Biomedical subjects

J Michael Edwardson

Publications and source records attributed to J Michael Edwardson.

26 records · Page 2Linked to original sources

PubMed2002

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy.

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 +/- 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.

Carbazoles↗

PubMed2002

Placental alkaline phosphatase is efficiently targeted to rafts in supported lipid bilayers.

Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.

Alkaline Phosphatase↗

PubMed2002

Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains.

Syncollin, a novel pancreatic zymogen granule protein, is present on the luminal side of the granule membrane. To address the function of syncollin, we searched for putative binding partners. Cross-linking experiments with purified syncollin, and granule content and membrane proteins revealed a direct interaction between syncollin and GP-2, a major glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. An interaction was also observed when cross-linking was performed with recombinant GP-2. In addition, syncollin could be cross-linked to itself, supporting the suggestion that it exists as a homo-oligomer. Cleavage of the GPI anchor of GP-2 by treatment of granule membranes with phosphatidylinositol-specific phospholipase C had no effect on the membrane attachment of syncollin, indicating that it is not mediated exclusively via an interaction with GP-2. Syncollin was found to be associated with detergent-insoluble cholesterol/glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose-density gradients and also contained GP-2, the lectin ZG16p, sulphated matrix proteoglycans and the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) syntaxin 3 and synaptobrevin 2. Our results indicate that membrane-associated syncollin is a component of lipid rafts, where it interacts both with GP-2 and membrane lipids. We suggest that the syncollin-GP-2 complex might play a role in signal transduction across the granule membrane.

Animals↗

PubMed2002

Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry.

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.

Acetylcholine↗

PubMed2002

The regulation of exocytosis in the pancreatic acinar cell.

The pancreatic acinar cell synthesises a variety of digestive enzymes. In transit through the secretory pathway, these enzymes are separated from constitutively secreted proteins and packaged into zymogen granules, which are localised in the apical pole of the cell. Stimulation of the cell by secretagogues such as acetylcholine and cholecystokinin, acting at receptors on the basolateral plasma membrane, causes the generation of an intracellular Ca(2+) signal. This signal, in turn, triggers the fusion of the zymogen granules with the apical plasma membrane, leading to the polarised secretion of the enzymes. This review describes recent advances in our understanding of the control of secretion in the acinar cell. In particular, we discuss the mechanisms underlying the sorting of digestive enzymes into the zymogen granules, the molecular components of the exocytotic "membrane fusion machine," the generation and propagation of the Ca(2+ signal and the development of new techniques for the visualisation of single granule fusion events.

Animals↗

PubMed2002

Post-testicular development of a novel membrane substructure within the equatorial segment of ram, bull, boar, and goat spermatozoa as viewed by atomic force microscopy.

Atomic force microscopy has been used to investigate changes in the plasma membrane overlying the head region of mammalian spermatozoa (bull, boar, ram, goat, stallion, mouse, and monkey) during post-testicular development, after ejaculation, and after exocytosis of the acrosomal vesicle. On ejaculated ram, bull, boar, and goat spermatozoa the postacrosomal plasma membrane has a more irregular surface than that covering the acrosome. The equatorial segment, by contrast, is relatively smooth except for an unusual semicircular substructure within it that has a coarse uneven appearance. This substructure (referred to as the equatorial subsegment) is situated adjacent to the boundary between the postacrosomal region and the equatorial segment itself and seems to be confined to the order Artiodactyla as it has not been observed on stallion, mouse, or monkey spermatozoa. The equatorial subsegment develops during epididymal maturation, and following induction of the acrosome reaction with Ca(2+) ionophore A23187, its topography changes from a finely ridged appearance to that resembling truncated papillae. A monoclonal antibody to the equatorial subsegment binds only to permeabilized spermatozoa, suggesting that the subsegment is related to the underlying perinuclear theca that surrounds the sperm nucleus. A role for the equatorial subsegment in mediating fusion with the oolemma at fertilization is discussed.

Acrosome↗

PubMed2002

Direct visualization of ligand-protein interactions using atomic force microscopy.

1. Streptavidin is a 60-kDa tetramer which binds four molecules of biotin with extremely high affinity (K(A) approximately 10(14) M(-1)). We have used atomic force microscopy (AFM) to visualize this ligand-protein interaction directly. 2. Biotin was tagged with a short (152-basepair; 50-nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105+/-3 nm(3)) was in close agreement with the value calculated from its molecular mass (114 nm(3)). Biotinylation increased the apparent size of streptavidin (to 133+/-2 nm(3)), concomitant with an increase in the thermal stability of the tetramer. 3. Images of streptavidin with one to four molecules of DNA-biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acute : obtuse angles (1 : 3) was lower than the expected value (1 : 2), indicating a degree of steric hindrance in the binding of the DNA-biotin. The slight under-representation of higher occupancy states supported this idea. 4. Streptavidin with a single molecule of DNA-biotin bound was used to tag biotinylated beta-galactosidase, a model multimeric enzyme. 5. The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the effect of complex formation on the structure of the protein. Furthermore, the use of DNA-biotin/streptavidin tags could potentially shed light on the architecture of multi-subunit proteins.

Binding Sites↗

PubMed2002

Acetylcholine-induced zymogen granule exocytosis: comparison between acini and single pancreatic acinar cells.

INTRODUCTION: Numerous studies have been carried out on the agonist-evoked calcium responses of single pancreatic acinar cells; however, several reports have shown that dissociation of the exocrine pancreas into predominantly single cells has an adverse effect on agonist-evoked amylase secretion. AIMS AND METHODOLOGY: To determine whether single acinar cells behave in an anomalous manner compared with cells within an intact acinus, we measured exocytosis in both single acinar cells and acini (2-5 cells) present in the same preparation. Exocytosis of individual zymogen granules was quantified in real-time by using the technique of continuous time-differential analysis of brightfield digital images. RESULTS: Basal rates of exocytosis were low in both single cells and intact acini. Application of 10 microM acetylcholine for 6 minutes stimulated a biphasic secretory response in acinar cells. Additionally, we found that exocytotic events occur repetitively in specific locations within the apical domain; i.e., there are exocytotic "hot spots." There were no statistically significant differences between the exocytotic rates, nor were there any differences in the characteristics of the exocytotic hot spots of single cells compared with those of acini. CONCLUSION: We conclude that time-differential analysis of brightfield images appears to be a useful tool for the investigation of the role of gap junctions in zymogen granule exocytosis and that single acinar cells provide a reasonable model for studies of acinar cell signaling and secretion.

Acetylcholine↗