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William G T Willats

Publications and source records attributed to William G T Willats.

11 recordsLinked to original sources
PubMed2006

Solid-phase chemical tools for glycobiology.

Techniques involving solid supports have played crucial roles in the development of genomics, proteomics, and in molecular biology in general. Similarly, methods for immobilization or attachment to surfaces and resins have become ubiquitous in sequencing, synthesis, analysis, and screening of oligonucleotides, peptides, and proteins. However, solid-phase tools have been employed to a much lesser extent in glycobiology and glycomics. This review provides a comprehensive overview of solid-phase chemical tools for glycobiology including methodologies and applications. We provide a broad perspective of different approaches, including some well-established ones, such as immobilization in microtiter plates and to cross-linked polymers. Emerging areas such as glycan microarrays and glycan sequencing, quantum dots, and gold nanoparticles for nanobioscience applications are also discussed. The applications reviewed here include enzymology, immunology, elucidation of biosynthesis, and systems biology, as well as first steps toward solid-supported sequencing. From these methods and applications emerge a general vision for the use of solid-phase chemical tools in glycobiology.

Adsorption↗
PubMed2006

The Arabidopsis co-expression tool (ACT): a WWW-based tool and database for microarray-based gene expression analysis.

We present a new WWW-based tool for plant gene analysis, the Arabidopsis Co-Expression Tool (ACT), based on a large Arabidopsis thaliana microarray data set obtained from the Nottingham Arabidopsis Stock Centre. The co-expression analysis tool allows users to identify genes whose expression patterns are correlated across selected experiments or the complete data set. Results are accompanied by estimates of the statistical significance of the correlation relationships, expressed as probability (P) and expectation (E) values. Additionally, highly ranked genes on a correlation list can be examined using the novel clique finder tool to determine the sets of genes most likely to be regulated in a similar manner. In combination, these tools offer three levels of analysis: creation of correlation lists of co-expressed genes, refinement of these lists using two-dimensional scatter plots, and dissection into cliques of co-regulated genes. We illustrate the applications of the software by analysing genes encoding functionally related proteins, as well as pathways involved in plant responses to environmental stimuli. These analyses demonstrate novel biological relationships underlying the observed gene co-expression patterns. To demonstrate the ability of the software to develop testable hypotheses on gene function within a defined biological process we have used the example of cell wall biosynthesis genes. The resource is freely available at http://www.arabidopsis.leeds.ac.uk/ACT/

Arabidopsis↗
PubMed2004

A monoclonal antibody to feruloylated-(1-->4)-beta-D-galactan.

We report the isolation and characterization of a monoclonal antibody, designated LM9, against feruloylated-(1-->4)-beta-D-galactan. This epitope is a structural feature of cell wall pectic polysaccharides of plants belonging to the family Amaranthaceae (including the Chenopodiaceae). Immuno-assays and immunofluorescence microscopy indicated that LM9 binding is specific to samples and cell walls obtained from species belonging to this family. In a series of competitive-inhibition enzyme-linked immunosorbent assays with potential oligosaccharide haptens, the most effective inhibitor was O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose (Gal2F). LM9 is therefore a useful antibody probe for the analysis of phenolic substitution of cell wall pectic polymers and of cell wall structure in the Amaranthaceae including sugar beet (Beta vulgaris L.) and spinach (Spinacia oleracea L.).

Amaranthaceae↗
PubMed2004

Novel cell wall architecture of isoxaben-habituated Arabidopsis suspension-cultured cells: global transcript profiling and cellular analysis.

The herbicide isoxaben is a highly specific and potent inhibitor of cellulose synthesis in plants. Nevertheless, suspension-cultured cells can be habituated to grow in high concentrations of isoxaben, and apparently compensate for the disruption of cellulose synthesis by the modulation of other cell wall components. We have habituated Arabidopsis cells to isoxaben and characterized the cellular and genetic consequences. Near whole-genome transcript profiling implicated novel genes in cell wall assembly and extended our understanding of the activity of known cell wall-related genes including glycosyltransferases involved in cellulose and pectin biosynthesis. Habituation does not appear to be mediated by stress response processes, nor by functional redundancy within the cellulose synthase (AtCesA) family. Uniquely, amongst the cellulose synthase superfamily, AtCslD5 was highly upregulated and may play a role in the biosynthesis of the novel walls of habituated cells. In silico analysis of differentially expressed genes with unknown functions identified a putative glycosyltransferase and collagen-like putative cell wall protein.

Adaptation, Biological↗
PubMed2003

A xylogalacturonan epitope is specifically associated with plant cell detachment.

A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified that is specifically associated with a plant cell separation process that results in complete cell detachment.

Antibodies, Monoclonal↗
PubMed2003

Synthetic methyl hexagalacturonate hapten inhibitors of anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7.

A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens provides insight into the structures of the partially methyl-esterified pectin epitopes of these widely used monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed2003

Isolation and characterisation of the homogalacturonan from type II cell walls of the commelinoid monocot wheat using HF-solvolysis.

In contrast to the typical type I cell wall of the dicot plants, the type II cell wall of the commelinoid monocot plants is known to be relatively poor in pectins. Assuming a critical role for the remaining pectins in terms of cell wall architecture and/or as a reservoir of signalling molecules, we have compared different protocols for the isolation of the main pectin polymer, homogalacturonan, from wheat leaf cell walls. Pectin was detected in these cell walls immunochemically using the monoclonal antibodies JIM5 and JIM7, and biochemically by monosaccharide analysis. The Ca(++)-chelators CDTA and imidazole extracted a pectin rich fraction from isolated cell walls which was however contaminated with significant amounts of hemicelluloses. Pretreatment of the cell walls with anhydrous hydrogen fluoride at controlled low temperatures followed by HF/ether- and water-extraction prior to imidazole-extraction of pectins yielded a purer homogalacturonan fraction. The near absence of rhamnosyl residues proved that the isolated homogalacturonan fraction was free of rhamnogalacturonans. If HF-solvolysis was performed at -23 degrees C, the resulting homogalacturonan had a degree of methyl esterification identical to that of the pectins in the initial wheat cell wall. The antibodies JIM5 and JIM7 as well as PAM1 and LM5 proved that the isolated homogalacturonan had a low methyl ester content, was polymeric and free of galactan side chains. We can thus isolate native homogalacturonan from the type II wheat cell walls with the original in muro pattern of methyl esterification still intact, to further investigate e.g., its degradability by plant or microbial pectic enzymes.

Cell Wall↗
PubMed2002

Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening.

The Cnr ( C olourless n on- r ipening) tomato ( Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were found in the solubility and composition of the pectic polysaccharides extracted from the CWM at both stages of development. In comparison with the wild type, the ripening-associated solubilisation of homogalacturonan-rich pectic polysaccharides was reduced in Cnr. The proportion of carbohydrate that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained larger amounts of galactosyl- and arabinosyl-containing polysaccharides that were tightly bound in the cell wall and could only be extracted with 4 M KOH, or remained in the insoluble residue. The complexity of the cell wall alterations that occur during fruit ripening and the significance of different extractable polymer pools from cell walls are discussed in relation to the Cnr phenotype.

Cell Adhesion↗
PubMed2002

Sugar-coated microarrays: a novel slide surface for the high-throughput analysis of glycans.

The development of DNA and protein microarrays represents a significant advance in transcriptomics and proteomics research. Such arrays allow the high-throughput, parallel analysis of protein occurrence and interactions and gene expression. However, this advance has not been matched by equivalent technology for analysis of glycomes. One reason for this is that compared to proteins, it is difficult to reliably immobilise populations of chemically and structurally diverse glycans. We describe the development of a new microarray slide surface to which diverse glycan structures can be directly immobilised without prior derivatisation of the slide surface or any modification of the arrayed samples. The slides can be used to produce comprehensive microarrays of carbohydrates, glycoproteins and proteoglycans using isolated samples or cell extracts. Using standard microarray equipment, a series of carbohydrate microarrays were generated and probed with a panel of monoclonal antibodies with specificities for glycan epitopes. The arrays were highly reproducible, stable, and could be stored dry for several months. Glycans play central roles in development, carcinogenesis, cell adhesion, and immunity and are increasingly the subject of therapeutic approaches. We anticipate that the development of carbohydrate microarrays will be important for the high-throughput analysis of glycans and their molecular interactions.

Animals↗
PubMed2002

Phage display: practicalities and prospects.

Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phage thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but also have the capacity to replicate. Using phage display vast numbers of variant nucleotide sequences may be converted into populations of variant peptides and proteins which may be screened for desired properties. It is now some seventeen years since the first demonstration of the feasibility of this technology and the intervening years have seen an explosion in its applications. This review discusses the major uses of phage display including its use for elucidating protein interactions, molecular evolution and for the production of recombinant antibodies.

Antibodies↗