Impact of CSF storage volume on the analysis of Alzheimer's disease biomarkers on an automated platform.

OBJECTIVES: To assess the potential influence of the ratio between the storage tube surface area and the volume of cerebrospinal fluid (CSF) (surface/volume) on the quantifications of four Alzheimer's disease (AD) biomarkers on the Lumipulse G600II automated platform. METHODS: CSF samples of 10 consecutive patients were stored in 2 ml polypropylene tubes containing four different CSF volumes: 1.5 ml, 1 ml, 0.5 ml and 0.25 ml. Concentration of CSF Aß1-42, Aß1-40, t-Tau and p-Tau were measured in all aliquots using the LUMIPULSE G600II automated platform from Fujirebio. RESULTS: Levels of CSF Aß1-42 and Aß1-40 were lower in samples stored with lower volumes (higher surface/volume ratios). This decrease was partly compensated by using the ratio Aß1-42/Aß1-40. Quantification of t-Tau and p-Tau were not influenced by this pre-analytical condition. CONCLUSION: The surface/volume ratio can potentially influence the results of amyloid AD biomarkers. It appears essential to take into account the surface/volume ratio of the storage tubes when quantifying CSF biomarkers in clinical routine.

SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross-talk and sheds light on regulation by effector molecules.

Surface plasmon resonance monitoring of the binding of transcription factors cAMP receptor protein (CRP) and nitrogen control factor of cyanobacteria (NtcA) from Synechocystis sp. PCC6803 to promoter fragments of glnA, glnN (NtcA regulon) and cccS (CRP regulon), revealed exclusive CRP binding to cccS, whereas NtcA was bound to all three promoters with different affinities, which were strongly increased by the NtcA activator 2-oxoglutarate. Effective NtcA affinity for 2-oxoglutarate varied with the promoter. High-affinity promoters and the NtcA-coactivating protein PII-interacting protein X (PipX) increased NtcA affinity towards 2-oxoglutarate, suggesting PipX-stabilization of the 2-oxoglutarate-bound NtcA conformation. PipX binding to NtcA required 2-oxoglutarate and was much tighter (Kd≈85 nM) than to the PipX-sequestering PII protein. NtcA appears to require more strongly PipX and 2-oxoglutarate (2OG) for estimulating gene expression at promoters having "imperfect" NtcA binding sites.

Histone deacetylase inhibitors stimulate mitochondrial HMG-CoA synthase gene expression via a promoter proximal Sp1 site.

The expression of mitochondrial HMG-CoA synthase in the colon has been correlated with the levels of butyrate present in this tissue. We report here that the effect of butyrate on mitochondrial HMG-CoA synthase gene expression is exerted in vivo at the transcriptional level, and that trichostatin A (TSA), a specific histone deacetylase inhibitor, also induces transcriptional activity and mRNA expression of the gene in human cell lines derived from colon carcinoma. Using chromatin immunoprecipitation assays, we show that histone deacetylase 1 (HDAC1) is associated with the endogenous mitochondrial HMG-CoA synthase promoter and that TSA induction correlates with hyperacetylation of H4 histone associated with the 5' flanking region of the gene. Overexpression of HDAC1 activity leads consistently to mitochondrial HMG-CoA synthase promoter hypoacetylation and reduces its transcriptional activity. The effect of butyrate and TSA maps to a single Sp1 site present in the proximal promoter of the gene, which is able to bind Sp1 and Sp3 proteins. Interestingly, the binding affinity of Sp1 and Sp3 proteins to the Sp1 site correlates with the TSA responsiveness of the promoter. Using a one-hybrid system (GAL4-Sp1 and GAL4-Sp3), we show that both proteins can mediate responsiveness to TSA in CaCo-2 cells employing distinct mechanisms.

Down-regulation of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene by insulin: the role of the forkhead transcription factor FKHRL1.

Normal physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation. This allows the use of ketone bodies for energy, thereby preserving the limited glucose for use by the brain. This adaptative response is switched off by insulin rapidly inhibiting the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS2) gene, which is a key control site of ketogenesis. We decided to investigate the molecular mechanism of this inhibition. In the present study, we show that FKHRL1, a member of the forkhead in rhabdosarcoma (FKHR) subclass of the Fox family of transcription factors, stimulates transcription from transfected 3-hydroxy-3-methylglutaryl-CoA synthase promoter-luciferase reporter constructs, and that this stimulation is repressed by insulin. An FKHRL1-responsive sequence AAAAATA, located 211 bp upstream of the HMGCS2 gene transcription start site, was identified by deletion analysis. It binds FKHRL1 in vivo and in vitro and confers FKHRL1 responsiveness on homologous and heterologous promoters. If it is mutated, it partially blocks the effect of insulin in HepG2 cells, both in the absence and presence of overexpressed FKHRL1. These results suggest that FKHRL1 contributes to the regulation of HMGCS2 gene expression by insulin.