In our study, administration of a small amount of fluid during the initial 24 h was not associated with a poor outcome. The need for a great amount of fluid during the initial 24 h was associated with a poor outcome; therefore, this group of patients must be carefully monitored.
SummaryWe have studied how the F-actin cytoskeleton is involved in establishing the heterogeneous intracellular Ca 2+ levels ([Ca 2+ ] i ) and in the organization of the exocytotic machinery in cultured bovine chromaffin cells. Simultaneous confocal visualization of [Ca 2+ ] i and transmitted light studies of the cytoskeleton showed that, following cell stimulation, the maximal signal from the Ca 2+ -sensitive fluorescent dye Fluo-3 was in the empty cytosolic spaces left by cytoskeletal cages. This was mostly due to the accumulation of the dye in spaces devoid of cytoskeletal components, as shown by the use of alternative Ca 2+ -insensitive fluorescent cytosolic markers. In addition to affecting the distribution of such compounds in the cytosol, the cytoskeleton influenced the location of L-and P-Q-type Ca 2+ channel clusters, which were associated with the borders of cytoskeletal cages in resting and stimulated cells. Indeed, syntaxin-1 and synaptotagmin-1, which are components of the secretory machinery, were present in the same location. Furthermore, granule exocytosis took place at these sites, indicating that the organization of the F-actin cytoskeletal cortex shapes the preferential sites for secretion by associating the secretory machinery with preferential sites for Ca 2+ entry. The influence of this cortical organization on the propagation of [Ca 2+ ] i can be modelled, illustrating how it serves to define rapid exocytosis.
BackgroundSoluble fragments of the amyloid precursor protein (APP) generated by α- and β-secretases, sAPPα and sAPPβ, have been postulated as promising new cerebrospinal fluid (CSF) biomarkers for the clinical diagnosis of Alzheimer’s disease (AD). However, the capacity of these soluble proteins to assemble has not been explored and could be relevant. Our aim is to characterize possible sAPP oligomers that could contribute to the quantification of sAPPα and sAPPβ in CSF by ELISA, as well as to characterize the possible presence of soluble full-length APP (sAPPf).ResultsWe employed co-immunoprecipitation, native polyacrylamide gel electrophoresis and ultracentrifugation in sucrose density gradients to characterize sAPP oligomers in CSF. We have characterized the presence of sAPPf in CSF from NDC and AD subjects and demonstrated that all forms, including sAPPα and sAPPβ, are capable of assembling into heteromers, which differ from brain APP membrane-dimers. We measured sAPPf, sAPPα and sAPPβ by ELISA in CSF samples from AD (n = 13) and non-disease subjects (NDC, n = 13) before and after immunoprecipitation with antibodies against the C-terminal APP or against sAPPβ. We demonstrated that these sAPP heteromers participate in the quantification of sAPPα and sAPPβ by ELISA. Immunoprecipitation with a C-terminal antibody to remove sAPPf reduced by ~30% the determinations of sAPPα and sAPPβ by ELISA, whereas immunoprecipitation with an APPβ antibody reduced by ~80% the determination of sAPPf and sAPPα.ConclusionsThe presence of sAPPf and sAPP heteromers should be taken into consideration when exploring the levels of sAPPα and sAPPβ as potential CSF biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/1750-1326-10-2) contains supplementary material, which is available to authorized users.
This study assesses whether C-terminal fragments (CTF) of the amyloid precursor protein (APP) are present in cerebrospinal fluid (CSF) and their potential as biomarkers for Alzheimer’s disease (AD). Immunoprecipitation and simultaneous assay by Western blotting using multiplex fluorescence imaging with specific antibodies against particular domains served to characterize CTFs of APP in human CSF. We demonstrate that APP-CTFs are detectable in human CSF, being the most abundant a 25-kDa fragment, probably resulting from proteolytic processing by η-secretase. The level of the 25-kDa APP-CTF was evaluated in three independent CSF sample sets of patients and controls. The CSF level of this 25-kDa CTF is higher in subjects with autosomal dominant AD linked to PSEN1 mutations, in demented Down syndrome individuals and in sporadic AD subjects compared to age-matched controls. Our data suggest that APP-CTF could be a potential diagnostic biomarker for AD.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.