas that possessed the bcl-2-Ig gene were found to express the APO-1/Fas antigen. Like normal activated lymphocytes, leukemia and lymphoma cells that expressed APO-1/Fas antigen were found to undergo apoptosis in vitro after incubation with 7C11. The APO-1/Fas antigen appears to regulate the growth of normal hematopoietic cells, and the marked upregulation of this antigen on activated normal lymphocytes contrasts sharply with the absence of APO-1/Fas on neoplastic cells of hematopoietic lineage. Defects in the apoptotic signal delivered through this antigen might contribute to the pathogenesis of hematopoietic neoplasms. Thus, the gene encoding APO-1/Fas can be considered a novel type of tumor suppressor gene, just as bcl-2 can be considered a cellular proto-oncogene.

Animals↗
PubMed1995

Pharmacological effects of GR138950, a novel angiotensin AT1 receptor antagonist.

The antagonist activity of GR138950 (1-[[3-bromo-2-[2-[[(trifluoromethyl)sulphonyl]amino]phenyl]-5- benzofuranyl]methyl]-4-cyclopropyl-2-ethyl-1H-imidazole-5-carboxamide) was investigated at angiotensin AT1 receptors and AT2 receptors in vitro and on blood pressure in conscious rats. GR138950 suppressed and displaced angiotensin II (AII) concentration-effect curves in the rabbit isolated aorta (pKb approximately 9.0-9.7) but had no effect against phenylephrine or serotonin induced-contractions. GR138950 competed with [3H]-AII for angiotensin AT1 receptors in rat liver membranes (pKi = 9.09). GR138950 had no apparent affinity for angiotensin AT2 receptors (bovine cerebellum; pKi < 6.0). GR138950 (1 mg/kg i.a. and p.o.) inhibited pressor responses to AII, but not phenylephrine, in conscious normotensive rats. Parallel displacements in AII dose-response curves occurred without any reduction in the maximum response to AII. The antagonist activity of GR138950 lasted for up to 24 h. GR138950 (> 0.03 mg/kg i.a., > 0.3 mg/kg p.o.) significantly reduced diastolic blood pressure (DBP) in renal artery ligated hypertensive rats. DBP was reduced maximally, 5 to 7 h after administration and the antihypertensive effect of GR138950 lasted for up to 48 h. Daily administration (5 days) of GR138950 to renal artery ligated hypertensive rats produced a sustained reduction in DBP. Acute administration of GR138950 (1 mg/kg i.a.) also significantly reduced DBP in spontaneously hypertensive rats but not in normotensive rats. Heart rate was little changed in renal artery ligated hypertensive rats, normotensive rats and spontaneously hypertensive rats. These experiments demonstrate that GR138950 is a potent, selective and specific angiotensin AT1 receptor antagonist that is orally active and reduces DBP in conscious hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1994

Interleukin 2 receptor gamma chain expression on resting and activated lymphoid cells.

The interleukin 2 receptor (IL-2R) is known to be comprised of at least three genetically distinct subunits termed alpha, beta, and gamma. These chains can be expressed individually or in various combinations resulting in distinct receptors with different affinities for IL-2. In contrast to alpha and beta, the cell surface expression of the gamma chain protein previously has not been well-characterized. To examine cell surface expression of IL-2R gamma on hematopoietic cells, we developed two new monoclonal antibodies (mAbs) specific for this protein. Both 1A11 (immunoglobulin [IgG1]) and 3G11 (IgM) specifically reacted with murine cells transfected with IL-2R gamma cDNA, and immunoprecipitation studies indicated that both antibodies precipitated a protein of approximately 62-65 kD. Scatchard analysis of IL-2 binding to murine cells transfected with cDNA-encoding combinations of IL-2R components demonstrated that neither beta nor gamma chain bind IL-2 with measurable affinity, but coexpression of both beta and gamma is sufficient to form an intermediate affinity receptor. In the absence of gamma chain, beta chain interacts with alpha chain to form a "pseudo-high" affinity receptor. In contrast, gamma chain does not appear capable of interacting with alpha in the absence of beta chain. Thus, gamma chain appears to interact only with beta, but beta chain is capable of interacting with both alpha and gamma. Using the newly developed mAbs to examine cell surface expression by immunofluorescence, resting T cells were found to express low levels of gamma chain without detectable alpha or beta. Early after mitogen stimulation, T cells expressed higher levels of alpha, beta, and gamma. However, at later time points, T cells expressed alpha and gamma in marked excess over beta. Thus, formation of high affinity IL-2R on activated T cells was primarily limited by beta chain expression. In contrast, resting natural killer (NK) cells constitutively expressed IL-2R beta without detectable alpha or gamma. After activation with either IL-2 or IL-12, expression of both alpha and gamma transiently increased and then returned to very low levels. Expression of functional IL-2R on resting and activated NK cells, therefore, appeared to be primarily limited by the expression of gamma chain. IL-2 binding studies with resting NK cells confirmed the results of immunofluorescence studies indicating the presence of very low numbers of intermediate affinity (beta gamma) receptors for IL-2 on these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

A phase II study of continuous infusion recombinant human granulocyte-macrophage colony-stimulating factor as an adjunct to autologous bone marrow transplantation for patients with non-Hodgkin's lymphoma in first remission.

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) clearly hastens myeloid recovery in patients with relapsed hematologic malignancies undergoing autologous bone marrow transplantation (ABMT). In efforts to further improve neutrophil engraftment and shorten hospital stay in ABMT patients, rhGM-CSF was administered by a potentially more potent route (continuous infusion) to non-Hodgkin's lymphoma (NHL) patients with better BM reserve (first remission). Time to myeloid engraftment was compared with that of NHL patients treated in first remission at our institution on a similar ABMT protocol but without growth factor support (controls). Median neutrophil engraftment (absolute neutrophil count, 500 cells/microL) in first remission patients treated with rhGM-CSF was 14 days, compared with 22 days in controls (P = .0001). Hospital stays were also significantly reduced for rhGM-CSF patients (P = .0003). Platelet engraftment did not differ between the two groups. Persistent fever and generalized serositis were the primary toxicities. rhGM-CSF, delivered by this route, was efficacious but more toxic than 2-hour rhGM-CSF infusions previously reported by other investigators. Future alterations in both dose and schedule may retain comparable efficacy yet diminish toxicity.

Adult↗
PubMed1994

Molecular cloning of the canine angiotensin II receptor. An AT1-like receptor with reduced affinity for DuP753.

Canine aortic strip studies revealed insensitivity of angiotensin II (AII)-induced aortic contraction to inhibition by the non-peptide antagonist DuP753 (pKB = 6.7 +/- 0.1). In order to determine the origin of this phenomenon we cloned the canine homologue of the AT1 AII receptor. Expression of this cDNA in COS-7 cells indicated a low affinity of DuP753 for the cloned receptor (KD = 92 nM). The predicted amino acid sequence is highly homologous to other mammalian AT1 receptors; sequence comparisons suggest the pharmacological difference may be the result of a threonine residue in position 163 in the IVth transmembrane domain.

Amino Acid Sequence↗
PubMed1994

Modulation of tachyphylaxis to angiotensin II in rabbit isolated aorta by the angiotensin AT1 receptor antagonist, losartan.

In rabbit aortic strip preparations, angiotensin II (AII) concentration-contractile response curves (0.3-100 nM) were highly reproducible. However, tachyphylaxis to the contractile response elicited by AII could be induced by repeated exposure to a supramaximal concentration (10 microM) of the peptide. In contrast, a correspondingly supramaximal concentration of the alpha 1-adrenoceptor agonist, phenylephrine (1 mM), did not cause the tissue to become tachyphylactic to phenylephrine. Furthermore, phenylephrine responses were unaffected in tissues previously made tachyphylactic to AII. When the non-peptide, competitive angiotensin AT1 receptor antagonist, losartan (300 nM), was administered before each supramaximal AII challenge, tachyphylaxis did not subsequently occur. Additionally, in tissues made tachyphylactic to AII, subsequent incubation with losartan (300 nM) reversed the AII tachyphylaxis. Thus, losartan may prevent the loss of contractility by preventing AII from interacting with its receptor in a manner which induces tachyphylaxis. However, since losartan can also completely reverse the loss of contractility, it appears capable of restoring the AT1 receptor to a state which allows subsequently administered AII to fully activate the contractile pathway.

Angiotensin II↗
PubMed1994

Agonist-antagonist interactions at angiotensin receptors: application of a two-state receptor model.

Interactions between agonists and antagonists at angiotensin receptors are characterized by a number of features: variation of antagonist dynamics between apparent simple competition, insurmountable antagonism and, occasionally, augmentation; the tendency for insurmountable antagonism to be saturable; slow recovery of agonist responses following agonist-induced tachyphylaxis; and the ability of competitive antagonists to accelerate recovery from the latter intervention. Some of these phenomena have also been observed in studies of 5-HT2 receptors where they were attributed to the operation of a two-state model with an allosteric site. In this article, Mark Robertson and colleagues propose that the properties of angiotensin AT1 receptors may be explained by a similar model, but without the need to evoke an allosteric site.

Angiotensin II↗
PubMed1994

Cardiovascular effects of GR117289, a novel angiotensin AT1 receptor antagonist.

1. The effect of GR117289, an angiotensin AT1 receptor antagonist, on diastolic blood pressure (DBP) was determined in angiotensin-dependent and angiotensin-independent models of hypertension in rats. In addition, the antagonist profile of GR117289 at angiotensin AT1 receptors was determined in conscious renal hypertensive rats and conscious normotensive rats, dogs and marmosets. 2. Intra-arterial and oral administration of GR117289 (0.3-3 mg kg-1, i.a.; 1-10 mg kg-1, p.o.) to 6-day left renal artery ligated hypertensive (RALH) rats (DBP > 140 mmHg) produced significant, dose-related reductions in DBP with little apparent effect on heart rate (< 15%). The antihypertensive effect of GR117289 developed progressively over several hours and with some doses persisted for 24-48 h after administration. 3. Administration of GR117289 (1 mg kg-1, i.a.) on 5 consecutive days to RALH rats reduced DBP on each day. The antihypertensive effect of GR117289 was not cumulative as DBP had almost returned to base-line values, 24 h after administration of each dose. 4. A dose of GR117289 (3 mg kg-1, i.a.), which produced a substantial reduction in DBP (about 70 mmHg) in RALH rats, was administered to rats in which blood pressure was elevated either by unilateral renal artery clipping, sustained infusion of angiotensin II (AII), DOCA-salt administration or genetic inbreeding. GR117289 reduced DBP in rats in which the renin-angiotensin system was activated by renal artery clipping or AII infusion but had little effect in normotensive rats, DOCA-salt rats and SHR. 5. Systemic administration of All to RALH rats and to normotensive rats, dogs and marmosets elicited reproducible pressor responses in all species. Systemic or oral administration of GR1 17289 (3 mg kg-1)inhibited the pressor responses produced by All, resulting in parallel, rightward displacements of All dose-response curves.6. Maximal displacements of All dose-response curves occurred 1 h and 1-7 h after systemic and oral administration, respectively. GR1 17289 produced a 32-246 fold displacement after systemic administration and a 4-12 fold displacement after oral administration. The effect in dogs was short lasting after systemic administration but the effect of GRI17289 lasted for up to 24 h in rats and marmosets and for up to 24 h after oral administration in all species. The antagonist activity appeared specific for angiotensin receptors as GRi17289 did not inhibit pressor responses to phenylephrine or vasopressin.7. These experiments demonstrate that GRI 17289 reduces blood pressure in conscious hypertensive rats after both systemic and oral administration, and is an effective antagonist at angiotensin AT1 receptors in conscious rats, dogs and marmosets.

Administration, Oral↗
PubMed1993

Fc gamma RIIIA-mediated signaling involves src-family lck in human natural killer cells.

The cell surface receptor for IgG Fc domain (Fc gamma RIIIA, CD16) is present as a multimeric complex on human NK cells and is essential for antibody-dependent cellular cytotoxicity. Although CD16 engagement induces tyrosine phosphorylation of several substrates, the intracytoplasmic domains of the CD16 molecule lack intrinsic protein tyrosine kinase consensus sequences. We report here that, in human NK cells, engagement of the CD16 receptor induces tyrosine phosphorylation of p56lck tyrosine kinase. CD16 triggering through anti-receptor antibody enhances in vitro kinase activity of phosphotyrosyl proteins and autophosphorylation activity of anti-lck immunoprecipitates. Similar results were obtained after stimulation with IL-12. In vivo labeling experiments showed that CD16 cross-linking primarily induces in vivo phosphorylation of p56lck at the tyr-394 autophosphorylation site. Using in vitro kinase assay of CD16 immunoprecipitates from lysates of human NK cells, we demonstrated that a phosphorylated protein of 62 to 65 kDa was co-precipitated with CD16. Reprecipitation experiments also suggested that a small amount of p56lck may interact with this complex. These results indicate that CD16 triggering involves p56lck and that this kinase is therefore involved in the signaling pathways regulating NK cell cytolytic functions. However, additional kinases not yet identified are also likely to play a role in the early signaling events through CD16.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

Developmental regulation of a mucinlike glycoprotein selectively expressed on natural killer cells.

Natural killer (NK) cells are CD3:TCR-, CD16+, CD56+ large granular lymphocytes capable of recognizing and eliminating a variety of virus-infected, malignant, and antibody-coated target cells. Two functionally distinct populations of peripheral blood NK cells can be differentiated by their surface expression of an isoform of the neural cell adhesion molecule (CD56). CD56bright NK cells have the attributes of an undifferentiated cell, in that they proliferate in response to exogenous cytokines, but exert poor cytolytic activity. CD56dim NK cells have the attributes of a more differentiated cell, in that they proliferate poorly in response to exogenous cytokines, but are potent cytolytic effector cells. Here we describe the molecular characterization of a NK cell restricted epitope (PEN5) that is selectively expressed on the functionally differentiated CD56dim NK cells. PEN5+ NK cells proliferate poorly in response to interleukin 2 (IL-2), but are potent cytolytic effectors, whereas PEN5- NK cells proliferate in response to IL-2, but are poor cytolytic effectors. Biochemical and immunochemical analyses reveal the PEN5 epitope to be an unusual sulfated poly-N-lactosamine carbohydrate related to keratan sulfate glycosaminoglycans. Immunoprecipitates prepared using a monoclonal antibody reactive with PEN5 include two polydisperse membrane-bound glycoproteins, PEN5 alpha (120-170 kD) and PEN5 beta (210-245 kD). Enzymatic deglycosylation reduces the apparent molecular weight of both PEN5 isoforms by 80-90%, and classifies PEN5 beta as a mucinlike glycoprotein. The surface expression of the PEN5 epitope is downmodulated by stimuli that induce NK cell proliferation, and it is absent from leukemic NK cells of patients with granular lymphocyte proliferative disorder. Taken together, these results indicate that PEN5 is a developmentally regulated poly-N-lactosamine epitope associated with a mucin-type glycoprotein, whose expression is restricted to the population of nonproliferative NK cells fully committed to cytolytic effector function.

Amino Sugars↗