Ulrich N. Wiesmann

Expression of antigenic markers during the development of oligodendrocytes in mouse brain cell cultures

The expression of myelin basic protein (MBP) and galactocerebroside (GC), two antigenic markers for oligodendrocytes, was checked on 7-, 14-, 21- and 28-day-old dissociated mouse brain cell cultures (BCC) by using the indirect immunofluorescence method with double staining. The number of GC positive cells increased between the 7th and the 14th day of culture before a steady state was reached. In contrast to this, the MBP-positive cells appeared only on the 14th day of culture, and their number increased with the age of the culture. In double staining, the serum produced against isolated oligodendrocytes shows the same picture as the anti-GC serum, while only a part of GC-positive cells showed also the presence of MBP. Our data suggest that the GC appears very early on the membrane of the oligodendrocytes during development while cells exhibiting both GC and MBP probably represent a more differentiated oligodendrocyte population.

Fatal cases of lipid storage myopathy with carnitine deficiency.

Three patients affected by a progressive myopathy with rapid lethal evolution are presented. Excessive lipid storage was found in type 1 fibres of muscle and in liver, kidney, and myocardium. Carnitine concentrations were markedly reduced in muscle, plasma, and heart, significantly lower in the liver, and normal in kidney. D-L carnitine replacement therapy was ineffective in the only case treated. The relationship of the present cases with the syndrome of lipid storage myopathy and carnitine deficiency is discussed.

Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases.

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster‐one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β‐Galactosidase activity was reduced to about 30‐50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4‐6‐4‐8 while arylsulphatase B had a pi of 7‐8 and 8‐1. When 4‐methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p‐nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes‐trone sulphatase I were solubilized by the addition of lysolecithin and Triton X‐100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6‐8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.

Restoration of the epidermal phenotype by follicular outer root sheath cells in recombinant culture with dermal fibroblasts

In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.

CHRONIC EXPOSURE OF HUMAN CELLS IN CULTURE TO THE TRICYCLIC ANTIDEPRESSANT DESIPRAMINE REDUCES THE NUMBER OF BETA-ADRENOCEPTORS

The effects of the antidepressant drug desipramine (DMI) on the density of beta-adrenoceptor sites were studied on intact cultured human cells: skin fibroblasts, lung fibroblasts and macrophages. Direct binding studies were performed with the radioligand 3H-CGP 12177, a hydrophilic beta-adrenergic antagonist. The confluent cell cultures were exposed to DMI and all three cell types showed a dose-dependent decrease in the number of beta-adrenergic binding sites. This receptor desensitisation was only seen after chronic exposure of the cells to DMI. The extent of desensitisation was comparable to that seen in brain following chronic treatment of rats with DMI. The affinity of the binding sites to the radioligand was not affected by the antidepressant drug action. From these results we suggest that the in vivo effect of antidepressant drugs on postsynaptic beta-adrenoceptor density, at least in part, reflects a primary drug action and not only an adaptive change to presynaptic events.

Systemic carnitine deficiency due to lack of electron transfer flavoprotein: ubiquinone oxidoreductase

A child with myopathy and systemic carnitine deficiency died at age 8 years in an acute metabolic attack. He had glutaric aciduria type II, and his cultured fibroblasts contained normal activity of four different acyl CoA dehydrogenases, but there was deficiency of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF-QO). This enzyme is thought to reduce coenzyme Q in the respiratory chain, funneling reducing equivalents from seven flavoproteins in the β-oxidation of acyl CoAs. There was massive urinary excretion of the short-chain acylcarnitines that accumulated in mitochondria as a result of the ETF-QO defect. Carnitine therefore acts as a buffer for excessive accumulation of intramitochondrial acyl CoAs, and defective β-oxidation can cause carnitine insufficiency.

CERAMIDE-GALACTOSYLTRANSFERASE AND CEREBROSIDE-SULPHOTRANSFERASE LOCALISATION IN GOLGI MEMBRANES ISOLATED BY A CONTINUOUS SUCROSE GRADIENT OF MOUSE BRAIN MICROSOMES

Abstract— A 17,000 g supernatant of mouse brain microsomes was subfractionated on a continuous sucrose gradient in order to localise ceramide galactosyltransferase (CGalt, EC 2.4.1.47) and cerebroside sulphotransferase (CST, EC 2.8.2.11), both enzymes involved in the synthesis of myelin lipids. The submicrosomal fractions were analysed for marker enzymes of myelin, plasma membranes, Golgi membranes, endoplasmic reticulum and lysosomes, and their protein distribution was studied. The results and EM studies give evidence that CGalT and CST are located in the Golgi membranes of the brain.

Organspecific, qualitative changes in the phospholipid composition of rats after chronic administration of the antidepressant drug desipramine

Rats were chronically treated with daily i.p. injections of 10 mg/kg desipramine for 21 days. A 30% decrease in the number of beta-adrenoceptors was observed in brain. A receptor desensitization of similar extent was noted in submaxillary glands and lung. No change in beta-adrenoceptor number was present in heart. Total phospholipid contents were not altered in these organs after chronic drug treatment. However, organ-specific changes were found in the phospholipid composition of submaxillary glands, lung and liver but not in whole brain and heart. The changes were variable but an increase in phosphatidylinositol and decreases in phosphatidylethanolamine and sphingomyelin were consistent. Possible alterations in the phospholipid composition of the brain might have been masked by the large and stable pool of myelin phospholipids. A casual relationship between changes in the phospholipid composition and beta-adrenoceptor desensitization is discussed.

CORRECTION OF THE DEFECTIVE SULFATIDE DEGRADATION IN CULTURED FIBROBLASTS FROM PATIENTS WITH META‐CHROMATIC LEUCODYSTROPHY1

A number of single hereditary lysosomal enzyme deficiencies result in severely disabling metabolic disorders for which no current therapy is known. Aside from the correction of the defective gene by bioengineering, the replacement of the missing lysosomal enzyme could prove to be an effective method in the treatment of single lysosomal enzyme deficiencies. Metachromatic leucodystrophy (MLD), a disease with a known enzyme deficiency which results in a well-established substrate degradation defect, provided us with the opportunity to investigate at the cellular level the effect of enzyme substitution upon the metabolic disorder. Late infantile MLD is a genetic disorder characterized by progressive demyelination and mental retardation. Large amounts of cerebroside sulfate (sulfatide) accumulate in the nervous system and in some visceral organs. The underlying defect is the absence of a lysosomal enzyme, arylsulfatase A (galactocerebroside sulfatase) which degrades sulfatide into cerebroside and sulfate (1, 2, 11). This enzyme is also missing in cultured fibroblasts of the patients. However, no accumulation of sulfatide takes place, since it is not synthesized in these cells. Exogenously administered 3%-

Cellular accumulation of amiodarone and desethylamiodarone in cultured human cells: Consequences of drug accumulation on cellular lipid metabolism and plasma membrane properties of chronically exposed cells

Amiodarone (AMIO), a potent antiarrhythmic drug, is clinically widely used despite its frequent side effects after chronic administration. These side effects coincide with an intralysosomal accumulation of AMIO and its main metabolite desethylamiodarone (DEA) and may be causally related to the drug-induced intracellular storage of phospholipids (PL). Kinetics of cellular uptake and release of radiolabelled AMIO and DEA were studied following single and multiple exposures of cultured human skin fibroblasts to 5 and 10 microM drug concentrations. AMIO and DEA were efficiently taken up into cultured cells. The rate of uptake was slower than that of other cationic amphiphilic drugs. The intracellular steady state concentrations were in the millimolar range suggesting a lysosomal trapping. Repetitive exposures of cultures resulted in a cumulative and partly saturable drug uptake. The accumulation of DEA was higher than that of AMIO throughout. AMIO and DEA previously taken up into the cells during a 2 hr exposure were completely released into the washing media, suggesting an exchangeable form of the accumulated drugs. Following repetitive exposures only part of the drugs was released. Under chasing conditions using washing media containing non-labelled AMIO and DEA respectively or ammonium chloride the release of the chronically accumulated 14C-labelled drugs was increased. This suggested a drug storage in the form of complexes in acidic compartments. Phospholipid (PL) content as well as individual PL fractions were changed in whole cells and in isolated plasma membranes. PL accumulation is assumed to occur by inhibition of PL degradation due to formation of non-degradable drug-PL complexes or by inhibition of phospholipase activities. Cellular PL accumulation seemed to interfere with PL recycling. Changes in PL composition of purified plasma membranes were in part complementary to the ones in whole cells. The alterations in membrane PL composition may explain the changes in membrane fluidity and the decrease in beta-adrenoceptor density and in isoproterenol-stimulated cAMP formation. The results obtained provide an explanation for the pharmacokinetic, and possibly for the pharmacodynamic and also toxicological behaviour of AMIO and DEA in vivo.