The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence

The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.     

Osteogenesis imperfecta: a heterogeneous morphologic phenotype in cultured dermal fibroblasts

Summary Osteogenesis imperfecta (OI) is a phenotype with clinical and biochemical heterogeneity. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n=6) and OI cell strains (n=10, representing the four major OI categories, Sillence classification) were compared by measuring the following: (i) days required in culture to reach confluence after plating at uniform density; (ii) cell density at confluence; (iii) width and length of cells (measured on phase contrast micrographs at 300xmagnification). Our results show that: (i) OI fibroblasts take longer (11–27 days, mean 20 days) than control cells (10–19 days, mean 16 days) to reach stationary phase; (ii) all OI phenotypes achieve a lower cell density (0.87x10⁶ cells/P60, range 0.3–1.6x10⁶) at stationary phase relative to control cells (2.2x10⁶ cells/P60, range 1.7–2.6x10⁶; F_4,77=56.1, p<0.01, indicating that OI cells are larger than normal). Cell shape (expressed as the width: length ratio) was also abnormal in OI cells. (F_4,730=37.6, p<0.01), types I and II OI cells have significantly increased ratios (p<0.01) relative to control, type III, and type IV cells. Intra-group phenotypic heterogeneity was also apparent in the OI categories and also within the control population. These findings confirm deviant morphologic phenotypes in OI dermal fibroblasts and further demonstrate interindividual heterogeneity in the expression of genes that determine size and shape of dermal fibroblasts in both OI and normal donors.Publication No. 84013 from the Montreal Children's Hospital Research Institute     

Effect of Cimetidine and other antihistaminics on the elimination of aminopyrine,phenacetin and caffein

Cimetidine is widely prescribed for the treatment of peptic ulcer disease and has recently been shown to inhibit the metabolism of warfarin, antipyrine and diazepam. To further examine this phenomenon we investigated the effect of various doses of cimetidine and other related drugs on ¹⁴C-aminopyrine, ¹⁴C-phenacetin and ¹⁴C-caffeine breath tests. Cimetidine caused a dose-related inhibition of the metabolism of aminopyrine and caffeine but had no effect on the phenacetin breath test. Metiamide, H₁-antihistamines, phenothiazines and local anesthetics also inhibited the aminopyrine breath test. Cyproheptadine had no effect on either phenacetin or caffeine elimination. This study demonstrates a complex drug-drug interaction which may have widespread clinical implications.     

New species of Passiflora subg. Plectostemma and subg. Tacsonia (Passifloraceae)

Five new species of Passiflora subgenera Plectostemma and Tacsonia from Ecuador are described, viz. P. discophora, P. monadelpha, P. subpurpurea, P. hirtiflora and P. sanctaebarbarae . Section Discophora of subg. Plectostemma is proposed.