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Biomedical subjects

N Crawford

Publications and source records attributed to N Crawford.

At least 37 records · Page 2Linked to original sources
PubMed1992

Fractionation of human neutrophils into subpopulations by countercurrent distribution: surface charge and functional heterogeneity.

Isolated blood neutrophils from normal healthy subjects were separated into fractions by sequential countercurrent distribution (CCD) in a charge-sensitive dextran/polyethylene glycol aqueous phase system. The neutrophils separated as a broad profile, and in a charged phase procedure the separation was based upon differences in cell surface electrokinetic properties, as confirmed by electrophoretic mobility measurements of fractions across the profile using analytical cytopherometry. The CCD cell fractions were generally pooled as three or four major subfractions for analysis of functional and metabolic differences. These included measurements of chemotaxis, phagocytosis, and respiratory burst. An inverse relationship was found between the electrophoretic mobility (EPM) of the subfraction pools and their functional competence, with the less electronegative cell fraction pools often as much as 2 to 3-fold more active than the more electronegative pools. This demonstration of electrokinetic and functional heterogeneity in 'resting' neutrophil subpopulations separated by CCD may reflect changes during their sojourn in the circulation that determine selective margination and recruitment of cells to inflammatory foci and sites of infection.

Cell Separation↗
PubMed1991

Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils.

After priming by a number of different host, bacterial and chemical agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone. Other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. We describe here a new extracellular role for inositol hexakisphosphate (InsP6) as a priming agent for a variety of human neutrophil functional responses. Preincubation of the cells with InsP6 alone (up to 250 microM) has no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus (FMLP, PMA or phagocytic particles) is substantially enhanced. Levels 100-200% higher than 'stimulus only' controls have been recorded. Peak enhancement of the FMLP-induced oxidative response occurs after 1-2 min preincubation with InsP6 and the effect is dose-dependent (maximum at approx. 100 microM InsP6). As others have shown FMLP stimulation of superoxide anion production has no external Ca2+ dependence but the presence of low levels of Ca2+ and Mg2+ (0.1 mM) during priming appears to be an essential requirement for full expression. Reports of intracellular concentrations of InsP6 in mammalian cells in the 30-100 microM range suggest that the local release of this inositol polyphosphate from damaged or effect cells could have a physiologically important modulatory role on neutrophil functions.

Calcium↗
PubMed1991

Dynamic changes in neutrophil cytoskeleton during priming and subsequent surface stimulated functions.

The naturally occurring inositol polyphosphate, inositol hexakisphosphate (InsP6, phytic acid) primes human neutrophils for enhanced responses to stimulatory agonists. Unlike other primers InsP6 has no effect at high doses (up to 500 microM) on the neutrophil's basal rate of superoxide production (O2-). Pretreatment of the cells for 2 min with InsP6 (100 microM) substantially enhances fMet-Leu-Phe (FMLP) (10(-7) M)-induced respiratory burst. In investigations of the dynamic aspect of neutrophil actin during priming and stimulation with FMLP the content of F-actin has been measured with and NBD (nitrobenz-2-oxa-1,3-diazole)-phallacidin assay. The induced assembly of F-actin is rapid (peaks at approximately 45 s) followed by depolymerization. In contrast, priming with InsP6, with or without FMLP stimulation, results in a sustained assembly of F-actin as visualized with a rhodamine-phalloidin staining technique. These changes in macromolecular status and distribution of F-actin during InsP6 priming occur in the absence of any other demonstrable functional responses and thus InsP6 may be a useful tool to follow early events in neutrophil functions or to monitor the presence of unprimed and preprimed subpopulations in the circulation. Since InsP6 is present in high concentrations in neutrophils (up to 100 microM), its release from damaged or effete cells may have a modulatory role on neutrophil functions.

Actins↗
PubMed1991

Enhancer, repressor, and promoter specificities combine to regulate the rat alpha-fetoprotein gene.

The upstream transcription control region of the rat alpha-fetoprotein (AFP) gene was analyzed using transient expression of CAT genes in HepG2 cells which express the gene; H4C3 cells which repress the AFP gene but express the albumin gene; and four nonexpressing cell lines. Deletion analysis based on the DNA sequence resolved three upstream enhancers corresponding to the mouse AFP enhancers, but showed additional weak effects from flanking sequences. Quantitative experiments demonstrated that the three enhancers were additive when acting through a single promoter and did not confirm the presence of a distal upstream repressor. All three enhancers stimulated the AFP, albumin, or thymidine kinase (tk) promoter in HepG2, but only the tk and albumin promoters in H4C3. Deletion of a proximal repressor region near the AFP promoter allowed expression in H4C3 cells with the AFP promoter. Thus, the liver-specific developmental repressor is near the AFP promoter, and H4C3 cells provide an in vitro system for analysis of this repressor in transfection assays. The repressor region also blocked expression of the SV40 enhancer through the AFP promoter in hepatic and nonhepatic cell lines, but when this enhancer was combined with an AFP promoter from which the repressor region was deleted, the combination showed expression in all six cell lines studied. AFP expression results from a combination of enhancer, promoter, and repressor activities, and the repressor is functional with a heterologous enhancer in a variety of cells.

Animals↗
PubMed1991

Electrokinetic properties and surface membrane sialic acid status of platelets in idiopathic thrombocytopenic purpura (ITP).

Continuous flow electrophoresis has been used to investigate heterogeneity in the circulating platelets from patients with idiopathic thrombocytopenia (ITP) and from age- and sex-matched control donors. The ITP patients' platelet counts ranged from 15 to 80 X 10(9)/l and they had much higher mean volumes and significantly higher (two-fold) total cell and neuraminidase-labile surface sialic acid contents expressed per unit cell than normal. After volume normalization the mean difference between the two groups was not statistically significant. The relationships of these findings to abnormal thrombopoiesis and to molecular recognition for reticuloendothelial system (RES) removal are discussed.

Adolescent↗
PubMed1990

Basal and induced prothrombinase expression in platelets from patients with essential thrombocythaemia (ET).

Both the basal and the collagen plus thrombin-stimulated prothrombinase activities have been measured in platelets from eight patients with essential thrombocythaemia (ET). On a mean basis these activities were significantly lower than the values recorded for a group of age and sex matched control subjects. When the Ca2+ ionophore A23187 was substituted for the agonist mixture the induced expression of prothrombinase activity was essentially the same for the patient and control groups. It is suggested that the defect in prothrombinase expression seen with ET platelets may reside in either membrane signal transduction processes concerned with Ca2+ mobilization or in Ca2+ sensitive cytoskeletal control of membrane phospholipid topography.

Adult↗
PubMed1989

Electrokinetic, analytical and functional heterogeneity of circulating human platelets: separation of subpopulations by continuous flow electrophoresis after taxol stabilization.

A continuous flow electrophoresis procedure has been developed to study platelet subpopulation heterogeneity with separations based upon surface electrical charge differences. Taxol at low concentrations has been used to transiently stabilize the cells during the separations. At a concentration of 10(-5) M taxol has no effect upon a wide range of physical, analytical and enzymatic properties and does not compromise agonist-induced activation responses (aggregation and secretion). A typical normal platelet subpopulation profile extends over 15-20 fractions with mobilities from -0.97 to -0.78 microns per s per volt per cm. Platelet size (resistive particle counter volumes) differed significantly across the profile, the most electronegative cells being the larger, and the least electronegative the smaller platelets. Total platelet sialic acid content and surface neuraminidase-labile sialic acid correlated positively with electronegativity, but the surface -SH group status had an inverse relationship with the least electronegative smaller platelets, having twice as many surface DTNB-titratable - SH groups as the most electrophoretically mobile and larger cells. Normalisation of analytical and enzymatic data to cell volumes revealed that the smaller less electronegative platelets were substantially richer in all constituents and properties than the larger more electronegative platelets. These smaller cells showed higher activities for lysosomal enzymes, and their functions (capacity to transport 5-hydroxytryptamine and adenosine across the plasma membrane and responsiveness to thrombin expressed by synthesis of thromboxane B2 (TXB2) or release of 5HT) were greater than the larger more electronegative cells. No significant differences were observed, however, in the subpopulations by optical aggregometry using six different agonists each at three different concentrations. This free flow electrophoresis separation of platelets, which can be carried out on a preparative scale, may have some advantages over the conventional density gradient separations of subpopulations for investigating clinical states affecting thrombopoietic regulation or platelet losses from the circulation due to vessel wall disease, prosthetic implants or during extracorporeal circuitry.

Alkaloids↗
PubMed1989

High incorporation of [3H]inositol into phosphoinositides of human platelets during reversible electropermeabilisation.

A new method for high incorporation of [3H]inositol into human platelets is described. The method involves incorporation of [3H]inositol during reversible electropermeabilisation by high voltage discharge, followed by resealing the cells during incubation at 37 degrees C. Between 10- and 20-fold increase of isotope uptake is achieved compared to control intact cells. Permeabilised resealed platelets maintain good responses to thrombin and collagen. Analysis of the incorporation of the label amongst the phosphoinositides shows 70% to be in PI, 20% in PIP, and 10% in PIP2. Stimulation with thrombin and analysis of the formation of IP1, IP2 and IP3 shows the labelling to occur in a hormone-sensitive pool. These studies indicate that reversible electropermeabilisation can be used to achieve good uptake of non-membrane penetrating substances such as inositol.

Blood Platelets↗
PubMed1989

Reversible electropermeabilisation of human and rat blood platelets: evaluation of morphological and functional integrity 'in vitro' and 'in vivo'.

A high-voltage discharge procedure has been developed for permeabilising the plasma membranes of both human and rat blood platelets. The cells can be resealed by incubation at 37 degrees C, show less than 4% loss of lactate dehydrogenase (LDH) implying minimal cell lysis and also have well maintained morphological and functional integrity. The prototype apparatus used at field strengths between 6 and 8 kV/cm produces membrane pores which allow free diffusion of low molecular weight substances such as adenine nucleotides, inositol phosphate and fluorescent dyes. Two properties, namely Ca2+-induced secretion of granule stored 5-hydroxytryptamine (5HT) and inositol 1,4,5-trisphosphate (IP3)-induced release of intracellularly sequestered 45Ca, which are both well expressed immediately after permeabilisation, are essentially abolished after resealing. The efficiency of permeabilisation and resealing can be simply monitored by shifts in 'apparent platelet volume' using a resistive particle counter (Coulter). Permeabilised platelets show a shift in modal volumes from a control range 4-7 fl to 10-15 fl. Resealing restores these modal volumes to the original control range. Encapsulation of the fluorochrome, Lucifer yellow (Mr 550), during permeabilisation revealed that after resealing greater than 85% of rat platelets, and close to 100% human platelets, contained the encapsulated dye. The initial rates and % aggregation responses of both human and rat platelets to collagen, thrombin and the thromboxane A2-mimetic U46619 remained essentially normal after permeabilisation and resealing further illustrating the maintenance of functional competence following treatment. Resealed rat platelets reinfused into the circulation after labelling with [111In]indium oxine gave survival curves similar to those of control platelets. Therefore, this reversible permeabilisation procedure may allow the use of autologous or heterologous platelets as carrier vehicles for the delivery of drugs and other agents 'in vivo'.

Adenine Nucleotides↗
PubMed1988

Action of guanosine 5'-[beta-thio]diphosphate on thrombin-induced activation and Ca2+ mobilization in saponin-permeabilized and intact human platelets.

The non-hydrolysable guanine analogues guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]) have been used extensively (as promoters and inhibitors respectively) to probe the importance of G-protein function. We report on the use of GDP[S] in permeabilized and intact platelets. The stimulatory analogue GTP[S] (9-60 microM) induces shape change, aggregation and 5-hydroxy[14C]-tryptamine secretion when added to saponin (12-14 micrograms/ml)-permeabilized platelets, but not to intact platelets. In line with the activation responses in permeabilized cells, GTP[S] induces an increase in [32P]-phosphatidic acid, which is indicative of phospholipase C activity. GDP[S] (greater than 400 microM) totally inhibits GTP[S] (90 microM)-stimulated phospholipase C activity and functional responses in saponized platelets. GDP[S] (1 mM) was also effective at inhibiting low-dose thrombin (0.1 unit/ml)-induced aggregation and secretion responses (without affecting shape change) in permeabilized platelets with inhibition of [32P]-phosphatidic acid formation. At higher doses of thrombin (greater than 0.5 unit/ml), both functional responses and [32P]phosphatidic acid formation are restored in the presence of GDP[S]. Studies on intact cells revealed that GDP[S] was as effective at inhibiting low-dose thrombin-induced functional responses as in the permeabilized cells, but there was no inhibition of [32P]phosphatidic acid formation, indicating that the agent is nonmembrane-penetrating. This reflected the fact that GDP[S] has additional inhibitory sites on the surface of platelets. In Fura-2-loaded cells GDP[S] inhibited thrombin-induced Ca2+ mobilization, as measured by Fura-2 fluorescence, in a dose-dependent manner. In studies with and without Ca2+ present on the outside, the effect of GDP[S] was to block Ca2+ influx. These studies indicate that, although GDP[S] is a valuable tool in studying G-protein function in permeabilized cells, it also has inhibitory activities on the surface of platelets, and one of these has been identified as an effect on the Ca2+-influx channel after agonist stimulation.

Blood Platelets↗
PubMed1988

A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase.

To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.

Adenosine Triphosphate↗