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We measured the endogenous plasma concentration of thrombopoietin (TPO) in 76 patients with acquired aplastic anaemia (AA) by a sensitive sandwich enzyme-linked immunosorbent assay (ELISA). The plasma TPO concentrations were significantly higher in AA patients when compared to healthy control subjects (P < 0.0001) and there was a significant negative correlation between plasma TPO concentrations and platelet counts in 54 AA patients who had not received any platelet transfusions prior to sampling. On the other hand, there was no statistically significant correlation between the TPO concentrations and platelet counts in 22 AA patients who had previously received platelet transfusions.

EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200–500 nm in diameter and were rich in both Cl−-ATPase and Na+,K+-ATPase activities. Such Cl−-ATPase-rich membrane vesicles accumulated Cl−in an ATP-dependent and osmotically reactive manner in the presence of 1 mMouabain. The Cl−uptake was maximally stimulated by ATP with a Kmvalue of 1.5 mM, GTP, ITP, and UTP partially stimulated Cl−uptake, but CTP,β,γ-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl−uptake was accelerated by an increase in the medium Cl−concentration with a Kmvalue of 7.4 mM. Such stimulation of Cl−uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37–42 °C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl−uptake with a concentration for half-maximal inhibition at 57 µM. N-Ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl−uptake. The membrane vesicles did not accumulate H+in the Cl−uptake assay medium. The ATP-dependent Cl−uptake profile agreed with that of Cl−-ATPase activity reported previously (Inagaki, C, Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705–1712), and this strongly supports the idea that Cl−-ATPase in the brain actively transports Cl−.

This nor Loposea. a nov construction for resource allocation problem(RA-Problem). A new PA-problem concidering both job sequence and Material flow in given and solved. Job sequence and material flow are expressed as a network model. This paper also proposes an identification method for PERT arrow-digram, which is the most simplified network model. The proposed identification method is heuristic, but it can evaluate the validity of identified values. An example is also given where the proposed methods were applied in the designing of n warehouse system.

Interferon (IFN)-gamma was produced with a high yield in cultures of human peripheral mononuclear cells by combined stimulation with OK-432 and staphylococcal enterotoxin B. Human mononuclear cells cultured in serum-free medium produced several times as much IFN as those in RPMI-1640 medium containing 10% fetal bovine serum. A synergistic effect of OK-432 and staphylococcal enterotoxin B on the production of IFN-gamma was demonstrated. Ultrogel AcA54 column chromatography of crude IFN showed a single peak with an apparent molecular weight of 43,000. Our production system for human IFN-gamma offers a feasible approach to preparation of large quantities of purified IFN-gamma for structure studies, antibody production, and clinical applications.

In a serum-free liquid culture, thrombopoietin (TPO) selectively stimulated the growth of megakaryocytic cells from CD34-positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 × 105) secreted > 1000 pg/ml of interleukin (IL)-8, in contrast to small amounts of IL-1β and IL-6. A time-course study showed that the IL-8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte-conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti-IL-8 antibody, suggesting the secretion of biologically active IL-8. The combination of TPO and IL-1α was required for a significant augmentation of the IL-8 secretion. Direct evidence for IL-8 synthesis in megakaryocytes was provided by reverse transcription–polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL-8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL-8 release by the megakaryocytic cells with the aid of IL-1.

Primary familial and congenital polycythaemia (PFCP) is a rare disease characterized by congenital erythrocytosis inherited in an autosomal dominant fashion. Recently, mutations in the erythropoietin receptor (EpoR) have been identified in PFCP families. We describe a Japanese family with an autosomal dominant inheritance of PFCP. An in vitro colony assay demonstrated hypersensitivity of erythroid progenitors to erythropoietin (Epo) in affected family members. Sequence analysis of RT-PCR products amplified from the C-terminal region of EpoR transcripts in affected family members revealed that they were all heterozygous for C and T bases at position 5986, which suggested a genetic mutation (C to T) on one allele of EpoR. This mutation gave rise to a translation termination codon TAG at amino acid 435. Thus, the resulting EpoR is a truncated protein product lacking all 74 amino acids downstream of the mutation. To date, all genetic mutations affecting a family with PFCP, including this one, have been located in the cytoplasmic negative regulatory region of the EpoR. All mutations gave rise to truncated Epo receptors between Tyrosine 427 and Tyrosine 455. The phosphotyrosines in this region of EpoR have been demonstrated to be binding sites for SHP-1 phosphatase. Therefore PFCP is presumably brought about as a result of genetic mutations which cause the loss of the SHP-1 binding site in the cytoplasmic region of EpoR.

Paroxysmal nocturnal haemoglobinuria (PNH), aplastic anaemia (AA) and myelodysplastic syndrome (MDS) are haemopoietic stem cell disorders. These disorders have some features in common, and a percentage of cases progress to acute leukaemia. We speculated that changes in gene stability are involved in the pathogenesis of these haemopoietic stem cell disorders. Therefore we investigated in vivo mutation frequencies in these disorders by erythrocyte glycophorin A (GPA) mutation assay. The assay enumerates NO or NN variant cells in 106 erythrocytes of the MN type using a flowcytometric technique. Patients undergoing chemotherapy known to be at risk of hypermutageneity were also studied. Events exceeding the 95th percentile of healthy donors (≧ 32 and 34 events, respectively for NO and NN variants) were defined as abnormal. Abnormal events in the NO variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, two out of seven patients with MDS, and four out of nine patients with PNH. Abnormal events in the NN variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, one out of seven patients with MDS, and two out of nine patients with PNH. These results suggest that not only PIG-A, but also other genes including the GPA gene, are hypermutable in haemopoietic stem cell disorders, and that mutagenic pressure and/or gene instability can contribute to the pathogenesis of these disorders.