Andrew Wallace

Aggregates from mutant and wild‐type α‐synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β‐sheet and amyloid‐like filaments

α‐Synuclein (α‐syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α‐syn gene have been reported in families susceptible to an inherited form of Parkinsons disease. We have shown that human wild‐type α‐syn, mutant α‐syn(Ala30Pro) and mutant α‐syn(Ala53Thr) proteins can self‐aggregate and form amyloid‐like filaments. Here we report that aggregates of NAC and α‐syn proteins induced apoptotic cell death in human neuroblastoma SH‐SY5Y cells. These findings indicate that accumulation of α‐syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.

Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α-synuclein protein implicated in Parkinson's disease

α‐Synuclein (α‐syn) protein has been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α‐syn gene have been reported in families susceptible to an inherited form of Parkinsons disease. We report here that human wild‐type α‐syn, PD‐linked mutant α‐syn(Ala30Pro) and mutant α‐syn(Ala53Thr) proteins can self‐aggregate and form amyloid‐like filaments. The mutant α‐syn forms more β‐sheet and mature filaments than the wild‐type protein. These findings suggest that accumulation of α‐syn as insoluble deposits of amyloid may play a major role in the pathogenesis of these neurodegenerative diseases.

mt4216C variant in linkage with the mtDNA TJ cluster may confer a susceptibility to mitochondrial dysfunction resulting in an increased risk of Parkinson's disease in the Irish

Polymorphism of the mtDNA genome has been implicated as playing a role in the development and pathogenesis of Parkinsons disease (PD). A PCR-RFLP methodology was employed to generate genetic haplotypes for a cohort of 90 PD sufferers. No association was observed between the various mtDNA haplotypes observed and PD in comparison to healthy aged controls. The longevity-associated European J haplogroup and T haplogroup were identified and were both found to be in tight linkage with the mt4216C polymorphism. The mt4216C variant was observed at a significantly increased frequency in the PD cases (28%) in comparison to the healthy aged controls (15%; p=0.014). However, when the frequency of the mt4216C variant was examined in a cohort of 200 young controls (18-45 years) a similar frequency to the PD cases (25%) was observed. The frequencies obtained for the two branches of the J haplogroup (J1 and J2) and the T haplogroup in the cohort of PD subjects also reflected those observed for the young controls used in the previous longevity study. These findings lead one to postulate that the mt4216C variant, in linkage with the mtDNA TJ cluster, may influence mitochondrial dysfunction, resulting in an increased risk of PD.

Mutation screening in exons 3 and 4 of alpha-synuclein in sporadic Parkinson's and sporadic and familial dementia with Lewy bodies cases.

Recently it has been reported that a missense G(88)C mutation within exon 3 and a missense G(209)A mutation within exon 4 of the alpha-synuclein gene were linked to familial Parkinsons Disease (PD). We decided to investigate if these and any other mutations in exons 3 and 4 of the alpha-synuclein gene could be detected in sixty two sporadic PD and dementia with Lewy bodies (DLB) patients. Four cases of familial DLB were also studied, two of which were from the same family. Single stranded conformational polymorphism, DNA sequencing analyses and PCR-RFLP of exons 3 and 4 failed to reveal any nucleotide changes. However, three nucleotide differences occurred in the intron 4 sequence compared to the published sequence. This study adds further support to the idea that these particular mutation in the alpha-synuclein gene are a rare case of PD and now, as we have shown here, also of DLB.

Fluorescent multiplex microsatellites used to define haplotypes associated with 75 CFTR mutations from the UK on 437 CF chromosomes

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal / carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for ΔF508, 217 for other mutations) from Northern Ireland and three English regions: the North‐West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, ΔF508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16‐7‐17, R117H with 16‐30‐13, 621 + 1G>T with 21‐31‐13, 3659delC with 16‐35‐13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes.

Ultramicrodetection of proteins in polyacrylamide gels

Here we report the development of a highly sensitive procedure to detect proteins within separation matrices which should facilitate the characterization of rare proteins. The procedure is based on photochemical reactions where very low amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. When this conversion is carried out in the presence of [35S]thiourea the resulting radioactive silver sulfide allows detection down to femtogram quantities of protein. In this work we applied the above principle to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, thus not influencing physical and chemical parameters which are important for separation. This procedure should find application in any technique where detection of very low or limited amounts of proteins are required.

A novel polymorphic triplet repeat in intron five of the α-synuclein gene: no evidence of expansion or allelic association with idiopathic Parkinson's disease in the Irish

The recent discovery of two mutations associated with autosomal dominant Parkinsons disease (PD) has led to the hypothesis that the &agr;-synuclein gene plays a role in the pathogenesis of PD. Here we report a novel triplet CAA repeat within the unusually large intron 5 sequence of the &agr;-synuclein gene. Microsatellite analysis revealed a high degree of polymorphism within the Irish population with seven alleles detected, ranging from eight to 17 CAA repeats. Analysis of the allele/genotype frequency differences observed between an Irish idiopathic PD cohort (nu2009=u200998) and a healthy aged control group (nu2009=u200992) revealed no strong association with either group. All PD subjects displaying homozygous profiles were examined for expansion of the trinucleotide repeat, but no expansion was observed. These results would suggest that polymorphism of the &agr;-synuclein gene may not play as significant a role in the pathogenesis of idiopathic PD as previously hypothesised.

Detection of Subpicogram Quantities of Protein in Polyacrylamide Gels

Publisher Summary This chapter discusses the detection of sub-picogram quantities of protein in polyacrylamide gels. The detection method is based on photochemical reactions in which very small amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. The first step of the method is staining with silver nitrate, which results in the diffusion of the silver salt throughout the gel matrix. In the second step, developing, silver nitrate is reacted with formaldehyde under basic conditions to produce metallic silver. Proteins act as catalysts in this reaction, leading to faster deposition of silver metal in the area of the protein bands, compared with that in the rest of the gel. The third step, known as bleaching, converts the deposited silver to silver bromide. Finally, the silver bromide is transformed to silver sulfide, resulting in a deposit of radioactive silver sulfide at the sites of proteins.

A novel approach to analysis of phage clones by reference strand mediated conformation analysis.

Declan P. Prendergasta,∗, M. Isla Hallidaya,∗∗, Neil V. McFerrana, Martin Curranb, Brian McIhattonb, Derek Middletonb, Mark T. Foxa and Andrew Wallacea Centre for Peptide and Protein Engineering, The Queen’s University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, N. Ireland Northern Ireland Histocompatability and Immunogenetics Laboratory, Blood Transfusion Building, Belfast City Hospital, Lisburn Road, Belfast, BT9 7TS, N. Ireland

Generation and screening of solution-phase synthetic peptide combinatorial libraries

Following the initial use of libraries of peptides displayed on filamentous bacteriophage [1] it was inevitable that the potential of solid-phase peptide synthesis should be harnessed to produce libraries of peptides by synthetic chemical methods. These libraries made their first appearance in the mid- 1980s following the development of multiple peptide synthesis by the groups of Geysen [2], Furka [3, 4] and Houghten [5], but it was not until the early 1990s that synthetic peptide combinatorial libraries (SPCLs) were described in the literature [6–8]. This was followed by a series of papers detailing the use of peptide libraries for the discovery of novel antibacterial and antinoistic compounds [9–12] (for a review, see [13]).