Inhibition of cytochrome c oxidase by psychosine (galactosylsphingosine)

Pi uptake and acetoacetate formation were suppressed by psychosine (galactosylsphingosine) in rat liver mitochondria. Besides, reduced form of cytochrome c increased in the reaction mixture which contained psychosine. Using reduced form of cytochrome c as substrate, less than 5 microM of psychosine (0.1 mumoles/mg of mitochondrial protein) inhibited cytochrome c oxidase by more than 50%. The inhibition was completely reversed by 1% human serum albumin. Thus, a lipid which is produced in the brain has a powerful and yet reversible inhibitory effect on an enzyme of cellular respiration.     

Globoid cell leukodystrophy (Krabbe's disease): isolation of myelin with normal glycolipid composition

Myelin was isolated from the brain of a patient with Krabbe's globoid cell leukodystrophy at 0.4% of the normal yield. Despite the exceedingly low yield, the fraction appeared morphologically clean, and consisted mostly of well-preserved myelin lamellae and few contaminating structures. Total lipid and cholesterol were slightly lower than in normal myelin. Total phospholipid was normal, but the ratio of ethanolamine phospholipid to lecithin was reversed. Total galactolipid was normal, and consisted only of cerebroside and sulfatide in normal proportions. The only sugar in cerebroside and sulfatide was galactose. The fatty acid composition of cerebroside and sulfatide was essentially normal with no deficiency of long-chain fatty acids and only with a reversed ratio of C(24:0) to C(24:1) in cerebroside. These data appear to exclude the previous postulate that abnormally rapid breakdown of myelin occurs in this disorder as the result of the formation of chemically abnormal myelin, deficient in sulfatide.     

Prenatal diagnosis of globoid cell leukodystrophy (Krabbe's disease)

Summary A case of globoid cell leukodystrophy (Krabbe's disease) was diagnosed prenatally by demonstrating a profound deficiency of cerebroside -galactosidase in cultured amniotic cells. The diagnosis was confirmed in the fetus aborted in the 19th week. In the cell-free amniotic fluid, normal enzyme activity was found. This finding, which had been demonstrated in a previous case, is discussed.     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

Galactosylceramide and galactosylsphingosine loading studies in cultured skin fibroblasts in human and murine globoid cell leukodystrophy

Cell level studies of 3H-galactosylceramide(GalCer) and 3H-galactosyl sphingosine (GalSph) have been carried out in cultured skin fibroblasts from human and murine globoid cell leukodystrophy (GLD). GalCer loading studies disclosed that the hydrolysis rates of GalCer in human control and GLD were 72% and 45%, respectively, and those from the murine control and GLD cells were 77% and 21%, respectively, on the 5th day of culture. On the other hand, GalSph loading studies showed that the hydrolysis rate of GalSph in the human control and GLD were 40% and 10%, respectively, and those from murine control and GLD cells were 38% and 10% on the 12th day of culture. These data suggest that both GalCer and GalSph degradations were impaired in cell level in human and murine GLD. Furthermore, when radioactive 3H-GalSph was loaded into cultured fibroblasts from murine and human GLD, 3H-GalCer band was formed via GalSph. These data strongly suggest that GalCer could be synthesized through the GalSph route as a minor pathway at least in cultured skin fibroblasts, although the major pathway to synthesize GalCer should be via ceramide.     

Krabbe's disease: globoid cell leukodystrophy

The clinical features of regression in mental and motor development of a 7-month-old child are reported, together with the demonstration of a profound deficiency of galactosylceramide beta-D-galactosidase in a liver biopsy. The diagnosis of Krabbe''s disease or globoid cell leukodystrophy (GLD) is therefore unequivocally established. The clinical features and morbid anatomical findings permitting the diagnosis of GLD in two of the child''s sibs are summarized. This is the first report from Newfoundland of this inborn error of sphingolipid metabolism.     

Genetic galactocerebrosidase deficiency (globoid cell leukodystrophy, Krabbe disease) in rhesus monkeys (Macaca mulatta)

Globoid cell leukodystrophy, or Krabbe disease, is a severe disorder of the peripheral and central nervous system myelin caused by deficient galactocerebrosidase (GALC) activity. This autosomal recessive disease affects humans and animals including dogs, mice, and rhesus monkeys. Cloning of the human and animal GALC genes opened opportunities for therapeutic trials using animal models. We describe the clinical, pathologic, and biochemical features of the affected rhesus monkey. Affected monkeys had very low GALC activity and a two base pair deletion in both copies of the GALC gene. Clinical signs of tremors, hypertonia, and incoordination led to humane euthanasia by 5 months of age. At necropsy, peripheral nerves were enlarged. Microscopically, the cerebral, cerebellar, and spinal cord white matter was infiltrated with periodic acid-Schiff-positive multinucleated globoid cells, and there was a striking lack of myelin. Peripheral nerve fibers were decreased in number and separated by Alcian blue- and safranin O-positive material. Myelin sheaths were greatly diminished. Lipid analysis of brains of 12-day-old and 158-day-old affected monkeys revealed a great excess of psychosine in white matter. The rhesus monkey model will be especially useful for exploring treatment options, including prenatal bone marrow transplantation and various approaches to gene therapy.     

Biosynthesis of galactosylsphingosine (Psychosine) in the twitcher mouse

In attempts to elucidate the origin of accumulated galactosylsphingosine in the twitcher mouse, a murine model of human globoid cell leukodystrophy (Krabbe's disease), UDP-galactose: sphingosine galactosyltransferase activity was assayed in tissues from normal and twitcher mice. Among several tissues from normal, 20 day postnatal mice, the highest galactosyltransferase activity was found in the brainstem and spinal cord, followed by cerebrum, kidney and liver, in that order. Chronologically, the enzyme activity in the central nervous tissue increased with age, reached a maximum at 25 postnatal days, and declined thereafter. In the kidney and liver, however, the activity remained much the same during development. In the twitcher mouse, developmental change in the enzyme activity was similar to that seen in control mouse, but the decrease in activity in the central nervous tissue after the 25 postnatal days was more rapid. The galactosyltransferase activity and the accumulation of galactosylsphingosine in the tissue of the twitcher mouse were closely related; where and when the enzyme activity was higher, the greater was the accumulation of galactosylsphingosine in the tissue of the twitcher mouse. These results strongly suggest that the accumulated galactosylsphingosine in the twitcher mouse is synthesized mainly by UDP-galactose: sphingosine galactosyltransferase.     

Adult onset globoid cell leukodystrophy (Krabbe disease): analysis of galactosylceramidase cDNA from four Japanese patients

We examined galactosylceramidase (GALC) cDNA in four Japanese patients with adult onset globoid cell leukodystrophy (Krabbe disease; AO-GLD) by polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis, subsequent sequence determination, and restriction enzyme digestion of PCR products. Initial symptoms were the onset of slowly progressive spastic paraplegia from the middle of the second decade, and all patients had diminished GALC activity in their leukocytes. We identified three missense mutations (I66M, G270D, L618S) and one exon-6 skipping (535– 573del). Two of the patients had only the I66M mutant mRNA, and one only the G270D mutant mRNA. The fourth patient carried a compound heterozygous mutation of 535–573del and L618S. To determine the enzymatic activities produced by these mutations, we constructed mutated GALC cDNAs and expressed them in COS-1 cells. Three mutations, viz., G270D, L618S, and exon-6 skipping (535–573del), produced diminished GALC activity as expected. The I66M mutation in the wild-type GALC cDNA(I289) had normal activity, but when this mutation and the V289 polymorphism were introduced into the same allele, it had decreased activity. Thus, the combination of a unique mutation and polymorphism causes conformational change in the GALC enzyme, resulting in low enzymatic activity. AO-GLD mutations, including those found here, are located in the N-terminus (I66M, G270D, 535–573del) or C-terminus (L618S) of the GALC enzyme, whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the clinical features of GLD. Received: 4 February 1997 / Accepted: 28 April 1997     

Prenatal enzymatic diagnosis of Krabbe disease (globoid-cell leukodystrophy) using chorionic villi

Summary Sixteen pregnancies in families with children enzymatically diagnosed as having Krabbe disease (KD) were monitored for prenatal KD using the assay of galactosyl ceramide -galactosidase (GCG) in uncultured chorionic villi (CV), cultured CV, or cultured amniotic fluid cells (AFC). Prenatal KD diagnoses were made for 5 pregnancies on the basis of lower than 10% normal GCG activity in cultured CV or AFC. Uncultured CV were studied in 3 out of the 5 KD embryos, although the GCG activities of 14%–23% as compared with control villi were diagnostically inconclusive; the relatively high activities were considered to be caused by maternal GCG contamination of these very small villus samples. Although the villi from 6 of the other pregnancies yielded more conclusive results, the use of uncultured CV alone is not recommended for prenatal KD diagnosis, this material being subject to possible uncontrolled contamination with maternal enzyme.     

Towards a molecular therapy for glycogen storage disease type II (Pompe disease)

Glycogen storage disease type II (GSD-II), also known as Pompe disease, is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. Currently, there is no treatment for this fatal disorder. However, several lines of research suggest the possibility of future treatment. Enzyme replacement strategies hold the greatest hope for patients currently affected by GSD-II, but future strategies could include in vivo or ex vivo gene therapy approaches and/or mesenchymal stem cell or bone-marrow transplantation approaches. Each of the approaches might eventually be combined to further improve the overall clinical efficacy of any one treatment regimen. The lessons learned from GSD-II research will also benefit a great number of individuals affected by other genetic disorders.     

An accumulation of galactocerebroside in kidney from mouse globoid cell leukodystrophy (twitcher)

The twitcher mouse is genetically determined mutant characterized by a deficiency of galactocerebroside beta-galactosidase. In this study, a significant accumulation of galactocerebroside was demonstrated in twitcher mouse kidney. The data suggest that mouse Krabbe's disease is not only involved in CNS, but also in visceral organs.     

Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data

Background

Single-molecule techniques have emerged as incisive approaches for addressing a wide range of questions arising in contemporary biological research [Trends Biochem Sci 38:30–37, 2013; Nat Rev Genet 14:9–22, 2013; Curr Opin Struct Biol 2014, 28C:112–121; Annu Rev Biophys 43:19–39, 2014]. The analysis and interpretation of raw single-molecule data benefits greatly from the ongoing development of sophisticated statistical analysis tools that enable accurate inference at the low signal-to-noise ratios frequently associated with these measurements. While a number of groups have released analysis toolkits as open source software [J Phys Chem B 114:5386–5403, 2010; Biophys J 79:1915–1927, 2000; Biophys J 91:1941–1951, 2006; Biophys J 79:1928–1944, 2000; Biophys J 86:4015–4029, 2004; Biophys J 97:3196–3205, 2009; PLoS One 7:e30024, 2012; BMC Bioinformatics 288 11(8):S2, 2010; Biophys J 106:1327–1337, 2014; Proc Int Conf Mach Learn 28:361–369, 2013], it remains difficult to compare analysis for experiments performed in different labs due to a lack of standardization.

Results

Here we propose a standardized single-molecule dataset (SMD) file format. SMD is designed to accommodate a wide variety of computer programming languages, single-molecule techniques, and analysis strategies. To facilitate adoption of this format we have made two existing data analysis packages that are used for single-molecule analysis compatible with this format.

Conclusion

Adoption of a common, standard data file format for sharing raw single-molecule data and analysis outcomes is a critical step for the emerging and powerful single-molecule field, which will benefit both sophisticated users and non-specialists by allowing standardized, transparent, and reproducible analysis practices.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0429-4) contains supplementary material, which is available to authorized users.     

Identification of a molecular target of psychosine and its role in globoid cell formation

Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.     

Sphingolipid profile in the CNS of the twitcher (globoid cell leukodystrophy) mouse: a lipidomics approach.

Globoid cell leukodystrophy (Krabbe disease) is caused by mutations in galactosylceramidase, a lysosomal enzyme that acts to digest galactosylceramide, a glycolipid concentrated in myelin, and psychosine (galactosylsphingosine). Globoid cell leukodystrophy has been identified in many species including humans and twitcher mice. Several studies on human tissue have examined the lipid profile in this disease by gas, liquid or thin layer chromatography. Electrospray ionization tandem mass spectrometry combined with reverse phase HPLC has become a powerful alternative strategy, used here to compare the sphingolipid profile of pons/medulla tissue from twitcher mice with control tissue. In this lipidomics LC-MS approach, we scanned for precursors of m/z 264 to obtain a semi-quantitative profile of ceramides and galactosylceramides. Sphingosine-1-phosphate, C18:0 ceramide, C22:0 ceramide and C24:0 ceramide levels were reduced in the pons/medulla of twitcher mice compared to levels in control mice at 31 and 35-37 days of age. The levels of C22:0 and C24:0 galactosylceramide were similar between twitcher and control specimens and there was a trend toward reduced levels of C24:1 galactosylceramide and C24:1 hydroxy-galactosylceramide in twitcher specimens. Psychosine, C 16:0 ceramide and C 18:0 galactosylceramide levels were increased in the CNS of twitcher mice compared to levels in control mice. These data indicate that there is a trend toward decreased levels of long chain fatty acids and increased levels of shorter chain fatty acids in galactosylceramides and ceramides from twitcher mice compared with control mice, and such changes may be due to demyelination characteristic of acute pathology.