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T Levade

Publications and source records attributed to T Levade.

At least 109 records · Page 6Linked to original sources
PubMed1988

Synthesis of pyrenesulfonylamido-sphingomyelin and its use as substrate for determining sphingomyelinase activity and diagnosing Niemann-Pick disease.

A new fluorescent derivative of sphingomyelin (PSA12-sphingomyelin) containing a pyrene-sulfonylamide residue was synthesized by covalently linking 12-((1-pyrenesulfonyl)amido)-dodecanoic acid (PSA12) to sphingosylphosphorylcholine. It was used as substrate for acidic and neutral human and murine sphingomyelinases permitting development of sensitive assays for these enzymatic activities. The product of the sphingomyelinase assay, PSA12-ceramide, could be detected in picomole quantities due to a fluorescence intensity which was 10-35-fold greater than that of other fluorescent ceramides (such as pyrene or nitrobenzoxadiazole derivatives). PSA12-sphingomyelin could be used in pure form or admixed with natural sphingomyelin; in the latter case, the enzyme hydrolyzed the fluorescent and non-fluorescent species at equal rates. Use of PSA12-sphingomyelin permitted determination of sphingomyelinase activity in cell extracts (eg human blood lymphocytes, lymphoid cell lines or cultured skin fibroblasts) as well as in hair follicles and urine. This new fluorescent derivative of sphingomyelin also permitted the detection of acid sphingomyelinase deficiency in cells derived from patients with Niemann-Pick disease.

Animals↗
PubMed1988

Evidence for both endogenous and exogenous sources of the sphingomyelin storage in lymphoid cell lines from patients with Niemann-Pick disease types A and B.

Epstein-Barr virus-transformed lymphoid cell lines from normal individuals and from patients with Niemann-Pick disease types A, B or C were subjected to various culture conditions in order to study the source of the characteristic lysosomal storage of sphingomyelin observed in the tissues of Niemann-Pick patients. The culture medium was supplemented with a serum substitute devoid of lipoproteins or with one of the following lipid sources: fetal calf serum, human low-density lipoprotein (LDL), or human high-density lipoprotein (HDL). Storage of sphingomyelin was demonstrated under all tested culture conditions in cells deficient in acid sphingomyelinase (Niemann-Pick disease types A and B). In contrast, the sphingomyelin concentration in the lymphoid cell line from a Niemann-Pick type C patient (not deficient in sphingomyelinase) was normal. After more than 30 days in a medium devoid of sphingomyelin, the Niemann-Pick types A and B lymphoid cell lines showed accumulation of sphingomyelin about twice control. The concentrations was higher when cells were grown in a medium supplemented with lipids, particularly human LDL or HDL. These results are consistent with the hypothesis that both exogenous and endogenous sphingomyelins participate in the lysosomal storage observed in lymphoid cell lines from patients with Niemann-Pick disease types A and B.

Cells, Cultured↗
PubMed1987

Enhancement of fluorescence of pyrene-containing lipids by polar media, detergents and phospholipids.

The fluorescence intensities of a medium-chain fatty acid and of several amphiphilic lipids, each containing pyrene in covalent linkage, were enhanced considerably by: 1) Dissolving in mixtures of a polar solvent (e.g. methanol, ethanol, tetrahydrofuran or dimethylsulfoxide) and water; for each individual compound, a certain ratio of solvent to water provided maximal fluorescence intensity. 2) Incorporating into micelles of reduced Triton X-100; an excess of detergent was used so that, statistically, only one molecule of lipid resided in one micelle of the Triton X-100. 3) Incorporating into liposomes of egg phosphatidylcholine; maximal fluorescence was observed using a large excess of phosphatidylcholine. When related to the fluorescence intensities in chloroform/methanol (2:1, by vol.) or water, the enhancement of fluorescence in the above three systems was about 2-6-fold or up to 60-fold, respectively.

Ceramides↗
PubMed1987

Uptake and intracellular degradation of fluorescent sphingomyelin by fibroblasts from normal individuals and a patient with Niemann-Pick disease.

Sphingomyelin, labelled with a fluorescent probe, pyrene, in the fatty acyl residue was associated with fetal calf serum; approx. 80% of the sphingomyelin was found in the low- and high-density lipoproteins. This was added to the growth medium of cultured human skin fibroblasts from normal individuals and a patient with Niemann-Pick disease type A, devoid of acid sphingomyelinase activity. The fluorescent sphingomyelin was taken up by both cell types, but only the former degraded it to produce fluorescent ceramide. Differences between normal and Niemann-Pick cells in sphingomyelin content or ceramide production were observed after several hours uptake. A more pronounced difference was noted when cells were incubated for 1 day with fluorescent sphingomyelin and then for two to three days in medium devoid of this compound. Under these conditions, the fluorescence intensity of the Niemann-Pick cells remained practically constant while that of their normal counterparts was almost completely eliminated from the cells. Comparison of fluorescence intensities of these two cell types could be made directly on aqueous suspensions of whole cells or, alternatively, on their lipid extracts. For evaluation of the degradation of fluorescent sphingomyelin to ceramide within the cells, several procedures were developed for the rapid isolation of the latter compound from the total lipid extract. The results suggest that when associated with the constituents of the fetal calf serum, sphingomyelin is taken up by the cells and transported into the lysosomal compartment where it is degraded to ceramide. Use of the fluorescent derivative of sphingomyelin provided a simple and rapid procedure for following the uptake by and degradation within the cultured cells. It also permitted the establishment of differences in the rates of degradation of the fluorescent sphingomyelin by cells with a normal metabolism and others lacking sphingomyelinase (i.e., Niemann-Pick disease type A cells).

Biological Transport, Active↗
PubMed1987

Chemical analysis of the bronchoalveolar washing fluid in the diagnosis of liquid paraffin pneumonia.

Lipid analyses of the bronchoalveolar washing fluid of three patients affected with chronic unidentified pneumonia showed the presence of a major hydrophobic compound identified as paraffin oil by thin layer chromatography, infrared spectroscopy and gas liquid chromatography (by comparison with various paraffin preparations as standards). Moreover the phospholipid and neutral lipid composition was determined. In conclusion, the usefulness of bronchoalveolar lavage and its biochemical analysis is discussed as a non-invasive method of diagnosis and therapeutic approach to liquid paraffin pneumonia (in the cases herein described, between 100 and 500 mg of paraffin oil were extracted from 100 ml of bronchoalveolar washing fluid.

Aged↗
PubMed1986

Biochemical and ultrastructural studies on an Epstein-Barr virus-transformed lymphoid cell line from a Niemann-Pick disease type C patient.

Human lymphoid cell lines established from normal subjects and from a Niemann-Pick disease type C patient were investigated from a triple point of view of enzymology, metabolism and ultrastructure: Sphingomyelinase activities, isoenzyme electrofocusing profiles and properties of the major enzyme were quite similar in type C and normal lymphoid cell lines. Similarly, no significant difference was observed in non-specific phosphodiesterases hydrolysing bis(methylumbelliferyl)phosphate and bis(methylumbelliferyl)pyrophosphate. The study of the lipid composition of type C cells showed no obvious accumulation of sphingomyelin or other phospholipid, but only a higher amount of glycolipids (mainly GlcCer and GbOse3Cer), as visualized by bidimensional thin-layer chromatography. Ultrastructural studies demonstrated, in type C cells, the presence of an obvious lysosomal storage of amphiphilic lipids quite similar to that observed in tissues of type C patients. These studies, which demonstrate the validity of lymphoid cell lines as an experimental model system for type C disease, agree with the current opinion that an impairment of sphingomyelin catabolism is not the primary defect in type C disease.

Cell Line↗
PubMed1986

Interindividual heterogeneity of molecular weight of human brain neutral sphingomyelinase determined by radiation inactivation method.

The molecular weight (Mr) of the membrane-bound neutral sphingomyelinase from human brain was determined using the radiation inactivation procedure. Previous studies on three human brains suggested a Mr of 165 +/- 25 kDa (J. Neurochem. 1985, 45:630-632). We now report that in another human brain the neutral sphingomyelinase had a Mr of 740 +/- 100 kDa; this higher Mr was not accompanied by differences in enzymatic properties nor heat-stability.

Adult↗
PubMed1986

Sphingomyelinases and Niemann-Pick disease.

In the first part the properties of normal mammalian sphingomyelinases are reviewed: The lysosomal acid sphingomyelinase is a polymeric glycoprotein (subunit Mr between 28 000 and 70 000) which hydrolyses natural sphingomyelin, coloured and fluorescent semi-synthetic analogues (trinitrophenyl-aminolauryl-sphingomyelin and pyrenedecanoyl-sphingomyelin) and the synthetic analogue 2-N-hexadecanoylamido-nitrophenyl-phosphorylcholine. The suitability of these substrates and of synthetic fluorescent derivatives of methylumbelliferone is discussed. The effect of lipids, detergents and other effectors on the enzyme activity is also described. The neutral sphingomyelinase from brain tissue, localized in cell membranes, has a high Mr (160 000 and 600 000), is heat labile, hydrolyses sphingomyelin and its coloured and fluorescent analogues, but not 2-N-hexadecanoylamido-nitrophenyl-phosphorylcholine. A new method is available for determining enzyme activity in the intact cell through the utilization of endogenous or exogenous sphingomyelin as substrate. In the second part of the review, the classification of Niemann-Pick disease, the characteristic features of each type and the biological tools used for the diagnosis are reported. Experimental models (animal models and cellular models in culture) are reviewed with a particular attention to a new model system, Epstein-Barr virus-transformed lymphoid cell lines.

Animals↗
PubMed1986

[Serum lysosomal hydrolases in various forms of scleroderma].

The activities of several lysosomal hydrolases (beta-galactosidase, beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase and acid phosphatase) were determined in serum and blood lymphocytes from patients affected with scleroderma. Statistical comparisons between means of patient and control groups (Student's t test) showed a significant difference in serum beta-galactosidase and acid phosphatase between patients and controls (serum beta-galactosidase: 36.5 +/- 22 nmol/h/ml in the scleroderma group versus 24 +/- 13 nmol/h/ml in the control group; serum acid phosphatase 853 +/- 345 nmol/h/ml in the scleroderma group versus 634 +/- 295 nmol/h/ml in the control group). These differences were significant (p less than 0.01). Both enzyme activities were also significantly increased in the group with systemic scleroderma, but the difference was less in the group with localized scleroderma (this is discussed in terms of statistics and pathophysiology). The other enzyme activities determined were not significantly modified. The validity of these results is discussed, together with their diagnostic value and with the pathophysiological hypotheses put forward to explain the high levels found.

Adult↗
PubMed1985

Molecular forms of sphingomyelinase and non-specific phosphodiesterases in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease types A and B.

Molecular forms of sphingomyelinase and phosphodiesterases from lymphocytes- and Epstein-Barr virus-transformed lymphoid cell lines were separated by preparative electrofocusing in granulated gels. In either type of cell derived from normal individuals, sphingomyelinase focused as a single peak (pI = 5.60 +/- 0.1) while phosphodiesterases hydrolyzing bis(4-methylumbelliferyl)phosphate and bis(4-methylumbelliferyl)diphosphate separated into seven and three molecular forms respectively; one of the latter showed sphingomyelinase as well as phosphodiesterase activities. Lymphoid cell lines derived from patients with Niemann-Pick disease, types A or B, were practically devoid of sphingomyelinase activity; this was not so for the phosphodiesterases which focussed essentially as normal. The protein peak, which in normal cells contained the three activities, had phosphodiesterase but no sphingomyelinase activity in the Niemann-Pick cells. In normal cells, sphingomyelinase and phosphodiesterase activities of this peak showed different responses to heating and several effectors. These data suggest that in lymphoid cell lines, which are a useful model for studies of Niemann-Pick disease, sphingomyelinase and phosphodiesterases are subject to separate genetic coding and that the latter activities are not a reliable measure for diagnosing Niemann-Pick disease.

Cell Line↗
PubMed1985

Molecular weight of human brain neutral sphingomyelinase determined in situ by the radiation inactivation method.

The radiation inactivation method was used to determine the molecular weight of membrane-bound neutral sphingomyelinase from normal human brain. Inactivation curves showed a molecular mass of 167,000 +/- 32,000. Molecular weights of two control enzymes, beta-N-acetylglucosaminidase and nonspecific beta-glucosidase, determined by the same procedure, were consistent with previous reports.

Acetylglucosaminidase↗
PubMed1985

Biochemical and ultrastructural findings in a lymphoid cell line from Niemann-Pick disease type A.

A lymphoid cell line (LCL) established by Epstein-Barr Virus (EBV)-transformation of blood B-lymphocytes from a patient affected with Niemann-Pick disease (NPD) Type A exhibited a severe deficiency of sphingomyelinase activity (less than 10% residual activity). Ultrastructural investigation showed in LCL from NPD type A, the presence of numerous osmiophilic, electron-dense inclusions with myelin-like figures characteristic of the accumulation of sphingomyelin (and other amphiphilic lipids) similar to those observed in tissues of patients affected with NPD.

Adult↗
PubMed1985

New tools for the study of Niemann-Pick disease: analogues of natural substrate and Epstein-Barr virus-transformed lymphoid cell lines.

Acid sphingomyelinase activity was determined in Epstein-Barr virus-transformed lymphoid cell lines (LCL) established from patients affected with Niemann-Pick disease (NPD) using several substrates: sphingomyelin derivatives, radiolabeled [14C]sphingomyelin (SM), fluorescent N-(10-(1-pyrene)decanoyl)sphingomyelin (P10-SM) or colored trinitrophenylaminolauryl-sphingomyelin, and the chromogenic non-natural substrate 2-N-(hexadecanoyl)amino-4-nitrophenylphosphoryl-choline. LCL from NPD Type A and Type B showed a severe deficiency of acid sphingomyelinase determined using either substrate, whereas LCL from normal subjects had an activity close to that of blood leukocytes. Sphingomyelinase in normal LCL had the same pH optimum (5.0-5.2) and molecular form (pI 5.8) as the enzyme from other sources; identical profiles and activity levels were obtained using the various analogues of sphingomyelin. However, among these derivatives, the assay using P10-SM appeared as the most useful and sensitive for enzymatic diagnosis of NPD. Electron microscopy of NPD LCL demonstrated the lysosomal storage. These results prove the validity of LCL as an experimental model system for NPD.

Cell Line↗
PubMed1985

[Alveolar paraffinosis: cytologic and biochemical study of bronchiolo-alveolar lavage fluid].

A case report of a diffuse alveolar lipoid pneumonia occurring in a patient incapable of giving a proper history underlines the diagnostic difficulty of lipoid pneumonias. Till now, unless the diagnosis was made at necropsy, one relied on a history of prolonged consumption of medications containing mineral oil, with evidence on open lung biopsy of excess fatty material in the pulmonary parenchyma. Chromatography on thin slices of the lipid extract of bronchiolo-alveolar lavage liquid fixed in silica gel, a simple technique, should rapidly give the diagnosis. In the alveolar form of the disease bronchopulmonary lavage returns a significant quantity of paraffin oil and may be considered as a therapeutic method.

Bronchi↗
PubMed1984

Sphingomyelinase and nonspecific phosphodiesterase activities in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease A, B and C.

Acid sphingomyelinase activity determined using the natural substrate, [choline-methyl-14C]sphingomyelin, or the chromogenic synthetic analogue, 2-N-(hexadecanoyl)amino-4-nitrophenylphosphorylcholine, was deficient in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease types A and B. In contrast, lines from Niemann-Pick disease type C and "sea-blue histiocyte syndrome" showed a sphingomyelinase activity within the normal range. Bis(4-methylumbelliferyl)phosphate and bis(4-methylumbelliferyl)pyrophosphate phosphodiesterase activities were not deficient in any Niemann-Pick disease cell line. These results demonstrate the validity of such cell lines as an experimental model system for enzymatic studies of Niemann-Pick disease.

Adult↗
PubMed1984

Ultrastructural investigations on two lymphoid cell lines from Niemann-Pick disease type B.

Lymphoid cell lines (LCL) were established by Epstein-Barr Virus (EBV) transformation of blood B-lymphocytes from two different patients affected with Niemann-Pick disease (NPD) type B. Those lines were severely deficient in sphingomyelinase activity (8% and 10% residual activity). Ultrastructural investigations showed in both these lines the presence of numerous osmiophilic, dense and pleiomorphic inclusions characteristic of lysosomal storage (due to the accumulation of amphiphilic lipids) similar to those observed in tissues from NPD.

Cell Line↗