The Scottish Structural Proteomics Facility: targets, methods and outputs.

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.

Specificity of elevated cerebrospinal fluid bilirubin in the investigation of subarachnoid haemorrhage.

BACKGROUND: The spectrophotometric examination of cerebrospinal fluid for bilirubin is an established investigation in patients with suspected subarachnoid haemorrhage. This study assesses the diagnostic specificity of an elevated cerebrospinal fluid bilirubin and how this may be influenced by the presence of oxyhaemoglobin and the concentration of cerebrospinal fluid total protein. METHODS: One thousand cerebrospinal fluid spectroscopy reports were reviewed. Electronic patient records were examined to determine the clinical outcome in patients with an elevated cerebrospinal fluid bilirubin. RESULTS: Forty-four out of 1000 cerebrospinal fluid scans showed an increase in cerebrospinal fluid bilirubin unrelated to elevated serum bilirubin concentrations. This was associated with subarachnoid haemorrhage in 16 (36%) cases. Subarachnoid haemorrhage was confirmed in 5/17 (29%) patients positive for cerebrospinal fluid bilirubin alone and in 11/27 (41%) patients positive for both cerebrospinal fluid bilirubin and oxyhaemoglobin. At cerebrospinal fluid total protein concentrations <1 g/L, the specificity for subarachnoid haemorrhage improved: 4/9 (44%) vs. 1/8 (13%) with an increase in cerebrospinal fluid bilirubin alone; 6/10 (60%) vs. 5/17 (29%) in patients with increases in both bilirubin and oxyhaemoglobin. CONCLUSION: While an increase in cerebrospinal fluid bilirubin may be considered consistent with subarachnoid haemorrhage, the likelihood that a subarachnoid haemorrhage has occurred is influenced by the presence of oxyhaemoglobin and the concentration of total protein in the cerebrospinal fluid.

The interaction between casein kinase Ialpha and 14-3-3 is phosphorylation dependent.

We have previously shown that casein kinase (CK) Ialpha from mammalian brain phosphorylates 14-3-3 zeta and tau isoforms on residue 233. In the present study, we show that CKIalpha associates with 14-3-3 both in vitro and in vivo. The interaction between CKIalpha and 14-3-3 is dependent on CKIalpha phosphorylation, unlike centaurin-alpha1 (also known as ADAP1), which binds to unphosphorylated CKIalpha on the same region. CKIalpha preferentially interacts with mammalian eta and gamma 14-3-3 isoforms, and peptides that bind to the 14-3-3 binding pocket prevent this interaction. The region containing Ser218 in this CKIalpha binding site was mutated and the interaction between CKIalpha and 14-3-3 was reduced. We subsequently identified a second phosphorylation-dependent 14-3-3 binding site within CKIalpha containing Ser242 that may be the principal site of interaction. We also show that both fission and budding yeast CKI kinase homologues phosphorylate mammalian and budding yeast (BMH1 and BMH2) 14-3-3 at the equivalent site.