Incubation of mutant Niemann-Pick C fibroblasts with low-density lipoprotein (LDL) resulted in excessive internalization of lipoprotein and extensive cellular over-accumulation of unesterified cholesterol. The uptake of LDL by the mutant cells appeared to occur through the classic LDL receptor pathway and internalized lipoprotein was processed in lysosomes. Lipoprotein uptake into mutant cells was associated with delays in the initiation of established cellular cholesterol homeostatic responses. Subcellular fractionation of mutant Niemann-Pick C fibroblasts accumulating LDL-cholesterol showed excess unesterified sterol to be localized in the light lysosome-light membrane region of a Percoll gradient, and revealed that cholesterol storage was associated with a specific alteration in the normal profiles of lysosomal marker enzymes.
We developed a sensitive and simple method to determine galactosylsphingosine and glucosylsphingosine as a 4-fluoro-7-nitrobenzofurazan autofluorescent compound, using HPLC equipped with a Showdex sugar column. Amounts of galactosylsphingosine were successfully measured in the picomole range. This novel procedure is more stable and simpler than the previous method using o-phthalaldehyde. It was applied to tissues from the twitcher mouse, an animal model of human globoid cell leukodystrophy. The amount of galactosylsphingosine was 34-102 micrograms/kg of wet tissues in control cerebrum and cerebellum, whereas in twitcher mice the range was 2,251-4,228 micrograms/kg of wet tissues. The psychosine concentration was also increased in the liver and kidney of twitcher mice, respectively, 1,513 micrograms and 1,106 micrograms/kg of wet tissue (normal liver, 125 micrograms; normal kidney, 74 micrograms/kg of wet tissue). This novel procedure is useful for the pathochemical evaluation of lysosphingolipids in various sphingolipidoses as well as in other neuropathological and cellular conditions.
Fluorescence microscopic examination of filipin-stained cultured skin fibroblasts derived from two brothers with group D Niemann-Pick disease revealed abnor-
To study structure-function relationships and molecular evolution, we determined the nucleotide sequence and chromosomal location of the gene encoding murine glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45). In the protein coding region of the murine cDNA, the nucleotide sequence and the corresponding deduced amino acid sequences were 82% and 86% identical to the respective human sequences. AU five amino acids presently known to be essential for normal enzymatic activity were conserved between mouse and man. The murine enzyme had a single deletion relative to the human enzyme at amino acid number 273. One ATG translation initiation signal was present in the mouse sequence in contrast to the human sequence, where two start codons have been reported. Nudeotide sequencing of a clone derived from murine genomic DNA revealed that the murine signal for translation initiation was located in exon 2. The locations of all 10 introns were conserved among mouse and man. We mapped the genetic locus for glucocerebrosidase to mouse chromosome 3, at a position 7.6 ± 3.2 centimorgans from the locus for the (3 subunit of nerve growth factor. Comparison of linkage relationships in the human and murine genome indicates that these closely linked mouse genes are also syntenic on human chromosome 1 but in positions that span the centromere.In the lysosomes of normal individuals, acid /-glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2
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