CYTOPLASMIC RESTORATION AND PERSISTENCE OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE ACTIVITY IN STABLE HYBRID MYOTUBES

In previous experiments, we obtained an in vitro restoration of a cyto solic enzyme glucose-6-phosphate dehydrogenase (G6PD) in hybrid myotub es formed between G6PD-deficient and human or murine myoblasts. In the present series of experiments, the degree of restoration was observed and the persistence of the restored activity was evaluated in the hyb rids formed. Quantitative measurements of enzymatic activity in single human-human or human-mouse myotubes, identified by using either fluor escent latex microspheres or Hoechst stain 33258, were made by using a computer-controlled Leitz photometer. Histospectrophotometry and stat istical analysis showed a 50% restoration of enzymatic activity in hum an-human hybrids compared to deficient and normal myotubes. The restor ed activity was uniformly distributed throughout the cytoplasm and per sisted in long-term cultures. No nuclear domain was observed for G6PD. Knowledge of the degree of restoration, of the extent of distribution of the products of ''competent'' nuclei and the demonstration that th e correction is not a transitory event in vitro, supports the potentia l usefulness of myoblast transfer therapy for this type of myopathy.