The Intracellular Transport and Distribution of Cysteamine Phosphate Derivatives

Radioautography and extractive techniques were used to analyze the transport of cysteamine phosphate and its derivatives in salamander oocytes. The quantitative relations among the processes involved — membrane permeation, enzymatic dephosphorylation, binding through mixed disulfide formation, and cytoplasmic diffusion — were elucidated. Within the detection limits, all of the intracellular material is present as dephosphorylated derivatives. Cytoplasmic diffusion is effectively slowed by binding (the “chromatographic” effect) and makes an appreciable contribution to cellular flux rates. As a consequence, one can observe by radioautography a cortical diffusion ring which spreads inward as a function of influx time, while also increasing in peak density because of the finite membrane permeability. Good agreement was found between the transport parameters determined by radioautography and those from influx data for the whole oocyte. The ratio of nuclear to cytoplasmic concentrations of the cysteamine phosphate derivatives at equilibrium is about 0.4. The nuclear membrane is, however, a negligible barrier to transport, and the asymmetry appears to arise primarily from the quantity and sulfhydryl content of the binding proteins in the two compartments

Correction of phenotype in a thalassemia mouse model using a nonmyeloablative marrow transplantation regimen

AbstractGene therapy, the replacement of normal human beta- or gamma-globin genes into the hematopoietic stem cells of patients with homozygous beta-thalassemia, is a promising therapy for the future. High-level lineage-specific stable globin expression in transduced cells reinfused into patients in an autologous transplantation setting could be curative, if successful. Previous studies have shown high-level donor chimerism in nonmyeloablated non-thalassemic hosts. We have now studied the conditions for stable long-term engraftment of normal cells into a thalassemia mouse model that lead to high-level donor chimerism and correction of the abnormal phenotype. Thalassemic female mice treated with 0 to 300 cGy whole-body irradiation received transplantations of donor cells harvested from wild-type males. Engraftment of male cells was quantitated by Y-chromosome polymerase chain reaction analysis of blood and marrow progenitors, and changes in hemoglobin levels, red cell morphology, and spleen size were measured at various times posttransplantation. High-level stable donor cell engraftment was achieved in mice given 200 cGy and receiving transplants of 2 x 10(7) or more donor cells. The anemia, abnormal peripheral blood smears, and splenomegaly improved in the thalassemic mice that had successful engraftment. These studies demonstrate that stable and successful levels of engraftment of normal cells can correct the thalassemic phenotype without fully myeloablating the host. This animal model should allow us to test the amount of cytoreduction required and the level of engraftment and beta-globin expression needed in autologous transplantation of beta-globin gene-transduced cells to correct the abnormal phenotype in thalassemic mice, and it may be relevant to human clinical trials, as well.Biol Blood Marrow Transplant 2002;8(8):453-61