Biomedical subjects

S Marchesini

Publications and source records attributed to S Marchesini.

At least 37 records · Page 2Linked to original sources

PubMed1993

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.

Alleles↗

PubMed1992

Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture.

We studied the metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with either N-lissamine-rhodaminyl-ceramide (LRh-Cer) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ceramide (NBD-Cer) to the cells cultured in a chemically-defined medium. With both probes the major fluorescent product turned out to be sphingomyelin (SM). Most of LRh-SM was not cell-associated but recovered from the culture medium, probably due to back-exchange to the lipid vesicles. The accumulation of LRh-SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefeldin A, whereas the production of NBD-SM was much less affected by these Golgi perturbing drugs. With LRh-Cer as substrate, LRh-labelled fatty acid (FA), galactosyl- and sulfogalactosyl-ceramides (GalCer and SGalCer) were also formed. NBD-Cer, however, was metabolized to glucosylceramide (GlcCer) and GalCer but not to SGalCer or NBD-FA. These data demonstrate that chemical modifications of ceramide alter its metabolism in oligodendrocytes and that the metabolites of LRh-Cer reflect the glycolipid composition of myelin more closely than those of NBD-Cer.

Animals↗

PubMed1992

Cultured oligodendrocytes metabolize a fluorescent analogue of sulphatide; inhibition by monensin.

It has been suggested that oligodendrocytes can actively phagocytose myelin debris during active myelination or after injury and experimental demyelination. Therefore, we have used a fluorescent analogue (N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulphate) to study the metabolic fate of sulphatide, a galactosphingolipid that is highly enriched in myelin membranes. The fluorescent sulphatide was incorporated in small unilamellar vesicles and administered to cultured oligodendrocytes. The association of the lipid probe to the cells in culture was saturable in time and with the concentration of the probe. The processes of association, internalization and subcellular distribution were followed by confocal scanning laser microscopy and appeared to be very rapid. Within 20 min a marked perinuclear staining was seen. After prolonged incubation the fluorescence distributed gradually over the cytoplasm and into cellular branches along structures suggestive of cytoskeletal elements. Lipid analysis demonstrated that ceramide was the major metabolite present in the cells but galactosylceramide, sphingomyelin and free fatty acid were also detected. In the culture medium only free fatty acid and sphingomyelin were found. Monensin did not affect the cellular association and internalization of the fluorescent sulphatide but markedly reduced its conversion to metabolic products. These results indicate that exogenous sulphatide is targeted to the Golgi apparatus prior to its lysosomal degradation.

Animals↗

PubMed1992

Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts.

The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.

Adolescent↗

PubMed1992

A novel fluorescent pH indicator for the acidic range.

Reaction of a commercial preparation of lissamine-rhodamine B sulfonyl chloride with primary amines gives rise to two sulfonamide derivatives, of which one behaves as a pH indicator with maximal fluorescence emission in the acidic range and a pKa of about 4.6. The structure of this derivative and the mechanism of fluorescence dependence on pH, elucidated by means of NMR, is presented.

Acids↗

PubMed1990

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes.

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.

Cerebroside-Sulfatase↗

PubMed1989

Synthesis of pyrene derivatives of cerebroside sulfate and their use for determining arylsulfatase A activity.

Two fluorescent derivatives of cerebroside sulfate ('sulfatide') have been synthesized and used as substrates for determining arylsulfatase A activity. These were 12-(1-pyrene)dodecanoyl cerebroside sulfate (P12-sulfatide) and 12(1-pyrenesulfonylamido)dodecanoyl cerebroside sulfate (PSA12-sulfatide). When incubated at pH 5.0 in the presence of 5 mM MnCl2 and 5.5 mM of taurodeoxycholate, either substrate was hydrolyzed by arylsulfatase A of human leukocytes. The rate of hydrolysis was proportional to the incubation time and concentration of enzyme; Michaelis-Menten type kinetics were observed with increasing concentrations of substrate. For determining the rate of hydrolysis, each of the two products (i.e., P12- and PSA12-cerebrosides) were separated from the bulk of respective unreacted sulfatide on small columns of DEAE-Sephadex A-25 and their fluorescence intensities read at 343-378 and 350-380 nm for the excitation and emission wavelengths for P12- and PSA12-cerebrosides, respectively. When extracts of skin fibroblasts derived from normal individuals and patients with Maroteaux-Lamy (lacking arylsulfatase B) or metachromatic leukodystrophy (lacking arylsulfatase A) were used as source of enzyme, P12-sulfatide was hydrolyzed by the former two but not by the latter cell extract. Several derivatives of cerebroside sulfate were also synthesized and found to inhibit the hydrolysis of pyrenesulfatide by leukocyte arylsulfatase A. The results demonstrate that these two pyrene containing sulfatides can be effectively used as specific substrates for the determination of arylsulfatase A activity in extract of cells and most probably also of tissues.

Animals↗

PubMed1989

Correlation of the dispersion state of pyrene cerebroside sulfate and its uptake and degradation by cultured cells.

This study aimed at increasing the efficiency and shortening the time required for administering cerebroside sulfate to cultured cells. For this purpose several modes of dispersion of a fluorescent derivative of cerebroside sulfate (sulfatide), in which the natural fatty acid has been replaced by pyrenedodecanoic acid (P12), were incubated with the cells. This fluorescent derivative of cerebroside sulfate (P12-CS) was introduced into the growth medium of the cells using the three following modes of dispersion: (1) P12-CS was dissolved in dimethylsulfoxide and added to the medium, (2) it was precomplexed with serum albumin or (3) incorporated into small, unilamellar vesicles (SUV) of phosphatidylcholine. With each of these respective modes of dispersion, the P12-CS was incubated for periods up to 48 h with cultured lymphoblasts or fibroblasts. Uptake by the cells could be determined by recording directly the cell-associated fluorescence, using a suspension of washed intact cells. The cell lipids were subsequently extracted with mixtures of chloroform/methanol and their fluorescence recorded. When related to the incubation time, uptake of P12-CS by the cells increased continuously using each of the above dispersions. The appearance of fluorescence at 475 nm ('excimer') and the ratio of this to the monomolecular fluorescence at 378 nm ('E/M') could be used as a measure for the presence of the internalized P12-CS in aggregated or fully dispersed states. These values (i.e., E/M), recorded on the suspensions of intact cells were rather high using the aqueous dispersions, intermediate values were observed using the SUV and rather low E/M values (0.5 or less) were observed using the preformed albumin-(P12-CS) complexes. Increasing the mole ratio of albumin to P12-CS (i.e., from 1:2 to 2:1 m/m), decreased the quantity of sulfatide which was taken up by the cells but also further decreased the E/M ratio, suggesting a fully dispersed state of the pyrene lipid within the cell. This indicated that, using an optimal albumin to P12-CS ratio of 1-2 (or its equivalent values in fetal calf serum) permitted an influx of single molecules of P12-CS into the cells. After 48 h, about 50% of the fluorescence of skin fibroblasts was found in metabolic degradation products of P12-CS. The parallel value for fibroblasts derived from a patient with metachromatic leukodystrophy was only about 5%. Appearance of the excimeric emission of a dispersion of P12-CS in water permitted estimation of its critical micellar concentration as being 7.5 x 10(-7) M.

Cell Line↗

PubMed1989

Rapid preparation of a distinct lysosomal population from myelinating mouse brain using Percoll gradients.

To study the vesicular lysosome-associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000-17,500-g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self-generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like beta-galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is approximately 100-fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains approximately 12-14% of the total acid hydrolase activities, with a protein yield of approximately 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an approximately 90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.

Animals↗

PubMed1988

N-pyrene dodecanoyl sulfatide as membrane probe: a study of glycolipid dynamic behavior in model membranes.

An N-linked pyrene-dodecanoyl sulfatide was employed to measure the ratio of excimer fluorescence to monomer fluorescence intensities (E/M). The E/M values provided information about both the dynamic behavior and the structural distribution of the labelled glycolipid in note dispersion of micellar sulfatides and multilamellar vesicles of different phospholipids. Most of the labelled sulfatide seems to be located in domains sequestered from the surrounding phospholipids still above the phase transition temperature of the vesicles. The glycolipids sequestered in these domain environments are less sensitive to the structural changes that the addition of cholesterol or Ca2+ can induce in the phospholipid regions during the phase transition.

Fluorescent Dyes↗

PubMed1987

Interactions of insulin with sulfatide-containing vesicles of phosphatidylcholine at different pHs.

Positively charged insulin is described to induce aggregation of phosphatidylcholine vesicles containing 10 mol% sulfatide at acidic pH. Techniques including light-scattering, Sepharose chromatography, centrifugation, trapped volume determination, circular dichroism and fluorescence polarization, demonstrate that large amounts of negatively charged insulin remain firmly associated to the vesicles upon raising the pH to 7. This is surprising, since only trace amounts of insulin associate to the sulfatide-containing vesicles upon direct incubation at pH 7. The possible molecular explanation of the phenomenon and the relevance of these findings to the actions of insulin in vivo are discussed.

Circular Dichroism↗

PubMed1987

Preparation of spin-labelled sulfatides for EPR studies on model membranes.

Using the N-hydroxysuccinimide ester of the fatty acids, galactosylceramide I3-sulfate containing a 5-or 16-doxyl-stearoyl residue was prepared in good yield by acylation of the galactosylsphingosine I3-sulfate (lysosulfatide) obtained from the saponification of the bovine brain sulfatide. The EPR behavior of the two semisynthetic sulfatides was analyzed in natural sulfatide micelles and in multilamellar vesicles of egg phosphatidylcholine. The evaluated parameters demonstrate that these spin-labelled sulfatides can be used for the study of sulfatide behavior in lipid structures.

Animals↗

PubMed1986

Fluorospectroscopic studies of mixtures of distearoylphosphatidylcholine and sulfatides with defined fatty acid compositions.

Simple study models characteristic for lamellar organization of distearoylphosphatidylcholine and sulfatide have been prepared for fluorospectroscopic investigations on the influence of these glycolipids on the chemico-physical properties of lecithin bilayers. The motion of 1,6-diphenyl-1,3,5-hexatriene in mixed lecithin-sulfatide bilayers changed with temperature, with the compositional ratio of the two lipids, with the presence of divalent cations such as Ca2+ and with the fatty acid composition of sulfatide moiety. Steady-state fluorescence measurements of the average motion of the fluorophore permit evaluation of the gel to liquid-crystalline phase transition in all these membrane models containing different sulfatides.

Fatty Acids↗

PubMed1986

Calorimetric properties of mixtures of distearoylphosphatidylcholine and sulfatides with definite fatty acid composition.

The thermotropic behavior of multilamellar liposomes prepared from mixtures of distearoyllecithin and sulfatide of definite fatty acid composition, has been studied by DTA calorimetry. The saturated stearoyl- and lignoceroyl-sulfatides do not modify significatively both the delta H and the Tm of the phase transition of the lecithins. These parameters, on the contrary, are decreased by all the sulfatides containing unsaturated fatty acids. A contrasting effect is at last exhibited by alpha-OH-stearoylsulfatide which promotes a decrease in delta H but not in Tm of the phase transition. The possibility that these sulfatide properties can influence the dynamic interactions and the sovramolecular organization of the phospholipid bilayers has been discussed.

Calorimetry↗

PubMed1984

A sensitive enzymatic assay for determination of cholesterol in lipid extracts.

A procedure for the determination of free and total cholesterol in lipid extracts is described. The method for free cholesterol employs cholesterol oxidase to generate H2O2 and peroxidase to catalyze the reaction of H2O2 with o-dianisidine to yield a colored product. For the determination of total cholesterol, cholesterol ester hydrolase is included.

Animals↗

PubMed1984

Human blood cells sialidases.

The conditions of a linear assay for 4-methylumbelliferyl-alpha-D-N-acetyl-neuraminic acid (4-MU-NeuAc) sialidase activity in human lymphocytes, granulocytes, platelets and red cells plasma membranes have been determined. Lymphocytes and granulocytes have the same pH curve with two maximums at pH 4.0 and 4.8; however lymphocytes show a higher specific activity and a higher Km value. Sialidase activity detected in red cells plasma membranes has again a double-peak pH curve with maximums at pH 4.2 and pH 4.6, and specific activity levels less than one/30 compared to the other blood cells preparations. Platelets have a sialidase activity of the same magnitude as granulocytes, with an optimum pH at 4.2.

Blood Platelets↗

PubMed1983

Electron spin resonance studies on the dynamics of phosphatidylcholine-sulfatide model membranes.

The effects of sulfatide on the fluidity and surface dynamics of bilayered and micellar model membranes of egg phosphatidylcholine containing sulfatide were studied by electron spin resonance (ESR). 5-Nitroxystearic acid and 15-nitroxystearic acid were employed as spin-label probes for the region close to the surface and that close to the hydrophobic core of lipid structures. In the vesicular structures, the signals generated by 5-nitroxystearic acid showed that the presence of sulfatide reduced the mobility of the hydrocarbon chains around the probe. The effect increased with increasing glycolipid concentration. The decrease in membrane fluidity was also monitored with the 15-nitroxystearic acid probe, although to a lesser extent. We think that sulfatide causes strong side-to-side head-group interactions on the bilayer surface, causing the lipid chains to assemble in a more rigid fashion, though this effect may be balanced in part by the disordered mechanical coupling of glycolipid acyl chains in the apposite faces of the hydrophobic core of the bilayer. Reduction of this mechanical coupling between apposite lipids when there was transition from a bilayered to a micellar structure resulted in a further increase in the order of the system.

Electron Spin Resonance Spectroscopy↗

PubMed1981

Influence of age and sex on five human plasma lysosomal enzymes assayed by automated procedures.

Automated fluorimetric procedures for the assay of five lysosomal glycohydrolases-beta-N-acetylglucosaminidase; beta-galactosidase; beta-glucuronidase; alpha-mannosidase; alpha-fucosidase-in human plasma were set up. A Carlo Erba autoanalyser CLA 1500, provided with a sampler refrigerating unit and connected with a recording Turner Mod 111 fluorimeter was employed. The automated procedures, under the established optimal conditions, proved to be highly accurate and reproducible. Using the automated assay procedures the effect of sex and age on the plasma levels of the same enzymes was studied. 1273 randomly selected health subjects were studied. No sex differences were observed for all the enzymes studied with the exception of beta-glucuronidase which displayed higher values (about 30%) in males from 25 to 60 years. The developmental profiles of all enzymes in females and males were similar and characterised by: (a) absolute maximum level in the umbilical cord blood; (b) absolute minimum level at 10-14 years; (c) decrease to a second minimum occurring around 35 years (not displayed by beta-galactosidase and by beta-glucuronidase in males); (e) slow further increase up to the elderly level which was then maintained till the oldest age examined, 74 years.

Acetylglucosaminidase↗