Changes in Sodium Concentration in Cardiac Myocytes from Diabetic Rats

The effects of streptozotocin induced diabetes on rats were studied. The animals showed an increase in blood glucose concentration and a loss of weight from both the body and the heart. Loss of weight from the heart was less severe leading to an increased heart to body weight ratio. Study of element concentrations by X-ray microanalysis showed that there was an increase in intracellular Na concentration in cardiac myocytes from the diabetic animals, but no change in Mg. These results agree with studies which show changes in Na/K ATPase after the onset of diabetes

Cells are Not Just Bags of Water: A Personal Appreciation of the Work of Dr. Brij L. Gupta and his Contribution to Biological X-Ray Microanalysis

This paper surveys the contribution made by the work of Dr. B.L. Gupta to the science of biological X-ray microanalysis. A brief biographical sketch of Brij\u27s early years is given, this is followed by considerations about the models for water transport across epithelia. The ultrastructural and histochemical studies carried out by Brij Gupta and colleagues are reviewed to introduce the historical need for the use of EPXMA for the study of ion and water transport in epithelia. The outstanding contribution made by Brij Gupta\u27s work in this field is outlined, and his understanding of the subcellular distribution ions in other cell types and in the pericellular environment is acknowledged

The Preparation of Cultured Cells for X-Ray Microanalysis

X-ray microanalysis of cells in culture is being used increasingly for the study of relationships between element (ion) content and cell function. There is, however, no one single method which can be used for the preparation of all different cell types for study by microanalysis. Cells in suspension are usually concentrated by centrifugation, before cryofixation, cryosectioning, and freeze drying. On the other hand cells grown as monolayers are more often studied as whole cell mounts, which are washed to remove the external medium before cryofixation and freeze drying. The alternative approach, sectioning of cell monolayers is rarely used. Some of the difficulties encountered in preparing and monolayers of smooth muscle cells for X-ray microanalysis are discussed here

Cryofixation of Heart Tissue for X-Ray Microanalysis

Cryofixation of tissues is necessary to be able to study the concentrations of elements by X-ray microanalysis. Simple dissection of heart tissue fragments of the size needed for optimum cryofixation by the conventional methods of plunge or slam freezing leads to the development of ischaemia in the tissue fragments and a consequent redistribution of the diffusible elements. Heart tissue can be frozen in vivo using liquid nitrogen cooled Cu clad pliers, but the morphological detail is preserved better if the cooled pliers are exposed to liquefied propane immediately before freezing, Concentrations of Na are lower and concentrations of K are higher in the tissue which has been frozen in vivo compared to tissue frozen after dissection

Concentrations of Elements in Dying Thymocytes from the Thymus Gland of Diabetic Rats

Atrophy of the thymus gland occurs in rats made diabetic by a single injection of the drug streptozotocin. Histological studies show the presence of thymocytes with pyknotic nuclei in thymus tissue taken from diabetic animals. Analysis of the elemental content of the pyknotic cells was carried out on freeze dried frozen sections of thymus tissue using the technique of X-ray microanalysis. There was no loss of elements from cells which had undergone the early morphological changes characteristic of pyknosis and which showed the condensed chromatin of pyknotic nuclei. However as the cells shrank and lost the clear distinction between nucleus and cytoplasm there was a loss of elements, particularly Mg and K from the cells. The changes in distribution of elements in the dying thymocytes suggest a method of cell death which does not involve disruption of the plasma membrane

Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease

Huntington's disease (HD) is a late-onset neurodegenerative disorder that is characterized neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA)-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy (AFM) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using AFM, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognize the ligand-captured material on SDS–PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit

Isolation of nano-meso scale detergent resistant membrane that has properties expected of lipid 'rafts'

This review assesses problems that confound attempts to isolate 'raft' domains from cell membranes, focusing in particular upon the isolation of detergent resistant membrane (DRM). Despite its widespread use, this technique is rightly viewed with skepticism by many membrane biochemists and biophysics for reasons that include the inability to isolate DRMs at 37 degrees C, the temperature at which their lipids are supposed to be ordered and so exclude detergents. If solubilization is done in an ionic buffer that preserves the lamellar phase of the metastable inner leaflet lipids, DRMs can readily be isolated at 37 degrees C, and these have many properties expected of lipid rafts. However, to date these DRMs have remained somewhat larger than current concepts of rafts. We describe an adaptation of this method that purifies nano-meso scale DRMs, and could be a significant step towards purifying the membrane of individual 'rafts'