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Biomedical subjects

A Maret

Publications and source records attributed to A Maret.

At least 55 records · Page 3Linked to original sources
PubMed1987

beta-Glucosidase isoenzymes in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and patients with type 1 Gaucher disease.

Lymphoid cell lines (LCL) from 3 adult patients with non-neuropathic Gaucher disease were established by Epstein-Barr virus (EBV) transformation and were investigated from the view of enzymology. Glucosylceramide-beta-glucosidase (GlcCer-beta-glucosidase) was present in soluble and particulate fraction of LCL from normal subjects and was deficient in type 1 Gaucher LCL; the deficiency of all molecular forms, shown by electrofocusing, indicates that they are coded by the same gene. The existence of two non-specific beta-glucosidases, one soluble (minor), the other membrane-bound (major), was demonstrated in leucocytes and LCL from normals; in Gaucher LCL, these were also present in a normal range. Characteristic properties of the non-specific membrane-bound beta-glucosidase were defined: lability at acidic pH and strong inhibitory effect by detergents. These properties allowed to discriminate it from the lysosomal GlcCer-beta-glucosidase and to define optimal assay conditions for determination of residual GlcCer-beta-glucosidase activity in Gaucher disease, using artificial substrate, without interference of non-specific membrane-bound beta-glucosidase. These results demonstrate that EBV-transformed LCL represent an accurate model system for enzymatic studies of Gaucher disease.

Adult↗
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

Lymphoid cell lines as a model system for the study of Wolman's disease: enzymatic, metabolic and ultrastructural investigations.

Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) were established from blood lymphocytes of a patient affected with Wolman's disease (WD) and from her parents. These LCL showed a severe deficiency in acid lipase activity using every substrate in comparison to LCL from normal subjects, in which acid lipase activity was similar to that in blood lymphocytes. In the LCL from Wolman's disease a major accumulation of neutral lipids was observed, mainly cholesteryl esters, CE (amount around 7 times higher than in normal cells and ratio of esterified/free cholesterol increased by 10 times) and to a lesser extent triglycerides, TG (amount increased by 1.5 times). Electron microscopy showed the storage vacuoles of neutral lipids quite characteristic of this lysosomal disease. The reported data demonstrated the validity of transformed LCL as a cellular model system in culture for experimental studies of Wolman's disease and for investigating the lysosomal metabolism of neutral lipids.

Cell Line↗
PubMed1986

[Wolman disease and cholesterol ester storage disease in adults. New means of study and diagnosis].

Two clinical forms of lysosomal storage of neutral lipids with deficiency of acid lipase are known: the severe infantile form is called Wolman disease, whereas the more benign adult form is called "polycorie cholesterolique de l'adulte" or cholesteryl ester storage disease (CESD). We have developed several new tools for the study of hereditary enzymopathies and we report here their use in the study of genetic defects in lysosomal acid lipase. Lymphoid cell lines established by Epstein-Barr Virus transformation represent a new experimental model system in culture. The validity of such cell lines is demonstrated from the triple point of view of enzymology, metabolism and morphology (microscopic studies). Those lines are characterized by the deficiency in acid lipase whereas other lipases and carboxylesterases are not deficient. The study of lipid composition demonstrated the accumulation of neutral lipids (cholesterylesters and triglycerides). The use of fluorescent lipids for enzymatic studies and for diagnosing acid lipase deficiencies is reported and the reliability of these new substrates is demonstrated by comparison with currently used natural or synthetic substrates. For the enzymatic assays, these fluorescent substrates showed similar advantages as the radioactively labelled substrates, but not their disadvantages: thus, they can be easily used in the clinical laboratories. A new area in the field of research on lysosomal genetic diseases, is the experimental study of the metabolism in the intact living cell. The lipoproteins route is the best way to the lysosomal compartment and fluorescent lipids incorporated into lipoproteins allow to examine the lysosomal metabolism of these lipids simultaneously, by microscopic and biochemical techniques.

Cells, Cultured↗
PubMed1985

[Neutral glycosphingolipids of Fabry's disease lymphoblastoid lines established by Epstein-Barr virus transformation].

Human lymphoid cell lines established by Epstein-Barr viral transformation of peripheral B-lymphocytes from normal subjects and from Fabry patients, were investigated for their ability to biosynthesize neutral glycosphingolipids from [14C]galactose and [14C]glucose as precursors. Galactose was taken up in the presence of high concentrations of glucose and selectively utilised by the cells in the synthesis of galactosphingolipids. The pattern of neutral glycosphingolipids labelled from [14C]galactose was slightly modified with time of labelling in either lymphoid cell line: the first labelled glycosphingolipid was lactosylceramide (LacCer) in the normal line and globotetraosylceramide (GbOse4Cer) in the Fabry line. After labelling for 96 h, a steady state was reached and the percentage of every type of labelled glycosphingolipid was stable in each cell line; however, differences in the neutral sphingolipid composition appeared between the various cell lines. When using radiolabelled glucose as precursor, the major part of the radioactivity was incorporated into neutral lipids and phospholipids; neutral sphingolipids were much less labelled than when using galactose. Catabolism of endogeneous labelled glycosphingolipids (synthesized by the cells during the 'pulse') was studied after cultivating the cells without radiolabelled precursors ('chase'). In the cells from normal subjects, all the neutral glycosphingolipids were slowly degraded (half-life time around 15-25 days for LacCer and GbOse3Cer). In contrast, in a lymphoid line from a Fabry patient, no appreciable degradation of GbOse3Cer occurred during 30 days. This block in the catabolism of GbOse3Cer is in good agreement with the previously reported deficiency of alpha-galactosidase A activity in this Fabry lymphoid cell line [Salvayre, R. et al. (1981) Biochim. Biophys. Acta 659, 445-456] and demonstrates that alpha-galactosidase B does not hydrolyze GbOse3Cer in the living cell (in contrast to the situation in vitro).

Adult↗
PubMed1985

Epstein-Barr virus transformed lymphoid cell lines as a new model system in culture for the study of GM2-gangliosidoses: Tay-Sachs and Sandhoff diseases.

Epstein-Barr Virus transformed cell lines (LCL) were established from blood B-lymphocytes of patients affected with GM2-gangliosidoses variant O (Sandhoff disease, SD) and variant B (Tay-Sachs disease, TSD). LCL from SD showed a severe deficiency of activity of the major lysosomal beta-N-acetylhexosaminidase isoenzymes, Hex A and B; the residual activity was due to Hex S and Hex C. In LCL from TSD, the whole Hex activity was not deficient but isoenzyme composition was completely abnormal. Ultrastructural investigations showed the presence of pleiomorphic enlarged lysosomes appearing as clear vacuoles containing a finely fibrillo-granular material characteristic of the visceral lysosomal storage of gangliosidoses.

Adult↗
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

Validity of lymphoid cell line for enzymatic studies of GM2-gangliosidosis variant 0 (Sandhoff disease).

A lymphoid cell line established by Epstein-Barr virus (EBV)-transformation of peripheral blood B-lymphocytes from a patient with Sandhoff disease showed a severe deficiency of beta-N-acetylhexosaminidase activity (residual activity around 10% of that in lymphoid cell lines from normals or other lipidotic patients). This residual beta-N-acetylhexosaminidase was completely heat-labile in contrast to that of normals. The molecular forms of residual beta-N-acetylhexosaminidase from Sandhoff lymphoid cell line were separated by Con A-sepharose and electrofocusing. Their properties and electrofocusing profiles were compared to those from Sandhoff fibroblasts and from fetal brain: this comparison permitted to identify the residual molecular forms with Hex S and Hex C. The microheterogeneity of Hex S and Hex C, demonstrated by electrofocusing, was discussed. 2-Acetamido-2-deoxy-D-galactonolactone (GalNAcLone) showed a strong inhibitory effect on lysosomal Hex A, B and S, but only a very slight effect on Hex C. Studies of the inhibition type (competitive on Hex A, B and S and mixed on Hex C) gave some informations about the enzymatic site. Elsewhere, differences in affinity of GalNAcLone for the various isoenzymes could be utilized to define optimal assay conditions for specifically determining Hex C (standard assay containing 400 mumol/l of GalNAcLone). These results demonstrated that EBV-transformed lymphoid cell lines represent an accurate model system for enzymatic studies of Sandhoff disease.

Acetylgalactosamine↗
PubMed1985

[Beta-glucosidases. Molecular bases of Gaucher's disease].

The first part of this review deals the biochemical data concerning human and mammalian beta-glucosidases. Glucosylceramide-beta-glucosidase and non-specific beta-glucosidase are characterized following their physical, functional, structural and genetical properties. The second part deals with the biochemical and pathophysiological data concerning the Gaucher disease.

Animals↗
PubMed1984

Modifications of the molecular weight of membrane-bound nonspecific beta-glucosidase in type 1 Gaucher disease determined in situ by the radiation inactivation method.

The radiation inactivation method has been used to compare the molecular weight of the nonspecific membrane-bound beta-glucosidase in situ in normal human spleen and in that of two patients with Gaucher disease type 1. We report, in type 1 Gaucher spleen, the presence of a high molecular weight component (557 000) in addition to the normal low molecular weight component (97 800). The various possible hypotheses explaining this high molecular weight component are discussed.

Adult↗
PubMed1984

Cytochemical and biochemical studies on neutrophil alkaline phosphatase in parents of trisomy 21 children.

A study of karyotypes and neutrophil alkaline phosphatase (NAP) was carried out for 66 parents (33 couples) of trisomy 21 children and for 60 control parents (30 couples). Enzyme activity was determined simultaneously by biochemical and cytochemical techniques. In the mothers of trisomy 21 children we found: (a) by biochemical techniques, a significant increase of NAP activity in polymorphonuclear leukocyte (PMN) homogenates (P less than 0.01) and a lower supernatant/pellet ratio (P less than 0.01); (b) by cytochemical techniques (with or without thermal and urea treatments) NAP activity was significantly higher (P less than 0.001) than in control mothers; in the fathers, the two techniques gave normal NAP activity results.

Alkaline Phosphatase↗
PubMed1984

[Alpha-galactosidases and alpha-N-acetylgalactosaminidase. Biochemical bases of Fabry's disease].

The first part of this review deals with the new biochemical and genetical data concerning alpha-galactosidase and alpha-N-acetylgalactosaminidase. Molecular forms of these both enzymes can be classified into two groups following their physical, enzymatic and genetical properties: - the 3 forms of the alpha-galactosidase A group differ by the number of sialyl residues and their isoelectric point. All the forms of this group are heat-labile, hydrolyse only alpha-galactosides and proceed from the same alpha-GalA, X-linked gene. - alpha-galactosidase B is an alpha-N-acetylgalactosaminidase with broad substrate specificity, in vitro, is heat-stable and proceed from the alpha- GalB or alpha- NAGA gene of the chromosome 22. Structural and enzymatic data concerning these enzymes and their functions in the catabolism of glycosphingolipids and glycoproteins are reviewed. The second part deals with the pathophysiology of Fabry disease. The more prominent genetical and biochemical data and their diagnostic uses are reported: isozymic determination, cell cloning, quantitative determination of accumulated glycolipids. At last, were pointed the new developments of the research on Fabry disease: cultured cells as experimental model (fibroblasts, lymphoid cell lines) and therapeutic attempts.

Chemical Phenomena↗
PubMed1983

Modification of subunit interaction in membrane-bound acid beta-glucosidase from Gaucher disease.

The radiation inactivation method has been used to determine the molecular mass of membrane-bound acid beta-glucosidase (EC 3.2.1.21) in situ, in normal human spleen and in that of two patients with type I Gaucher disease: the molecular mass in Gaucher spleen is about double (125 000 +/- 8900) of that found in the normal spleen (67 000 +/- 7700) which is compatible with the existence of subunit coupling in the muted acid beta-glucosidase. From the results, we conclude that subunit interaction is altered in mutant acid beta-glucosidase and that this may be due to a direct effect of the mutation.

Cell Membrane↗
PubMed1983

Molecular forms of beta-N-acetylhexosaminidase in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and patients with Tay-Sachs disease.

In whole leukocytes and in lymphocytes from normal subjects, the percentage activity of heat-stable beta-N-acetylhexosaminidase (30 +/- 5% and 45 +/- 5%, respectively) was higher than in the transformed lymphoid cell line (19 +/- 3%). In Tay-Sachs transformed cells as well as non-transformed beta-N-acetylhexosaminidase was almost completely heat-stable (95 - 98%). In the transformed cells from normal subjects, the beta-N-acetylhexosaminidase B (Hex B) activity (5% of total) was significantly lower than in blood lymphocytes (average 25 - 30% of total activity), whereas Hex A and Hex I were similar in the either cell type. Blood lymphocytes and lymphoid cell lines established from a Tay-Sachs patient lacked heat-labile Hex A and expressed high heat-stable Hex I and Hex B activities (3-6-fold). After neuraminidase treatment, Hex A peak sharpened while Hex I peaks switched to higher pI than normal Hex I, in the region of Hex B. PreHex A/S pI was not affected. Hydrolytic properties using the both substrates (4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside and 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-galactopyranoside) of each molecular form were similar in transformed and non-transformed cells. Data derived from the use of a mixture of substrates were consistent with the model which proposes a common active site for either substrate in the case of preHex A, Hex B and Hex I, but not for Hex A. Thus Epstein-Barr virus-transformed lymphoid cell lines represent an accurate model system for studies on Tay-Sachs disease.

Cell Line↗
PubMed1983

Substrate specificity of the human splenic non-specific soluble beta-glucosidase.

The soluble non-specific 4-methylumbelliferyl-beta-D-glucosidase, one of the three splenic groups previously reported [Maret, A., Salvayre, R., Nègre, A., and Douste-Blazy, L. (1981) Eur. J. Biochem. 115, 455-461], was partially purified by gel filtration and heat-inactivation (which inactivated the contaminating acid beta-galactosidase). Its substrate specificity was accurately demonstrated by comparing the enzymic properties towards each substrate and two-substrate mixed assays: 4-methylumbelliferyl beta-D-galactopyranoside, beta-D-glucopyranoside, beta-D-fucopyranoside, alpha-L-arabinopyranoside and beta-D-xylopyranoside (relative rates 100, 95, 70, 15, 5, respectively in the standard conditions used) were competitively hydrolysed. Information about the enzymic site was given by the effect of various substrate analogs: generally we observed a greater inhibition of beta-galactosidase activity than beta-glucosidase and beta-fucosidase activities. Moreover, p-nitrophenyl-beta-D-mannopyranoside showed a strong inhibitory effect on the three main activities of this enzyme, similar to D-glucono-1,5-lactone inhibition. A selective inhibition by Triton X-100 on beta-D-galactosidase activity and in a lesser degree on beta-D-fucosidase activity was discussed. The non-specific soluble beta-glucosidase from a Gaucher patient and from normal subjects showed identical properties. Thus this enzyme is not affected by the mutation in the Gaucher type I spleen.

Adult↗