Biomedical subjects

Kathryn Lilley

Publications and source records attributed to Kathryn Lilley.

9 recordsLinked to original sources

PubMed2007

2-D DIGE analysis of liver and red blood cells provides further evidence for oxidative stress in schizophrenia.

The molecular disease mechanisms associated with schizophrenia remain largely unknown. Although primarily considered a disorder of the brain, there is evidence of a peripheral component to schizophrenia. In this study, we investigated liver tissue and red blood cells (RBC) from schizophrenia patients and controls using 2-D DIGE proteomic analysis. Fourteen proteins were significantly altered in liver samples from schizophrenia patients (n = 15) compared to healthy controls (n = 15). Analysis of the schizophrenia RBC proteome revealed 8 proteins significantly altered in samples from schizophrenia patients (13 antipsychotic-treated and 7 drug-naïve) compared to controls (n = 20). Six of the altered proteins in the liver and four of the altered RBC proteins are related to oxidative stress. These results corroborate our earlier findings obtained from post-mortem brain studies and substantiate our hypothesis that metabolic alterations leading to oxidative stress are linked to the schizophrenia disease process. Our results also suggest that at least some of the pathological processes associated with the schizophrenia disease process can be traced in peripheral tissue. If peripheral cells can be used as a disease surrogate, promising new investigative avenues could be explored.

Adult↗

PubMed2006

Differentiation of isomeric N-glycan structures by normal-phase liquid chromatography-MALDI-TOF/TOF tandem mass spectrometry.

The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins.

Amination↗

PubMed2005

Confident protein identification using the average peptide score method coupled with search-specific, ab initio thresholds.

Perhaps the greatest difficulty in interpreting large sets of protein identifications derived from mass spectrometric methods is whether or not to trust the results. For such experiments, the level of confidence in each protein identification made needs to be far greater than the often used 95% significance threshold to avoid the identification of many false-positives. To provide higher confidence results, we have developed an innovative scoring strategy coupling the recently published Average Peptide Score (APS) method with pre-filtering of peptide identifications, using a simple peptide quality filter. Iterative generation of these filters in conjunction with reversed database searching is used to determine the correct levels at which the APS and peptide quality thresholds should be set to return virtually zero false-positive reports. This proceeds without the need to reference a known dataset.

Computational Biology↗

PubMed2005

Identification of a new quorum-sensing-controlled virulence factor in Erwinia carotovora subsp. atroseptica secreted via the type II targeting pathway.

Two-dimensional polyacrylamide gel electrophoresis of the secreted proteins of Erwinia carotovora subsp. atroseptica revealed a low-abundance protein that was identified by mass spectrometry as a homologue of a Xanthomonas campestris avirulence protein with unknown function. The predicted Svx protein has an N-terminal signal sequence and zinc binding-region signature, and the mature protein is post-translationally modified. A 2D difference gel electrophoresis (DIGE) showed that the protein is secreted by the type II (out) secretion apparatus, which is also responsible for the secretion of the major known virulence factors, PelC and CelV. Transcription of the svx gene is under N-acyl-homoserine lactone-mediated quorum-sensing control. The svx gene was inactivated by transposon insertion. The mutant showed a decrease in virulence in potato plant assays, demonstrating a role for Svx in the pathogenicity of E. carotovora subsp. atroseptica. These results show that Svx is a previously unidentified virulence determinant which is secreted by the out machinery and is regulated by quorum sensing, two systems employed by several other virulence factors. Thus, the type II secretory machine is a conduit for virulence factors other than the main pectinnases and cellulase in E. carotovora subsp. atroseptica.

Bacterial Proteins↗

PubMed2004

Functional specialization amongst the Arabidopsis Toc159 family of chloroplast protein import receptors.

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

Arabidopsis↗

PubMed2004

Specific changes in the Arabidopsis proteome in response to bacterial challenge: differentiating basal and R-gene mediated resistance.

Alterations in the proteome of Arabidopsis thaliana leaves during early responses to challenge by Pseudomonas syringae pv. tomato DC3000 (DC3000) were analysed using two-dimensional (2D) gel electrophoresis. Protein changes characteristic of the establishment of basal resistance and R-gene mediated resistance were examined by comparing responses to DC3000, a hrp mutant and DC3000 expressing avrRpm1 respectively. The abundance of selected transcripts was also analysed in GeneChip experiments. Here we present data from the soluble fraction of leaf protein, highlighting changes in two antioxidant enzyme groups; the glutathione S-transferases (GSTs F2, F6, F7 and F8) and peroxiredoxins (PrxA, B and IIE). Members of both enzyme groups showed signs of specific post-translational modifications, represented by multiple spots on gels. We suggest that oxidation of specific residues is responsible for some of the spot shifts. All forms of the GST proteins identified here increased following inoculation with bacteria. GSTF8 showed particularly dynamic responses to pathogen challenge, the corresponding transcript was significantly up-regulated by 2 h after inoculation, and the protein showed post-translational modifications specific to an incompatible interaction. Differential changes were observed with the peroxiredoxin proteins; PrxIIE and to a lesser extent PrxB, no change was observed with PrxA, but a truncated form PrxA-L was greatly reduced in abundance following bacterial challenges. Our data suggest that bacterial challenge generally induces Prxs and the antioxidants GSTs, however individual members of these families may be specifically modified dependent upon the virulence of the DC3000 strain and outcome of the interaction. Finally, proteomic and transcriptomic data derived from the same inoculation system are compared and the advantages offered by 2D gel analysis discussed in light of our results.

Arabidopsis↗

PubMed2003

The Arabidopsis ppi1 mutant is specifically defective in the expression, chloroplast import, and accumulation of photosynthetic proteins.

The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins.

Arabidopsis↗

PubMed2002

Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures.

The structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapie-associated prion protein, PrP(Sc). These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results.

Carbohydrate Sequence↗

PubMed2002

GARNet, the Genomic Arabidopsis Resource Network.

GARNet, the Genomic Arabidopsis Resource Network, was created to establish UK-based facilities for functional genomic research on Arabidopsis thaliana. In addition, GARNet provides a platform for international Arabidopsis research and for research on other plant species. To use the GARNet facilities apply via the website: http://garnet.arabidopsis.org.uk. All GARNet services and resources are publicly available, and data created using the GARNet resources will be freely distributed via databases held at the Nottingham Arabidopsis Stock Centre and the John Innes Centre.

Arabidopsis↗