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N Crawford

Publications and source records attributed to N Crawford.

At least 55 records · Page 3Linked to original sources
PubMed1988

Introduction of antibody (PL/IM 430) to a 100 kDa protein into permeabilised platelets inhibits intracellular sequestration of Ca2+.

A monoclonal antibody (PL/IM 430), previously found to inhibit the uptake of Ca2+ into highly purified platelet intracellular membrane vesicles (Hack, N., Wilkinson, J.M. and Crawford, N. 1988, Biochem. J. 250, 355-361) has been introduced into saponin-permeabilised platelets. At a saponin concentration (20-25 micrograms/ml) commensurate with total LDH release, sequestration of Ca2+ into intracellular non-mitochondrial stores is inhibited by the antibody (approximately 50% inhibition at 20 micrograms/ml IgG). At higher saponin concentrations when intracellular binding of 125I-labelled mAb is maximum, inhibition of Ca2+ sequestration approaches 70%. The inhibition is specific, control studies with non-platelet directed mouse IgG and mAbs which immunoblot platelet antigens other than the 100 kDa protein did not affect the Ca2+ sequestration. No effect of the antibody were observed against IP3-induced release of prestored Ca2+, either in permeabilised platelets or with isolated intracellular membrane vesicles. The mAb PL/IM 430 appears to bind only to the Ca2+ translocating channel protein associated with the intracellular membrane (Ca2+ + Mg2+) ATPase and not to Ca2+ channels responsive to IP3.

Antibodies, Monoclonal↗
PubMed1988

Albumin and alpha-fetoprotein gene expression and DNA methylation in rat hepatoma cell lines.

To define systems for the study of gene control and differentiation in vitro, we analyzed albumin and alpha-fetoprotein (AFP) gene expression and gene methylation in a series of rat hepatoma-derived cell lines and controls. These cell lines had several specific phenotypes: adult (high albumin and low AFP mRNA), fetal (high albumin, high AFP), embryonic (low albumin, high AFP), or undifferentiated (no albumin or AFP). The adult hepatocyte phenotype is marked by a novel 2.2-kb AFP gene transcript and high DNA methylation. In general, tumor cell lines had higher albumin and AFP gene methylation than hepatocytes in vivo. Levels of total DNA methylation did not determine the methylation patterns of specific genes, except for one cell line with hypermethylated and one with hypomethylated DNA. 5'-Hypomethylation of the AFP gene correlated with gene activity in all cases; the albumin gene showed a similar relationship, but with some exceptions. Only adult hepatocytes, not cell lines, have a unique 3'-region of AFP gene demethylation.

Albumins↗
PubMed1988

Characterization of a monoclonal antibody ITI-Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes.

A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.

Animals↗
PubMed1988

Platelet surface charge heterogeneity: characterization of human platelet subpopulations separated by high voltage continuous flow electrophoresis.

Washed formol-fixed normal human platelets have been separated into surface charge-dependent subpopulations using high voltage continuous flow electrophoresis. The procedure is highly reproducible and the heterogeneity profile extends over 20-25 fraction tubes on the anodal side of the entry port to the separation chamber. Fractions have been subdivided into subpopulation pools A, B and C which have mean mobilities by analytical cytopherometry extending over the range 0.81-0.91 micron/s/V/cm from the least (C) to the most (A) electronegative cells. Coulter volume differences across the profile from 5.0 to 12.8 fl correlated well with electrophoretic mobilities whereas buoyant density appeared to be an independent parameter. Analysis of surface neuraminidase-labile sialic acid of the platelets in pools A and C correlated well with differences in electrophoretic mobility, whereas a similar relationship for the alkaline phosphatase-labile phosphate moieties (also believed to be contributory to cell surface electrokinetic properties) could not be established even though in both cases the profiles of the enzyme-treated platelets showed significant shifts towards the cathode when compared with untreated cells. Titration of surface DTNB-reactive sulphydryl (-SH) groups revealed an inverse relationship between electronegativity and membrane -SH group status. This electrophoretic expression of subpopulation heterogeneity within the circulating platelet pool may have advantages in studying clinical conditions where the profiles may reflect cell surface interactions 'in vivo'.

Blood Platelets↗
PubMed1987

Inositol 1,4,5-trisphosphate (IP3) induced rapid formation of thromboxane B2 in saponin-permeabilised human platelets: mechanism of IP3 action.

The mechanism of IP3-induced activation of saponin-permeabilised platelets has been examined. Saponin permeabilization resulted in the leakage of low-Mr substances into and from the cells without loss of cytoplasmic proteins. Addition of IP3 rapidly induced a dose-related formation of thromboxane B2 and release into the medium, leading to the responses of shape change, aggregation and [14C]5HT release. These responses were inhibited by the thromboxane A2 receptor antagonist AH23848. The IP3-induced release of 45Ca from intracellular stores was not affected by indomethacin. Synthesis of thromboxane was inhibited if Ca2+ elevation was prevented by using Ca-EGTA buffers during permeabilization. These studies indicate that IP3-induced activation was due to Ca2+ mobilisation leading to phospholipase activation and thromboxane synthesis.

Biphenyl Compounds↗
PubMed1987

Actin in B16 melanoma cells of differing metastatic potential. Effects of trypsin and serum.

Actin is present in cells in monomeric and polymeric (filamentous) forms. Filamentous actin is distributed in Triton-soluble (cytosolic) and Triton-insoluble (cytoskeletal core) fractions. We have used the DNase 1 inhibition assay and immunofluorescence to investigate the distribution of actin in monomeric and polymeric forms in cloned B16 murine melanoma cell lines of low and high metastatic capacity. The protease trypsin caused rounding up and detachment of both cell lines within 5 min. This was associated with almost complete depolymerization of cytosolic actin filaments but the Triton-insoluble cytoskeleton was not quantitatively affected by trypsin treatment. There were quantitative differences between the clones in their response to incubation in the presence or absence of 10% serum. The highly metastatic cell line contained 35% more actin when incubated in the presence of 10% serum, almost completely distributed to the Triton-insoluble cytoskeleton, an effect not seen in the low metastatic cells.

Actins↗
PubMed1987

Changes in the distribution and organization of platelet actin induced by diamide and its functional consequences.

Exposure of blood platelets to diamide (azodicarboxylic acid-bis-dimethylamide) results in oxidation of sulphydryl groups present in the cytoskeleton and other proteins. This results in dramatic changes in functional behaviour of the cells. The distribution and level of organization of the major cytoskeletal protein actin has been studied analytically by the DNase-I inhibition assay and morphologically by electron microscopy (EM) of Triton X-100 treated platelets adherent to EM grids. Exposure to diamide results in a redistribution of actin within the cell reflected in an increase in cytoskeletal F-actin and a concomitant decrease in cytosolic actin. The magnitude of these changes depends upon the concentration of diamide and the time of exposure. Diamide also alters platelet aggregatory functions in response to certain stimuli. Treatment of normal human platelets with 0.1 mM diamide proceeds via disaggregation (5 min exposure to diamide), inhibition of aggregation (30 min exposure), to finally a normalization of the aggregation response after 60-120 min incubation with diamide. In parallel with the return to full functional response the distribution of F-actin between the cytoskeleton and cytoplasmic compartments returns to the control pattern. Incubation of the platelets with 0.5 mM diamide for 60 or more minutes leads to total inhibition of the aggregatory ability. In these cells the cytoskeleton associated F-actin remains significantly elevated and the structural organization of the cytoskeleton is markedly altered. In contrast to the network of filaments subadjacent to the surface membrane seen in unstimulated platelets, the cytoskeleton now shows electron dense zones in the more central parts of the cytoplasm. This diamide-induced structural reorganization of platelet cytoskeletal elements, associated with the inhibition of functional responses, emphasizes the dynamic nature of the membrane-cytoskeletal axis and its importance in the expression of shape changes and aggregatory phenomena in response to surface stimuli.

Actins↗
PubMed1987

Tumour-cell-induced platelet aggregation: studies with cloned metastatic and non-metastatic variants.

Blood platelets have been suggested to play an important role in modulating the development of experimental metastases. Tumour cells can induce platelet aggregation in vitro and a number of mechanisms have been proposed to explain the in vivo and in vitro observations. In the present study, we used tumour cells cloned from B16 melanoma and mouse mammary tumour virus (MMTV) carcinoma polyclonal populations to check whether tumour cells with low- and high-metastatic behaviour in vivo had different quantitative and qualitative platelet-aggregating activity in vitro. We found no significant quantitative difference between platelet aggregation induced by the low- and the high-metastatic clones. Indeed both the high and the low metastatic B16 melanoma clones poorly aggregated platelets, while both the high and low metastatic MMTV carcinoma clones efficiently aggregated platelets. Both the B16 melanoma and the MMTV carcinoma parental cell lines, which can be classed as intermediate metastatic, aggregated platelets well. However, based on the results with heparin and creatine phosphate/creatine phosphokinase, it appeared that qualitative differences might exist in the mechanism of platelet aggregation by tumour cells. For the parental lines and highly metastatic clone C1 a thrombin-linked component was more important than an ADP-like component, which was nevertheless present, to promote platelet aggregation. For the low-metastatic clone C2, the ADP-like component appeared to be the most important.

Adenosine Diphosphate↗
PubMed1986

Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.

Adenosine Triphosphatases↗
PubMed1986

Metabolic and functional consequences of introducing inositol 1,4,5-trisphosphate into saponin-permeabilized human platelets.

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.

Adenine↗
PubMed1986

Formation of diacyl- and alkylacylphosphatidylcholine by the membranes of human platelets.

We have investigated the distribution and fatty acid preference of two acyl-CoA transferase activities in a human platelet mixed membrane fraction and in well-characterised surface and intracellular membrane subfractions prepared from it by high-voltage free-flow electrophoresis. One transferase inserts long-chain unsaturated fatty acids into 1-acyllysophosphatidylcholine (1-acyl-LPC) and the other into lyso-platelet-activating factor (LPAF). Both transferase activities were approx. 4-fold enriched in the intracellular membranes with respect to their specific activities in the mixed membranes. The surface membrane activities were correspondingly depleted. Using 1-acyl-LPC as the acceptor, all the intracellular membrane preparations showed transferase preference for the CoA ester of 8,11,14-eicosatrienoic acid. In contrast when LPAF was the acceptor the CoA esters of linoleic and arachidonic acid were the preferred donors.

Acyltransferases↗
PubMed1986

A sensitive procedure for quantifying the phagocytic competence of blood granulocytes.

The phagocytic activity of blood granulocytes can be quantitatively assayed by ingestion of opsonised paraffin oil droplets containing the dye "Oil Red-O" (Stossel, T.P., Mason, R.J., Hartwig, J., and Vaughan, M. (1972) J. Clin. Invest. 51: 615-624). We have modified this assay by incorporating [3H]glycerol into the oil droplets which allows a more sensitive and reproducible measurement of the phagocytic competence of blood granulocytes even at very low cell counts. Comparative studies after one day storage of the blood at 4 degrees C is feasible since they retain 84% of the phagocytic capacity measured when isolated from fresh blood.

Animals↗
PubMed1986

Effect of transformation by Rous sarcoma virus on the character and distribution of actin in Rat-1 fibroblasts: a biochemical and microscopical study.

Actin has been measured in subcellular fractions from Rat-1 fibroblasts and in Rous sarcoma virus-transformed Rat-1 cells (VIT), using the DNase 1 inhibition assay. The transformed cells showed a significant shift in the actin monomer (G)in equilibrium with polymer (F) equilibrium within the cell cytosol, and a significant increase in actin in the Triton-insoluble cytoskeletal core in comparison with untransformed cells. This incorporation of actin into the cytoskeletal core fraction is associated with a change in filamentous actin assemblies from 'stress fibre' patterns to punctate filament aggregates. These differences have been correlated with changes in morphology, in actin, vinculin and alpha-actinin distribution, in adhesion plaque formation and with the production of pp60v-src-associated protein kinase activity in the transformed cells. Changes in actin distribution and its polymerization in response to src-gene expression may play an important role in the determination of the transformed cell characteristics.

Actins↗
PubMed1985

Inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ from highly purified human platelet intracellular membranes.

Evidence has accumulated in support of a role for intracellularly generated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in raising cytosol [Ca2+] when various hormones, neurotransmitters, growth factors and other stimulants act on cell surfaces. The increase in [Ca2+] that follows stimulant-receptor interaction is accompanied by rapid hydrolysis of phosphoinositides. One product, Ins(1,4,5)P3, arising from the breakdown of phosphatidylinositol 4,5-bisphosphate was shown to promote the release of Ca2+ from non-mitochondrial stores in a variety of cells. Although platelet intracellular membranes have been implicated in the control of cytosol [Ca2+] and we previously characterized a Ca2+-sequestering mechanism associated with them, we have as yet no knowledge of how this Ca2+ store is mobilized after a stimulus-receptor interaction at the platelet surface. Using free-flow electrophoresis, we isolated and purified human platelet intracellular membranes. They show high enrichment and exclusive localization of the endoplasmic-reticulum marker NADH:cytochrome c reductase, and they sequester Ca2+ by an ATP-dependent process, reaching steady-state values in 10-12 min. Saturation with Ca2+ occurs at around 10-30 microM external Ca2+. When Ins(1,4,5)P3 is added to the 45Ca-loaded vesicles, a rapid release of Ca2+ occurs (approx. 35% in 15-30s). The magnitude of the release depends upon external [Ca2+], being maximum in the range 0.3-0.8 microM and low at external [Ca2+] greater than 1 microM. After release there is a rapid re-uptake of Ca2+, with restoration of the former steady-state values within 1 min. Half-maximal release occurs at approx. 0.25 microM-Ins(1,4,5)P3. This release and re-uptake pattern is not observed with ionophore A23187 or arachidonic acid, both of which liberate Ca2+ irreversibly. Inositol 1,4-bisphosphate was ineffective in releasing Ca2+ from these intracellular membranes. The results support the role of Ins(1,4,5)P3 as a specific intracellular mediator, transducing the action of excitatory agonists acting on the platelet surface into metabolic, mechanochemical and other functional events, known to occur during platelet activation.

Arachidonic Acid↗
PubMed1985

Redistribution of membrane-bound and cytosolic action in rabbit polymorphonuclear leucocytes during phagocytosis.

In the analysis of highly purified surface membrane from both resting and phagocytosing neutrophils an increase in the surface membrane associated actin has been demonstrated. This change at the cell periphery is associated with a coincident increase in the F-actin content of the cells following stimulation of the cells by exposure to opsonized Oil Red O droplets. The actin which is newly associated with the surface membrane of the phagocytosing cells was more susceptible to removal by detergent than the membrane-associated actin in resting cells and it was also noted that the F-actin associated with phagosomes was readily disrupted by detergent. A redistribution of the surface membrane glycoprotein 5'-nucleotidase was observed during phagocytosis, but no change in distribution of a 125I-labelled Lens culinaris lectin was observed during the entire phagocytic process.

5'-Nucleotidase↗
PubMed1985

Diacylglycerol lipase activity in human platelet intracellular and surface membranes. Some kinetic properties and fatty acid specificity.

Diacyl glycerol lipase activity has been examined of intracellular and surface membranes isolated from human blood platelets by free flow electrophoresis. Enzyme activity is present on both membranes but is activated at different substrate concentrations (Km 14 microM and 140 microM for intracellular and surface membrane, respectively). Both enzyme activities are stimulated by EGTA and GSH, and inhibited by added Ca2+. The specificity of the intracellular membrane enzyme has been investigated using a range of diacylglycerol substrates differing only in their '2' position fatty acid. Arachidonic acid is clearly the preferred '2' position moiety with activities towards eicosatrienoic, linoleic, oleic and palmitic acid-containing substrates, all substantially lower.

Blood Platelets↗