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A simple and rapid method for the preparation of plasma membranes.Maeda T, Balakrishnan K, Mehdi SQBiochim. Biophys. Acta , (731), 115-20, 1983. Pubmed A simple and rapid method for preparing plasma membranes from isolated cells or tissues is described. The membranes were characterised (a) biochemically by an analysis of specific marker enzymes, (b) by quantitation of cell surface receptors, and (c) immunologically by their ability to elicit specific allogeneic responses from cytotoxic T cells in secondary in vitro stimulations. Based on both biochemical and immunologic criteria, plasma membranes prepared by the method described here are of equal or greater 'purity' compared to those prepared by two other methods that are most widely used to date and the yields are several-fold higher.
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Fusion and biochemical expression of membrane receptors in foreign living cells.Balakrishnan K, Lewis J, Mehdi SQ, McConnell HMBiochim. Biophys. Acta , (731), 121-6, 1983. Pubmed Successful transplantation of cell surface molecules from the membranes of one cell type to recipient cells of a different type is described. Plasma membranes purified from donor cells were fluoresceinated and fused to recipient cells using poly(ethylene glycol) and the fate of the transplanted membrane components was followed by fluorescence microscopy. In approximately 100 min the 'foreign' membrane components were seen to cluster and internalise. During this time, judged by the criteria of hormonal stimulation and immune cytotoxic killing, the cell surface of the recipient cell mimicked the cell surface phenotype of the donor cell.
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Lipid hapten containing membrane targets can trigger specific immunoglobulin E-dependent degranulation of rat basophil leukemia cells.Balakrishnan K, Hsu FJ, Cooper AD, McConnell HMJ. Biol. Chem. , (257), 6427-33, 1982. Pubmed We have studied the binding of liposomes containing dinitrophenylated lipid to rat basophil leukemia cells armed with monoclonal anti-dinitrophenyl IgE. The liposomes were either "fluid" at 37 degrees C (dimyristoylphosphatidylcholine or an equimolar binary mixture dipalmitoylphosphatidylcholine and cholesterol) or "solid" (dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, or dibehanoylphosphatidylcholine). We have also studied the immune mediated degranulation of these cells induced by the above lipid membrane targets. In some cases both studies were carried out with liposomes containing various surface densities of lipid haptens. From these studies we conclude that freely mobile nonaggregated lipid haptens in bilayer membrane targets can trigger efficient serotonin release from rat basophil leukemia cells in the presence of specific antihapten IgE. Solid target membranes are also effective as stimulators of serotonin release. The release of serotonin depends strongly on the surface density of lipid haptens over a narrow range of surface densities. These studies with lipid membrane targets having well defined physical properties indicate the need for generalized molecular models of receptor-mediated cell triggering.
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Studies on the reaction of isocyanides with haemproteins. II. Binding to normal and modified human haemoglobins.Brunori M, Talbot B, Colosimo A, Antonini E, Wyman JJ. Mol. Biol. , (65), 423-34, 1972. Pubmed |
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Expression of melatonin receptors in arteries involved in thermoregulation.Viswanathan M, Laitinen JT, Saavedra JMProc. Natl. Acad. Sci. U.S.A. , (87), 6200-3, 1990. Pubmed Melatonin binding sites were localized and characterized in the vasculature of the rat by using the melatonin analogue 2-[125I]iodomelatonin (125I-melatonin) and quantitative in vitro autoradiography. The expression of these sites was restricted to the caudal artery and to the arteries that form the circle of Willis at the base of the brain. The arterial 125I-melatonin binding was stable, saturable, and reversible. Saturation studies revealed that the binding represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 3.4 x 10(-11) M in the anterior cerebral artery and 1.05 x 10(-10) M in the caudal artery. The binding capacities (Bmax) in these arteries were 19 and 15 fmol/mg of protein, respectively. The relative order of potency of indoles for inhibition of 125I-melatonin binding at these sites was typical of a melatonin receptor: 2-iodomelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine. Norepinephrine-induced contraction of the caudal artery in vitro was significantly prolonged and potentiated by melatonin in a concentration-dependent manner, suggesting that these arterial binding sites are functional melatonin receptors. Neither primary steps in smooth muscle contraction (inositol phospholipid hydrolysis) nor relaxation (adenylate cyclase activation) were affected by melatonin. Melatonin, through its action on the tone of these arteries, may cause circulatory adjustments in these arteries, which are believed to be involved in thermoregulation.
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Changes in expression of angiotensin receptor subtypes in the rat aorta during development.Viswanathan M, Tsutsumi K, Correa FM, Saavedra JMBiochem. Biophys. Res. Commun. , (179), 1361-7, 1991. Pubmed Quantitative autoradiography was used to characterize angiotensin AT1 and AT2 receptors, in the rat aorta at three developmental ages; embryonic day 18 (E18), and postnatal weeks 2 and 8. The expression of angiotensin receptors was higher in the aorta of E18 and 2-week-old rat. A major proportion of the angiotensin receptors expressed in the aorta at these two ages was AT2 (84 and 81% respectively). Conversely, in the aorta of 8-week-old rats, AT1 was the predominant angiotensin receptor subtype (71%). In 8-week-old rats, the AT2 subtype was also present (28%). In pre- and postnatal rats, [125I]Sar1-angiotensin II binding to AT1 receptors was sensitive to GTP gamma S whereas binding to AT2 receptors was not. AT2 receptors may serve an important role during stages of rapid growth of the aorta, and also have a significant function in the adult vasculature.
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Balloon angioplasty enhances the expression of angiotensin II AT1 receptors in neointima of rat aorta.Viswanathan M, Strömberg C, Seltzer A, Saavedra JMJ. Clin. Invest. , (90), 1707-12, 1992. Article Pubmed Angiotensin II is a vasoactive peptide and may act as a growth factor in vascular smooth muscle cells. Experimental injury of the rat aorta causes rapid migration of medial smooth muscle cells and their proliferation resulting in the formation of neointima. We have examined, using quantitative autoradiography, the expression of angiotensin II receptor subtypes AT1 and AT2, and angiotensin-converting enzyme, in the neointima formed in the rat thoracic aorta 15 d after balloon-catheter injury. In contrast to the normal aortic wall, which contained both AT1 and AT2 receptors (80% and 20%, respectively), neointimal cells expressed almost exclusively angiotensin II AT1 receptors. The apparent number of these receptors was fourfold higher in the neointima compared to that in the normal aortic wall. The affinities of the neointimal receptors to angiotensin II or to the AT1 receptor antagonist, losartan, were not different from those in the normal aortic wall. Angiotensin-converting enzyme binding in the neointima was not different from that in the media of the uninjured aorta. Our data suggest that angiotensin II AT1 receptors may have a significant role in injury-induced vascular smooth muscle proliferation and migration.
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