Effect of recombinant human erythropoietin on the anemia of chronic renal failure

Phase I and Phase II studies of recombinant human erythropoietin (rhEpo) were conducted in normal volunteers and in anemic patients with chronic renal failure on maintenance hemodialysis. Three hundred U/person of rhEpo was administered intravenously to healthy normal volunteers in the Phase I study, resulting in no subjective or objective changes. In the Phase II study, 66 patients with chronic renal failure on maintenance hemodialysis with less than 20% hematocrit values were treated with rhEpo in doses of 50 U/kg to 200 U/kg two or three times a week. Hematocrit values increased significantly during the 12 weeks, and the patients' conditions improved. Patients previously requiring blood transfusions became transfusion-independent during our study. There were no obvious side effects, thus indicating the safety and efficacy of rhEpo in the anemia of chronic renal failure.     

Synthesis and properties of the box 9R and 9R' sequences of Tetrahymena rRNA

The chloroethoxyethyl (CEE) group is completely stable under the acidic conditions required to remove the 5'-protecting groups in the oligoribonucleotide synthesis, but can be cleaved under the similar condition to that of the tetrahydropyranyl (THP) group in region of pH 2-3. The oligoribonucleotides were synthesized by the phosphoramidite method on solid supports.     

Burst-promoting activity in anemia and polycythemia

Burst-promoting activity (BPA) in the sera of patients with various types of anemia and polycythemia was compared with that of normal subjects by an in vitro method using mouse bone marrow cells. The control culture contained normal human AB serum instead of sample materials. Results were expressed as a percentage of burst numbers in control cultures. Serum erythropoietin (Epo) levels were determined by a radioimmunoassay. Serum BPA in patients with aplastic anemia (155.4 +/- 56.7%, mean +/- SD) was significantly higher than that in normal subjects (112.1 +/- 29.1%, Wilcoxon's rank sum test, P less than 0.05). However, serum BPA in patients with uremic anemia (122.2 +/- 26.5%), polycythemia vera (101.9 +/- 19.5%) and stress polycythemia (115.5 +/- 25.6%) was not significantly different from normal subjects. There was a correlation between serum BPA and Epo titers in patients with aplastic anemia and paroxysmal nocturnal hemoglobinuria (r = 0.81, t test, P less than 0.001).     

Purification of human lung angiotensin-converting enzyme by high-performance liquid chromatography: properties and N-terminal amino acid sequence

Angiotensin-converting enzyme from the human lung was purified to apparent homogeneity, using high-performance liquid chromatography following trypsin treatment of the detergent-extract. A 1,750-fold purification was achieved with a 26% yield. The specific activity of the enzyme was 105 units per mg protein with the substrate hippuryl-L-histidyl-L-leucine (HHL) at 37 degrees C, and the Km value for HHL was 1.9 mM. The molecular weight was estimated to be 170,000 by sodium dodecyl sulfate gel electrophoresis, and the isoelectric point was about 4.8, by chromatofocusing. The N-terminal amino acid sequence was (NH2)-X-X-Pro-Gly-Leu-Glu-Pro-Gly-X-Phe-Ser-Ala-Arg-Glu-Ala-Gly-Ala. This is highly homologous to the corresponding sequences of the enzymes from bovine and rabbit lung and from pig, bovine, and mouse kidney, but significantly different from that of the human kidney enzyme.     

Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1.

Vpr, an accessory gene of HIV-1, induces cell cycle abnormality with accumulation at G2/M phase and increased ploidy. Since abnormality of mitotic checkpoint control provides a molecular basis of genomic instability, we studied the effects of Vpr on genetic integrity using a stable clone, named MIT-23, in which Vpr expression is controlled by the tetracycline-responsive promoter. Treatment of MIT-23 cells with doxycycline (DOX) induced Vpr expression with a giant multinuclear cell formation. Increased micronuclei (MIN) formation was also detected in these cells. Abolishment of Vpr expression by DOX removal induced numerous asynchronous cytokinesis in the multinuclear cells with leaving MIN in cytoplasm, suggesting that the transient Vpr expression could cause genetic unbalance. Consistent with this expectation, MIT-23 cells, originally pseudodiploid cells, became aneuploid after repeated expression of Vpr. Experiments using deletion mutants of Vpr revealed that the domain inducing MIN formation as well as multinucleation was located in the carboxy-terminal region of Vpr protein. These results suggest that Vpr induces genomic instability, implicating the possible role in the development of AIDS-related malignancies.     

The enhanced cellular uptake of very-low-density lipoprotein enriched in apolipoprotein E.

We have recently reported an increased clearance of plasma very-low-density lipoprotein (VLDL) after intravenous injection of apolipoprotein (apo) E in Watanabe heritable hyperlipidemic (WHHL) rabbits. In the present study, we have investigated the cellular uptake of VLDL enriched in apo E (VLDL-E) which had been incubated with purified rabbit apo E. VLDL-E was taken up approx. 2-fold more than VLDL in human skin fibroblast, human monocyte-derived macrophage and Hep G2 cell and its degradation was least in macrophage. To characterize the binding of VLDL-E, we performed a binding assay using hepatic endosome isolated from estradiol-treated rats and we observed both increased EDTA-sensitive and -resistant binding of VLDL-E on endosome. Ligand blotting of hepatic endosome demonstrated two major bands of LDL receptor (130 and 260 kDa protein) and a minor band of LDL receptor-related protein (580 kDa protein) with a ligand of VLDL-E. These results suggested that VLDL-E was endocytosed in liver through a similar pathway among three cell types, and enrichment of apo E in VLDL enhanced the uptake of VLDL not only via an EDTA-sensitive binding site (classical LDL receptor) but also via other binding sites including an EDTA-resistant binding site and an LDL receptor-related protein.