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839 records · Page 47Linked to original sources
PubMed1996

Analysis of a splice-site mutation in the sap-precursor gene of a patient with metachromatic leukodystrophy.

Sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins required for the lysosomal degradation of various sphingolipids with a short oligosaccharide chain by their exohydrolases. Four of the five known activator proteins (sap-A-sap-D), also called "saposins," are derived from a common precursor by proteolytic processing. sap-B stimulates hydrolysis of sulfatides by arylsulfatase A in vivo. Its recessively inherited deficiency results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is caused by a defect of arylsulfatase A. Here we report on a patient with sap-B deficiency. Reverse-transcription-PCR studies on the patient's mRNA revealed the occurrence of two distinct mutant species: one with an in-frame deletion of the first 21 bases of exon 6, the other with a complete in-frame deletion of this exon. The patient was homozygous for the underlying mutation, which was found to be a G-->T transversion within the acceptor splice site between intron e and exon 6, abolishing normal RNA splicing. Allele-specific oligonucleotide hybridization revealed that the parents and both grandfathers of the patient were carriers of this mutation. In order to analyze the fate of the mutant precursor proteins, both abnormal cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments showed that the deletion of 21 bp had no effect on the transport and the maturation of the encoded precursor. All sap forms except sap-B were detectable by immunochemical methods. The cDNA bearing a complete deletion of exon 6 encoded a shortened precursor of only 60 kD, and no mature SAPs were detectable. The carbohydrate chains of this polypeptide were of the high-mannose and hybrid type, indicating no transport of the mutant precursor beyond early Golgi apparatus. An endoplasmic-reticulum localization of this polypeptide was supported by indirect immunofluorescence analysis.

Animals↗
PubMed1977

[Oligozoospermia in Klinefelter's syndrome].

Case report of a 31 year old male with a 47-XXY-Klinefelter-syndrome which showed a maximum of 4,0 million spermatozoa/ml with a motility of up to 51% in his ejaculate. No rise of spermatozoa counts could be observed after 3x25 mg mesterolone/day over 8 weeks.

Adult↗
PubMed1996

Restoration of hexosaminidase A activity in human Tay-Sachs fibroblasts via adenoviral vector-mediated gene transfer.

Tay-Sachs disease (TSD) is a lysosomal storage disease due to hexosaminidase A deficiency caused by mutations in the gene for alpha-chain (Hex alpha). A human Hex alpha cDNA was subcloned into the adenoviral plasmid pAdRSV. Hex alpha. Replication-deficient adenovirus was generated by homologous recombination in 293 cells. Human fibroblasts from a patient suffering from TSD were infected with the recombinant adenovirus. TSD fibroblasts expressing the recombinant alpha-chain had an enzyme activity on the natural substrate ranging from 40 to 84% of the normal. The corrected cells secreted up to 25 times more Hex alpha than control fibroblasts. The Hex alpha encoded by the adenovirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of GM2 ganglioside in TSD fibroblasts.

Adenoviridae↗
PubMed1996

Molecular analysis of a GM2-activator deficiency in two patients with GM2-gangliosidosis AB variant.

Lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (hex A) requires the presence of the GM2 activator protein (GM2AP) as an essential cofactor. A deficiency of the GM2 activator causes the AB variant of GM2 gangliosidosis, a recessively inherited disorder characterized by excessive neuronal accumulation of GM2 and related glycolipids. Two novel mutations in the GM2 activator gene (GM2A) have been identified by the reverse-transcriptase-PCR method--a three-base deletion, AAG262-264, resulting in a deletion of Lys88, and a single-base deletion, A410, that causes a frameshift. The latter results in substitution of 33 amino acids and the loss of another 24 amino acid residues. Both patients are homoallelic for their respective mutations inherited from their parents, who are heteroallelic at the GM2A locus. Although the cultured fibroblasts of both patients produce normal levels of activator mRNA, they lack a lysosomal form of GM2AP. Pulse/chase labeling of cultured fibroblasts of the patients, in presence and absence of brefeldin A, indicates a premature degradation of both--mutant and truncated--GM2APs in the endoplasmic reticulum or Golgi. These results were supported by in vitro translation experiments and expression of the mutated proteins. When the mutated GM2APs were expressed in Escherichia coli, both mature GM2AP forms turned proved to exhibit only residual activities in an in vitro assay.

Adult↗
PubMed1997

[Juvenile GM2 gangliosidosis with progressive spinal muscular atrophy onset].

GM2 gangliosidosis are caused by a beta-hexosaminidase A enzyme deficiency. Mutations in the gene leaving residual enzyme activity give rise to juvenile and adult forms of the disease which have a great clinical heterogeneity. We report three cases which have been considered for some time as Kugelberg-Welander disease. beta-hexosaminidase A was determined with the sulfated synthetic substrate, 4-méthylumbelliferyl-N-acetylglucosamine 6-sulfate (4-MUGS), which allowed the diagnosis. Two of these cases from one family had normal values of hexosaminidase A in serum as found in the B1 variant. Compound mutations were detected. The B1 variants had a classical B1 mutation (G533-->A) and a new mutation located on exon 11. The patient of the second family had the classical mutation of adult GM2 gangliosidosis (Gly269-->Ser) and a new mutation on exon 1, at the initiation codon.

Adult↗
PubMed1997

Turnover of endogenous ceramide in cultured normal and Farber fibroblasts.

De novo synthesis and turnover of endogenous ceramide in cultured skin fibroblasts from patients affected with Farber lipogranulomatosis were studied by biosynthetical labeling of cellular sphingolipids with [14C]serine. The cellular uptake of [14C]serine and incorporation into de novo synthesized ceramide was similar in normal and Farber fibroblasts, with a half life of newly synthesized ceramide of 2.7 h in normal and diseased cells. Newly synthesized ceramide was found to be channeled directly into biosynthesis of complex sphingolipids rather than contributing to the pool of accumulated ceramide in Farber fibroblasts. The degradation of ceramide generated by the catabolism of complex sphingolipids in Farber cells was greatly delayed compared with control fibroblasts, with differences in the amount of radiolabeled cellular ceramide becoming evident after 6 h chase time. Individual Farber cell lines differed from each other in the amount of accumulated ceramide; however, no correlation was found between ceramide accumulation and residual acid ceramidase activity as determined in vitro. In addition, the amount of radiolabeled sphingomyelin was significantly increased in Farber fibroblasts suggesting a delayed degradation of this compound in this ceramide storage disorder. We propose biosynthetical labeling of endogenous ceramide with [14C]serine, in addition to other established methods, as a highly sensitive and reliable method for the diagnosis of Farber disease, allowing semiquantitative measurement of ceramide accumulation in cultured skin fibroblasts of patients affected with Farber lipogranulomatosis.

Acid Ceramidase↗
PubMed1998

Processing of sphingolipid activator proteins and the topology of lysosomal digestion.

Plasma membrane derived glycosphingolipids (GSLs) destined for digestion are internalized through the endocytic pathway and delivered to the lysosomes. There, GSLs are degraded by the action of exohydrolases, which are supported, in the case of GSLs with short oligosaccharide chains, by sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins) are synthesized from a single precursor protein (pSAP). Intracellular routing of pSAP and of the GM2 activator protein is only in part dependent on mannose-6-phosphate residues. Their endocytosis occurs in a carbohydrate-independent manner. The inherited deficiencies of individual activators, the GM2 activator, SAP-B, and SAP-C, as well as the deficiency of the precursor pSAP give rise to different, neuronal, white matter or visceral sphingolipid storage diseases. The analysis of cultured fibroblasts from corresponding patients suggests a new model for the topology of endocytosis and lysosomal digestion. It supports the hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures, that are then digested within the lysosomes. In combined activator protein deficient cells nondegradable GSLs on the surface of intralysosomal vesicles protect them against lysosomal digestion. Mice with disrupted genes for activator proteins (SAP precursor -/-, GM2A -/-) as well as disrupted genes for ganglioside GM2 degrading hexosaminidases (HEXA -/-, HEXB -/-) turned out to be useful models for known human diseases whereas double knock out mice (HEXA -/- and HEXB -/-) show a new phenotype of both mucopolysaccharidosis and gangliosidosis.

Animals↗
PubMed1999

Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry.

A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.

Animals↗