Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets

U73122 ({1-[6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)exyl]-1H-pyrrole-2,5-dione}) is generally used as a selective inhibitor of phospholipase C (PLC) and the related rise in cytosolic Ca2+. Recently, by using hepatocytes, it was suggested that its action sites are different for PLC activation and increase in Ca2+ concentration. To verify whether U73122 has different sites for inhibiting PLC activation and calcium responses in human platelets, aggregation, Mn2+ influx, cytosolic Ca2+ increase and PLC activation were studied in response to thrombin and the synthetic agonist of the thromboxane receptor U46619 (9,11-dideoxy-9 alpha,11 alpha-methanoepoxyprostaglandin F-2 alpha). With both agonists, U73122 inhibited aggregation, Mn2+ influx and the enhancement of cytosolic calcium at concentrations of 2 mu M or lower, while 10 mu M was necessary to inhibit PLC activation. Our results suggested that U73122 is much more active in antagonizing Ca2+ channels, both the intracellular ones, which are activated by formation of inositol 1,4,5 P-3 and those present on plasma membrane, than in reducing the activation of PLC. BIOCHEM PHARMACOL 56;11:1481-1484, 1998. (C) 1998 Elsevier Science Inc

Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression

BACKGROUND: In mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated. RESULTS: In the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance. CONCLUSIONS: Sequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter

Additional file 1: of Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression

Table S1.A lists the 45,713 loci identified using the ERE1 consensus sequence deposited at the RepBase database as query for a BLAST search against the horse genome reference sequence. Table S1B reports the 34,131 ERE1 loci with sizes similar to the ERE1 consensus (225 ± 10 bp) and with minimum identity to the consensus of 84 %. Table S1.C lists the 27,396 ERE1 loci that are not located inside other repetitive elements. (XLSX 3293 kb