Long-term asymmetrical acceleration of protein evolution after gene duplication.

Rapid divergence of gene copies after duplication is thought to determine the fate of the copies and evolution of novel protein functions. However, data on how long the gene copies continue to experience an elevated rate of evolution remain scarce. Standard theory of gene duplications based on some level of genetic redundancy of gene copies predicts that the period of accelerated evolution must end relatively quickly. Using a maximum-likelihood approach we estimate preduplication, initial postduplication, and recent postduplication rates of evolution that occurred in the mammalian lineage. We find that both gene copies experience a similar in magnitude acceleration in their rate of evolution. The copy located in the original genomic position typically returns to the preduplication rates of evolution in a short period of time. The burst of faster evolution of the copy that is located in a new genomic position typically lasts longer. Furthermore, the fast-evolving copies on average continue to evolve faster than the preduplication rates far longer than predicted by standard theory of gene duplications. We hypothesize that the prolonged elevated rates of evolution are determined by functional properties that were acquired during, or soon after, the gene duplication event.

Plasma stimulation of prostacyclin production by rat smooth muscle cells requires previous induction of phospholipase activity.

Human normal platelet poor plasma (PPPn) stimulates prostacyclin (PGI2) production in a dose-dependent manner and after 15 to 60 min of incubation time when confluent rat smooth muscle cells (RSMC) were preincubated for 24 hours with fresh culture medium. This PGI2 production was independent of new protein synthesis, and was not observed in the cells maintained only in exhausted medium. The serum of fresh culture medium also induced a significant and transient increase of prostaglandin endoperoxide synthase (PGHS) activity as a function of preincubation time, which was dependent of protein synthesis. However, neither PGHS activity nor arachidonic acid availability limited the PPPn induced PGI2 synthesis in RSMC. Moreover, the previous addition of phorbol 13-myristate acetate also allowed the PPPn to induce PGI2 synthesis, that was significantly inhibited by a specific phospholipase A2 inhibitor. Furthermore, we found that PPPn induced a significant increase of intracellular calcium, and also stimulated PGI2 production at short incubation times due to its effect on phospholipases, and not by a direct supply of substrate. We conclude that a previous activation of phospholipase A2 was necessary to observe a significant and sustained PGI2 synthesis induced by PPPn in RSMC, and that the increase of intracellular calcium observed with PPPn might stimulate these previously activated phospholipases.

Effects of linoleic and oleic acid anilides on prostacyclin synthesis and fibrinolytic profile of human endothelial cells in culture: relevance to the toxic oil syndrome.

We evaluated the effects of fatty-acid anilides (FAA) on prostacyclin (PGI2) synthesis and on the fibrinolytic properties of human umbilical vein endothelial cells. Preincubation of endothelial cells with oleic- and linoleic-anilides (OAA and LAA, respectively) resulted in a time- and concentration-dependent inhibition of ionophore A23187- and thrombin-induced PGI2 synthesis. However, no significant effects of FAA on arachidonic acid-induced PGI2 synthesis were found, except with 1000 microM LAA which inhibited cyclooxygenase activity after 24 h. In general terms, OAA showed similar inhibitory effects on PGI2 production as did LAA, but with a shifted time course, since the production of PGI2 at 24 h for OAA was similar to that observed for LAA at 2 h. The release of labeled arachidonic acid from cell membranes was significantly reduced (75-85%), after 24 h, with both FAA. The effect of 100 microM LAA on thrombin-induced PGI2 production was rapid (within 15 min) and irreversible after 60 min. The recovery of PGI2 synthesis after LAA treatment was blocked by cycloheximide, suggesting a decrease of phospholipase(s) activity or cessation of enzyme synthesis. Moreover, this reduced PGI2 synthesis was not associated with [3H]adenine release. Our data indicate that FAA induce a significant impairment of stimulated PGI2 synthesis and arachidonic acid release in endothelial cells, acting primarily as inhibitors of phospholipase(s) rather than of cyclooxygenase. Finally, both LAA and OAA induce an anti-fibrinolytic activity in these cells where major changes are observed in the plasminogen activator inhibitor and the urine-type plasminogen activator.

Influence of fatty acid anilides present in toxic oils on the metabolism of exogenous arachidonic acid in cultured human endothelial cells.

The effect of fatty acid anilides (FAA) on the exogenous arachidonic acid (AA) metabolism and toxicity of isolated human endothelial cells was studied to clarify their possible role in the etiology of toxic oil syndrome. Confluent cells were incubated with and without linoleic acid anilide (LAA), oleic acid anilide (OAA) and two unrelated samples for 2-24 h prior to the addition of [l-14C]AA alone or with calcium ionophore A-23187. The eicosanoids produced were analyzed by RP-HPLC. A dual stimulatory and inhibitory effect on the conversion of exogenous AA as a function of preincubation time with anilides (100 and 1000 microM) was observed. Treated cells significantly increased (1-3-fold) the production of the main cyclooxygenase-derived prostanoids (6-keto-PGF1 alpha and PGF2 alpha) formed by these cells, with a maximum stimulatory effect after 2-3 h, only when AA was used alone. However, afterwards a time- and dose-dependent decrease in prostanoid formation was observed with LAA (P < 0.05 at 24 h), either in the absence or presence of ionophore A-23187 in the incubation mixture. This inhibitory effect on cyclooxygenase was not observed with OAA, which still stimulate after 24 h of treatment. The changes in prostanoid synthesis were not followed with a parallel release in the lactate dehydrogenase activity in the medium (except with unrelated samples). Moreover, anilide treatment increased the appearance of cytosolic lipid droplets or vacuoles after 2 and 5 h of contact with LAA and OAA, respectively. From these results, it was suggested that anilides impair prostanoid synthesis in endothelial cells; their stimulatory effect could be explained by an unspecific effect on cell membrane, not related to cell toxicity and the inhibitory effect by an inhibition of the cyclooxygenase activity. These observations further contribute to our understanding of the possible role of anilides in the etiology of the toxic oil syndrome.

[Importance of antiphosphatidylserine antibodies in patients with lupus anticoagulant. Analysis of 30 cases]. / Importancia de los anticuerpos antifosfatidilserina en pacientes con anticoagulante lúpico. Análisis de 30 casos.

Anti-phosphatidylserine (APS) and anticardiolipin (ACL) antibodies were measured in 30 patients with lupus anticoagulant. In 17 cases there were clinical features of thrombosis (56.7%). The number of patients with IgG APS and ACL levels higher than seven standard deviations from the normal mean was significantly higher in the group with thrombosis (p less than 0.017 and p less than 0.02, respectively). The IgG APS level was higher in the group without systemic lupus erythematosus (p less than 0.029). There was a positive correlation between IgG APS and the activated thromboplastin time (r = 0.654, p less than 0.001). The correlation between IgG APS and IgG ACL was rho = 0.61. Two of the 5 patients with increased IgG APS and normal IgG ACL had thrombosis. We think that, in this group of patients, IgG APS titer may be, like IgG ACL, a biological marker of thrombosis risk.

Serial measurements of a plasma prostacyclin inhibitor in a patient with recurrent abortions and lupus anticoagulant; a case report.

Defective plasmatic stimulation of prostacyclin (PGI2) production by vascular cells has been described in patients with lupus anticoagulant (LAC). A young woman with recurrent abortions, LAC and evidence for deficient PGI2 production was studied. Serial measurements of a plasma PGI2 inhibitor, LAC and anticardiolipin antibodies (ACA) have been performed before and throughout her fourth pregnancy. Antenatal care and treatment with prednisone and heparin started at 10 weeks gestation. The plasma of our patient continued to inhibit PGI2 production by vascular cells despite treatment. The presence of inhibitor(s) of PGI2 release was confirmed by mixing the patient's plasma with normal plasma. In addition, an IgM lupus anticoagulant fraction (but not the IgG fraction) interfered with the release of arachidonic acid in human endothelial cells induced by thrombin. Despite prednisone and heparin treatment we did not find a complete correction of the LAC activity and the ACA (IgM type) still remained positive before the detection of a fetal death at 26 weeks. The placenta showed abundant infarcts and areas of ischaemic necrosis. We suggest that the defect in vascular PGI2 release could compromise fetal outcome.