Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F1 alpha formation was significantly increased 3-fold. Since this increase was greater (P less than 0.01) than that seen with either 7-(1-imidazolyl)heptanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F1 alpha was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F1 alpha synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.     

Mortality among male anaesthetists in the United Kingdom, 1957-83.

A cohort of 3769 male anaesthetists resident in the United Kingdom between 1957 and 1983 was followed up for a total of 51,431 person years of observation. All subjects were fellows of the Faculty of Anaesthetists and held full registration with the General Medical Council. With all men in social class I being taken as the standard, the standardised mortality ratio among anaesthetists for all causes of death was 68 (95% confidence interval 59 to 77) and the standardised mortality ratio for all cancers was 50 (95% confidence interval 36 to 67). There was no significant excess mortality from lymphomas or leukaemias, but 16 of the 221 deaths in anaesthetists were due to suicide, giving a standardised mortality ratio of 202 (95% confidence interval 115 to 328). When anaesthetists were compared with all doctors the standardised mortality ratio for suicide was only 114, a nonsignificant excess. These findings confirm that the risk of suicide among anaesthetists is twice as high as among other men in social class I but suggest that the risk does not differ significantly from that among doctors as a whole. There was no evidence of a significant excess risk of cancer, and, in particular, the small excess of cancer of the pancreas reported previously could not be confirmed.     

Water relations of the parasite: host relationship between the mistletoe Amyema linophyllum (Fenzl) Tieghem and Casuarina obesa Miq

Summary Seasonal and diurnal gas exchange and water relations of Amyema linophyllum and its host Casuarina obesa were studied at Gingin Western Australia. As recorded elsewhere for other species of mistletoe, stomatal conductances and transpiration rates were consistently higher in parasite than host, but assimilation rates did not differ significantly between partners, and water use efficiency was accordingly substantially lower in the parasite. Parallel responses of the species to environmental conditions suggested closely coordinated stomatal behaviour. However, sunlit and artifically shaded clumps of Amyema maintained high leaf conductances even when foliage fell below turgor loss point, yet their tissue capacitance values indicated maintenance of greater tissue water reserves during stress than in the host. Pressure-volume relationships indicated that differences in tissue water relations were unlikely to contribute significantly to the observed gradient in leaf water potential between partners. An experiment measuring changes in water potential of freshly detached host: parasite systems cut with the host shoot end immersed in water indicated that the haustorial junction was the principal site of resistance to transpiration-driven water flow into the parasite. A parallel experiment on intact attached shoots with mistletoe clumps enclosed and darkened just before dawn, demonstrated that, once the host commenced rapid transpiration, the water potential gradient between partners became reversed.     

Phospholamban is a good substrate for cyclic GMP-dependent protein kinase in vitro, but not in intact cardiac or smooth muscle.

Protein residualizing labels facilitate localization of tissue sites of protein catabolism and the quantification of protein accumulation because of their prolonged intracellular retention of protein accumulation because of their prolonged intracellular retention times. Radioiodinated residualizing labels have been used to define the metabolism of a wide variety of proteins, but this has necessitated destructive analysis. Here we describe the implementation and validation of a novel 19F-containing residualizing label for protein, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), that permits the non-invasive assessment of protein accumulation and catabolism by n.m.r. spectroscopy in vivo. DLBA comprises a reporter molecule containing six equivalent 19F atoms. 19F is strongly n.m.r.-active, has 100% natural abundance, and is present in minimal background concentrations in soft tissues. We validated the use of DLBA as a protein-labelling compound by coupling to asialofetuin (ASF), a protein that is recognized exclusively by hepatic tissue via a saturable receptor-mediated process. Coupling of DLBA to ASF by reductive amination had no effect on the physiological receptor-mediated uptake of the protein in rat liver in vivo. The 19F-n.m.r. spectrum of DLBA exhibited a single peak that was subject to a small chemical-shift change and broadening after coupling to ASF. Pronase digestion of DLBA-ASF was performed to simulate intracellular degradation products, and resulted in a narrower set of resonances, with chemical shifts intermediate between those of uncoupled DLBA and DLBA-ASF. Intravenous administration of DLBA-ASF to rats followed by quantification of 19F in homogenates of liver tissue indicated that the half-life of residence time of degradation products from DLBA-ASF in liver was approx. 2 days. This intracellular half-life was comparable with that described for similar residualizing labels that contain radioiodide as a reporter. Similar results for the half-life of retention were obtained non-destructively and non-invasively in situ with the use of a whole-body radio-frequency antenna to acquire sequential spectra over 80 h after intravenous administration of DLBA-ASF. Quantification of these spectra demonstrated an initial accumulation of DLBA-ASF in liver followed by an expected gradual loss of 19F-labelled degradation products. The approach developed offers promise for the sequential and longitudinal characterization of metabolism of specific proteins in individual experimental animals and ultimately in human subjects.     

Some pharmacological activities of novel adenine-related compounds isolated from a marine sponge Agelas mauritiana

Agelasimine A and agelasimine B, two novel compounds related to adenine, have been isolated from the orange sponge, Agelas mauritiana, and have been tested for a variety of biological activities. Both compounds inhibited proliferation of cultured L1210 leukemia cells at nanomolar concentrations with accumulation in the G1 stage of the cell cycle. However, no prolongation of life was observed in mice bearing P388 leukemia treated with these compounds. In the rat isolated aorta, micromolar concentrations of agelasimines were very effective in inhibiting contractions elicited by potassium chloride but had little or no effect on responses for prostaglandin F2 alpha and had modest effects on the responses to noradrenaline and significant effects on 5-hydroxytryptamine. Agelsamines A and B appeared to be equipotent in causing relaxation in rabbit jejunum and bovine coronary artery, and they also inhibited nucleoside transport into rabbit erythrocytes in micromolar concentrations.     

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.     

Lysophosphatidylcholine as an intermediate in phosphatidylcholine metabolism and glycerophosphocholine synthesis in cultured cells: an evaluation of the roles of 1-acyl- and 2-acyl-lysophosphatidylcholine

Previous studies in our laboratory have shown that the principal pathway of phosphatidylcholine (PtdCho) degradation in cultured mouse N1E-115 neuroblastoma, C6 rat glioma, primary rat brain glia and human fibroblasts is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----glycerophosphate plus choline (Morash, S.C. et al. (1988) Biochim. Biophys. Acta 961, 194-202). GroPCho is the first quantitatively major degradation product in this pathway, and could be formed by phospholipases A1 or A2, followed by lysophospholipase, or by a co-ordinated attack releasing both fatty acids by phospholipase B. The quality and quantities of lysoPtdCho present in cells reflect the nature of the initial hydrolysis step (A1 or A2), specificities of the lysophospholipases, and activities of acyltransferases that form PtdCho from lysoPtdCho. The present study was undertaken to elucidate the relative importance of these pathways by examining the fate of exogenous 1-acyl and 2-acyl-lysoPtdCho incubated with N1E-115 and C6 cells in culture. By fatty acid composition, endogenous lysoPtdCho was found to be mainly 1-acyl in both cell types based on a predominance of saturated acyl species; this suggested either preferential further deacylation or reacylation of 2-acyl-lysoPtdCho, or that 2-acyl-lysoPtdCho was not formed. Exogenous 1- and 2-acyl-lysoPtdCho specifically radiolabelled with choline and/or fatty acid were incubated either singly or as equimolar mixtures with cells. Cell association was rapid and not reversible by washing and both species were taken up at similar rates. The 2-acyl species was acylated to PtdCho faster than the 1-acyl species in both cell lines. Acylation of both lyso species was higher in C6 compared to N1E-115 cells. Hydrolysis of lysoPtdCho to GroPCho was higher in N1E-115 cells and with 1-acyl-lysoPtdCho. Transacylation between two molecules of lysoPtdCho was a minor pathway. These results document the variety and relative importance of reactions of lysoPtdCho metabolism; under similar conditions, 1- and 2-acyl-lysoPtdCho are handled differently. Both species turn over actively, but only the 1-acyl species accumulates while 2-acyl-lysoPtdCho is likely to be reacylated to form PtdCho.