Loss of estrogen receptor β decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice

Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females

Physical map of the genome of Vibrio cholerae 569B and localization of genetic markers.

A combined physical and genetic map of the genome of the classical O1 hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. The enzymes NotI, SfiI and CeuI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis. The digests produced 37, 22, and 7 fragments, respectively. The CeuI maps of the genomes of strains 569B and O395, constructed by partial restriction digestion, were identical, and the data are consistent with the concept of circular chromosomes. The genome size of each of the strains was estimated to be about 3.2 Mb. The NotI and SfiI digestion profiles of the genomic DNAs of strains 569B and O395 exhibited distinct restriction fragment length polymorphism. The linkages between the 37 NotI fragments of the genome of strain 569B were determined by combining three approaches: isolation of linking clones, analysis of partial digestion fragments, and identification of NotI fragments in isolated CeuI and SfiI fragments. To align linked fragments precisely, NotI-digested genomic DNA was end labeled and separated in the same gel with the NotI-digested DNA to be probed with linking clones. This also allowed the identification of smaller restriction fragments that are not visible in ethidium bromide-stained gels. The presence of repetitive DNA sequences in the V. cholerae 569B genome has been demonstrated. Twenty cloned homologous and heterologous genes and seven rrn operons have been positioned on the physical map. The two copies of the Ctx genetic element in the genome of strain 569B are located about 1,000 kb apart