Peter Estibeiro

A candidate marsupial PrP gene reveals two domains conserved in mammalian PrP proteins

The normal function of the pathogenicity-related protein, PrP (or prion protein), is unknown. To shed light on functionally important domains, we have characterized a candidate marsupial PrP gene. The deduced marsupial PrP has an overall identity of about 80% to eutherian PrP at the amino acid (aa) level. This similarity is not equally distributed and two regions (aa 118-142 and 177-223) are particularly highly conserved. In contrast, a repeat region in the N-terminal half of the marsupial PrP shows dipeptide inserts not described in other PrP. Another particular feature of the marsupial gene is the lack of a continuous ORF on the antisense strand, as is found in most eutherian PrP. We propose that antisense ORFs found in other species are artefactual. Comparison with all known PrP argues that the molecule characterised is the true marsupial PrP orthologue.

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function.

Enhanced long-term potentiation in the hippocampus of rats expressing mutant presenillin-1 is age related

Electrophysiological recordings were made from Fischer rats engineered to express the human presenilin 1 gene carrying the M146V mutation. Extracellular recordings of field excitatory post-synaptic potential (EPSPs) were made to investigate EPSP properties, paired pulse responses, posttetanic potentiation, and long-term potentiation in the stratum radiatum and dentate gyrus of hippocampal slices maintained in vitro. Transgenic rats aged approximately 6 months showed no differences from their wild-type littermates in any of these properties. However, at 18 months, long-term potentiation in the CA1 was facilitated in the transgenic rats with a different pattern of synaptic enhancement. No changes were observed in paired pulse facilitation (PPF) or post-tetanic potentiation (PPT) and no changes were seen in the dentate gyrus. Field potential amplitudes were significantly greater and PPF was enhanced in the CA1 of all older rats. Intracellular recordings from CA1 pyramidal cells of the older group of rats revealed no differences in the passive or active membrane properties of cells in the two groups, but intracellularly recorded EPSPs were significantly longer.

The use of nucleic acid tools for target validation in central nervous system therapy.

The main challenge facing target validation today comes from the ongoing genomics revolution, which is generating an unprecedented number of potential targets. Existing technologies, such as mouse knockouts, are struggling to provide the throughput now required. Nucleic acid tools including antisense, RNA interference, ribozymes and aptamers offer a potentially higher throughput means of manipulating gene expression and thus validating targets in complex biological systems such as the central nervous system.