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1.
Alexander Koshkaryev Gregory Barshtein Saul Yedgar 《Cell biochemistry and biophysics》2010,56(2-3):109-114
Red blood cell (RBC) adhesion to vessel wall endothelium is a potent catalyst of vascular occlusion and occurs in oxidative stress states such as hemoglobinopathies and cardiovascular conditions. These are often treated with vitamin E (VitE), a “classic” antioxidant. In this study, we examined the effects of VitE on RBC adhesion to vascular endothelial cells (EC), and on translocation of phosphatidylserine (PS) to RBC surface, known as a potent mediator of RBC/EC adhesion, facilitating thrombus formation. Treatment of RBC with VitE strongly induces (up to sevenfold) PS externalization and enhances (up to 20-fold) their adherence to EC. The VitE hydrophilic analogue—Trolox—does not incorporate into cell membranes. Trolox did not exhibit any of these effects, implying that the VitE effect is due to its known ability to incorporate into cell membranes. The membrane-incorporated VitE significantly reduced the level of reactive oxygen species in H2O2-treated RBC, demonstrating that VitE elevates RBC/EC adhesion despite acting as an anti-oxidant. This study demonstrates for the first time that contrary to the common view of VitE as a beneficial supplement, VitE may introduce a circulatory risk by inducing flow-disturbing RBC adherence to blood vessel wall and the pro-thrombotic PS exposure. 相似文献
2.
Chemical modification of the Na+/H+ exchanger of thymic lymphocytes. Inhibition by N-ethylmaleimide 总被引:2,自引:0,他引:2
A Na+/H+ exchanger is involved in the regulation of cytoplasmic pH and cellular volume in a variety of cells. Little is known about the molecular nature of this exchanger. The purpose of this study was to survey a variety of group-specific covalent reagents as potential inhibitors of the exchanger. Na+/H+ countertransport activity was assayed as the amiloride-sensitive rate of Na+-induced alkalinization in acid-loaded lymphocytes, or as the rate of swelling in cells suspended in sodium propionate medium. Activity was not affected by proteinases or by carboxyl-group and amino-group specific reagents. A significant inhibition was produced by diethylpyrocarbonate, a histidine-specific reagent and by N-ethylmaleimide, a sulfhydryl group reagent. A similarly reactive but nonpermeating sulfhydryl agent, glutathione-maleimide, failed to inhibit Na+-H+ exchange. Moreover, the reaction with N-ethylmaleimide was sensitive to changes in the cytoplasmic pH. The data suggest that the chemically reactive groups of the Na+/H+ exchanger of lymphocytes have limited exposure to the extracellular medium but that an internally located sulfhydryl group is critical for the cation-exchange activity. 相似文献
3.
4.
Summary An in situ hybridization method was developed for detecting single or low copy number genes in metaphase chromosomes of plants. Using as a probe 3H-labelled plasmid pABDI, which confers kanamycin resistance (Kmr) to transformed cells. DNA introduced into the plant genome by direct gene transfer was detected with a high efficiency: about 60% to 80% of interphase and metaphase plates showed a strong signal. The insertion site of the Kmr gene in two independent transformants was localised on different homologous chromosome pairs. This result independently confirmed previous genetic data which had indicated that transformed DNA was integrated into plant chromosomes in single blocks. 相似文献
5.
Asymmetry of the Na+/H+ antiport of dog red cell ghosts. Sidedness of inhibition by amiloride 总被引:2,自引:0,他引:2
Amiloride is a potent inhibitor of the Na+/H+ antiport. Inhibition is generally competitive with extracellular Na+ and therefore believed to result from binding to the outward-facing transport site. It is not known whether amiloride can interact with the internal aspect of the antiport. This question was addressed by trapping the drug inside resealed dog red cell ghosts. The antiport, which is quiescent in resting ghosts, was activated by acid-loading the cytoplasm. This was accomplished by exchanging extracellular Cl- for internal HCO-3 through capnophorin, the endogenous anion exchanger. The activity of the Na+/H+ antiport was detected as an increase in cell volume, resulting from the net osmotic gain associated with coupled Na+/H+ and Cl-/HCO-3 exchange, or as the uptake of 22Na+. Intracellular amiloride, at concentrations in excess of 100 microM, failed to inhibit Na+/H+ exchange. This is approximately 10 times higher than the concentration required for half-maximal inhibition when amiloride is added externally. Independent experiments demonstrated that failure of internal amiloride to inhibit exchange was not due to leakage of the inhibitor, to differences in pH, or to binding or inactivation of amiloride by the soluble contents. It was concluded that the antiport is functionally asymmetric with respect to amiloride. This implies that the transport site undergoes a conformational change upon translocation across the membrane or, alternatively, that a second site required for amiloride binding is only accessible from the outside. 相似文献
6.
Calcium-specific ionophores are used widely to stimulate Ca2+-dependent secretion from cells on the assumption that permeabilization of the cell membranes to Ca2+ ions leads to a rise in concentration of cytosolic Ca2+ ([Ca2+]i), which in turn serves as a signal for secretion. In this way, events that precede mobilization of Ca2+ ions via receptor stimulation are bypassed. One such event is thought to be the rapid hydrolysis of membrane inositol phospholipids to form inositol phosphates and diacylglycerol. Accordingly, rat leukemic basophil (2H3) cells can be stimulated to secrete histamine either with the ionophores or by aggregation of receptors for IgE in the plasma membrane. We find, however, that ionophore A23187 stimulates secretion of histamine only at concentrations (200-1000 nM) that stimulate hydrolysis of membrane inositol phospholipids. The extent of hydrolysis of inositol phospholipids was dependent on the concentration of ionophore and the presence of external Ca2+ ions and correlated with the magnitude of the secretory response. A similar correlation between secretion and hydrolysis of inositol phospholipids was observed in response to the Ca2+-specific ionophore, ionomycin. Although this hydrolysis (possibly a consequence of elevated [Ca2+]i) was less extensive than that induced by aggregation of receptors, it may govern the secretory response to A23187. The studies revealed one paradox. The rise in [Ca2+]i depended on intracellular ATP levels, when either an ionophore or antigen was used as a stimulant irrespective of whether hydrolysis of inositol phospholipids was stimulated or not. The concept of how the ionophores act, therefore, requires critical reevaluation. 相似文献
7.
Transmembrane signaling by the B subunit of cholera toxin: increased cytoplasmic free calcium in rat lymphocytes 总被引:8,自引:2,他引:6 下载免费PDF全文
It has previously been shown that the B subunit of cholera toxin, which binds solely to the plasma membrane ganglioside GM1, stimulates the proliferation of rat thymic lymphocytes (Spiegel, S., P. H. Fishman, and R. J. Weber, 1985, Science [Wash. DC], 230:1285-1287). The purpose of this study was to identify which transmembrane signaling system(s) are activated by the B subunit of cholera toxin. We compared the effects of B subunit and concanavalin A (Con A), a potent mitogenic lectin, on a number of second messenger systems that are putative mediators of T cell activation. Changes in the fluorescence of quin2-loaded cells revealed that mitogenic doses of either B subunit or Con A induced rapid and sustained increases in cytoplasmic free Ca2+ ([Ca2+]i). Within 5 min, [Ca2+]i increased from a basal level of 69 +/- 4 to 136 +/- 17 and 185 +/- 24 nM, respectively. The effects of B subunit and Con A were additive and largely dependent on the presence of extracellular Ca2+, though release of Ca2+ from intracellular stores could be detected for Con A, but not B subunit, using indo-1. The B subunit had no effect on either inositol phosphate levels or on the distribution of protein kinase C, indicating that, unlike Con A, the B subunit does not activate phosphoinositide hydrolysis. Fluorimetric measurements on cells loaded with bis(carboxyethyl)-5,6-carboxyfluorescein revealed that Con A induced a rapid cytoplasmic alkalinization via activation of Na+/H+ exchange, whereas B subunit had no effect on intracellular pH. Finally, by monitoring bis-oxonol fluorescence, we found that Con A induced a small hyperpolarization of the membrane potential, whereas B subunit had no acute effect. These data suggest that the biological effects of B subunit are mediated by an increase in [Ca2+]i resulting from a net influx of extracellular Ca2+. 相似文献
8.
Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane 总被引:6,自引:3,他引:3 下载免费PDF全文
R J Howard J A Lyon S Uni A J Saul S B Aley F Klotz L J Panton J A Sherwood K Marsh M Aikawa 《The Journal of cell biology》1987,104(5):1269-1280
The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage. 相似文献
9.
Susan E. Jensen Saul Wolfe Donald W. S. Westlake 《Applied microbiology and biotechnology》1989,30(2):111-114
Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains. 相似文献
10.
G P Downey C K Chan S Grinstein 《Biochemical and biophysical research communications》1989,164(2):700-705
Polymerization of microfilaments, one of the responses triggered in neutrophils by stimuli such as the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), involves the conversion of actin from the monomeric to the filamentous form. The exact sequence of events responsible for this conversion remains to be defined, but its susceptibility to inhibition by pertussis toxin provides indirect evidence that GTP-binding proteins (G-proteins) are involved. In this report, electropermeabilized cells were used to obtain more direct evidence of a role for G-proteins in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin and flow cytometry were used to monitor the formation of filamentous actin. GTP-gamma-S, a nonhydrolyzable analogue of GTP and aluminum fluoride, which in combination with GDP can activate G-proteins, stimulated actin assembly in electropermeabilized cells but had only marginal effects on intact cells. fMLP-induced actin polymerization in permeabilized cells was inhibited by pretreatment with GDP-beta-S, an analogue of GDP that stabilizes the inactive form of G-proteins. In contrast, stimulation by phorbol 12-myristate 13-acetate (PMA) was largely unaffected by GDP-3-S. These observations indicate that activation of G-proteins is essential for actin assembly induced by receptor-dependent stimuli such as fMLP. Moreover, GTP-binding proteins do not seem to be required in the late stages of the signalling cascade, i.e. after stimulation of protein kinase C. 相似文献