Search PubMed⌕ Search

Biomedical subjects

Paul Webster

Publications and source records attributed to Paul Webster.

At least 55 records · Page 3Linked to original sources
PubMed2003

A simpler way of comparing the labelling densities of cellular compartments illustrated using data from VPARP and LAMP-1 immunogold labelling experiments.

Quantitative immunoelectron microscopy of gold label in intracellular compartments often involves calculating labelling densities (LDs). These are related to antigen concentrations and usually refer gold particle counts to the sizes of compartments on sections (for example, golds per microm(2) of organelle profile area or per microm of membrane trace length). Here, we show how LD values can be estimated more simply (without estimating areas or lengths) and also how observed and expected LD values can be used to calculate a relative labelling index (RLI) for each compartment and then test statistically for preferential (non-random) labelling. For random labelling, RLI=1. Compartment size is estimated stereologically by superimposing random test points (which hit organelle profiles in proportion to their area) or test lines (which intersect membrane traces in proportion to their length). By this means, the observed LD of a compartment (LD(obs)) can be expressed simply as golds per test point (organelles) or per intersection (membranes). Furthermore, the LD obtained by dividing total golds (on all compartments) by total points or intersections (on all compartments) is the value to be expected (LD(exp)) when compartments label randomly. For each compartment, RLI=LD(obs)/LD(exp). Statistical analysis is undertaken by comparing observed distributions of golds with predicted random distributions (calculated from point or intersection counts). A compartment is preferentially labelled if two criteria are met: (1) its RLI>1 (i.e. LD(obs) is greater than LD(exp)) and (2) its partial chi-squared value makes a substantial contribution to total chi-squared value. This approach provides a simple and efficient way of comparing LDs in different compartments. Its utility is illustrated using data from VPARP and LAMP-1 labelling experiments.

Animals↗
PubMed2003

Optical recordings of Ca2+ signaling activities from identified inner ear cells in cochlear slices and hemicochleae.

One of the major obstacles hindering the progress of studies on mammalian cochlear physiology is the inaccessibility of inner ear cells located in a complex structure of the bony labyrinth. We describe here a technique to record cellular fluorescent signals from any identified inner ear cells in cochlear slices and hemicochleae. Cochlear slices were obtained from postnatal rats (P0-P7) before the cochlea completely ossify, and hemicochleae were cut from older animals (P7-adult). Individual inner ear cells were visually identified using infrared differential interference contrast or oblique illumination optics. Techniques were developed for either bulk-loading cells or loading selected single cells with Ca(2+) indicator dyes, and for maintaining functional viability of cochlear slices/hemicochleae for recordings. Robust and reliable responses of ligand-gated receptors were recorded from individual inner ear cells (e.g. hair cells, spiral ganglion neurons etc.) for at least 24 h after slices/hemicochleae were cut by an oscillating tissue slicer. The technique described here allowed direct observations of [Ca(2+)](i) activities from multiple cells simultaneously in situ, thus providing a feasible way to study the intercellular communication or networking activities from identified cells in the inner ear.

Action Potentials↗
PubMed2003

Cytoplasmic bacteria can be targets for autophagy.

Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components. The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes. In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy. If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles. The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria. These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER. Internalization of metabolically arrested cytoplasmic L. monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin. Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway. Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.

Animals↗