Structural studies of urinary oligosaccharides from patients with mannosidosis.
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Adult↗
Biomedical subjects
G A Grabowski
Publications and source records attributed to G A Grabowski.
Advances in the treatment of inherited metabolic diseases.
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Blood Transfusion↗
Chronic GM2 gangliosidosis masquerading as atypical Friedreich ataxia: clinical, morphologic, and biochemical studies of nine cases.
A progressive spinocerebellar degenerative disorder was characterized in nine patients, aged 11 to 37 years, from four unrelated Ashkenazi Jewish families; affected individuals had markedly deficient beta-hexosaminidase A activity. Symptoms included early onset of cerebellar signs (tremor, incoordination, and dysarthia) and, with maturity, the development of upper and lower motor neuron disorders, marked dysarthia, and ataxia. Three older patients, aged 26, 32, and 37 years, had dementia or recurrent psychotic episodes. Membrane-bound lamellar cytoplasmic inclusions, consistent with lysosomal ganglioside accumulation, were observed in rectal ganglia. The activity of beta-hexosaminidase A was markedly deficient in all sources analyzed. Parents had activities consistent with heterozygosity, confirming autosomal-recessive transmission of the beta-hexosaminidase A-deficient gene and the adult variant disorder. Residual beta-hexosaminidase A activity, partially purified by anion-exchange chromatography from cultured skin fibroblasts of the affected individuals, was heat-labile and co-electrophoresed with normal beta-hexosaminidase A. These findings suggest that these patients were allelic for a new beta-hexosaminidase A mutation and may represent a genetic compound of this allele and the allele causing Tay-Sachs disease.
Adolescent↗
The M-mode echocardiogram in Fabry's disease.
Fabry's disease results from deficient activity of the enzyme alpha-galactosidase A. Cardiac abnormalities result from glycosphingolipid deposition in the myocardium, valvular tissue, and vessel walls. A noninvasive method to examine these abnormalities would be useful in the evaluation of patients. We examined the echocardiograms of 32 patients, 25 hemizygous and seven heterozygous, for Fabry's disease. The aortic root diameter was measured in each hemizygote. In nine patients under 26 years it was 33.2 +/- 1.2 mm. and in two it was dilated. In 16 patients over 26 years it was 38.8 +/- 1.2 mm. (p < 0.01), and in 12 it was dilated. The left ventricular posterior wall was measured in the echocardiogram of 21 normotensive hemizygotes. The difference in thickness between nine patients under 26 years (9.6 +/- 1.3 mm.) and 12 patients over 26 years (12.4 +/- .07 mm.) was not statistically significant. Only two of the nine younger patients had left ventricular wall thickness greater than normal compared to eight of the 12 older patients. The mean left ventricular shortening fraction of 22 hemizygous patients was normal. One hemizygote had echocardiographic evidence of mitral valve prolapse. Four of the seven heterozygotes had normal echocardiograms. Among the other three, one had increased left ventricular wall thickness, a second had disproportionate ventricular septal thickness, and a third had both abnormalities. The echocardiographic aortic root size was normal in each heterozygote. Abnormal echocardiographic findings were more common in older hemizygous patients, a distribution similar to that of the age of onset of cardiac dysfunction. Increased left ventricular wall thickness probably reflects glycosphingolipid deposition in the myocardium. Dilatation of the aortic root may result from degenerative changes of the aortic media. Abnormalities of mitral valve echoes were uncommon.
Adolescent↗
Comparative physical, kinetic and immunologic properties of the acidic and neutral alpha-D-mannosidase isozymes from human liver.
Human alpha-mannosidases A, B and C were partially purified from liver by conventional and affinity chromatographic procedures. The kinetic, physical and immunologic properties of the A, B and C isozymes were determined including pH optima, Km, effects of various inhibitors and activators, thermal stabilities, electrophoretic mobilities, native molecular weights and antigenic relationships. By gel filtration, the apparent native molecular weights were 240,000, 300,000 and 420,000 for the A, B and C isozymes, respectively. By polyacrylamide gel electrophoresis, the A and B isozymes had native molecular weights of approximately 150,000 and appeared to be charge isomers. Antibodies to the purified A and B isozymes were produced; cross-reactivity between the A and B isozymes against anti-A and anti-B immune sera were observed by double immunodiffusion and immunoprecipitation. In contrast, no reactivity of the C isozyme was detected with either immune serum. Based on these studies, models are presented for the molecular interrelationship of alpha-mannosidases A and B.
Electrophoresis, Polyacrylamide Gel↗
Enzyme therapy XVII: metabolic and immunologic evaluation of alpha- galactosidase A replacement in Fabry disease.
A pilot trial of enzyme replacement using splenic and plasma forms of alpha-galactosidase A was undertaken in 2 brothers with Fabry disease, an X-linked glycosphingolipid storage disease. Partially purified preparations of alpha-galactosidase A from human spleen and plasma Cohn fraction IV-1 were prepared aseptically for in vivo administration. The disappearance of enzymatic activity from plasma, levels of circulating substrate, and potential immune response were evaluated following IV administration of 6 unentrapped doses (2,000 U/kg) of each enzyme form to the respective recipient during a 117-day period. Repeated injections were well tolerated. The circulating half-life of the splenic form was about 10 min whereas that for the plasma form was approximately 70 min. No immune response was detected by skin and immunodiffusion tests or by alterations in the maximal activity or clearance kinetics for either enzyme following successive administrations. After each dose of the splenic form, the concentration of the accumulated circulating substrate globotriaosylceramide, decreased maximally (approximately 50% of initial values) in 15 min and returned to preinfusion levels by 2-3 hr. In marked contrast, injection of the plasma form decreased the circulating substrate levels 50-70% by 2-6 hr; the concentrations of globotriaosylceramide gradually returned to preinfusion values by 36-72 hr. Two consecutive doses of the plasma form, administered on days 1 and 3, reduced the circulating substrate concentration to normal levels. Prior to the 6th enzyme administration, circulating substrate was stable-isotope labeled by the infusion of dideutero-glucose, and the effects of each enzyme form on circulating substrate degradation and reaccumulation were determined. The results of this study indicated that labeled (newly synthesized) substrate reaccumulated following injection of the splenic enzyme whereas both unlabeled (previously stored?) and labeled substrate reaccumulated in the circulation after administration of the plasma form. These studies demonstrated the differential disappearance kinetics of the splenic and plasma forms of alpha-galactosidase A, their differential effects on circulating substrate degradation and reaccumulation, as well as the lack of an immune response to repeated administrations of these homologous, unentrapped enzymes.
Adult↗
Human mannosidosis: in vitro and in vivo studies of cofactor supplementation.
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Adolescent↗
Evidence for carrier proteins in bile acid synthesis. The effect of squalene and sterol carrier protein and albumin on the activity of 12alpha-hydroxylase.
The possibility that carrier proteins are involved in bile acid synthesis was investigated using rat liver homogenates. The 105 000 X g supernatant fraction was found to contain heat stable proteins that bound the bile acid precursor, 7alpha-hydroxy-4-cholesten-3-one, and increased the amount of 7alpha, 12alpha-dihydroxy-4-cholesten-3-one formed by the microsomal enzyme, 12alpha-hydroxylase. Subsequent studies were carried out to determine if squalene and sterol carrier protein or albumin, two lipid binding proteins present in the 105 00 X g supernatant fraction of rat liver homogenates, may be responsible for the effects seen with this fraction. Squalene and sterol carrier protein bound several water insoluble bile acid precursors, including 7alpha-hydroxy-4-cholesten-3-one, and increased the apparent activity of 12alpha-hydroxylase. Squalene and sterol carrier protein, however, did not bind either cholic acid or chenodeoxycholic acid. Rat serum albumin also bound 7alpha-hydroxy-4-cholesten-3-one and increased the apparent activity of 12alpha-hydroxylase. Kinetic analysis indicated that the apparent stimulation of 12alpha-hydroxylase by squalene and sterol carrier protein and albumin was due to increased solubilization of the substrate, 7alpha-hydroxy-4-cholesten-3-one. Thus, these studies indicate that bile acid precursor carrier proteins are present in the 105 000 Xg supernatant fraction of rat liver homogenates and suggest that squalene and sterol carrier protein or albumin may participate as carrier proteins in bile acid synthesis.
Animals↗
Mannosidosis: clinical, morphologic, immunologic, and biochemical studies.
The primary metabolic defect in mannosidosis is the deficiency of the acidic alpha-mannosidase A and B activites which results in the lysosomal accumulation of mannose-rich substrates. Out studies demonstrate that the enzymatic diagnosis of suspect homozygotes can be made reliably using plasma, isolated leukocytes, or cultured skin fibroblasts assayed carefully at the appropriate acidic pH. Immunologic studies of a mannosidosis homozygote revealed significant abnormalities of neutrophil function; these included a depressed chemotactic responsiveness and impaired phagocytosis of bacteria. Lymphocyte transformation studies showed a 20% of normal response to purified phytohemagglutinin and a 25% of normal response to concanavalin A. Three major components of alpha-mannosidase activity in normal human liver were resolved by ion exchange chromatography on DEAE-cellulose and electrophoresis on cellulose acetate gels. Electrophoresis of the liver extract from homozygote I with mannosidosis revealed only one band of activity which coelectrophoresed with the alpha-mannosidase C isozyme partially purified from normal liver. However, ion exchange chromatography revealed the presence of residual hepatic acidic activities; the residual A isozyme was eluted in a position corresponding to that of normal alpha-mannosidase A whereas the residual B activity was eluted at a slightly more electronegative position than that of normal B isozyme. The apparent Km values for alpha-mannosidase activity as determined from Linweaver-burk plots were 1.1 mM for normal liver and 0.9 mM for normal leukocytes. In contrast, the residual activity in these sources from homozygote 1 could not be saturated within the solubility range of the substrate; the apparent Km value was estimated at 15.4 mM in liver extracts. Zinc significantly lowered the apparent Km value of the acidic activity in normal liver (from 1.2 to 0.24 mM), whereas this metallic ion had little effect on the values for mannosidosis hepatic activity (from 15.4 to 12.3 mM). Unlike zinc, cobalt had its major effect on the acidic activity in the mannosidosis liver extract, lowering the apparent Km from 15.4 to 3.9 mM, whereas the apparent Km for the normal activity was increased from 1.2 to 1.9 mM. The residual acidic activities were markedly stimulated by zinc in both leukocytes (approximately 300%) and plasma ( approximately 400%) from the homozygotes and to a lesser extent in those sources from normal individuals. In contrast, cobalt enhanced the residual acidic activities in leukocytes (approximately 500%) and plasma (approximately 200%) from the homozygotes while inhibiting these acidic activities (78.9% and 47.7%, respectively) in normal individuals.
Carbohydrate Metabolism, Inborn Errors↗
Heterogeneity of mutations in the acid beta-glucosidase gene of Gaucher disease patients.
Gaucher disease is inherited in an autosomal recessive manner and is the most prevalent lysosomal storage disease. Gaucher disease has marked phenotypic variation and molecular heterogeneity, and several simple and complex alleles of the acid beta-glucosidase gene have been identified as causal to this disease. Certain combinations of alleles have been shown to correlate well with the severity of the disease, but many Gaucher disease patients exist whose disease is not explained by any of the published mutations. This study was undertaken to identify mutant alleles in such incompletely characterized Gaucher disease, in an attempt to find further correlations between clinical phenotype and the presence of acid beta-glucosidase alleles. RNA was isolated from Gaucher cell lines and converted to cDNA, the cDNA was amplified by PCR and cloned, and several clones for each allele were sequenced. Several new singly mutated and multiply mutated alleles were identified, and sequence-specific oligonucleotide hybridization was used to verify the presence of these mutations in the genome of these patients. All newly identified mutations occurred only rarely in the Gaucher disease population, making it difficult to determine whether inheritance of a particular combination of alleles always correlates with the clinical manifestations seen in the test patients. Three of the newly described alleles were single missense mutations in exon 8, one was a single missense mutation in exon 5, and the fifth was a complex allele, comprising a series of different point mutations scattered throughout exons 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS)
Alleles↗
Silicon nephropathy mimicking Fabry's disease.
Percutaneous renal biopsy was performed in a surface coal miner with radiographic and histopathologic pulmonary changes consistent with acute silicolipoproteinosis who developed proteinuria and hematuria. Electron microscopic evaluation of the renal tissue specimen revealed a diffusely thickened glomerular basement membrane, foot process effacement, and dense lamellar inclusions in swollen glomerular epithelial cells, similar to those seen in Fabry's disease. However, normal levels of plasma alpha-galactosidase A, normal urinary sediment glycosphingolipids and the absence of the clinical characteristics of Fabry's disease excluded this diagnosis. This case illustrates that electron-dense lamellar inclusions, similar to those seen in Fabry's disease, may be seen in other entities such as nephropathy associated with silicosis.
Adult↗
Oculomotor abnormalities in chronic GM2 gangliosidosis.
The clinical and electro-oculographic eye abnormalities in chronic GM2 gangliosidosis have been described. The most prominent oculomotor disturbances in our patients involved the pursuit system. This was evident during performance of eye tracking and vestibulo-ocular reflex suppression. Saccadic eye movements were dysmetric but were normal in velocity. These findings point to disturbances of cerebellar oculomotor control. While the oculomotor defects are nonspecific in this chronic form of beta-hexosaminidase A deficiency, they broaden the spectrum of clinical features considered to be typical ocular manifestations in many storage diseases. Disturbances in the oculomotor system may be the only ocular sign of chronic GM2 gangliosidosis.
Adolescent↗