Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) characterized by the severe deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity (< 10%). Rapid ADAMTS13 testing is crucial for an early diagnosis and optimal management of acute TTP. We evaluated the performance of the HemosIL AcuStar ADAMTS13 activity assay (Instrumentation Laboratory, Bedford, Massachusetts, United States), a fully automated chemiluminescent immunoassay with an analytical time of 33 minutes. A method comparison study was performed on 176 samples from 49 healthy donors and 127 TMA patients (109 TTP, 7 atypical hemolytic uremic syndrome, 11 other TMAs), comparing this new assay with an in-house FRETS-VWF73 assay and a commercial enzyme-linked immunosorbent assay (ELISA) (TECHNOZYM ADAMTS-13 Activity, Technoclone GmbH, Vienna, Austria). Agreement between methods was assessed with focus on ADAMTS13 activity less than 10%, the medical decision level relevant for TTP diagnosis. The HemosIL AcuStar ADAMTS13 Activity showed good correlation with both the FRETS-VWF73 (r = 0.96) and ELISA (r = 0.96) methods. Slope of the Passing–Bablok regression was 1.05 for FRETS-VWF73 and 1.02 for ELISA, and absolute bias at the medical decision level was +0.1 and +0.3%, respectively. The study also revealed high agreement with FRETS-VWF73 (kappa 0.97) and ELISA (kappa 0.98) methods in classifying TTP patients with a severe deficiency of ADAMTS13 activity. Because of its short turnaround time and full automation, the HemosIL AcuStar ADAMTS13 activity assay might become the assay of choice to rapidly test ADAMTS13 activity in plasma and thus establish the diagnosis of acute TTP in emergency settings.

Epidermal growth factor (EGF) interferes with beta-adrenergic receptor (beta-AR) signaling in adipocytes and hepatocytes, which leads to decreased lipolytic and glycogenolytic responses, respectively. We studied the effect of EGF on the heart. EGF interfered with the cAMP signal generated by beta-AR agonists in cardiac myocytes. In perfused hearts, EGF decreased inotropic and chronotropic responses to epinephrine but not to 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Sustained epinephrine infusion induced heart contracture, which resulted in altered heart function as demonstrated by decreased inotropy and increased heart rate variability. EGF prevented all these alterations. In the whole animal (anesthetized mice), EGF administration reduced the rise in heart rate induced by a single epinephrine dose and the occurrence of Bezold-Jarisch reflex episodes induced by repeated doses. Sialoadenectomy enhanced the response to epinephrine, and EGF administration restored normal response. All these results suggest that, by interfering with beta-AR signaling, EGF protects the heart against the harmful effects of epinephrine.

Abstract-The tumor suppressor protein p53 plays an important role in the cell-cycle G 1 and G 2 checkpoints. In response to DNA damage, p53 can induce the transcription of p21, which inhibits the activation of various G 1 cyclin/cyclindependent kinase complexes. It is not known whether p53 plays a role in the initial migration of vascular smooth muscle cells from the arterial tunica media (mVSMCs). In this study, we have investigated whether mVSMC migration from healthy tunica media of young pigs and proliferation are regulated by p53. After 6 hours of incubation in mitogen-rich medium, explanted porcine tunica media tissue showed complete downregulation of p53 protein and p53 mRNA. The blockage of gene activity was not due to DNA methylation at the 5Ј control region of the gene. The mVSMC outgrowth did not show p53 expression. Mitogen-depletion of cultured p53 Ϫ /mVSMCs did not restore p53 expression. Incubation of explanted porcine tunica media tissue in mitogen-deprived medium increased p53 protein content and blocked mVSMC outgrowth from the explant. As in p53-deficient rodent cells, mVSMCs incubated with colcemid overrode the spindle-dependent checkpoint, giving polyploidy and chromosomal pairing. UV-induced DNA damage in mVSMCs incubated with mitogen-free medium induced p53 expression and apoptotic cell death showing DNA nucleosomal laddering. However, UV-irradiated mVSMCs incubated in mitogen-rich medium did not express p53 and did not show cell death. In conclusion, our results demonstrate that early mVSMC migration from the tunica media requires mitogen-induced suppression of p53 that is highly expressed in contractile mVSMCs residing in the healthy vessel wall.

We describe a cell-free chromatin assembly system derived from the yeast Saccharomyces cerevisiae, which efficiently packages DNA into minichromosomes in a reaction dependent on exogenous core histones and an ATP-regenerating system. Both supercoiled and relaxed plasmid DNA serve as templates for nucleosomal loading in a gradual process that takes at least 6 h for completion at 30 degrees C. Micrococcal nuclease digestion of the assembled minichromosomes displays an extended nucleosomal ladder with a repeat length of 165 bp. The purified minichromosomes contain the four core histones in stoichiometric proportion and exhibit phased nucleosomes over the mouse mammary tumour virus (MMTV) promoter. The progesterone receptor and NF1 synergize on these minichromosomes resulting in efficient cell-free transcription. The ease of manipulation and the potential use of yeast strains carrying mutations in the chromatin handling machinery make this system suitable for detailed mechanistic studies.