Helen C. Baxter

Immunolocalisation of 14-3-3 isoforms in normal and scrapie-infected murine brain

The appearance of 14-3-3 proteins in the cerebrospinal fluid is characteristic of some neurodegenerative conditions which include sporadic Creutzfeldt-Jakob disease. Although 14-3-3 proteins are physiochemically well characterised and are known to be present in neuronal cells little is known of the neuroanatomical localisation of the individual isoforms. Using 14-3-3 isoform specific antibodies we have examined the distribution of the isoforms in normal murine brain and the changes observed during neurodegeneration as a result of ME7 scrapie infection. In normal brain there are two major patterns of immunolabelling. The beta, gamma, eta and zeta isoforms which exhibit a similar distribution pattern showing labelling of neuronal cell bodies often in particular anatomical nuclei. However the individual isoforms exhibit variation revealing subtle differences in location. The tau isoform was found only in the hippocampus and medulla, and the epsilon isoform was found throughout grey matter of the CNS. In the scrapie-infected murine brain, where severe pathological changes occur during the course of the disease, significant differences in the 14-3-3 isoform distribution were observed in the hippocampus and in the thalamus. Importantly, both the 14-3-3 eta isoform and prion protein were seen in the same neurones in both the cerebellar roof nuclei and in the lateral hypothalamic nuclei. Our study of 14-3-3 isoform distribution in adult murine brain clearly demonstrates a heterogeneous pattern of neurolocation for specific 14-3-3 isoforms. The fact that isoform labelling in terminal scrapie CNS is lost in some brain areas, but increases in others, suggests that the processing of these proteins during neurodegeneration may be much more complex than previously recognised.

Unchanged Survival Rates of 14-3-3γ Knockout Mice after Inoculation with Pathological Prion Protein

ABSTRACT The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) is based on typical clinical findings and is supported by a positive 14-3-3 Western blot of cerebrospinal fluid. However, it is not clear whether 14-3-3 indicates general neuronal damage or is of pathophysiological relevance in CJD. The fact that the 14-3-3 isoform spectrum in cerebrospinal fluid does not correspond to that found in the brain points to a regulated process. To investigate a possible role of 14-3-3 proteins in transmissible spongiform diseases, we generated a 14-3-3γ-deficient mutant mouse line by using a classical knockout strategy. The anatomy and cage behavior of the mutant mice were normal. Western blot analyses of brain homogenates revealed no changes in the protein expression of other 14-3-3 isoforms (ε, β, ζ, and η). Proteomic analyses of mouse brains by two-dimensional differential gel electrophoresis showed that several proteins, including growth hormone, 1-Cys peroxiredoxin, CCT-zeta, glucose-6-phosphate isomerase, GRP170 precursor, and α-SNAP, were differentially expressed. Mutant and wild-type mice were inoculated either intracerebrally or intraperitoneally with the Rocky Mountain Laboratory strain of scrapie, but no differences were detected in the postinoculation survival rates. These results indicate that 14-3-3γ is unlikely to play a causal role in CJD and related diseases.

Binding of 14-3-3 proteins to a single stranded oligodeoxynucleotide aptamer.

A synthetic library of ca. 10(13) single stranded oligodeoxynucleotides, each comprising a randomized 40mer sequence and homogeneous 10mer flanking regions, was screened for binding to recombinant human 14-3-3gamma. A single aptamer, which showed similar affinities (K(D) approximately 10(-8)M) for six isoforms of the protein, has been shown to bind to undenatured 14-3-3 protein in the cerebral spinal fluid of scrapie infected sheep.

The mechanism of Escherichia coli dethiobiotin synthetase—the closure of the ureido ring of dethiobiotin involves formation of a carbamic-phosphate mixed anhydride

The final intermediate in the enzymatic synthesis of the ureido ring of D-dethiobiotin 1 from (7R,8S)-7,8-diaminononanoate 2 catalysed by E. coli dethiobiotin synthetase is the phosphoric acid anydride 4 of the carbamate 3.

Mechanism of dethiobiotin synthetase—characterisation of the 8-aminocarbamate of (7R,8S)-7,8 diaminononanoate as an enzyme-bound intermediate

Enzymatically catalysed formation of the ureido ring of dethiobiotin 2 from (7R, 8S)-7,8-diaminononanoate 3 by purified E. coli dethiobiotin synthetase involves the formal hydrolysis of one equivalent of ATP, a combination of substrate loading and trapping experiments show that the carbamate 5 is an intermediate in the synthesis of dethiobiotin from 3.

Dental handpiece contamination: a proteomics and surface analysis approach

Dental handpieces (DHPs) become biofouled internally with patient derived material that is difficult to access for removal and inactivation. This study undertook a quantitative and qualitative investigation of protein contamination of internal components from three different types of DHP: the turbine, slow speed contra-angle and surgical. Eluates from the high speed turbine, low speed spray channels and surgical gear were assayed for protein using an orthophthaldehyde assay. Eluates concentrated by Amicon ultrafiltration were also analysed by SDS-PAGE, mass spectroscopy, Western blotting and ELISA. The surfaces of handpiece components were also investigated by SEM, EFSCAN and EDAX microscopy. Surgical gears contained highest levels of protein (403 μg), followed by low speed spray channels (17.7 μg) and the high speed turbine (<5 μg). Mass spectroscopy of surgical gears demonstrated mostly serum derived proteins. Decontamination of the DHPs using an automated washer disinfector and handpiece irrigator showed a significant reduction in residual protein levels.

Toward fluorescence detection of protein residues on surgical instruments

Prion proteins are the infectious agents that cause Creutzfeldt-Jakob Disease (CJD) in humans. These proteins are particularly resistant to normal sterilization procedures, and the theoretical risk of prion transmission via surgical instruments is of current public and professional concern. We are currently investigating fluorescence methods for the detection of proteins on surfaces, with a view to developing an optical-fiber-based system for routine, online monitoring of residual protein contamination on surgical instruments, in hospital sterilization departments. This paper presents preliminary results on the detection of femtomole amounts of fluorescently labelled protein on surgical steel and discusses some of the problems involved in the detection of fluorescence from metal samples.

An overview of new technologies for the decontamination of surgical instruments and the quantification of protein residues: low-pressure radiofrequency (RF) gas-plasma decontamination and epifluorescence scanning (EFSCAN) detection

Abstract: In this chapter we examine two quite different – but linked – aspects of surgical instrument decontamination. The first of these focuses on high-level decontamination using low-pressure radiofrequency (RF) gas-plasmas. The second topic is a description of the principles and use of the EFSCAN (epifluorescence scanning) technology which has been developed to enable rapid automated quantification of low levels of residual protein residues on ‘decontaminated’ instruments.

Quantification of protein contamination on surfaces

Summary form only given. Gas-plasma techniques are capable of destroying and removing both small organic molecules and larger biomolecular structures adhering to, or adsorbed on, a variety of surfaces without causing significant damage to the surfaces themselves. This has led to a large number of decontamination applications. However, while gas-plasma cleaning is capable of very significant reductions in the surface load of organic contamination, the development of robust methods to quantify the efficacy of particular treatments lags far behind the development of the processes themselves.