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Biomedical subjects

M T Vanier

Publications and source records attributed to M T Vanier.

At least 127 records · Page 7Linked to original sources
PubMed1983

Rat pancreas actin: purification and characterization.

Isolation of rat pancreas actin was performed with three different technics: polymerization-depolymerization method, affinity chromatography on DNase I-Sepharose 4B or ion exchange chromatography on DEAE-cellulose. Inhibition of DNase I activity, localization by SDS polyacrylamide slab gel electrophoresis and presence of microfilaments allowed its identification. Affinity process led us to obtain actin which kept inhibitory activity (30,000 U per mg) on DNase I when using vacuum dialysis. Actin eluted from DEAE-cellulose associated reversibly in 50-70 A microfilaments in the presence of phalloidin, was pure at 95% and had a satisfactory inhibitor activity (77,000 U per mg).

Actins↗
PubMed1983

Chromatofocusing of skin fibroblast sphingomyelinase: alterations in Niemann-Pick disease type C shared by GM1-gangliosidosis.

Sphingomyelinase activity of cultivated skin fibroblast extracts from normal individuals was resolved by chromatofocusing in the pH range 8-5 into three major components with pI's of 7.3, 6.3 and 5.9, respectively. Chromatofocusing proved a more efficient and reproducible separation technique than preparative flat-bed isoelectric focusing and it gave a constant profile even when detergent concentration varied. In skin fibroblasts from five patients with Niemann-Pick disease type C, a varying degree of reduction in the proportion of the 7.3 peak was observed. In a patient with clinical features of Niemann-Pick disease type C, the finding of such a profile would thus be a good argument for the diagnosis, but it is not pathognomonic as we found similar changes in two cases with GM1-gangliosidosis, while some cases of Niemann-Pick disease type C have borderline normal profiles. These results challenge the concept of a specific sphingomyelinase isoenzyme deficiency as the basic defect in Niemann-Pick disease type C.

Fibroblasts↗
PubMed1983

Effect of tricyclic antidepressants on sphingomyelinase and other sphingolipid hydrolases in C6 cultured glioma cells.

Cationic amphiphilic drugs, which include tricyclic antidepressants, have been shown to give rise to lipidoses under experimental conditions, with a general increase of lipids especially phospholipids. We report here an early and important decrease in sphingomyelinase activity in C6 glioma cells cultured in the presence of imipramine or desipramine at final concentrations of 0.01 and 0.05 mM. The effect was both dose-dependent and time-dependent and was observed before any lipid accumulation. Cerebroside beta-glucosidase and cerebroside beta-galactosidase had normal activities under the same experimental conditions and thus there was no general effect on membrane-bound sphingolipid hydrolases. A decrease of sphingomyelinase activity has been previously reported for two amphiphilic compounds, perhexiline maleate and AY 9944. These results suggest a potential function of sphingomyelinase in the mode of action of these drugs.

Animals↗
PubMed1983

Biochemical studies in Niemann-Pick disease. I. Major sphingolipids of liver and spleen.

In liver and spleen specimens of 12 patients with Niemann-Pick disease types A or B, sphingomyelin was increased 15-45-fold, total phospholipids 4-10-fold and cholesterol 3-6-fold over the normal values. The storage pattern was qualitatively similar in both types but the degree of accumulation was less in type B. In Niemann-Pick disease type C (16 cases), sphingomyelin was increased 3.5-fold in liver and 6-fold in spleen. In all forms of Niemann-Pick disease, bis(monoacylglycero)phosphate was markedly elevated. Glycosphingolipids were studied in six cases with type C, three cases with type B and two cases with type A. Glucosylceramide showed the largest increase from the normal pattern in all types of Niemann-Pick disease. Highest values were recorded in type C, 14- and 35-fold normal concentrations in liver and spleen, respectively. Other neutral glycosphingolipids, particularly lactosylceramide, were also elevated, and a 2-4-fold increase of ganglioside GM3 occurred. The fatty acid profiles of the sphingolipids showed only minor alterations. In contrast to the largely dominating sphingomyelin storage found in liver and spleen of Niemann-Pick disease types A and B, the major characteristic of the lipid storage in Niemann-Pick disease type C was the absence of any prevailing accumulation and, thus, the concept of this disorder as a primary sphingomyelin storage disease is not founded.

Adolescent↗
PubMed1983

Chromatofocusing of purified placental sphingomyelinase.

To establish the profile of the multiple forms of placental lysosomal sphingomyelinase, we have developed in a preliminary step, a new sequence of purification. This protocol has a good yield such that the final preparation is representative of the whole enzyme. Using chromatofocusing, purified sphingomyelinase is resolved into 4 major forms whose pI's are 7.2, 6.5, 5.9 and 5.2. Chromatofocusing is preferable to isoelectric focusing because it is more rapid, more reproducible and above all it provides a constant profile of activity which is not influenced by experimental conditions, in particular, detergent concentration.

Chromatography↗
PubMed1981

Lipid metabolism and oxygen consumption in a hereditary demyelinating neuropathy, the trembler mouse: an in vitro study.

Sciatic nerves from 15-day-old trembler and control mice were maintained in vitro up to 53 h and the metabolism of myelin lipids and the oxygen consumption were investigated [35S]Sulfate was incorporated into sulfatides at a higher rate and turned over more rapidly in trembler nerves than in controls. [14C]Galactose was incorporated into cerebrosides of trembler nerves at a lower rate and turned over like the controls. In contrast, synthesis of sulfatides labeled with [14]galactose was increased in mutants and no significant turnover was observed for both trembler and control nerves during the whole incubation period. Similar results were obtained using [3H]serine as a precursor and no significant differences were observed in the turnover rates of sphingomyelin and phosphatidylcholine between trembler and control nerves. These data suggest the presence of two different pools of cerebrosides, a small one formed by the fast recycling of sulfatides and which does not mix with a second, larger one. The rate of oxygen consumption did not change significantly during the incubation period and was 2-3-fold higher in trembler nerves than in controls, reflecting, at least partly, the increased sulfatide metabolism.

Animals↗
PubMed1981

Use of leukocytes in diagnosis of Krabbe disease and detection of carriers.

The optimal conditions for the assay of the cerebroside-beta-galactosidase deficiency in Krabbe disease were studied. Labelled galactosylceramides and lactosylceramides isolated from natural sources were both suitable substrates provided that the lipophilic moieties were identical in labelled and unlabelled portions. Galactosylceramides and lactosylceramides with uniform fatty acid compositions were also tested. The highest specific cerebroside-beta-galactosidase activities were obtained with N-palmitoyl galactosyl- and lactosylceramides. The chromogenic substrate 2-hexadecanoyl-amino-4-nitrophenyl-beta-D-galactopyranoside (HNGal) was shown to be an unreliable substrate requiring large amounts of enzyme protein and giving false normal results. Krabbe disease was diagnosed in 45 patients. With galactosylceramides as substrates the residual cerebroside-beta-galactosidase activity was 7%, with lactosylceramides 8%, and there was no overlap in enzyme activity between the 45 patients and 42 parents. Approximately 50% of the obligate carriers had values within the range for the normal controls, independently of which substrate was used.

Adolescent↗
PubMed1981

Prenatal diagnosis of Krabbe disease.

Krabbe disease was diagnosed prenatally in Göteborg (Sweden) and Lyon (France) by assaying the cerebroside-beta-galactosidase activity with galactosylceramides and lactosylceramides as substrates in cultivated amniotic fluid cells. Altogether, 48 pregnancies at risk were monitored between 1972 and 1980. Ten pregnancies at risk were terminated because of a predicted affection of the fetus. Biochemical examination of material available from 7 of the 10 abortuses confirmed the diagnoses. All the remaining 36 pregnancies ended in the birth of a healthy infant. The study showed that prenatal diagnosis of Krabbe disease is difficult because of the relatively high residual cerebroside-beta-galactosidase activity in some affected fetuses. Except for the large biological variation, the enzyme activity was sensitive to variation in cultivation conditions and differed strikingly between morphologically different cell types. These two factors were controlled by including control cell samples cultivated under identical conditions and by relating the cerebroside-beta-galactosidase activity to that of two marker enzymes. The biological variation was investigated further by measuring the cerebroside-beta-galactosidase activity in cultured skin fibroblasts from infants with Krabbe disease and from their parents. Results obtained in 18 unrelated patients with Krabbe disease, 26 obligate heterozygotes and 63 controls showed a wide range of variation in enzyme activity in the controls, a large overlap between the controls and obligate heterozygotes, and a high residual activity in some patients. Nevertheless, a high residual activity in a patient was combined with a relatively high enzyme activity in the two parents. In the light of the above findings and deliberations, it appears warranted to conclude that laboratories with experienced personnel can make a reliable prenatal diagnosis of Krabbe disease and that the examination should be offered to all known couples at risk.

Cells, Cultured↗
PubMed1980

Sphingomyelinase activities of various human tissues in control subjects and in Niemann-Pick disease - development and evaluation of a microprocedure.

A micromethod was elaborated for the assay of sphingomyelinase activities with native labelled substrate in leukocytes, cultivated skin fibroblasts, liver tissue and cultivated amniotic fluid cells. The optimal assay conditions and specific activities in control samples were investigated for each enzyme souce. No significant difference was found between results obtained either with the micromethod or with our previous procedure. Findings obtained in pathological material from 62 patients with the various forms of Niemann-Pick disease and 21 obligate heterozygotes by one or another method are reported. A generalized severe sphingomyelinase deficiency was observed in all cases with Niemann-Pick disease type A or B, while in Niemann-Pick disease type C, sphingomyelinase activities were normal in leukocytes, elevated in liver tissue and partially deficient in cultivated skin fibroblasts. Six pregnancies at risk were monitored.

Amniotic Fluid↗
PubMed1980

Krabbe disease: a galactosylsphingosine (psychosine) lipidosis.

The primary genetic defect underlying Krabbe disease or globoid cell leukodystrophy is considered to be a deficiency of galactosylceramide-beta-galactosidase. In the present study of the brains from 18 patients who had died from Krabbe disease at 7-37 months of age, the concentration of galactosylceramide of cerebral and cerebellar white matter was severely reduced to 10-20% of that in age-matched controls. The lowest values were found in the most long-standing cases. Lactosylceramide was reduced to about 50% of normal, while globotriaosylceramide, blobotetraosylceramide and III3-alpha-fucosylneolactotetraosylceramide were increased 10 to 100-fold. Two glycosphingolipids, which have never before been isolated from normal human brains were now isolated and characterized: galactosylsphingosine (psychosine) and galactosyl beta 1 leads to 4 galactosylceramide. We were unable to identify galactosylsphingosine in normal human brains with certainty. We estimate its concentration in the cerebral white matter in Krabbe disease to be increased at least 100-fold (higher than normal). Psychosine was isolated also from the cerebral cortex in Psychosine was isolated also from the cerebral cortex in Krabbe disease after derivatization to the N-acetyl form. Its concentration there was 1 nmol/g tissue compared with 6-10 nmol/g in the white matter. All the neutral glycosphingolipids were isolated and their structure proved by the quantitative determination of their components, degradation by acid and specific glycohydrolases and permethylation and gas-liquid chromatographic-mass spectrometric assay of the methylated sugars. The paradoxical findings of a severely reduced concentration of galactosylceramide and a primary deficiency of cerebroside-beta-galactosidase can be explained by the present finding of the accumulation of galactosylsphingosine in the brains from patients who had died from Krabbe disease. The enzyme has a broad specificity and it normally also degrades galactosylsphingosine. Because of competitive inhibition by the accumulated galactosylceramide its lysosomal hydrolysis will be blocked. The concentration of psychosine will steadily increase and reach toxic levels and kill the oligodendroglial cells. This results in an arrest of the galactosylceramide biosynthesis. Therefore, we feel that galactosylsphingosine and not galactosylceramide is the primary storage substance in the brain in Krabbe disease that the disease is a psychosine lipidosis.

Brain Chemistry↗
PubMed1979

The assay of sphingolipid hydrolases in white blood cells with labelled natural substrates.

Methods were elaborated for the assay on leukocytes of sphingolipid hydrolases with native and artificial substrates. The optimal conditions for the isolation of leukocytes and the preparation of labelled sphingolipids were investigated. The KM and Vmax values for all substrates are given as the specific activities of the sphingolipidhydrolases in normal males and females. It is concluded that leukocytes are the enzyme source of choice for the enzymic diagnosis of neurolipidoses.

Adult↗
PubMed1978

Lipid and fatty acid composition of human cerebral myelin during development.

A development study of major lipid fatty acids in human brain myelin was undertaken and compared to those in cerebral white matter of the same region. The myelin was isolated from 23 subjects at ages from newborn to old age. The proportions of cholesterol and galactolipids increased in myelin during the first 6 months of age and up to 2 years of age in cerebral white matter. During the same periods the individual phospholipids also showed marked variations. Serine phosphoglycerides and especially sphingomyelins increased, and choline phosphoglycerides decreased. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) and sphingomyelins underwent the largest maturation changes. The proportions of saturated fatty acids in EPG diminished rapidly with a corresponding increase of monoenoic acids. Fatty acids of the linoleic acid series showed a peak between 4 months and 12 months of age, and then their proportion slowly diminished to old age. The fatty acid changes in serine phosphoglycerides were much less pronounced than in EPG but of similar type. In sphingomyelin the proportion of saturated long-chain fatty acids diminished while the proportion of monoenoic acids increased--this increase continued at least to the age of 15 years. The same fatty acid changes occurred in cerebrosides and sulfatides as in the sphingomyelins, but they were less pronounced. The fatty acid changes during development were much more pronounced in white matter than in myelin but already from 1-2 years of age the lipids of myelin and white matter had the same patterns--in the galactolipids from 2 months of age. The individual variations of the lipid fatty acid patterns were small except for at the youngest ages and the variations found for this period might depend on the difficulties in determining the gestational age.

Adolescent↗
PubMed1978

Neurovisceral storage disorder simulating Niemann-Pick disease. A new form of oligosaccharidosis?

Two sisters are described with demonstrable splenomegaly already from infancy and, after the age of 2--4 years, signs of slowly progressive encephalophy, vacuolated lymphocytes in the peripheral blood, and peculiar foam cells in the bone marrow aspirates. The died at 7 3/4 and 6 1/2 years. Widely spread in the brain, the nerve cell bodies were found to show extensive ballooning. It was most striking in the brain stem and spinal cord, while cerebellar structures were remarkably well preserved. The cytoplasm of the ballooned nerve cells was filled with finely granular storage material stainable as a readily soluble glycolipid. The spleen, liver and intestinal wall contained numerous foamy PAS-positive macrophages. Chemical assays showed a ten-fold increase of lactosylceramide and a modest one of minor gangliosides brain cortex. No accumulation sphingomyelin could be revealed, and the sphingomyelinase activity was found to be normal. The ganglioside GM1 beta-galactosidase activity of leucocytes was reduced to 20--25% of normal, which indicated a disturbance of the glycosaminoglycan metabolism. The tissue content of glycosaminoglycans was, however, normal, but an accumulation of lactose was demonstrated in the spleen. It is postulated that the primary enzymic defect is a disturbance of a lysosomal beta-galactosidase with a substrate specificity for lactose and other oligosaccharides with a terminal beta-galactosidic linkage.

Bone Marrow↗