Evaluation and Application of Screening Strategies for Point Mutations in the Retinoblastoma Gene

The present study aimed to evaluate two screening strategies, single strand conformation polymorphism (SSCP) and amplification mismatch detection (AMD) analysis, for the detection of point mutations in the retinoblastoma gene (RB). SSCP was optimised and applied to exons 12-22 of the RB gene which constitute the most important functional domain. Leukocyte DNA from 20 patients with bilateral retinoblastoma (Rb), tumour DNA from 40 patients with bladder carcinoma and tumour DNA from 39 patients with breast carcinoma were subjected to SSCP analysis. SSCP band shifts were found in 4 of 20, 1 of 40 and none of 39 patients respectively. AMD was optimised and applied to exons 12-16 of the RB gene and also to reverse-transcriptase PCR in the 20 patients with bilateral Rb. Cleavage was found in 2 patients; one was found in a cDNA segment and the other was found in genomic DNA. Neither of these patients corresponded to the 4 with SSCP band shifts. Thus in total, 6 patients with Rb and one with bladder carcinoma had mutations detected and proof was sought by sequencing. Amplification of segment C of the cDNA of patients with bilateral Rb has revealed that patient EAS showed an additional band indicating either a deletion or a splice mutation. Analysis of exon 17 and the flanking intron of the same patient with AMD showed a cleavage with hydroxylamine. Sequencing of the exon revealed that the mutation is a C substitution of the A at position -2 of the acceptor site of intron 16, impairing normal splicing of the RNA. The mutation results in skipping of exon 17, because the short transcript band on the agarose gel was approximately 196 bp shorter than the original band and exon 17 was 196 bp in size. This leads in turn to the production of a truncated RB protein. By analogy to other published mutations, this aberrant, destabilised protein might not be able to bind the E1A oncoprotein. In addition, the mutant RB protein may fail to complex with SV40 large T antigen. Analysis of segment C of the cDNA from patient PC with bilateral Rb showed a cleavage with hydroxylamine reaction. Sequencing of the segment revealed the mutation to be a T→G transversion at nucleotide position 1587 within exon 16 causing a substitution of histidine to glycine. The missense mutation may or may not have a functional effect. However, this residue lies within an RB domain (aminoacids 393-572) identified recently by in vitro deletion mutants to be required for oncoprotein binding. This mutation creates a restriction site for Nde I. Sequencing of exon 21 from patient MH who had an SSCP band shift, revealed that an insertion of a G at nucleotide position 2251 within exon 21 resulted in a novel stop codon (TAA) at codon 719 (nucleotide position 2295) within exon 21 thus deleting the domain interacting with the SV40 T antigen. The translated protein is most probably too short to be functional. To confirm whether the observed SSCP pattern in the region of exon 21 of the RB gene was a new germ line mutation or inherited from one of the parents, heteroduplex analysis of the parents revealed either the mutation was de novo or one of the parents had germ line mosaicism. In addition, the change creates a restriction site for the restriction enzyme FokI. Another mutation detected (from patient AR) by SSCP analysis was an A to C transversion at position 1636 in exon 16 causing AGA to CGA codon change, both coding for the same amino acid: Arginine, this mutation abolishes a restriction site for the restriction enzyme CvijI. The mutation site was located in the last base of the exon 16, although it has not been shown in this study in mRNA, it could affect splicing of the mRNA of RB gene. Two mutations found were considered to be silent mutations, because they do not cause any amino acid change. Their RNA transcripts were found to be normal. These mutations were a T to C transition at position 1617 in exon 16 (Patient GM) which alters the codon GTT (GUU) to GTC (GUC) both coding for the same amino acid; Valine and an A→G transition in intron 19 (patient EAS) which abolishes a restriction site for the restriction enzyme Tsp 509 I which may be useful in tracking this mutation in affected family members. Analysis of 100 samples (patients with bilateral Rb and other tumours) for this restriction site revealed that none of them has same change. The mutation found from the patient with bladder carcinoma was a G to C transversion at position +1 of the donor site of intron 12, probably impairing normal splicing of the RNA. The second mutation was assumed to lie in a part of the retinoblastoma gene that was not analysed, since in somatic cases two hits in the RB gene are expected. Intact RNA could not always be recovered from the clinical material used in this study, therefore the diagnostic strategy for bladder carcinoma was chosen not to be based on the analysis of RNA transcript. The change creates a recognition site for the restriction enzymes MaeII, Bpu101, DdeI. The seven mutations detected in this study were all novel and emphasise the heterogeneity of the molecular pathology in this gene

Sensing at nanostructures for agri-food and enviromental applications

With a predicted population increase of 2.3 billion people, by 2050, agricultural productivity must be vastly improved and made sustainable. Globally, agriculture must deliver a 60% increase in food production to cope with the population demand. Moreover, this needs to be achieved against a changing climate, an exploitation of natural resources, and growing water and land scarcities. New digital technologies can optimise production efficiency and ensure food security and safety while also minimising waste within the production systems and the supply chain. To this end, new sensor technologies are being developed for applications in animal health diagnostics and environmental issues related to the global population, such as food & crop protection, pathogen and toxin detection, and environmental remediation. In this thesis, two new nanosensing diagnostic devices are developed and presented; surface enhanced Raman sensing and electrochemical sensing. Surface-enhanced Raman spectroscopy (SERS) substrates were fabricated by templating a flexible thermoplastic polymer against an aluminium drinks can followed by coating with a silver film, to produce a rough nanostructured metallic surface. SERS is used for both qualitative (molecular fingerprint) and quantitative detection of dye molecules and food toxins. In addition, the SERS technique is also applied in combination with nanoelectrochemical square wave voltammetry to detect nano-concentrations of neonicotinoid pesticides. The enhanced sensitivity and minimum sample preparation requirements provide tremendous opportunities for food safety and security sectors. An impedimetric immunosensor device (with a micro SD style pin-out) was also developed for the serological diagnosis of viruses and antibodies associated with bovine respiratory disease and bovine liver fluke. The silicon chip devices consist of six on-chip nanoband electrodes which can be independently modified with a polymer layer for covalent immobilisation of capture and target biomolecules. This electrochemical biosensor technology provides label-free and cost-efficient sensing capability in a compact size, and demonstrates the potential development of immunoassay-based point-of-use devices for on-farm diagnosis or therapeutic monitoring in animal health applications

Modulation of kainic acid neurotoxicity in rats

1. Systemically administered kainic acid causes a dose dependent increase in the amount of peripheral benzodiazepine receptor in the rat hippocampus, as assessed by [3H]PK11195 binding. 2. This increase in binding is due to an increase in Bmax and not K0. The increase in PK11195 binding indicates that reactive gliosis has occurred and, by inference, neuronal loss. 3. The kainic acid induced elevation in binding is blocked by the non-NMDA antagonist GYKI 52466 and the NMDA antagonists MK801 and CPP. 4. The adenosine A1 receptor agonist R-phenylisopropyladenosine (R-PIA) is able to attenuate the kainic acid induced neuronal loss in a dose dependent and time dependent manner. 5. The R-PIA response is blocked by 8-cyclopentyl,-l ,3- dipropylxanthine (DPCPX) in a dose dependent manner, although DPCPX is unable to potentiate kainate induced neurotoxicity. 6. 8-p-sulfophenyltheophylline (8-SPT), is unable to cross the blood-brain barrier, and is unable to block the R-PIA induced neuroprotection indicating that the R-PIA effect is centrally mediated. 7. Kynurenine, but not kynurenic acid or tryptophan is able to attenuate kainic acid induced neurotoxicity in a dose dependent manner. 8. Kainic acid and potassium chloride (KCl) are able to release [3H] glutamate from hippocampal slices in a dose dependent manner. 9. The kainic acid induced elevation induces a period of heightened release after the first, but not second or third stimulations, and this is not seen after either KCl or kainic acid/KCl stimulations. 10. The adenosine A1 agonist 2-chloroadenosine (5muM) is unable to block the kainic acid induced release of [3H] glutamate. 11. DPCPX (5nM) is able to induce a significant decrease in KCl stimulated release of [3H] glutamate but not the kainic acid induced release. 12. In conclusion, kainic acid is causing neurotoxicity in a dose dependent manner that is mediated through both non-NMDA and NMD A receptors, and can be attenuated by R-PIA, and ascorbate