Nicholas A. Hall

Recent Biochemical and Genetic Advances in Our Understanding of Batten's Disease (Ceroid-Lipofuscinosis)

Protein is the major component of the intra-lysosomal storage material which characteristically accumulates in Battens disease. In the late-infantile, juvenile and adult forms of the disease, and in a form affecting sheep, this protein is principally composed of a single polypeptide, subunit c of mitochondrial ATP synthase. Subunit c is not stored in the infantile form of Battens disease, supporting recent genetic data which suggest this is a distinct disease. Nor is subunit c found in storage material within other lysosomal storage diseases or in lipofuscin of old age. Subunit c storage, therefore, is specific for the later-onset forms of Battens disease and indeed may be central to their aetiology.

Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.

Accumulation of phosphorylated dolichol in several tissues in ceroid-lipofuscinosis (Batten disease).

The concentrations of phosphorylated dolichol (P-dolichol) and of non-phosphorylated dolichol were estimated in a number of different tissues from ceroid-lipofuscinosis (CL) and control cases. The P-dolichol contents of four tissues, brain, pancreas, muscle and kidney were significantly greater than control values. Liver and spleen showed little or no increase in P-dolichol content. Though non-phosphorylated dolichol levels were higher in CL tissues than in control tissues, the differences were significant only for brain and pancreas. In control muscle, a linear age-related increase in non-phosphorylated dolichol content was observed. The relative amounts of the major homologs of P-dolichol and of non-phosphorylated dolichol were estimated. Each tissue has a characteristic homolog size-distribution, and there is close similarity between the size-distribution profiles of P-dolichol and of non-phosphorylated dolichol for each tissue.

A high-performance liquid chromatography method for the analysis of picomole amounts of oligosaccharides

A method for the high-performance liquid chromatography separation of tritium-reduced, acetylated oligosaccharides is described. Their highly sensitive detection in column eluant is facilitated by the use of a flow radioactivity detector. The method differentiates some structural isomers and provides resolution of high-mannose oligosaccharides comparable or superior to that of other high-performance liquid chromatography methods. The detection limit is 0.3 pmol of oligosaccharide. For the detection of radioactive oligosaccharides this method is much less laborious than scintillation counting of collected peak fractions. Generation of a continuous chromatographic trace offers a particular advantage in the detection of partially resolved peaks and the visualization of peak shape. A study of some of the factors influencing acetylation and reduction has led to the development of a robust analytical method.Abstract A method for the high-performance liquid chromatography separation of tritium-reduced, acetylated oligosaccharides is described. Their highly sensitive detection in column eluant is facilitated by the use of a flow radioactivity detector. The method differentiates some structural isomers and provides resolution of high-mannose oligosaccharides comparable or superior to that of other high-performance liquid chromatography methods. The detection limit is 0.3 pmol of oligosaccharide. For the detection of radioactive oligosaccharides this method is much less laborious than scintilation counting of collected peak fractions. Generation of a continuous chromatographic trace offers a particular advantage in the detection of partially resolved peaks and the visualization of peak shape. A study of some of the factors influencing acetylation and reduction has led to the development of a robust analytical method.

A high performance liquid chromatography method for the analysis of 35S-cystine: application to the diagnosis of cystinosis.

An HPLC method for the measurement of radioactively labelled cystine is described. This method has been applied to studies of the uptake and retention of 35S-cystine by cultured cells. Radioactive cystine was measured, as a proportion of the non-protein labelled products in cultured cells incubated with medium containing 35S-cystine. Cells from healthy individuals contained less than 7% cystine whereas cells from cases of cystinosis contained at least 19% cystine. The method has been applied to the prenatal diagnosis of cystinosis. The use of flow radioactivity detection provides the advantages of rapid diagnosis and quantitation of metabolites.