Lysosomal hydrolases in macrophages exposed to swainsonine.

Swainsonine reversibly inhibits macrophage lysosomal acid alpha-mannosidase in vitro. When supplied to cultured cells for periods of up to 24 h, swainsonine penetrates the cells and produces a dose- and time-dependent inhibition of cellular alpha-mannosidase. Exposure of macrophages to swainsonine for 24 h, followed by continued incubation in the absence of this agent, produces elevated cellular activity of alpha-mannosidase, relative to unexposed controls; prolonged incubation of macrophage cultures with swainsonine for 1-2 weeks results also in significant increases in cell protein, lactate dehydrogenase activity and in that of another lysosomal enzyme, beta-hexosaminidase.

Cardiomyopathy in generalised glycogenosis type II in cattle.

The clinical, electrocardiographic and postmortem findings in five cattle affected by generalised glycogenosis type II are described. Three of the affected animals showed generalised muscle weakness for some months before being killed at 11, 15 and 16 months of age. Of the remaining two, one developed severe respiratory distress when 3 months old and died within 6 h of first being noticed to be ill. The other animal showed respiratory distress on exertion at 5 months of age, became recumbent and died when 7 months old. The sum of the QRS complex amplitudes in ECG leads I, II, a VR, aVL and aVF of the affected animals was significantly increased from the control or carrier animals, but only the two affected animals which showed clinical and pathological signs of congestive heart failure had increased heart weight to body weight, left and right ventricular weight to body weight ratios. In view of the lack of correlation between the increased QRS amplitudes and the presence of cardiac enlargement, it is suggested that the increased QRS amplitudes are a reflection of a conduction disorder. It is further suggested that QRS complex abnormalities in generalised glycogenosis in man, particularly in the late onset form may be due to a similar phenomenon.

Specific inhibition of human beta-D-glucuronidase and alpha-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 beta-D-glucuronidase and alpha-L-iduronidase. No other glycosidases are inhibited. The inhibition of beta-D-glucuronidase is competitive, with a Ki of 8 X 10(-5) M and is pH-dependent. This inhibitor may be useful to induce a mucopolysaccharidosis or to investigate the function of microsomal beta-D-glucuronidase.

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

acid alpha-glucosidase (EC 3.2.1.20) was purified from fetal bovine muscle by affinity chromatography on concanavalin A and Sephadex G-100 and added to the culture medium of mature muscle cultures from animals affected by glycogenosis type II. The enzyme activity in homogenates of treated cultures was significantly increased within 4 h of the addition of enzyme, was maximal by 18 h and the internalised activity was stable for at least 48 h after the removal of the enzyme from the culture medium. The acid alpha-glucosidase activity was internalised with an uptake constant of 300 nM and a Vmax of uptake of 133 nmol/h per mg protein. The glycogen concentration in affected cultures treated with exogenous acid alpha-glucosidase for 24 h had decreased by 20% and after a further 24 h of culture was comparable to the concentration observed in cultures from non-affected animals.

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 alpha-D-mannosidase II as well as lysosomal alpha-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.

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.

Natural bone marrow transplantation in cattle with Pompe's disease.

Adding acid alpha-glucosidase to cultures of Pompe's 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 other's 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 Pompe's 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 Pompe's disease.

Alpha-naphthyl-isothiocyanate-induced cholestasis in the rat: studies of liver plasma membrane enzymes.

Oral administration of a single dose of alpha-naphthyl-isothiocyanate (ANIT) to rats produced a conjugated hyperbilirubinaemia, maximal at 2 days and which subsided by 7. The activities of 3 liver plasma membrane enzymes, Mg-2+-ATPase, (Na-+-K-+)-ATPase and 5-nucleotidase, and serum bilirubin levels were studied for up to 7 days after treatment. Activities of the 3 enzymes were significantly decreased at 2 days after treatment and returned to normal by 7, thus varying inversely with the degree of hyperbilirubinaemia. Enzyme histochemistry used to demonstrate canalicular localization of Mg-2+-ATPase in sections of whole liver and of isolated plasma membrane pellets showed that the reduction in activity was not a uniform partial loss, but represented a range of reductions in most canaliculi with a few retaining normal staining intensity. The results suggest that after ANIT intoxication there is a membrane lesion which may be responsible for the observed hyperbilirubinaemia due to the failure of secretion of biliary constituents into the canaliculus. However, more direct studies are necessary to determine whether any one of these enzymes is directly involved in the transport of biliary constituents across the bile canalicular membrane.

Effect of the ingestion of heliotrope and copper on the concentration of zinc, selenium and molybdenum in the liver of sheep.

Merino wethers were used to examine the effect of feeding heliotrope, with and without additional copper (Cu), on the development of clinical signs, lesions in the liver and concentration in the liver of Cu, zinc (Zn), selenium (Se) and molybdenum (Mo). The feeding of heliotrope reduced liver concentrations of Zn (P < 0.05) and Mo (P < 0.01), but Cu ingestion had no effect on these parameters. The concentration of Se in the liver was only increased when heliotrope was given with copper (P < 0.01); it had previously been shown that Cu and heliotrope ingestion is followed by an interaction which results in a marked increase in the concentration of Cu and the production of extensive lesions in the liver. The increase in liver Se may therefore have represented a passive or active response to liver necrosis, whereas effects on Zn and Mo represented specific metabolic disturbances attributable to heliotrope feeding.

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.

Swainsonine toxicosis suppresses appetite and retards growth in weanling rats.

Two groups of weanling rats were treated with swainsonine, the toxin responsible for 'pea-struck' and locoism in grazing animals, for 21 days. The initial dose rate was 46 mg kg-1 d-1 in one group, and 7.6 mg kg-1 d-1 in the other. Food and water intake, urinary volume and bodyweight gain were recorded for each rat and compared with those for pair-fed and ad libitum fed control individuals. At both dose rates, swainsonine caused marked retardation of growth consequent to profound suppression of appetite. In intoxicated rats, intake of water was also diminished.

The effect of copper and heliotrope on the composition of bile in sheep.

The effects of interrupting the enterohepatic circulation (EHC) of bile salts for seven hours and of feeding copper and heliotrope alone and combined for 13 weeks, on bile flow and excretion of copper, zinc, iron and alpha-mannosidase were studied in sheep. Interruption of EHC reduced bile flow rate and increased the concentration of copper, zinc, iron and bile acids while alpha-mannosidase's activity remained stable. Changes in concentration were related to changes in bile volume for copper and zinc only. Total output per hour was not changed. Biliary concentration of copper correlated with alpha-mannosidase's activity in control sheep and those given copper or heliotrope, supporting the hypothesis that lysosomes are involved in biliary secretion of copper in sheep. Increasing the intake of copper increased the rate of excretion of copper in bile. Copper output was lower when heliotrope was fed alone.

Suspected ergotism in two heifers.

A syndrome similar to gangrenous ergotism was seen in 2 Friesian heifers grazing meadow hay containing perennial rye grass seed heads parasited by an ergot presumed to be Claviceps purpurea. Clinical signs were bilateral hind limb lameness and gangrene. There was angiographic evidence of vasoconstriction in the lower hind limbs with necrosis of all tissues in the distal region of both hind limbs.

Identification of galegine, an isoprenyl guanidine, as the toxic principle of Schoenus asperocarpus (poison sedge).

An isoprenyl guanidine, galegine, was isolated from the Western Australian sedge Schoenus asperocarpus (Cyperaceae). Synthetic galegine was shown to reproduce the clinical and pathological features of poisoning by this plant. Preliminary results suggest that the massive thoracic effusion observed in sedge poisoning is the result of a direct effect on pulmonary vascular permeability.

Stypandra imbricata ("blindgrass") toxicosis in goats and sheep - clinical and pathologic findings in 4 field cases.

Two goats and 2 sheep from field outbreaks of Stypandra imbricata toxicosis had severe lesions in the retina, optic nerves and white matter of the central nervous system. Clinical, ophthalmoscopic, fluorescein angiographic, electroretinographic and histologic atrophy, necrosis and degeneration of axons in the optic nerves and optic tracts and status spongiosus of cerebral white matter.