Anne Dubart

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.

Identification of a new mutation responsible for hepatoerythropoietic porphyria

Abstract. A deficiency in the activity of uroporphyrinogen decarboxylase (URO‐D), the fifth enzyme of the haem biosynthetic pathway, is found in two hereditary diseases, familial porphyria cutanea tarda (PCT) and hepatoerythropoietic porphyria (HEP). Little is known about the genetic relationship between those two diseases and it has been postulated that HEP is the homozygous form of PCT. A URO‐D cDNA was cloned from an HEP patient and the comparison between the mutant and the wild‐type sequences showed a single base difference within the coding sequence leading to the replacement of a glutamic acid by a lysine at codon 167 of the mutant protein. This replacement produced a protein which is rapidly degraded in the presence of cell lysate. On the basis of hybridization of synthetic oligomers to amplified genomic DNA, we demonstrated that this patient is homozygous for this single base mutation. In order to look for any relationship between HEP and PCT, we tested six unrelated patients with familial PCT and could not detect the codon 167 mutation in any of them. These results indicate an heterogeneity in the mutations responsible for the PCT and HEP phenotypes.

Assignment of human uroporphyrinogen decarboxylase (URO-D) to the p34 band of chromosome 1

SummaryA cDNA probe corresponding to mRNA encoding human uroporphyrinogen decarboxylase (URO-D) was used to determine the chromosomal localization of the URO-D gene in the human genome. In agreement with previous studies, we have found that the locus for URO-D is located on chromosome 1 in hybrid cell mapping panels. The use of in sity hybridization allowed us to map the URO-D locus to band 1p34.

Hemoglobin Synthesis in 7-Day and 14-Day-Old Erythroid Colonies from the Bone Marrow of Normal Individuals

Bone marrow cells from hematologically normal subjects were obtained by aspiration in 19 cases and by surgery in two cases; erythroid colonies were subsequently cultured from these cells in plasma clots. The hemoglobins synthesized in culture were studied either by incubation of the cells at the 6th-7th day of culture, or at the 13th-l4th day of culture with 3H-leucine, in order to determine whether the reactivation of fetal hemoglobin (Hb F) in vitro was related to the stage of differentiation of the erythroid precursors. Hemoglobins synthesized in culture were specifically purified from other proteins by affinity chromatography on Sepharose-haptoglobin and were analyzed by separation of globin chain on carboxy-methyl-cellulose in urea. In 14-day-old cultures, reactivation of Hb F synthesis was observed in all cases but one (mean value 8.2%, range 2.3% to 17.7%). In the 7-day-old cultures, a lower proportion of Hb F synthesis was observed (mean value 4.2%, range 1.6% to 7.6%).These results indicate that ...

Cell-free translation of messenger RNA for human bisphospho-glyceromutase

Abstract mRNAs prepared from different human tissues were translated in a cell-free reticulocyte lysate system and, when present, the neosynthesized bisphosphoglyceromutase (BPGM) was specifically isolated by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. Analysis of the translation products showed that bisphosphoglyceromutase was synthesized in vitro with its mature molecular weight and messenger RNA specifying the synthesis of BPGM exhibited a sedimentation coefficient of 12 S in human reticulocytes. This synthesis seems to be highly tissue specific since we could not evidence any synthesis of this enzyme using mRNA obtained from non erythroid tissue. The BPGM synthesis represents 0.1% of the total neosynthesized non heme proteins in human reticulocyte and ten times less (0.01%) in human fetal liver.

Beta°-Thalassemia/Hb E Association

Hemoglobin synthesis in a 34-year-old man of Laotian ancestry with Hb E/β°- thalassemia was studied. Hemoglobin electrophoresis exhibited only Hb F and Hb E. Reticulocytes and bone m

Cell-free translation of human uroporphyrinogen decarboxylase mRNAs

Uroporphyrinogen decarboxylase was synthesized in a reticulocyte lysate cell-free system under the direction of messenger RNAs isolated from human fetal liver and from human reticulocytes. The enzyme was specifically isolated by immuno affinity chromatography. Analysis of the translation products showed that uroporphyrinogen decarboxylase was synthesized in vitro with its mature molecular weight. This enzyme represented 0.04% of the total neosynthesized proteins under the direction of fetal liver mRNA and about ten times less (0.005%) with reticulocyte mRNA.

Lasting Hb F Reactivation and Hb A2 Reduction Induced by the Treatment of Hodgkin’s Disease in a Woman Heterozygous for Beta-Thalassemia and the Swiss Type of the Heterocellular Hereditary Persistence of Hb F

A remarkable augmentation of Hb F and a reduction of Hb A2 were observed in a Sicilian woman during and after a course of treatment for Hodgkins disease. An inverse correlation between the proportion of Hb F and Hb A2 was found over an 8-year period, as well as in populations of red blood cells fractionated by density gradient. She exhibited two genetic defects, the Swiss type of heterocellular hereditary persistence of fetal hemoglobin and a beta-thalassemia trait, which were confirmed by the study of the hemoglobin synthesis and by a family study. The lasting reactivation of Hb F synthesis is attributable to the interaction of several acquired and inherited factors.