Cholesterol sulfate is not degraded but does not accumulate in Epstein-Barr virus-transformed lymphoid cells from patients with X-linked ichthyosis.

The metabolism of cholesterol sulfate (CS) was investigated in immortalized, Epstein-Barr virus-transformed lymphoid cell lines derived from normal individuals and patients affected with recessive X-linked ichthyosis (XLI). Normal lymphoid cells expressed arylsulfatase C and steroid sulfatase (including cholesterol sulfatase) activities, and these two sulfohydrolases showed the same enzyme properties as in other human cells, e.g., leukocytes or skin fibroblasts. XLI-derived lymphoid cell lines exhibited extremely deficient activity of both arylsulfatase C and steroid sulfatase. While normal and XLI intact, living lymphoid cells could take up exogenous radiolabelled CS through a non-receptor-mediated process. XLI cells were completely unable to degrade CS to cholesterol. However, despite their defect in CS degradation, steroid sulfatase-deficient cells did not accumulate CS because of outflux of this sterol. The potential implications of these findings to the pathogenesis of increased CS content in plasma and epidermis of XLI patients are discussed. This study also demonstrates that immortalized lymphoid cell lines may represent a useful experimental model system for the study of XLI.

Sulfatide and sphingomyelin loading of living cells as tools for the study of ceramide turnover by lysosomal ceramidase--implications for the diagnosis of Farber disease.

The ceramide turnover by lysosomal ceramidase in intact, living cells was investigated by loading radiolabeled sulfatide or sphingomyelin in situ on skin fibroblasts and lymphoid cells. The cells originated from normal individuals and from patients with acid ceramidase deficiency (Farber disease). While fibroblasts from individuals with Farber disease exhibited some impairment in the degradation of the ceramide produced by sulfatide hydrolysis, lymphoid cells from individuals with Farber disease metabolized the ceramide as readily as did normal cells, suggesting the existence in lymphoid cells of a non-lysosomal degradation pathway for the sulfatide-derived ceramide. In contrast, sphingomyelin loading in the presence of serum showed a considerably decreased turnover of ceramide in both fibroblasts and lymphoid cells from individuals with Farber disease. Further methodologic variation led to the use of LDL-associated radioactive sphingomyelin; LDL-association promoted the targeting of exogenous sphingomyelin to lysosomes. As a result, an almost complete deficiency of ceramide degradation was found in cells from severely affected patients with Farber disease. Our data with this novel method show that sphingomyelin loading of intact living cells is a simple, alternative means for determining ceramide degradation by lysosomal ceramidase and for diagnosing Farber disease.

Functional compartments of sulphatide metabolism in cultured living cells: evidence for the involvement of a novel sulphatide-degrading pathway.

The modes of uptake and degradation of radiolabelled cerebroside sulphate (CS or sulphatide) were investigated in cultured living skin fibroblasts and Epstein-Barr virus-transformed lymphoblastoid cell lines established from control individuals and patients affected with metachromatic leucodystrophy (cerebroside sulphatase deficiency), multiple sulphatase deficiency and low-density-lipoprotein-receptor-negative familial hypercholesterolaemia. In both cell types, CS was taken up through a non-receptor-mediated process. In fibroblasts, CS degradation occurred intralysosomally as was evident from the findings that fibroblasts from metachromatic leucodystrophic patients accumulated the sulphatide and that chloroquine inhibited its degradation by normal cells. In contrast, under similar conditions of CS availability, lymphoblastoid cell lines from patients with metachromatic leucodystrophy could degrade the incorporated sulphatide exactly as their normal counterparts. This metabolic pathway was also fully active in lymphoblastoid cells from patients with multiple sulphatase deficiency and was not inhibited by chloroquine treatment. These data are consistent with a non-lysosomal type of hydrolysis. In addition to the lysosomal and non-lysosomal compartments, a third compartment was identified in the two cell types which is probably formed by the pool of the sulphatide molecules incorporated into the plasma membrane. This is the first report on the existence of a CS-degrading pathway in intact cells with deficient lysosomal cerebroside sulphatase activity.

The in situ degradation of ceramide, a potential lipid mediator, is not completely impaired in Farber disease.

The time course of degradation of a radiolabelled natural ceramide has been studied in intact, living lymphoid cells and skin fibroblasts from normal individuals and from patients affected with Farber disease, an inborn disorder of ceramide metabolism due to deficient activity of lysosomal ceramidase. The hydrolysis of ceramide in lysosomes was selectively followed by examining the turnover of an LDL-associated radioactive sphingomyelin. This permitted to estimate accurately the effective lysosomal ceramidase activity and to demonstrate: (i) a very active catabolism of ceramide in normal cells; and (ii) the absence of a complete block of ceramide degradation in Farber cells. The possible implication of ceramide as a lipid mediator of the pathogenesis of Farber disease is discussed.

Arylsulfatases A and B in EBV-transformed lymphoid cell lines: studies on their molecular forms in cells from patients with inborn sulfatase deficiencies. Comparative diagnostic value of enzymatic assays.

The enzyme activity of arylsulfatase A and arylsulfatase B was studied in Epstein-Barr virus-transformed lymphoid cell lines established from control individuals and patients affected with metachromatic leukodystrophy, mucopolysaccharidosis type VI (or Maroteaux-Lamy syndrome) and multiple sulfatase deficiency. Lymphoid cells derived from patients with metachromatic leukodystrophy showed a severe deficiency in cerebroside sulfatase activity, as measured using radiolabelled sulfatide, but some residual activity of arylsulfatase A when measured with the chromogenic substrate, para-nitrocatechol sulfate. Lymphoid cells from mucopolysaccharidosis type VI had virtually no arylsulfatase B activity. In cells from patients with multiple sulfatase deficiency, the activities of lysosomal sulfatases as well as steroid sulfatase were deficient. Study of the molecular forms of arylsulfatases confirmed the complete deficiency of arylsulfatase A and arylsulfatase B activities in metachromatic leukodystrophy and mucopolysaccharidosis type VI lymphoid cells, respectively. The arylsulfatase A defect in metachromatic leukodys-lymphoid cells, respectively. The arylsulfatase A defect in metachromatic leukodystrophy cells could be demonstrated on focused fractions even using the artificial substrates, para-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. To investigate the discrepancy of the arylsulfatase A activity data observed between whole cell homogenates and focused fractions when using the synthetic substrates, assays were tentatively performed for optimizing the determination of arylsulfatase A on crude homogenates of lymphoid cells. Although this work has indicated methodological limitations of the enzymatic assay of arylsulfatase A in lymphoid cells using methylumbelliferyl sulfate, it emphasizes the validity of lymphoid cell lines as an experimental model for the study of inborn deficiencies of arylsulfatases A and B.

[Surveillance of Neisseria gonorrhoeae strains isolated in Toulouse. In vitro antibiotic sensitivity and plasmid content]. / Surveillance des souches de Neisseria gonorrhoeae isolées à Toulouse. Sensibilité aux antibiotiques in vitro et contenus plasmidiques.

The minimal inhibitory concentration (MIC) of twelve antibiotics was determined by the method of dilution in agar for 50 non penicillinase-producing and 15 penicillinase-producing Neisseria gonorrhoeae (PPNG) strains. The commonly used antibiotics are active in vitro against all the strains. For the non PPNG stains, no difference was observed between the MICs with a previous investigation, in 1980. New quinolones are highly active against all the strains tested but two, with decreased sensitivity. Since 1980, 16 PPNG strains were isolated (2.2%) and analysed for plasmid content. Asian type plasmid (7.4 kb) was present in 9 strains and african type (5.3 kb) in 7 strains. The conjugative plasmid (39 kb) was present in 5 strains and the cryptic plasmid (4.4 kb) in all the strains.

In vitro activity of six quinolone derivatives against Neisseria gonorrhoeae.

The in vitro activities of six quinolone derivatives, rosoxacin, pefloxacin, ofloxacin, ciprofloxacin, A-56619 and A-56620, were compared with those of penicillin, cefotaxime, spectinomycin, chloramphenicol, tetracycline and erythromycin against 50 nonpenicillinase-producing and 15 penicillinase-producing Neisseria gonorrhoeae strains. Ciprofloxacin was the most active compound in vitro (MIC50, 0.004 mg/l) followed by ofloxacin and A-56620 (MIC50, 0.008 mg/l), A-56619 and cefotaxime (MIC50, 0.016 mg/l). The six quinolones are highly active against all the strains tested but 2, with decreased sensitivity.