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Query: UMLS:C0016632 (Fox)
 1,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously demonstrated that insulin stimulates glycerolipid synthesis and phospholipid hydrolysis in BC3H-1 myocytes, resulting in the generation of membrane diacylglycerol, a known cellular mediator. This led us to the original proposal that diacylglycerol may contribute to the mediation of insulin action, especially stimulation of glucose transport. The fact that agents such as phenylephrine and phorbol esters, which increase or act as membrane diacylglycerols, are fully active in stimulating glucose transport in this tissue lent further support to this proposal. In this paper, we demonstrate that the diacylglycerol analogues PMA (4 beta-phorbol 12-myristate 13-acetate) and mezerein (both possessing 12 beta- and 13 alpha-O-linked substituents as well as a 4 beta-hydroxyl group) each increase the Vmax of the glucose transporter as does insulin. Diacylglycerol generated by the addition of phospholipase C also stimulates glucose uptake to a maximum which is equal and nonadditive to that of insulin, while addition of the narrowly active phosphatidylinositol-specific phospholipase C which generates the putative phosphoinositol-glycan mediator of Saltiel et al. (Saltiel, A., Fox, J., She Lin, P., and Cutrecasas, P. (1986) Science 233, 967-972) stimulates pyruvate dehydrogenase in these cells without any effect on glucose uptake. Pretreatment of the myocytes with PMA resulted in desensitization of subsequent glucose uptake to stimulation by phenylephrine, but had no effect on stimulation of glucose uptake by phospholipase C or by insulin, indicating that PMA pretreatment primarily desensitizes agonist-induced polyphosphoinositide hydrolysis which, as we have previously shown, is not involved in the insulin-induced generation of diacylglycerol. This was confirmed by the absence of intracellular Ca2+ mobilization during insulin administration, as measured by the sensitive fluorescent probe fura-2 in attached monolayer BC3H-1 myocytes. Furthermore, we have shown that insulin-generated diacylglycerol satisfies several criteria for a mediator of insulin action, including the demonstration that insulin-stimulated endogenous diacylglycerol generation is antecedent to glucose transport and has an identical insulin dose-response curve and moreover that the magnitude and time course of subsequent stimulation of glucose transport is reproduced by the addition of the simple exogenous diacylglyerol, dioctanoylglycerol, in the complete absence of the hormone. These results establish a central role for insulin-induced glycerolipid metabolism in mediating insulin-stimulated glucose transport in BC3H-1 myocytes.
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PMID:Insulin-induced glycerolipid mediators and the stimulation of glucose transport in BC3H-1 myocytes. 328 20

Secretion of platelet granule constituents is closely associated with the phosphorylation of a cytosol polypeptide of Mr = 47,000 that we have called P47 (Haslam, R. J., Lynham, J. A., and Fox, J. E. B. (1979) Biochem. J. 178, 397-406). This polypeptide is a substrate of Ca2+-activated phospholipid-dependent protein kinase (Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., and Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309-317). Two-dimensional gel electrophoresis of protein from human platelets that had been preincubated with 32Pi demonstrated the presence under control conditions of 2-3 major forms of P47 that contained very little 32P (pI values, 6.6-6.8) and, after induction of secretion with thrombin, their replacement by 7-9 highly labeled phosphorylated forms of P47 (pI values, 6.1-6.5). Native phosphorylated P47 was purified from thrombin-stimulated 32P-labeled platelets by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxylapatite. The final 32P-labeled product was obtained in a yield of 20-25% and was purified about 400-fold relative to platelet lysate. This material was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but, like the starting material, contained 7-9 separable phosphorylated components with different pI values. Purified phosphorylated P47 had a sedimentation coefficient (s20,w) of 3.57 S and a Stokes radius of 3.33 nm from which an Mr = 49,000 and a frictional ratio (f/f0) of 1.4 were calculated. These findings and failure to detect multimers after treatment of the protein with dimethyl suberimidate indicate that P47 normally exists as a monomer. The 32P-labeled phosphate present in purified P47 had the chemical stability of serine or threonine phosphoesters and analysis indicated the presence of 83% phosphoserine and 17% phosphothreonine. Limited proteolysis of purified 32P-labeled P47 by Staphylococcus aureus V8 protease generated a major unlabeled fragment (Mr = 23,500) and up to six labeled fragments (Mr = 24,700-14,800), the relative amounts of the latter depending on the extent of proteolysis. The same labeled fragments were obtained after proteolysis of each of the major phosphorylated components of P47, suggesting that these represent different phosphorylation states of variants of the same protein and that most or all of the phosphorylation sites are on a single 14,800-Da segment of the protein. The availability of pure native phosphorylated P47 should facilitate investigation of the physiological role of this protein in platelets.
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PMID:Purification and characterization of the 47,000-dalton protein phosphorylated during degranulation of human platelets. 688 23

Changes in ligand binding ability of the integrin alpha IIb beta 3 can be monitored by the concomitant expression of ligand-inducible binding sites (LIBS). A new LIBS, the hexapeptide sequence GPNICT (residues 1-6) at the amino terminus of beta 3 recognized by the murine monoclonal antibody (mAb) AP5, is sensitive both to the binding of ligand and to micromolar differences in divalent cation levels. Calcium or magnesium can completely inhibit the binding of AP5 to alpha IIb beta 3 on platelets, with ID50 values of 80 and 1500 microM, respectively. The inhibitory effect of calcium plus magnesium is cumulative. In the presence of 1 mM calcium plus 1 mM magnesium, the peptide RGDW overcomes this inhibition and induces maximal binding of AP5. Maximal AP5 binding is also induced by a molar excess of EDTA. The unique location of the AP5 LIBS was determined by comparing the binding of LIBS-specific mAb to recombinant human-Xenopus beta 3 chimeras produced in a baculovirus expression system. AP5 defines one region at the amino terminus beta 3 1-6. A second region, defined by mAb D3GP3, is probably located within beta 3 422-490, confirming the finding of Kouns et al. (Kouns, W. C., Newman, P.J., Puckett, K. J., Miller, A. A., Wall, C. D., Fox, C. F., Seyer, J. M., and Jennings, L. K. (1991) Blood 78, 3215-3223). The third region, encompassing at most residues 490-690, and perhaps more precisely located within 602-690 (Du X., Gu, M., Weise, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H. (1993) J. Biol. Chem. 268, 23087-23092), is recognized by the four mAb, anti-LIBS2, anti-LIBS3, anti-LIBS6, and P41. Since its exposure is uniquely regulated by both divalent cations and ligand, the amino terminus of beta 3 may be involved in control of ligand binding by divalent cation mobilization.
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PMID:Topography of ligand-induced binding sites, including a novel cation-sensitive epitope (AP5) at the amino terminus, of the human integrin beta 3 subunit. 753 28

The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine.
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PMID:CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells. 754 43

Calpain is distributed ubiquitously in virtually every tissue (Croall, D. E., and DeMartino, G. N. (1991) Physiol. Rev. 71, 813-846), but its physiological role remains to be determined. The identification of its natural endogenous substrates would be of great interest. Since pp60src, a major tyrosine kinase in platelets, is known to be easily cleaved during purification from cells (Feder, D., and Bishop, J. M. (1990) J. Biol. Chem. 265, 8205-8211), we examined the possibility that it is an endogenous substrate of calpain. In the whole cell lysate from resting platelets, which was analyzed by Western blotting with monoclonal antibody 327, we found pp60src almost exclusively in a 60-kDa form, with a trace of 52-kDa form. Addition of A23187 (a calcium ionophore) or dibucaine, which are known to be activators of platelet calpain (Croall and DeMartino, 1991; Fox, J. E., Reynolds, C., Morrow, J. S., and Phillips, D. R. (1987) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990b) J. Cell Biol. 111, 483-493), caused dose- and time-dependent cleavage of actin-binding protein and p235 protein (talin). At the same time, loss of the 60-kDa species of pp60src and generation of the 52-kDa (occasionally seen as doublets) and 47-kDa species were detected by the Western blotting. In platelets aggregated by 1 unit/ml thrombin, apparently identical cleavage products were found. The cleavage of pp60src was inhibited by calpeptin (20 microM), an inhibitor of calpain (Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N., Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153, 1201-1208; Tsujinaka, T., Ariyoshi, H., Uemura, Y., Sakon, M., Kambayashi, J., and Mori, T. (1990) Life Sci. 46, 1059-1066; Fox, J. E., Clifford, C. C., and Austin, C. D. (1990) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990) J. Cell. Biol. 111, 483-493; Fox, J. E., Austin, C. D., Clifford, C. C., and Steffen, P. K. (1991) J. Biol. Chem. 266, 13289-13295). Addition of EGTA (3 mM) to the extracellular media completely inhibited the cleavage of actin-binding protein, talin, and pp60src in response to A23187 (1 microM). Intact pp60src was distributed in both cytosolic and particulate (membrane) fractions. Cleaved species were found exclusively in the cytosolic fraction. pp60src-associated enolase kinase activity was reduced. Thus, pp60src is an endogenous substrate for calpain, the cleavage of which may have regulatory effects on the kinase.
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PMID:pp60src is an endogenous substrate for calpain in human blood platelets. 768 44

We have studied the inactivation of high-voltage-activated (HVA), omega-conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal root ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced inward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of approximately 100 ms and > 1 s). As reported previously, upon depolarization of the holding potential to -40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowycky, M. C., A. P. Fox, and R. W. Tsien. 1985. Nature. 316:440-443; Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987a. Journal of Physiology. 394:149-172; Swandulla, D., and C. M. Armstrong. 1988. Journal of General Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. Hess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bean. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal of Neuroscience. 12:906-914). Such kinetic properties might be explained by a model in which N channels inactivate by both fast and slow voltage-dependent processes. Alternatively, kinetic models of Ca-dependent inactivation suggest that the biphasic kinetics and holding-potential-dependence of N current inactivation could be due to a combination of Ca-dependent and slow voltage-dependent inactivation mechanisms. To distinguish between these possibilities we have performed several experiments to test for the presence of Ca-dependent inactivation. Three lines of evidence suggest that N channels inactivate in a Ca-dependent manner. (a) The total extent of inactivation increased 50%, and the ratio of rapid to slow inactivation increased approximately twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was reduced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally increased when the extracellular Ca2+ concentration was raised from 0.5 to 5 mM. (c) Substituting Na+ for Ca2+ as the permeant ion eliminated the rapid phase of inactivation. Other results do not support the notion of current-dependent inactivation, however. Although high intracellular EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid phase inactivation, they did not eliminate it. Increasing the extracellular Ca2+ from 0.5 to 5 mM had little effect on this residual fast inactivation, indicating that it is not appreciably sensitive to Ca2+ influx under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inactivation of N-type calcium current in chick sensory neurons: calcium and voltage dependence. 780 51

Basic fibroblast growth factor (FGF) stimulates the proliferation, differentiation, and motility of multiple cell types. Signal transduction by FGF is mediated by high affinity FGF receptors that have autophosphorylating tyrosine kinase activity and also elicit the release of low molecular weight signaling molecules, including inositol 1,4,5-trisphosphate, diacylglycerol, and arachidonate. We have shown previously that basic FGF-stimulated, phospholipase A2 (PLA2)-mediated arachidonate release regulates endothelial cell (EC) motility (Sa, G., and Fox, P.L. (1994) J. Biol. Chem. 269, 3219-3225). Here we identify the phospholipase responsible for basic FGF-mediated arachidonate release as cytosolic PLA2 (cPLA2) by demonstrating in EC lysates a requirement for micromolar Ca2+, dithiothreitol insensitivity, and inactivation by anti-cPLA2 antiserum. The role of cPLA2 is also indicated by the observed mechanisms of activation which show a requirement for p42 mitogen-activated protein kinase activity, cPLA2 phosphorylation, and cPLA2 translocation from cytosol to membranes. Phosphorylation of cPLA2, arachidonate release from prelabeled intact cells, and cell motility all have similar concentration dependencies on basic FGF. Since arachidonate release is required for basic FGF-stimulated motility of EC, our results show that p42 mitogen-activated protein kinase activation of cPLA2 may be a regulatory event in stimulation of cellular release of this important eicosanoid precursor during cellular responses to basic FGF.
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PMID:Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. 783 70

In this review, we have concentrated on the parallels between the cellular properties of the NMDA receptor and a variety of functional properties within sensory and motor systems. Of course, the NMDA channel exists within the cell in conjunction with a variety of other channels, including non-NMDA channels. Although the NMDA receptor is unique in a cellular sense--it is the only ligand-gated channel that is also voltage dependent and calcium permeable--it is not unique in a functional sense. A cell that has non-NMDA receptors and voltage-sensitive channels will also exhibit nonlinear behavior. Moreover, Buhrle & Sonnhof (1983) demonstrated some time ago that calcium flows into frog motor neurons through more than one type of calcium channel. The contribution to the inflow of calcium from NMDA channels may vary from cell to cell and could easily be a minor proportion of the total. Many authors have pointed out that the NMDA channel has a low conductance at a resting potential of -70 mV. However, many cells in the nervous system are depolarized from -70 mV by excitatory input. Thus, as pointed out above. NMDA receptors make a contribution to the tonic or spontaneous activity of cells in both visual cortex and spinal cord. In practice, many cells are probably working in a range of membrane potentials where the NMDA channels are always open to some extent. Even in the hippocampal slice where a substantial amount of afferent input is removed, NMDA receptors contribute to spontaneous activity (Sah et al 1989). Does the NMDA receptor act as a switch? Does it act as an AND gate? The suggestion that it may act as a switch comes from work on LTP in the hippocampus, which is readily produced by high-frequency stimulation and is abolished by APV. However, activation of the NMDA receptor is only the first in a sequence of reactions leading to LTP: In theory, switch-like behavior could also be produced by calcium-buffering systems within dendritic spines, or by enzymatic processes (Lisman 1985; Zador et al 1990). Fox & Daw (1992) have modeled the action of NMDA and non-NMDA receptors that are activated in parallel with each other, and shown that the occurrence of switch-like behavior depends on the relative density of NMDA versus non-NMDA receptors. Switch-like behavior is not seen in the visual cortex, but might be seen in the hippocampus if the relative density of NMDA receptors there was higher than in the visual cortex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The role of NMDA receptors in information processing. 846 Aug 91

Interferon-gamma (IFN-gamma), in the presence of tumor necrosis factor-alpha (TNF-alpha), decreases proliferation of a human salivary gland ductal cell line, HSG (Wu, A., R. Kurrasch, J. Katz, P. Fox, B. Baum, and J. Atkinson. J. Cell. Physiol. 161:217-226, 1994). We examined the possible effects of these cytokines (1,000 U/ml IFN-gamma +/- 20 U/ml TNF-alpha for 7 days) on Ca2+ mobilization in HSG cells. In HSG cells, fetal bovine serum (10%) or carbachol (100 microM) stimulated rapid increases in cytosolic Ca2+ concentration ([Ca2+]i), apparently mobilized from different thapsigargin-sensitive intracellular Ca2+ stores. Serum induced a proliferative effect on HSG cells, which was suppressed (> 90%) by treatment with IFN-gamma +/- TNF-alpha, but not with TNF-alpha alone. Serum-, carbachol-, and thapsigargin-stimulated [Ca2+]i elevations were reduced by 90, 60, and > 65%, respectively, in cells treated with IFN-gamma +/- TNF-alpha and 30, 45, and 45%, respectively, in cells treated with TNF-alpha. Removal of the cytokines from the growth medium induced recovery of both cell proliferation and Ca2+ mobilization responses within 7 days. Treatment of HSG cells with thapsigargin (0.02-2 nM) induced a dose-dependent decrease in cell proliferation. Additionally, acute treatment (< 10 min) of cells with IFN-gamma did not affect [Ca2+]i or alter carbachol-, thapsigargin-, or serum-induced changes in [Ca2+]i. These data demonstrate that prolonged treatment of HSG cells with IFN-gamma +/- TNF-alpha leads to a persistent depletion of intracellular Ca2+ stores. We suggest that this may have a role in cell growth.
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PMID:Interferon-gamma induces persistent depletion of internal Ca2+ stores in a human salivary gland cell line. 877 14

Fifteen randomly selected nasopharyngeal (NP) swab specimens (culture-negative for influenza A virus) were spiked with influenza A virus and the nucleic acids were extracted and subjected to PCR amplification with Thermus aquaticus (Taq) and T. thermophilus (Tth) DNA polymerases. Products of the expected size, and giving equivalent band intensities, were obtained from four specimens with both polymerases. Fox six specimens, less products were obtained with Taq DNA polymerase than with Tth DNA polymerase. Products were detected from five NPs only by PCR with Tth DNA polymerase. The transport medium and the calcium alginate swab fibre of the specimens were shown not to be the source of the inhibitors. The incorporation of 32P-dCTP into cDNA, and the yield of PCR products of cDNA made from control RNA template (purified from H2O spiked virus suspension) were decreased in the presence of inhibitory extracts, showing that both the reverse transcription (RT) and PCR steps in amplification with Taq DNA polymerase were sensitive to the inhibitors. In contrast, Tth DNA polymerase was more resistant to the inhibitors and viral nucleic acid from all the specimens examined could be amplified and detected in a single step by RT-PCR with Tth DNA polymerase.
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PMID:Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus. 985 50


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