Research
-Diabetes Research
-Collaboration with Ann Simpson, University of Technology, Gore Hill (genetic engineering of human hepatoma cell lines, biochemical studies); Don Martin, UTS, Sydney (patch clamping studies).
Type I diabetes is an autoimmune disease in which the beta cells of the pancreas are destroyed, causing severe insulin deficiency. Because this affects children it is also known as Juvenile diabetes. Our group is the first in the world to successfully engineer two liver cell lines to store and secrete insulin to the metabolic stimulus glucose. The aim of the research is to use somatic gene therapy to permanently correct patient blood glucose concentrations. Because the cells would be engineered from the patient€s own liver cells, immunosuppressive drugs would not be required.
Before artificial beta cells can be used clinically, we need to know more about the insulin storage and secretion mechanisms of these cells, and their response to glucose.
We are studying this in the Anatomy department, Sydney University, using confocal microscopy to study the secretion process in live cells. We are using immunoelectron microscopy to determine where insulin is stored in these cells.
This work is supported by the Australian Research Council and Australian Diabetes research grants.
Improved ultrastructural preservation of cells using betaine-containing fixatives
The most important step in processing specimens for transmission electron microscopy is the primary fixative. The development of an ideal fixative which causes minimal damage to the specimen is not a trivial exercise, and it cannot be assumed that a fixative which is ideal for a tissue from one species will be suitable for all other tissues or cells from the same species. Similarly, a fixative which is ideal for a particular cell from one species of animals will not usually be suitable for the same cell type from a different but related species.
Provided sufficient aldehydes are present in the fixative, the next most important factor is the osmolality. Our laboratory has shown that adding an osmoprotectant, betaine, to a fixative will reduce osmotic damage during fixative penetration. We have shown this to be the case with immersion fixation of rat epididymis, and for fixation of various cell suspensions such as human hepatoma cells, and human and ram spermatozoa.
The importance of this work is that it allows the effect of experimental treatments to be studied at an ultrastructural level, without the counfounding influence of artifactual cellular damage.