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Biomedical subjects

B G Winchester

Publications and source records attributed to B G Winchester.

At least 19 recordsLinked to original sources
PubMed1995

Late-infantile Batten disease: purification of the subunit c of the mitochondrial ATP synthase from storage material.

The accumulation of subunit c of the mitochondrial ATP synthase in late-infantile neuronal lipofuscinosis (LINCL) and juvenile neuronal lipofuscinosis (JNCL) is well documented. The purification of the subunit from diverse sources has been reported previously, although not from the brain of Batten disease patients. This proteolipid has now been purified from late-infantile Batten disease brain. The procedures used were an original combination of the conventional solubilisation, differential centrifugation, organic solvent extractions, preparative gel electrophoresis, and FPLC. Gel filtration of the purified protein indicated molecular mass equal to or greater than 2 x 10(6) Da; however, electrophoresis of this pure protein suggested a molecular mass of approximately 3,500 Da, which is a characteristic of subunit c. The pure protein may be solubilised in aqueous buffer containing < 1% lithium dodecyl sulphate (LDS). The protein binds dicyclohexylcarbodiimide (DCCD) and shows immunoreactivity to antibodies raised against ovine storage bodies.

Blotting, Western↗
PubMed1995

A new form of ovine GM1-gangliosidosis.

Neurological signs were observed in 3 lambs at approximately 1 month of age, in a flock of 1 ram and 29 ewes with 43 lambs. Deterioration occurred such that the lambs had either died or been killed by 4 months of age. Necropsies of two of these lambs revealed a diffuse encephalopathy in which the most prominent feature was ballooned neurons. Sections of frozen brain showed PAS-positive, oil red O-negative, and weak Sudan Black-positive material in the swollen neuronal cytoplasm. The ultrastructure of the neuronal inclusions showed characteristic whorled membranes, suggesting diagnosis of a gangliosidosis. The underlying enzymic defect was investigated by assaying 11 lysosomal enzymes in extracts of kidney from an affected lamb and from normal lambs. A deficiency (90%) of acidic beta-D-galactosidase was found in the affected lamb. All other activities, including N-acetylneuraminidase, were normal. A specific deficiency of lysosomal beta-D-galactosidase was demonstrated by separating the lysosomal and cytosolic beta-D-galactosidase by chromatography on concanavalin A-Sepharose. Diagnosis of GM1-gangliosidosis, analogous to the severe infantile form of the human disease, was made on the basis of the pathology and enzymology. The beta-D-galactosidase activity in the white blood cells of the ram and several of the ewes was consistent with their being heterozygotes. This disorder is different from a previously described lipidosis in sheep, in which there was a combined deficiency of beta-D-galactosidase and alpha-neuraminidase.

Animals↗
PubMed1993

Sequence variations in the first exon of alpha-galactosidase A.

The alpha-galactosidase A gene (GALA), which is deficient in males with Anderson-Fabry disease, is shown to be remarkably polymorphic in the 5' untranslated region. GALA contains seven exons. The first exon contains 60 bp of 5' untranslated sequence before the methionine initiation codon. Single strand conformation polymorphism (SSCP) screening has shown three polymorphic variants from the published sequence within the 60 base pairs. The sequence changes involved are C to T at -10, G to A at -12 (which removes an MspI site), and G to A at -30 (which removes a SacII site). The combined frequency of these is 10%. A further insertion-deletion polymorphism is detected by SSCP of a 400 bp fragment including exon 3. Both polymorphisms can be easily detected using small polyacrylamide gels and ethidium bromide staining. Nine of 20 women were informative for one of these polymorphisms and this simple SSCP analysis should be of great assistance in family studies of Anderson-Fabry disease. Such a high level of polymorphism has not been previously reported in the 5' untranslated region of a human gene and is unusual in any such short stretch of DNA.

Base Sequence↗
PubMed1992

Substrate specificity of the bovine and feline neutral alpha-mannosidases.

Neutral alpha-mannosidases were prepared from bovine and cat liver. The activities were distinguished from lysosomal and Golgi alpha-mannosidases by their neutral pH optima, relatively low Km for their synthetic substrate p-nitrophenyl alpha-D-mannoside, inhibition by Zn2+ and absence of inhibition by Co2+, EDTA, low concentrations of swainsonine, or deoxymannojirimycin. The cytosolic alpha-mannosidases were not retained by concanavalin A-Sepharose. They were able to degrade efficiently a variety of oligosaccharides with structures corresponding to certain high-mannose glycans or the oligomannosyl parts of hybrid and complex glycans. However, unlike lysosomal alpha-mannosidases from the same species these enzymes were not able to degrade Man9GlcNAc2 efficiently, and the bovine neutral alpha-mannosidase was not able to degrade a hexasaccharide with a structure analogous to Man5GlcNAc2-PP-dolichol. Sharp differences were noted for the bovine and cat enzymes with regard to the specificity of degradation. The bovine neutral alpha-mannosidase degraded the substrates by defined pathways, but the cat neutral alpha-mannosidase often produced complex mixtures of products, especially from the larger oligosaccharides. Therefore the bovine enzyme resembled the rat and human cytosolic alpha-mannosidases, but the cat enzyme did not. The bovine and cat neutral alpha-mannosidases, unlike the corresponding lysosomal activities, did not show specificity for the hydrolysis of the (1----3)- and (1----6)-linked mannose residues in the N-linked glycan pentasaccharide core.

Animals↗
PubMed1992

Hypertrophic obstructive cardiomyopathy in a neonate with the carbohydrate-deficient glycoprotein syndrome.

The carbohydrate-deficient glycoprotein (CDG) syndrome in its most severe form (neonatal olivopontocerebellar atrophy) is a life-threatening multisystem disease. We report a neonate who was referred for cardiological assessment because of respiratory distress, a murmur and episodes of desaturation. After initial spontaneous improvement he presented at 9 weeks with evidence of a severe hypertrophic obstructive cardiomyopathy (HOCM). The diagnosis of CDG syndrome was suggested by the characteristic dysmorphic features, hypotonia, visual inattention and severe failure to thrive; it was confirmed by electrophoresis of serum transferrin. HOCM can be a feature of the CDG syndrome, in addition to the (previously reported) pericardial effusions.

Blood Protein Electrophoresis↗
PubMed1991

The substrate specificity of bovine and feline lysosomal alpha-D-mannosidases in relation to alpha-mannosidosis.

Lysosomal alpha-mannosidases were partially purified from bovine and feline liver and employed to digest a large number of oligosaccharides with structures corresponding to the oligomannosyl parts of complex, hybrid, and high-mannose glycans. The incubation products were identified by high pressure liquid chromatography with reference compounds of defined structure and by acetolysis. For all classes of substrates, the lysosomal alpha-mannosidases displayed a high degree of in vitro specificity with regard to the hydrolysis of mannose residues. Thus, in each case, 1 or at most 2 residues were always preferentially cleaved so that the degradative process proceeded down a well defined pathway. A comparison of the relative efficiency with which lysosomal alpha-mannosidases catalyzed the hydrolysis of particular oligosaccharides and of the structures of the resulting intermediates with those of the compounds accumulated in alpha-mannosidosis allows conclusions to be drawn regarding the nature of the enzymatic defect. In bovine alpha-mannosidosis, the oligosaccharides are those expected for a partial deficiency of normal lysosomal alpha-mannosidase, so that they correspond to intermediates in the normal catabolic pathway. In feline alpha-mannosidosis, in which the alpha-mannosidase deficiency is more severe than in cattle, the accumulated oligosaccharides primarily represent intact oligomannosyl moieties of N-linked glycans rather than the products of residual alpha-mannosidase activity.

Animals↗
PubMed1990

The structural basis of the inhibition of human glycosidases by castanospermine analogues.

A series of epimers and deoxy derivatives of castanospermine has been synthesized to investigate the contribution of the different chiral centres to the specificity and potency of inhibition of human liver glycosidases. Castanospermine inhibits all forms of alpha- and beta-D-glucosidases, but alteration to any of the five chiral centres in castanospermine markedly decreases the inhibition. 6-Epicastanospermine, which is related to D-pyranomannose in the same way as castanospermine is to D-pyranoglucose, does not inhibit lysosomal (acidic) alpha-mannosidase, but is a good inhibitor of the cytosolic or neutral alpha-mannosidase. Conversely, 1-deoxy-6-epicastanospermine inhibits acidic alpha-mannosidase strongly, but not the neutral alpha-mannosidase. An explanation of this different inhibition based on preferential recognition of different configurations of mannose by the different forms of alpha-mannosidase is postulated. All derivatives of 6-epicastanospermine also have the minimum structural feature for the inhibition of alpha-L-fucosidase, but those with a beta-anomeric substituent do not inhibit the enzyme, or do so very weakly. 1-Deoxy-6,8a-diepicastanospermine, which has four chiral centres identical with alpha-L-fucose, is, however, a potent inhibitor of alpha-L-fucosidase (Ki 1.3 microM).

Alkaloids↗
PubMed1986

GM1 gangliosidosis (type 1) in a cat.

A kitten with clinical and morphological symptoms of a neurovisceral lysosomal-storage disease has been shown to have a marked deficiency of acidic beta-D-galactosidase in the brain, kidney and spleen. Chromatography on concanavalin A-Sepharose and inhibition studies with 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, a selective inhibitor of the neutral broad-specificity beta-D-galactosidase, have shown that the residual beta-D-galactosidase at pH 4.0 in the tissues of the affected cat is due to the neutral beta-D-galactosidase and that there is a complete deficiency of the acidic (lysosomal) beta-D-galactosidase. There is marked accumulation in all tissues and excretion in the urine of neutral oligosaccharides. Analysis of these oligosaccharides by fast-atom-bombardment mass spectrometry and g.l.c. suggests that they arise from the incomplete catabolism of N-glycans of glycoproteins. The ganglioside content of all the tissues is elevated, and it has been shown by t.l.c. that the concentration of a ganglioside fraction with a mobility similar to that of GM1 ganglioside is particularly increased. There is also some evidence of accumulation of glycosaminoglycans in the brain. The clinical symptoms, the complete deficiency of acidic beta-D-galactosidase and the storage products in visceral organs all suggest that this is a case of feline GM1-type gangliosidosis comparable with the severe infantile (Type 1) form of the disease in humans.

Alkaloids↗
PubMed1983

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.

Alkaloids↗
PubMed1980

Biochemical studies on a case of feline mannosidosis.

Evidence is presented for the biochemical diagnosis of the first case of feline mannosidosis. A marked deficiency of acidic alpha-D-mannosidase in the brain, kidney and liver and excessive excretion of mannose-rich oligosaccharides in the urine were found in a kitten suffering from a nervous disorder. Residual acidic alpha-D-mannosidase, ranging from 2 to 5.5% of the normal activity, was observed in the tissues of the affected kitten. It has similar kinetic and physicochemical properties to the normal activity. The amount of mannose in the urine of the affected kitten was 19-fold greater than in a comparable control, and the molar ratio of mannose to N-acetylglucosamine was approx. 6 : 1. High concentrations of neutral oligosaccharides were detected in the urine. The predominant oligosaccharide appeared to be a hexasaccharide. The biochemical features of bovine, feline and human mannosidosis are compared, and it is concluded that feline mannosidosis may be a useful animal model for studying the human disease.

Animals↗
PubMed1980

Comparison of the alpha-mannosidases in fibroblast cultures from patients with mannosidosis and mucolipidosis II and from controls.

The intracellular and extracellular acidic alpha-mannosidase in cultures of fibroblasts from mucolipidosis-II patients has normal kinetics. The extracellular activity in cultures of cells from mannosidosis patients is normal, but a mutant enzyme is associated with the cell surface and intracellular fraction. The results support the involvement of membrane cycling and recognition markers in lysosomal enzyme localization.

Cells, Cultured↗
PubMed1978

Characterization of the mutant alpha-mannosidase in bovine mannosidosis.

Residual acidic alpha-mannosidase, varying in amount up to approx. 15% of normal values, can be measured in various organs of a calf with mannosidosis. The highest specific activity and relative proportion of residual activity were found in the liver. Chromatography on DEAE-cellulose showed that the residual activity was associated with two components, which were eluted at comparable positions with those found in normal tissues. The residual activity had a lower thermal stability and a higher K(m) value for a synthetic substrate than did the normal enzyme. No differences in molecular weight or electrophoretic mobility between normal acidic alpha-mannosidase and the residual activity were observed by gel filtration and electrophoresis on cellulose acetate respectively. The isoelectric focusing profiles for the alpha-mannosidase in the normal and pathological livers were very similar. It is suggested that a mutant enzyme, resulting from a mutation in a structural gene, accounts for the residual acidic alpha-mannosidase in mannosidosis. The mutant enzyme, which cross-reacts with antiserum raised against normal bovine acidic alpha-mannosidase, is present at a decreased concentration compared with the normal enzyme. There is a correlation between the concentrations of residual activity and cross-reacting material in mannosidosis. alpha-Mannosidase with a pH optimum of 5.75 and which is activated by Zn(2+) was also detected in the liver of the calf with mannosidosis. However, it is probably not a product of the defective gene because addition of Zn(2+) indicated that it was also present in normal tissues.

Animals↗