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Journal of Inherited Metabolic Disease

, Volume 8, Issue 3, pp 95–99 | Cite as

A method for enrichment of hybrid somatic cells: Complementation studies in certain lysosomal enzymopathies

  • P. V. Nelson
  • W. F. Carey
Article

Abstract

An improved method, which combines a number of published techniques, is described for the polyethyleneglycol-induced fusion of mononuclear human skin fibroblasts in the presence of phytohaemagglutinin-P and for the subsequent isolation of polynuclear cells by Ficoll gradient sedimentation. Enriched cultures contain between 60 and 75% multinucleated cells and may be maintained in culture without fetal calf serum for up to 14 days without significant overgrowth by the few contaminating mononuclear parental cells. Complementation appears not to occur between GM1 gangliosidosis and mucopolysaccharidosis, type VIB (Morquio) cell strains; this experimental observation provides support for the earlier hypothesis that the mutations for these conditions are allelic. Earlier observations that complementation does not occur between selected phenotypic variants (viz., neuronopathic forms and those without neurological involvement) of sphingomyelin storage (Niemann-Pick) disease or Gaucher's disease are confirmed.

Keywords

Enrich Culture Skin Fibroblast Multinucleated Cell Neurological Involvement Complementation Study 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Besley, G. T. N., Hoogeboom, A. J. M., Hoogeveen, A., Kleimjer, W. J. and Galjaard, H. Different gene mutations in Niemann-Pick variants.Hum. Genet. 54 (1980) 409–412PubMedGoogle Scholar
  2. Bettger, W. J., Boyce, S. T., Walthall, B. J. and Ham, R. G. Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin and dexamethasone.Proc. Natl. Acad. Sci. (Wash). 78 (1981) 5588–5592Google Scholar
  3. Brady, R. O. Sphingomyelin lipidosis: Niemann-Pick disease. In Stanbury, J. B., Wyngaarden, J. B., Frederickson, D. S., Goldstein, J. L. and Brown, M. S. (eds.)The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1983, pp. 831–841Google Scholar
  4. Cantz, M., Gehler, J. and Spanger, J. Mucolipidosis I: Increased sialic acid content and deficiency of α-N-acetylneuramidase in cultured fibroblasts.Biochem. Biophys. Res. Commun. 74 (1977) 245–252Google Scholar
  5. Carey, W. F. and Pollard, A. C. Variability of fibroblast lysosomal acid hydrolases with reference to the detection of enzyme deficiencies.Aust. J. Exp. Biol. Med. Sci. 55 (1977) 247–252Google Scholar
  6. D'Azzo, A., Halley, D. J. J., Hoogeveen, A. and Galjaard, H. Correction of I-cell defect by hybridisation with lysosomal enzyme deficient human fibroblasts.Am. J. Hum. Genet. 32 (1980) 519–528PubMedGoogle Scholar
  7. Eisenberg, L. R. and Migeon, B. R. Enrichment of human heterokaryons by Ficoll gradient for complementation analysis of iduronate sulphatase deficiency.Somatic Cell Genet. 5 (1979) 1079–1089PubMedGoogle Scholar
  8. Ginns, E. I., Brady, R. O., Pirrucello, S., Moore, C., Sorrell, S., Furbish, F. S., Murray, G. J., Tager, J. and Barranger, J. A. Mutations of glucocerebrosidase: Discrimination of neurologic and non-neurologic phenotypes of Gaucher disease.Proc. Natl. Acad. Sci. USA 79 (1982) 5607–5610PubMedGoogle Scholar
  9. Hamet, M., Bonissol, C. and Cartier, P. Enzymatic activities in purine and pyrimidine metabolism in nine mycoplasma species contaminating cell cultures.Clin. Chim. Acta 103 (1980) 15–22PubMedGoogle Scholar
  10. Hopwood, J. J., Muller, V., Harrison, J. R., Carey, W. F., Elliott, H., Robertson, E. F. and Pollard, A. C. Enzymatic diagnosis of the mucopolysaccharidoses. Experience of 96 cases diagnosed in a 5 year period.Med. J. Aust. 1 (1982) 257–260PubMedGoogle Scholar
  11. Kennett, R. H. Cell fusion. In: Jakoby, W. B. and Paston, I. H. (eds.)Methods of Enzymology, Vol. 58, Academic Press, New York, 1978, pp. 345–359Google Scholar
  12. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. Protein measurement with the folin phenol reagent.J. Biol. Chem. 193 (1951) 265–275PubMedGoogle Scholar
  13. Mercer, W. E. and Schlegel, R. A. Phytohemagglutinin enhancement of cell fusion reduces polyethylene glycol cytotoxicity.Exp. Cell Res. 120 (1979) 417–421PubMedGoogle Scholar
  14. McKusick, V. A. and Neufeld, E. F. The mucopolysaccharide storage diseases. In Stanbury, J. B., Wyngaarden, J. B., Frederickson, D. S., Goldstein, J. L. and Brown, M. S. (eds.)The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1983, pp. 751–777Google Scholar
  15. Mueller, O. T., Honey, N. K., Little, L. E., Miller, A. L. and Shows, T. B. Mucolipidosis II and III. The genetic relationships between two disorders of lysosomal enzyme biosynthesis.J Clin. Invest. 72 (1983) 1016–1023PubMedGoogle Scholar
  16. Pollard, A. C., Carey, W. F., Nelson, P. V., Poulos, A. and Hill, G. N. Enzymological diagnosis of a group of lysosomal storage diseases. Review of a 5-year experience with 1600 patient sample referrals.Med. J. Aust. 2 (1980) 549–553PubMedGoogle Scholar
  17. Poulos, A. and Beckman, K. The bile salt activation of leucocyte sphingolipid hydrolase activity and the modifying effects of Triton X-100.Clin. Chim. Acta 107 (1980) 27–35PubMedGoogle Scholar
  18. Poulos, A. and Pollard, A. C. A rapid method for the estimation of β-galactocerebrosidase, β-glucocerebrosidase and sphingomyelinase activities in leucocytes.Clin. Chim. Acta 72 (1976) 327–335PubMedGoogle Scholar
  19. Wiebel, F. and Baserga, R. Early alteration in amino acid pools and protein synthesis of diploid fibroblasts stimulated to synthesise DNA by addition of serum.J. Cell. Physiol. 74 (1969) 191–202PubMedGoogle Scholar

Copyright information

© SSIEM and MTP Press Limited 1985

Authors and Affiliations

  • P. V. Nelson
    • 1
  • W. F. Carey
    • 1
  1. 1.Department of Chemical PathologyThe Adelaide Children's HospitalNorth AdelaideAustralia

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