SPECIFIC BINDING OF NERVE GROWTH FACTOR (NGF) BY MURINE C 1300 NEUROBLASTOMA CELLS

Murine C 1300 neuroblastoma cells bind with high avidity on their membrane surface the nerve growth factor (NGF), a protein capable of inducing differentiation of sympathetic nerve cells. The total binding capacity of NGF by the cells was quantitatively measured by a radioimmunoassay technique, using 125I-labeled NGF. An average number of about 106 molecules of NGF could be bound, at saturation, by each cell with an average relative association constant of about 107 liters/mol. Using synchronized cells, it was found, however, that either the number of molecules of ligand bound or the avidity of the binding interaction between NGF and cells varied depending upon their growth cycle, the maximal-binding occurring during the G1 and early S phase. Binding of [125I]NGF was suppressed by trypsin treatment of the cells, however new receptor sites were rapidly replaced onto the membrane surface within 1–2 h. Cells exposed to 3 M KCl released into the supernate a protein product exhibiting similar high avidity for NGF. Acrylamide gel electrophoresis suggested a restricted molecular heterogeneity of this product, with a major component in the 52,000 mol wt region. Antibodies made specific to this protein were capable, in the absence of the complement, of inhibiting the binding of [125I]NGF by the cells and in the presence of the complement they killed them

Ultrastructural studies of spontaneous in vitro transformation of cultured marrow monocyte-macrophage cells from a patient with congenital hypoplastic anemia

The CM-S cell line was established from the bone marrow of a patient suffering from congenital hypoplastic anemia (syndrome of Diamond-Blackfan). The cells grew in suspension in liquid culture and were dependent for their continuous replication in vitro on growth factors produced by the same cells seeded at high density. Initially, undifferentiated blasts, immature myeloid, megakaryocytic and, rarely, erythroid cells were observed. Eventually, a population of cells with characteristics of monocyte-macrophage precursors predominated. These cells could be induced to terminal macrophage differentiation by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. During this period (over 150 continuous passages), the cells failed to form colonies in agar and to give rise to tumors when inoculated into athymic mice. On prolonged passages, however, the cells gradually increased their growth capacity in liquid culture and became capable of forming colonies in agar and tumors in animals. Ultrastructural studies revealed that the expression of differentiated traits markedly changed as a function of time: after 277 passages, the transformed cells, although displaying characteristics of monocyte precursors, appeared blocked at this stage and no longer responded to 12-O-tetradecanoylphorbol-13-acetate

Ultrastructural studies of spontaneous in vitro transformation of cultured marrow monocyte-macrophage cells from a patient with congenital hypoplastic anemia

The CM-S cell line was established from the bone marrow of a patient suffering from congenital hypoplastic anemia (syndrome of Diamond-Blackfan). The cells grew in suspension in liquid culture and were dependent for their continuous replication in vitro on growth factors produced by the same cells seeded at high density. Initially, undifferentiated blasts, immature myeloid, megakaryocytic and, rarely, erythroid cells were observed. Eventually, a population of cells with characteristics of monocyte-macrophage precursors predominated. These cells could be induced to terminal macrophage differentiation by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. During this period (over 150 continuous passages), the cells failed to form colonies in agar and to give rise to tumors when inoculated into athymic mice. On prolonged passages, however, the cells gradually increased their growth capacity in liquid culture and became capable of forming colonies in agar and tumors in animals. Ultrastructural studies revealed that the expression of differentiated traits markedly changed as a function of time: after 277 passages, the transformed cells, although displaying characteristics of monocyte precursors, appeared blocked at this stage and no longer responded to 12-O-tetradecanoylphorbol-13-acetate

Ultrastructural studies of spontaneous in vitro transformation of cultured marrow monocyte-macrophage cells from a patient with congenital hypoplastic anemia

The CM-S cell line was established from the bone marrow of a patient suffering from congenital hypoplastic anemia (syndrome of Diamond-Blackfan). The cells grew in suspension in liquid culture and were dependent for their continuous replication in vitro on growth factors produced by the same cells seeded at high density. Initially, undifferentiated blasts, immature myeloid, megakaryocytic and, rarely, erythroid cells were observed. Eventually, a population of cells with characteristics of monocyte-macrophage precursors predominated. These cells could be induced to terminal macrophage differentiation by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. During this period (over 150 continuous passages), the cells failed to form colonies in agar and to give rise to tumors when inoculated into athymic mice. On prolonged passages, however, the cells gradually increased their growth capacity in liquid culture and became capable of forming colonies in agar and tumors in animals. Ultrastructural studies revealed that the expression of differentiated traits markedly changed as a function of time: after 277 passages, the transformed cells, although displaying characteristics of monocyte precursors, appeared blocked at this stage and no longer responded to 12-O-tetradecanoylphorbol-13-acetate

Metabolic activation of benzo(alpha)pyrene in fetal mouse hepatocyte lines: Induction of DNA adducts and micronuclei

The 32P-postlabelling assay and the micronucleus test have been used to evaluate spontaneous and induced levels of DNA damage in cultured mammalian cells (C6 and C2.8) following treatment with benzo[a]pyrene and [3H]anti-benzo[a]pyrene diolepoxide

NGF IS RELEASED INTO PLASMA DURING HUMAN-PREGNANCY - AN OXYTOCIN-MEDIATED RESPONSE

THE presence of biologically active nerve growth factor (NGF) in the peripheral circulation of women during pregnancy, labour and lactation was investigated. Using a sensitive immunoenzymatic assay (ELISA), we found an approximately five-fold increase in plasma NGF levels during labour and lactation compared with the concentrations found at the term of gestation or in control healthy women. Since labour and lactation are characterized by activation of the hypothalamo-pituitary-adrenal axis and by high plasma levels of the neurohypophyseal hormone oxytocin, and since the intravenous injection of oxytocin in female rats causes a 176% increase in the hypothalamic levels of NGF, it is possible that the increased amount of circulating NGF is correlated with one or both of these events

Expression Cloning and Biochemical Characterizations of Recombinant Cyclophilin Proteins from Schistosoma mansoni

Recombinant Schistosoma mansoni cyclophilin proteins of the A and the B subtypes (SmCYP A and B) were expressed in bacterial cells as histidine- and maltose-binding fusion proteins and also as nonfused proteins. In addition, S. mansoni CYPs were produced in Sf9 insect cells in their natural forms. Purified recombinant SmCYP B was found to possess a peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 8.2 x 10(5) M-1 s-1. The SmCYP B isoform is approximately two to three times more active than SmCYP A. SmCYP B-specific RNA appears to be more abundant in adult schistosomes than SmCYP A RNA in Northern blots. These results support the conclusion that SmCYP B represents the major schistosomal CYP. The PPIase-associated activity of both CYPs was inhibitable by the immunosuppressive drug cyclosporin A (CsA). We attempt to explain differences in PPIase activities and in CsA inhibition by examining models of the two CYPs complexed to Cs