Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin

Fucoidan, poly(L-fucopyranose) linked primarily alpha 1----2 with either a C3- or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G. F., Wurzel, H. A., McNeal, G. M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc. Soc. Exp. Biol. Med. 94, 404-409). We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin III and heparin cofactor II. Fucoidan enhances the heparin cofactor II-thrombin reaction more than 3500-fold. The apparent second-order rate constant of thrombin inhibition by heparin cofactor II increases from 4 x 10(4) (in the absence of fucoidan) to 1.5 x 10(8) M-1 min-1 as the fucoidan concentration increases from 0.1 to 10 micrograms/ml and then decreases as fucoidan is increased above 10 micrograms/ml. The fucoidan reaction with heparin cofactor II-thrombin is kinetically equivalent to a "template model." Apparent fucoidan-heparin cofactor II and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively. The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor II or of thrombin. The fucoidan-antithrombin III reactions with thrombin and factor Xa are accelerated maximally 285- and 35-fold at fucoidan concentrations of 30 and 500 micrograms/ml, respectively. Using human plasma and 125I-labeled thrombin in an ex vivo system, the heparin cofactor II-thrombin complex is formed preferentially over the antithrombin III-thrombin complex in the presence of 10 micrograms/ml fucoidan. Our results indicate that heparin cofactor II is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor II and not by antithrombin III.     

A new interpretation of the involvement of serotonin in delayed-type hypersensitivity. Serotonin-2 receptor antagonists inhibit contact sensitivity by an effect on T cells

5-HT is a neuromediator and a vasoactive amine released by platelets and murine mast cells at sites of inflammation. A role for 5-HT has been proposed in murine DTH and has been attributed to its 5-HT2R-dependent vasoactive properties. We have tested the hypothesis that the role of 5-HT in DTH is related to an interaction of 5-HT with DTH effector T cells. In vivo treatment of sensitized mice with the 5-HT2R antagonists methysergide or ketanserin inhibited both their capacity to elicit DTH and the ability of their lymphoid cells to transfer DTH. In vitro treatment of lymphoid cells, or of nylon wool-purified T cells from sensitized mice, with 10(-7) to 10(-9) M of the 5-HT2R antagonists methysergide, ketanserin, ritanserin, or LY 53857, followed by three washings, inhibited as strongly their ability to transfer DTH, both systemically or locally. Systemic and local co-transfer experiments of 5-HT2R antagonist-treated and untreated cells indicated that this inhibition was not related to the induction of suppression. 5-HT2R antagonist treatment was nontoxic to T cells; did not affect the in vitro response of T cells to mitogen; selectively inhibited the efferent, but not the afferent limb of DTH; and in the efferent T cell cascade, affected the late-acting (24 h) inflammatory DTH T cells, but not the early-acting, DTH-initiating T cells. 5-HT2R selectivity was suggested by the absence of effect of an alpha-adrenergic R antagonist, and by prevention of the inhibitory effect of a 5-HT2R antagonist by prior incubation with the selective 5-HT2R agonist 1-(2,5-dimethoxy phenyl-4-methyl)-2 aminopropane. In summary, inhibition of DTH effector T cell function appeared sufficient, independently of any vascular effect, to account for the in vivo inhibitory effect of 5-HT2R antagonists on the elicitation of DTH. Our data suggest that late-acting DTH effector T cells might express functional 5-HT2R, and that these receptors might require in vivo activation in order for the T cells to locally produce the inflammatory lymphokine-dependent aspects of DTH.     

Effects of genetic and environmental host plant variation on the susceptibility of two noctuids toBacillus thuringiensis

TwoApium graveolens var.rapaceum (L.) cultivars that differ in their suitability for the survival and growth ofSpodoptera exigua (Hübner) andTrichoplusia ni (Hübner) were used to examine the effect of genetic and seasonal environmental variation in host plant suitability on the efficacy ofBacillus thuringiensis subsp.kurstaki (Berliner). The effects of host plant genotype andB. thuringiensis were generally independent, so thatB. thuringiensis efficacy was greatest on the resistant host plant cultivar. Host plant suitability varied within growing season for both insect species but, while host plant suitability decreased with increasing plant age forT. ni, the response ofS. exigua to plant age was not as clear. Within season variation in host plant suitability affectedB. thuringiensis efficacy and the interaction betweenB. thuringiensis and host plant cultivar forS. exigua but not forT. ni. Soluble protein and Folin-Denis phenolic concentrations of host plant tissue were not correlated with changes in host plant suitability to either insect species.     

Use of personal medical records for research purposes.

The established practice of doctors using medical records for research purposes is threatened by the recent proposed guidelines from the Department of Health, the BMA, and the European Commission. The European Commission has proposed that explicit consent should be obtained from each patient before his or her medical records can be used; the proposals from the Department of Health and the BMA would require all research that needs access to personal medical records to be submitted to an ethics committee. We believe that these proposals would seriously impair an entire category of research and suggest therefore that another set of guidelines, proposed by a Royal College of Physicians'' working group, should be used to modify the proposals. The guidelines of the working group encourage the use of medical records for research and ensure that such use can be made in a confidential manner without causing harm.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Aminoimidazole carboxamide ribonucleoside toxicity: a model for study of pyrimidine starvation

Aminoimidazole carboxamide ribonucleoside (AIC-R), a purine precursor, has biphasic effects on the growth of Chinese hamster fibroblasts. At 200 microM AIC-R cell growth is almost completely arrested, while at 50 and 700 microM AIC-R cell growth is comparable to that observed in the absence of nucleoside. The growth inhibition produced by AIC-R is the consequence of inhibition of the orotate phosphoribosyltransferase-orotidylic decarboxylase (OPRT-ODC) reactions, as evidenced by a 87% reduction in the intracellular concentrations of UTP and CTP, accumulation of orotate in the medium, and restoration of normal growth by inclusion of 100 microM uridine in the medium. Inhibition of pyrimidine nucleotide synthesis at 200 microM AIC-R is associated with an 82% reduction in the intracellular concentration of PP-ribose-P and a 150% increase in the concentration of purine nucleotides. Restoration of cell growth to a normal rate at 700 microM AIC-R--a condition under which PP-ribose-P remains depressed and purine nucleotide concentrations are also depressed (40% of control)--and absence of toxicity at 50 microM AIC-R--a condition under which purine nucleotide concentrations are increased by 150% and PP-ribose-P concentration is normal--suggest that the inhibition of OPRT-ODC observed at 200 microM AIC-R is caused by the combination of the reduction in PP-ribose-P and increase in purine nucleotides. These studies provide a better understanding of the control of the OPRT-ODC reactions in the cell and provide additional insight into the basis of pyrimidine starvation induced by purine nucleosides.     

Characteristics affecting fibrinolytic activity and plasma fibrinogen concentrations.

As part of a study to determine the extent to which the haemostatic system is implicated in the onset of clinically manifest ischaemic heart disease, characteristics influencing fibrinolytic activity (FA) and plasma fibrinogen concentrations were examined in 1601 men aged 18-64 and 707 women aged 18-59 in several occupational groups in North-west London. In men FA noticeably decreased till the age of about 58, when there was a small rise. In women a small increase in FA between 18 and about 40 was followed by a slightly larger fall between 40 and 59. There was a pronounced negative association of FA with obesity. FA was significantly less in smokers than non-smokers, though the effect was not large. FA increased with alcohol consumption. FA in men appeared to be greatest in the lower social classes, and men on night shift had poorer FA than those on day work. FA was greater in women using oral contraceptives than in those not using these preparations. In both sexes FA increased with exercise, but there were no associations between any of the characteristics studied and the increase. Plasma fibrinogen concentrations increase with age and obesity, are higher in smokers than non-smokers, and fall with alcohol consumption. In women the concentrations are higher in those using oral contraceptives. The general epidemiology of FA and plasma fibrinogen concentrations suggests that they may well be implicated in the pathogenesis of ischaemic heart disease.