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1.
Thromb Haemost ; 119(11): 1767-1772, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31587247

RESUMO

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.


Assuntos
Proteína ADAMTS13/sangue , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Imunoensaio/métodos , Púrpura Trombocitopênica Trombótica/diagnóstico , Proteína ADAMTS13/deficiência , Síndrome Hemolítico-Urêmica Atípica/sangue , Síndrome Hemolítico-Urêmica Atípica/enzimologia , Automação Laboratorial , Biomarcadores/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Medições Luminescentes , Valor Preditivo dos Testes , Púrpura Trombocitopênica Trombótica/sangue , Púrpura Trombocitopênica Trombótica/enzimologia , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de Trabalho
2.
Nucleic Acids Res ; 32(13): e111, 2004 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-15282330

RESUMO

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.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Animais , Extratos Celulares , Cromatina/química , Cromatina/metabolismo , Cromossomos/química , Humanos , Camundongos , Nucleossomos/química , Nucleossomos/metabolismo , Plasmídeos , Ratos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo
3.
Am J Physiol Heart Circ Physiol ; 283(5): H1887-95, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12384466

RESUMO

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.


Assuntos
Agonistas Adrenérgicos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Epinefrina/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , AMP Cíclico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Miócitos Cardíacos/efeitos dos fármacos , Perfusão , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/efeitos dos fármacos
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