P.R. Dorling

Specific inhibition of human β-D-glucuronidase and α-L-iduronidase by a trihydroxy pipecolic acid of plant origin

The glucuronic acid analogue of 1‐deoxynojirimycin, 2(S)‐carboxy‐3(R), 4(R), 5(S)‐trihydroxypiperidine, recently isolated from seeds of Baphia racemosa, is a novel specific inhibitor of human liver β‐D‐glucuronidase and α‐L‐iduronidase. No other glycosidases are inhibited. The inhibition of β‐D‐glucuronidase is competitive, with a K i of 8 × 10−5 M and is pH‐dependent. This inhibitor may be useful to induce a mucopolysaccharidosis or to investigate the function of microsomal β‐D‐glucoronidase.

Onset and regression of neuroaxonal lesions in sheep with mannosidosis induced experimentally with swainsonine

SummaryA group of young sheep were fed a diet containing theα-mannosidase inhibitor swainsonine, which resulted in the induction of a neuronal lysosomal mannoside storage disease. Sheep were killed at various intervals during and following the treatment period and the nature and distribution of neuronal and axonal lesions in the brain were assessed by routine light and electron microscopy and by the rapid Golgi impregnation technique. Neuronal mannoside storage, axonal dystrophy and meganeurite formation were induced by 80 days of treatment and the lesions had regressed by 40 days after the end of treatment. The results are discussed in relation to their relevance to the current widespread interest in the pathobiology of neuronal lysosomal storage.

Swainsonine affects the processing of glycoproteins in vivo

Rats, sheep and guinea pigs treated with swainsonine excrete ‘high mannose’ oligosaccharides in urine. The major rat and guinea pig oligosaccharide is (Man)5GlcNAc whereas sheep excrete a mixture of oligosaccharides of composition (Man)2–5GlcNAc2 and (Man)3–5GlcNAc. The presence of these oligosaccharides suggests that Golgi α‐D‐mannosidase II as well as lysosomal α‐D‐mannosidase is inhibited by swainsonine resulting in storage of abnormally processed asparagine‐linked glycans from glycoproteins. Altered glycoprotein processing appears to have little effect on the health of the intoxicated animal, but the accompanying lysosomal storage produces a disease state.

Experimental copper and heliotrope intoxication in sheep: Morphological changes

Young Merino wethers were used to determine the effects of copper and heliotrope, fed together or separately, on the development of toxicity and the concentration of trace elements in the liver and kidney. In one experiment copper and heliotrope were given concurrently, in a second experiment heliotrope was fed for 12 weeks and copper administration commenced 8 weeks later. The 10 sheep fed heliotrope alone did not show signs of clinical illness but one died and was found to have severe liver damage. Eleven sheep were given copper alone and three developed the clinical signs and lesions of haemolysis. Fourteen sheep were given copper and heliotrope and 13 became ill. Of these, three developed haemolysis, eight became jaundiced and two became weak without developing jaundice. The concentrations of copper in the livers of control and heliotrope-treated sheep, were comparable. In the animals given copper alone, the concentration of copper in the liver was twice as high as that in controls and in those given heliotrope and copper, it was three times as high as in the liver of control sheep. Feeding heliotrope alone induced the histological changes of pyrrolizidine alkaloid toxicity in the liver, but this was not associated with an excessive accumulation of copper or the development of clinical illness. However, it did predispose the animals to the effects of a second toxin since giving heliotrope and copper concurrently, or giving copper subsequent to feeding heliotrope, markedly enhanced the toxicity of the two substances and caused an excessive accumulation of copper in the liver.

Mannoside storage and axonal dystrophy in sensory neurones of swainsonine-treated rats: Morphogenesis of lesions

SummaryYoung rats were treated with swainsonine for up to 200 days at a dose rate that restricted neuronal mannoside storage to neurones not protected by the blood/brain barrier. In lumbar dorsal root ganglion neurones, mannoside storage in the cell body developed in parallel to dystrophic changes at the extremities of peripherally and centrally directed axons. The dystrophic process involved the accumulation of autophagic structures. In the CNS, axonal dystrophy was confined to areas receiving long processes from affected neurones. The results suggest that axonal dystrophy is a direct consequence of the lysosomal storage process in parent cell bodies. The possible relationship of axonal dystrophy to neuronal lysosomal function is discussed.

Dianellidin, stypandrol and dianellinone: An oxidation-related series from dianella revoluta

Dianellidin, stypandrol and dianellinone were isolated from Dianella revoluta and were considered with respect to their biosynthetic relationship and to the toxicity of Stypandra imbricata.

Bovine generalised glycogenosis type II: Uptake of lysosomal α-glucosidase by cultured skeletal muscle and reversal of glycogen accumulation

Glycogenosis type II a‐Glucosidase Enzyme uptake Pompes disease Cultured muscle

Natural bone marrow transplantation in cattle with Pompe's disease

Adding acid alpha-glucosidase to cultures of Pompes disease muscle has resulted in enzyme uptake and reduction in concentration of glycogen. However, bone marrow transplantation has been unsuccessful as a treatment. Immune rejection may have contributed to this failure. Twin calves share a placenta and carry lymphoreticular cells of each others type, they become lymphoreticular chimeras in utero and immune rejection does not occur. One natural and three sets of twins produced by embryo transfer were studied in Pompes disease cattle. Chimerism persisted throughout life and the situation was analogous to a transplant of histocompatible bone marrow stem cells. The activity of acid alpha-glucosidase in leucocytes and in biopsies of the semitendinosus muscle and the mean activity in diaphragm, spleen and lymph node obtained after death from affected twins were significantly higher than in single affected calves. Glycogen concentration was lowered in liver, spleen and lymph node but not in muscles. The affected twins showed clinical signs and changes in muscle similar to those seen in affected single calves. It is concluded that bone marrow transplantation is unlikely to be a successful treatment for Pompes disease.

Bovine glycogenosis type II: The molecular defect in shorthorn cattle

The molecular defect in Shorthorn cattle affected with glycogenosis type II was studied. Polyclonal and monoclonal antibodies specific for bovine skeletal muscle acid alpha-glucosidase were raised and used to study the molecular and biochemical defect in seven affected animals. Cultured normal bovine fibroblasts pulsed and chased with [3H] leucine produced a 130 kDa precursor form of acid alpha-glucosidase which was processed via several 100 kDa intermediate forms to the 65 kDa mature form within 26 h. Fibroblasts from affected animals were labelled in vitro and were shown to produce a cross-reactive protein which was identified as the precursor form of the enzyme. The mature form of the enzyme was not found. The precursor form of the enzyme was demonstrated in Western blots of muscle tissue extracts from affected animals. Glycogenosis type II in Shorthorn and Brahman cattle must be caused by a different, independent maturation, since Brahman cattle lack the cross-reactive protein for acid alpha-glucosidase.

BOVINE GLYCOGENOSIS TYPE II. BIOCHEMICAL AND MORPHOLOGICAL CHARACTERISTICS OF SKELETAL MUSCLE IN CULTURE

The biochemical and morphological properties of cultured skeletal muscle from calves born into a herd of cattle, which are heterozygous for glycogenosis type II, were studied over 17 days. Muscle was cultured by a modification of the explant technique in which the mononucleated cells that grew from the explants were subcultured. Skeletal muscle from animals up to 15 months of age grew in culture to produce mature, cross‐striated and spontaneously contractile myotubes. The creatine kinase activity was 310 (±45.4) mU/mg protein on day 7 when fusion was complete, and 210 (±15.1) mU/mg protein on day 17 of culture. Mature muscle cultures from animals affected by glycogenosis type II showed the characteristic biochemical and morphological abnormalities previously observed in vivo. Acid α‐glucosidase activity was absent whereas the activities of neutral α‐glucosidase, lysosomal a‐mannosidase and creatine kinase were the same as in cultures of unaffected muscle. The concentration of glycogen was higher in cultured affected muscle than in cultured unaffected muscle. On days 7, 9 and 17 of culture the glycogen concentrations were 66.7 (±2.7), 89.0 (±5.5) and 120.3 (±34.2) μg/mg protein respectively in affected muscle and 51.8 (±3.6), 59.9 (±5.4) and 55.4 (±1.0) μg/mg protein respectively in non‐affected muscle. Electron microscopic studies showed that the glycogen accumulated within the lysosomes. These results indicate that bovine glycogenosis type II is expressed in tissue culture since the cultured skeletal muscle from affected animals shows the same abnormalities as skeletal muscle in vivo.