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M J Fisher

Publications and source records attributed to M J Fisher.

At least 37 records · Page 2Linked to original sources
PubMed1999

Intracellular signalling proteins as smart' agents in parallel distributed processes.

In eucaryotic organisms, responses to external signals are mediated by a repertoire of intracellular signalling pathways that ultimately bring about the activation/inactivation of protein kinases and/or protein phosphatases. Until relatively recently, little thought had been given to the intracellular distribution of the components of these signalling pathways. However, experimental evidence from a diverse range of organisms indicates that rather than being freely distributed, many of the protein components of signalling cascades show a significant degree of spatial organisation. Here, we briefly review the roles of 'anchor' 'scaffold' and 'adaptor' proteins in the organisation and functioning of intracellular signalling pathways. We then consider some of the parallel distributed processing capacities of these adaptive systems. We focus on signalling proteins-both as individual 'devices' (agents) and as 'networks' (ecologies) of parallel processes. Signalling proteins are described as 'smart thermodynamic machines' which satisfy 'gluing' (functorial) roles in the information economy of the cell. This combines two information-processing views of signalling proteins. Individually, they show 'cognitive' capacities and collectively they integrate (cohere) cellular processes. We exploit these views by drawing comparisons between signalling proteins and verbs. This text/dialogical metaphor also helps refine our view of signalling proteins as context-sensitive information processing agents.

Models, Biological↗
PubMed1998

Isoprenylation of polypeptides in the nematode Caenorhabditis elegans.

Covalent modification of eucaryotic proteins, involving addition of isoprenyl groups, is a widespread phenomenon. Here we provide direct evidence for this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of [3H]mevalonolactone, specific C. elegans polypeptides became labelled in both aqueous and detergent (Triton X-114)-enriched extracts. Chemical and GC-MS analysis of modifying groups, cleaved from C. elegans polypeptides, revealed that geranylgeranylation and, to a lesser extent, farnesylation of target polypeptides occurred. Immunoblot analysis provided preliminary evidence that the ras-like let-60 polypeptide was a target for isoprenylation in C. elegans.

Animals↗
PubMed1998

Fatty acylation of polypeptides in the nematode Caenorhabditis elegans.

Covalent modification of eucaryotic proteins, involving addition of fatty acyl groups, is a widespread phenomenon. Here we describe the occurrence of this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of either [3H]-myristic acid or [3H]-palmitic acid, specific C. elegans polypeptides became labelled. Chemical analysis revealed that following incubation of C. elegans with [3H]-myristic acid, polypeptides became labelled with myristoyl, palmitoyl or stearoyl moieties; after incubation with [3H]-palmitic acid, palmitoyl or stearoyl moieties were incorporated into polypeptides. Fatty acyl groups were linked to target polypeptides, predominantly through alkali-labile thioester or ester linkages and acid-labile amide linkages. Where myristoylation involved an amide linkage, the modified amino acid was usually glycine. Preliminary immunological evidence indicated that heterotrimeric GTP-binding protein alpha subunit(s) are possible target(s) for acylation in C. elegans.

Acylation↗
PubMed1998

Antenatal glucocorticoid therapy suppresses angiotensin-converting enzyme activity in rats with nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Neonates with congenital diaphragmatic hernia (CDH) have a high morbidity and mortality rate caused by pulmonary hypoplasia associated with pulmonary hypertension (PH). In experimental CDH, antenatal glucocorticoid therapy improves surfactant biochemical immaturity, enhances lung compliance, and induces morphological maturation in CDH rats. The effects of steroid therapy on preventing or treating PH in this condition have not been studied. Angiotensin converting enzyme (ACE), which is produced by the vascular endothelium, is implicated in the pathogenesis of pulmonary hypertension. The aim of this study was to evaluate the effect of antenatal glucocorticoid therapy on ACE activity and expression in CDH rat lungs. METHODS: CDH was induced in fetal rats by the maternal administration of 100 mg nitrofen on day 9.5 of gestation (term, day 22). Dexamethasone (Dex) (0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 before delivery of the fetuses by cesarean section on day 21.5. Control animals received olive oil (OO) by gavage and normal saline (NS) as vehicle injection. ACE activity was measured spectrophotometrically in the lungs of rats from four treatment groups: CDH-NS, non-CDH-NS, CDH-Dex, and OO-NS controls. Total lung ACE activity (mU per lung) was significantly lower in CDH-NS (P = .002) and CDH-Dex (P = .004) rats compared with non-CDH-NS and OO-NS controls (9.1 +/- 1.0 and 10.7 +/- 1.3 v 16.2 +/- 1.6 and 15.4 +/- 1.7). When specific ACE activity (mU/mg protein) was derived by expressing ACE activity per milligram of lung protein, CDH-NS animals showed elevated specific ACE activity (P = .05) compared with OO-NS controls (6.31 +/- 1.1 v 4.4 +/- 0.4). CDH-Dex animals had a significantly lower specific ACE activity (P = .01) compared with CDH-NS and Non-CDH-NS rats (4.0 +/- 0.4 v 6.31 +/- 1.1 and 5.83 +/- 0.54). The specific ACE activity levels of CDH-Dex rats were equivalent to those seen in the lungs of OO-NS controls (P = .24). CONCLUSION: Antenatal steroid therapy, by suppressing pulmonary ACE activity, may reduce the risk of pulmonary hypertension developing in human newborns with antenatally diagnosed CDH.

Animals↗
PubMed1998

Diadenosine polyphosphate-mediated activation of phospholipase D in isolated rat liver cells.

Diadenosine polyphosphates (ApnAs) can, through interaction with appropriate purinoceptors, affect a range of cellular activities. Ap4A, the most prominent naturally occurring diadenosine polyphosphate, stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that Ap4A, and other naturally occurring diadenosine polyphosphates, also stimulates phospholipase D (PLD) activity in isolated rat liver cells. The characteristics of Ap4A-mediated activation of PLD are similar to those for the activation of PLD by extracellular ATP. These results are discussed in the context of the relation between diadenosine polyphosphate- and adenine mononucleotide-mediated cellular signalling processes.

Animals↗
PubMed1998

Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304.

1. To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca2+, ([Ca2+]c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists. 2. Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC50 values of 4.2 microM for ATP, 2.5 microM for UTP and 14 microM for adenosine-5'-O-(3-thio)triphosphate (ATPgammaS). EC50 values for 2-methylthioATP, ADP, adenosine-5'-O-(2-thio)diphosphate (ADPbetaS) and AMP were 0.5 microM, 3.5 microM, 15 microM and 4.7 microM respectively, but maximal [Ca2+]c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and beta,gamma,methyleneATP. 3. Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor. 4. ECV304 [Ca2+]c responses to 2-methylthioATP were inhibited in the presence of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), whereas [Ca2+]c responses to UTP were unaffected by this treatment. 5. ECV304 cells responded to the diadenosine polyphosphate Ap3A with rises in [Ca2+]c. Apparent responses to Ap4A, Ap5A and Ap6A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase. 6. ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y2 receptor insensitive to the diadenosine polyphosphates and a P2Y1 receptor sensitive to Ap3A. In addition, ECV304 cells respond to AMP with increases in [Ca2+]c via an as yet uncharacterized receptor.

Adenosine Triphosphate↗
PubMed1998

Effect of dexamethasone on endothelial nitric oxide synthase in experimental congenital diaphragmatic hernia.

AIMS: To study the effect of prenatal glucocorticoid treatment on endothelial nitric oxide synthase (eNOS) expression in rats with congenital diaphragmatic hernia (CDH). METHODS: CDH was induced in fetal rats by the maternal administration of nitrofen on day 9.5 of gestation. Dexamethasone was administered on days 18.5 and 19.5 before delivery of the fetuses on days 20.5 and 21.5. Pulmonary eNOS protein expression was studied by western immunoblotting and immunohistochemistry. RESULTS: On day 20.5, eNOS expression was significantly reduced in CDH pups compared with normal control rats. Dexamethasone treated CDH pups had eNOS concentrations equivalent to those of normal animals. By day 21.5, however, there was no detectable difference in eNOS expression between the experimental groups. CONCLUSIONS: eNOS is deficient in near term (day 20.5) CDH rats. Dexamethasone restores eNOS expression in these animals to that seen in normal rat lungs. At term, the precise role of eNOS in the pathophysiology of CDH remains uncertain.

Animals↗
PubMed1997

N-Myristoylation of the catalytic subunit of cAMP-dependent protein kinase in the free-living nematode Caenorhabditis elegans.

N-Myristoylation of the catalytic subunit (C-subunit) of cAMP-dependent protein kinase is widespread in animal cells. Some invertebrates express non-myristoylated isoforms of C-subunit but these co-exist with at least one myristoylated isoform. The generality of this observation implies an indispensable function for myristoylated C-subunit, but notwithstanding this, neither of the C-subunit isoforms hitherto described in C. elegans is apparently N-myristoylated. In light of this anomaly, the myristoylation status of the C-subunit has been examined in adult C. elegans. Evidence is presented for the presence of an N-myristoylated isoform.

Animals↗
PubMed1997

Non-peptide RGD surrogates which mimic a Gly-Asp beta-turn: potent antagonists of platelet glycoprotein IIb-IIIa.

Cyclic heptapeptide 1, which contains an Arg-Gly-Asp sequence, has good affinity for the platelet receptor GPIIb-IIIa and was chosen for study by 1H NMR techniques. The key RGD sequence of this molecule was found to reside in a conformationally defined type II' Gly-Asp beta-turn, and this information was used in the design of simple non-peptide RGD mimics. Disubstituted isoquinolones, bearing an acidic side chain at position 2 and a basic side chain at position 6, were prepared and were found to have modest affinity for GPIIb-IIIa. Systematic modification of the basic residue contained in these molecules yielded compounds with high affinity for GPIIb-IIIa.

Aspartic Acid↗
PubMed1997

Diadenosine polyphosphate-stimulated gluconeogenesis in isolated rat proximal tubules.

Diadenosine polyphosphates released into the extracellular environment influence a variety of metabolic and other cellular activities in a wide range of target tissues. Here we have studied the impact of these novel nucleotides on gluconeogenesis in isolated rat proximal tubules. Gluconeogenesis was stimulated following exposure of isolated proximal tubules to a range of adenine-containing nucleotides including ADP, ATP, Ap3A, Ap4A, Ap5A and Ap6A. The concentration-dependence of ATP-, Ap3A- and Ap4A-mediated stimulation of gluconeogenesis was similar and was consistent with a role for these agents in the physiological control of renal metabolism. Nucleotide-stimulated gluconeogenesis was diminished in the presence of agents that interfere with phospholipase C activation or intracellular Ca2+ metabolism, indicative of a role for polyphosphoinositide-mediated Ca2+ mobilization in the mechanism of action of ATP, Ap3A and Ap4A. The characteristics of binding of [2-3H]Ap4A to renal plasma-membrane preparations suggest that Ap4A mediates its effects on proximal tubule gluconeogenesis via interaction with P2y-like purinoceptor(s) also recognized by extracellular ATP.

Adenine Nucleotides↗
PubMed1997

Induction of enzymes involved in molting hormone (ecdysteroid) inactivation by ecdysteroids and an agonist, 1,2-dibenzoyl-1-tert-butylhydrazine (RH-5849).

Molting in insects is regulated by molting hormones (ecdysteroids). The major active hormone, 20-hydroxyecdysone, is formed by ecdysone 20-monooxygenase-catalyzed hydroxylation of ecdysone. During times of decreasing hormone titers, inactivation occurs by several routes including (i) 26-hydroxylation and further oxidation to the 26-oic acid, (ii) formation of various conjugates (e.g. phosphates), and (iii) in Lepidoptera in particular, ecdysone oxidase-catalyzed formation of 3-dehydroecdysteroid, which is reduced to 3-epiecdysteroid, followed by phosphotransferase-catalyzed formation of phosphate conjugates. Administration of the nonsteroidal ecdysteroid agonist RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), but not 20-hydroxyecdysone, to tobacco hornworm (Manduca sexta) resulted in induction of midgut cytosolic ecdysone oxidase and ecdysteroid phosphotransferase activities. In addition, both 20-hydroxyecdysone and RH-5849 caused induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes, whereas 20-hydroxylase was induced to a lesser extent by 20-hydroxyecdysone in mitochondria and by either RH-5849 or 20-hydroxyecdysone in microsomes. Commensurate with induction of the enzymes by ecdysteroid and RH-5849 is a requirement for RNA and protein synthesis, without precluding indirect mechanisms. These results indicate that molting hormone stimulates at least one universal route of its own inactivation by inducing ecdysteroid 26-hydroxylase activity and are discussed in relation to an analogous phenomenon observed for vitamin D inactivation in vertebrates.

Animals↗
PubMed1997

Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

Animals↗
PubMed1997

Thrombomodulin expression in bovine brain capillaries. Anticoagulant function of the blood-brain barrier, regional differences, and regulatory mechanisms.

Thrombomodulin (TM), a key cofactor of the TM-protein C pathway, is of major biologic significance for the antithrombotic properties of endothelial cells. Yet, there is uncertainty whether TM is expressed in brain and what mechanisms govern brain endothelial anticoagulant activity. In this study, bovine brain capillaries were used as an in vitro model of the blood-brain barrier to determine factors involved in the regulation of TM expression in cerebral vasculature. Quantitative competitive-polymerase chain reaction assay revealed significant regional differences in the amount of brain capillary TM mRNA, i.e., cortical > cerebellar > pontine, consistent with the reverse transcription-polymerase chain reaction findings in which the abundance of TM mRNA was analyzed relative to beta-actin mRNA. Regional differences in TM mRNA brain capillary level correlated well with differences in protein C activation. The TM mRNA and activity were not detectable in brain parenchyma. Pathogenic mediators of ischemic stroke, interleukin 1 beta (10 U/mL), and tumor necrosis factor alpha (10 U/mL), produced a time-dependent decrease in brain capillary TM mRNA (t1/2 of 2.1 and 3.9 hours, respectively) and reduced endothelial TM activity. Incubation of brain capillaries with retinoic acid (10 mumol/L) and dibutyryl cAMP (3 mmol/L) resulted in a 4-fold increase in TM mRNA at 4 and 8 hours, respectively, followed by an increase in protein C activation. We conclude that TM at the blood-brain barrier is likely to be an important physiologic anticoagulant in brain microcirculation. Its downregulation by cytokines may contribute to ischemic brain damage and potentially could be counteracted by retinoic acid and cAMP.

Animals↗
PubMed1996

p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2.

The Ca2+-sensitive 85-kDa cytosolic phospholipase A2 (cPLA2) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA2, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA2 is not required for its receptor-mediated activation.

Arachidonic Acid↗
PubMed1996

Characterization of the binding of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) to rat liver cell membranes.

Diadenosine polyphosphates present in the extracellular environment can, through interaction with appropriate purinoceptors, influence a range of cellular activities. Here we have investigated the nature of the ligand:receptor interactions involved in diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A)-mediated stimulation of glycogen breakdown in isolated rat liver cells. [2-3H]Ap4A showed specific binding to both intact isolated liver cells and plasma membrane fractions prepared from isolated liver cells. HPLC analysis confirmed that binding was mediated by intact Ap4A and not by potential breakdown products (e.g. ATP, adenosine, etc.). Binding of [2-3H]Ap4A, to isolated liver cell plasma membrane preparations, was successfully displaced by a range of both naturally occurring and synthetic diadenosine polyphospates with the rank order potency Ap4A > or = Ap5A > Ap6A > Ap3A > Ap2A. [2-3H]Ap4A binding was not displaced by P1 effectors but was successfully displaced by a range of P2 effectors with the rank order potency 2-methylthio-ATP > ATP > ATP > or = adenosine 5'-[alpha beta-methylene]triphosphate > adenosine 5'-[beta gamma-methylene]triphospate. These findings are consistent with the interaction of Ap4A with a P2y-like subclass of purinoceptor and are discussed in relation to (1) the known purinoceptor populations in liver cell plasma membranes and (2) observations concerning the binding of diadenosine polyphosphates to purinoceptors in other tissues.

Animals↗
PubMed1996

Differential effects of streptozotocin-induced diabetes on phenylalanine hydroxylase protein and mRNA abundance in isolated rat liver cells.

Phenylalanine hydroxylase catalyzes the major regulatory step of the phenylalanine degradation pathway. In view of the glucogenic nature of phenylalanine breakdown, and hence its potential contribution to glucose homeostasis, we have investigated the impact of streptozotocin-induced diabetes upon the expression of rat phenylalanine hydroxylase. Northern blot analysis revealed that induction of diabetes was associated with an increase in the in vivo abundance of hepatic phenylalanine hydroxylase-specific mRNA. This increase in mRNA abundance was maintained for at least 8 hr in liver cells isolated from diabetic animals. In contrast, phenylalanine hydroxylase immunoreactivity and enzymic activity decreased, over the 8 hr incubation period, to levels similar to those observed in liver cells from normal animals. These changes were retarded, but not prevented, by the presence of dexamethasone in incubation media. In liver cells from normal animals the abundance of phenylalanine hydroxylase-specific mRNA, immunoreactivity and enzymic activity, were largely insensitive to treatment with dexamethasone and/or glucagon over an 8 hr incubation period. It is concluded that, whereas diabetes-related alterations in phenylalanine hydroxylase-specific mRNA abundance persist after isolation of liver cells, changes in phenylalanine hydroxylase protein abundance do not. Additionally, in contrast to certain other enzymes (e.g. phosphoenolpyruvate carboxykinase) it is not possible to mimic diabetes-related alterations in the expression of phenylalanine hydroxylase, in liver cells from normal animals, by simple hormonal manipulation of incubation media. This implies that other additional factors must also contribute to diabetes-related alterations in hepatic enzyme expression.

Animals↗