RESUMEN
Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)-Golgi secretory pathway. FGF1 is released through a Cu(2+)-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu(2+)-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu(2+) to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu(2+) and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu(2+) in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb(3+) show that there are two Cu(2+)-binding pockets on the C2B domain, and one of these is also a Ca(2+)-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu(2+). The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.
RESUMEN
OBJECTIVE: This paper provides the first systematic examination of the content and clinical utility of psychiatric advance directives, which are documents that specify treatment preferences in advance of periods of compromised decision making. METHODS: Directives were completed by 106 community mental health center outpatients with at least two psychiatric hospitalizations or emergency department visits within two years. Participants used AD-Maker software in groups of up to six people led by peer trainers. Clinical utility was defined as the degree to which instructions are clinically feasible, useful, and consistent with standards of care. RESULTS: Fifty-five percent of participants were female, and 24 percent were nonwhite. Their mean+/-SD age was 42+/-9.1 years. Primary diagnoses included schizophrenia spectrum disorders (44 percent), bipolar disorders (27 percent), major depression (22 percent), and other disorders (7 percent). Eighty-one percent of participants listed preferred medications, most often antidepressants and second-generation antipsychotics, and 64 percent listed medications they would refuse, most commonly first-generation antipsychotics. Sixty-eight percent preferred hospital alternatives over hospitalization, 89 percent specified methods of de-escalating crises, and 72 percent indicated that they would refuse electroconvulsive therapy (ECT). Forty-six percent appointed a surrogate decision maker. Fifty-seven percent desired a directive that is irrevocable during periods of incapacity. Instructions were rated as feasible, useful, and consistent with practice standards for at least 95 percent of the advance directives, with the exception of instructions about the willingness to use medications not specifically listed in the directive. CONCLUSIONS: Results suggested that psychiatric advance directives provide a wealth of treatment preference information that is almost uniformly considered clinically useful. Although the utility of advance directives may vary depending on the circumstances of specific crisis episodes, the information provided can expedite and strengthen clinical care.