Measurements from normal umbilical cord blood of four lysosomal enzymatic activities: alpha-L-iduronidase (Hurler), galactocerebrosidase (globoid cell leukodystrophy), arylsulfatase A (metachromatic leukodystrophy), arylsulfatase B (Maroteaux-Lamy).
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Umbilical cord blood (UCB) has received increasing attention as a source of unrelated hematopoietic stem cells for transplantation. Lysosomal diseases have been effectively treated and normal enzymatic activity has occurred subsequent to engraftment using UCB. The use of donor cells with normal amounts of enzyme, rather than those from carriers whose level may be 50% or less, is an obvious goal. The frequency of such heterozygotes varies from 1:10 to 1:140 or lower depending upon the disease at issue. We assayed the levels of lysosomal enzymes in normal UCB in random samples as well as those used for transplantation. We measured the following enzymatic activities: alpha-l-iduronidase (Hurler), galactocerebrosidase (globoid cell leuko- dystrophy) and arylsulfatase A (metachromatic leukodystrophy). For the latter, levels of activity in UCB are comparable to those found in adult blood. In the case of arylsulfatase B (Maroteaux-Lamy) a level lower than adult level was found. An informed choice by the transplanting physician based on the activity of the relevant enzyme in the UCB donor will provide a better opportunity for an improved prognosis for more complete correction of the recipient's primary disease. Bone Marrow Transplantation (2000) 25, 541-544. (+info)
Paradoxical influence of acid beta-galactosidase gene dosage on phenotype of the twitcher mouse (genetic galactosylceramidase deficiency).
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We have cross-bred twitcher mice (galactosylceramidase deficiency) and acid beta-galactosidase knockout mice (G(M1) gangliosidosis) and found that the acid beta-galactosidase gene dosage exerts an unexpected and paradoxical influence on the twitcher phenotype. Twitcher mice with an additional complete deficiency of acid beta-galactosidase have the mildest phenotype with the longest lifespan and nearly rescued CNS pathology. In contrast, twitcher mice with a single functional acid beta-galactosidase gene have the most severe disease with the shortest lifespan, despite the fact that G(M1) gangliosidosis carrier mice with an otherwise normal genetic background are phenotypically normal. A significant proportion of these galc(-/-), bgal(+/-) mice clinically develop additional extreme hyper-reactivity and generalized seizures not seen in any other genotypes. Consistent with the clinical seizures, widespread neuronal degeneration is present in the galc(-/-), bgal(+/-) mice, most prominently in the CA3 region of the hippocampus. The double knockout mice show a massive accumulation of lactosylceramide in all tissues. The brain inexplicably contains only a half-normal amount of galactosylceramide, which may account for the mild clinical and pathological phenotype. On the other hand, brain psychosine level is increased in all twitcher mice, but galc(-/-), bgal(+/-) mice show a significantly higher level than other genotypes. The reduced galactosylceramide in the brain of the double knockout mice and the significantly higher psychosine in the brain of the galc(-/-), bgal(+/-) mice cannot readily be explained from the genotypes of these mice. These observations are contrary to the expected outcome of Mendelian autosomal recessive single gene disorders and may also be interpreted as that the acid beta-galactosidase gene functions as a modifier gene for the phenotypic expression of genetic galactosylceramidase deficiency. (+info)
Globoid cell leukodystrophy: the first case with antemortem diagnosis in Japan.
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A Japanese boy was diagnosed as globoid cell leukodystrophy on the basis of a marked decrease in the galactocerebroside beta-galactosidase activity in the leukocytes and the serum when one year and two months old. At autopsy when 1 year and 10 months, microscopic findings were characteristic for those of globoid cell leukodystrophy. Galactocerebroside beta-galactosidase activities of leukocytes and sera of his father and mother were found to be half those of control subjects, thus it suggested the parents being heterozygotes of the disease. (+info)
Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry.
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BACKGROUND: Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside beta-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample. METHODS: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions. RESULTS: The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39-70 nmol. mg(-1). h(-1) compared with 3.7-5.1 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0-6.8 nmol. mg(-1). h(-1) in cell lysates from healthy patients compared with 0.1-0.2 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 microg) for both ASM and GCG. CONCLUSIONS: Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample. (+info)
A mutation in the saposin A domain of the sphingolipid activator protein (prosaposin) gene results in a late-onset, chronic form of globoid cell leukodystrophy in the mouse.
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Sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins derived from a common precursor protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A(-/-) mice developed slowly progressive hind leg paralysis with clinical onset at approximately 2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency. (+info)
Intraventricular administration of recombinant adenovirus to neonatal twitcher mouse leads to clinicopathological improvements.
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Twitcher mouse is a murine model of human globoid cell leukodystrophy (Krabbe disease), which is characterized by a genetic deficiency in galactocerebrosidase (GALC) activity. The nervous system is affected early and severely by demyelination in the white matter. So far, there is no effective treatment for Krabbe disease except bone marrow transplantation (BMT). However, BMT has inherent limitations such as unavailability of donors and graft-versus-host disease. In this study, we injected recombinant adenovirus encoding GALC into the lateral ventricle of twitcher mice at postnatal day 0 (PND 0) and the therapeutic effects were evaluated. Our results showed slight, but significant improvements in motor functions, body weight and twitching and a prolonged life span. In brain, GALC activity was increased to 15% that of normal littermates and psychosine concentration was decreased to 55% that of untreated twitcher mice at PND 15. The number of PAS-positive globoid cells in brain stem was also reduced significantly at PND 35. In contrast, when adenoviruses were injected to the twitcher mice at PND 15, almost no improvements were observed. These results demonstrate that the timing of treatment may be of great importance in Krabbe disease. (+info)
Characterization of the functional domains of galactosylceramide expression factor 1 in MDCK cells.
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We previously reported that GalCer expression factor 1 (GEF-1), a rat homologue of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), induced GalCer expression, morphological changes, and cell growth inhibition in COS-7 cells. In this study, we describe the characterization of GEF-1 in MDCK cells. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as dramatic morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of UDP-galactose:ceramide galactosyltransferase (CGT) increased significantly in MDCK/GEF-1 cells compared with control cells. GEF-1 molecule is composed of four domains; a zinc-finger (Z), a proline-rich (P), a coiled-coil (C), and a proline/glutamine-rich (Q) domain. MDCK cells transfected with various GEF-1 deletion mutants were examined for morphology and for glycolipid expression. MDCK cells transfected with Z-domain deletion mutant (MDCK/PCQ) and those with both Z- and P-domains deletion mutant (MDCK/CQ) were similar to those with a wild-type GEF-1 (MDCK/ZPCQ) in shape, exhibiting fibroblast-like cells, whereas those with the other deletion mutants showed no morphological changes, exhibiting typical epithelial-like cells. On the other hand, MDCK/ZPCQ, MDCK/PCQ, MDCK/CQ, and MDCK/Q cells expressed sulfatide, whereas those with the other deletion mutants that did not include the Q-domain showed neither GalCer nor sulfatide expression. Thus, the correlation between fibroblast-like cells in shape and the glycolipid expression was good in these deletion mutants except MDCK/Q cells, which showed epithelial-like cells, but expressed sulfatide. The glycolipid expression paralleled CGT mRNA levels. Taking these results together, it is suggested that only the Q-domain may be essential for the role of GEF-1 in inducing CGT mRNA, whereas the Q-domain together with the C-domain may be required for the induction of morphological changes in MDCK cells. (+info)
Saposins (sap) A and C activate the degradation of galactosylsphingosine.
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As previously shown for [(3)H-galactosyl]ceramide, the breakdown of [(3)H-galactosyl]sphingosine was reduced in prosaposin-deficient skin fibroblast homogenates. Galactosylsphingosine hydrolysis was also deficient in cell homogenates from Krabbe's disease (beta-galactocerebrosidase-deficient) patients, but not acid beta-galactosidase-deficient patients. Moreover, hydrolysis of galactosylsphingosine in the prosaposin-deficient cell homogenates could be partially restored by adding pure saposin A or C, thereby identifying these saposins as essential facilitators of galactosylsphingosine hydrolysis. By contrast, saposins B and D had little effect on galactosylsphingosine hydrolysis in the prosaposin-deficient cells. The reduced galactosylsphingosine turnover in prosaposin-deficiency suggests that there could be a pathogenetic cerebral accumulation of galactosylsphingosine in this disorder. (+info)