The use of CFD modelling to optimise measurement of overflow rates in a downstream-controlled dual-overflow structure.

The measurement of the flow through complex combined sewer overflow structures in the frame of automated monitoring remains difficult. In this paper, a methodology based on the use of computational fluid dynamics (CFD) modelling in order to improve the instrumentation of a downstream-controlled dual-overflow structure is presented. The dual-overflow structure is composed of two combined sewer overflows (CSOs) connected by a rectangular channel and controlled by a downstream gate located at the entry of the Meyzieu waste water treatment plant (close to Lyon, France). The analysis of the CFD results provides: (i) a better understanding of the interaction between the two CSOs--that means the hydraulic operation, the hydrodynamic behaviour, the backflow effect--and (ii) an ability to optimise the location of the water depth sensor. The measured water depth is used to assess the overflow rate by means of a numerical relationship. Uncertainties are also assessed.

Involvement of the p38 mitogen-activated protein kinase pathway in tissue inhibitor of metalloproteinases-1-induced erythroid differentiation.

We examined the role of the mitogen-activated protein (MAP) kinase pathway in tissue inhibitor of metalloproteinases-1 (TIMP-1)-mediated cellular effects in a human erythroleukemic cell line UT-7. We show that TIMP-1 induced both UT-7 cell erythroid differentiation and proliferation and tyrosine phosphorylation of many intracellular proteins. Using a panel of phosphospecific antibodies, we also demonstrate that phosphorylation of the p38 and c-Jun N-terminal kinases is increased by TIMP-1 whereas phosphorylation of extracellular signal-regulated kinase 1/2 is not induced. Moreover, inhibition of the p38 activity by SB203580 significantly reduces erythroid differentiation induced by TIMP-1, suggesting that the p38 MAP kinase pathway is involved in TIMP-1-induced erythroid differentiation.

Erythropoietin induction of tissue inhibitors of metalloproteinase-1 expression and secretion is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

In the present study, we demonstrate that erythropoietin (Epo) induces the expression and the release of tissue inhibitors of metalloproteinase-1 (TIMP-1) in a time- and dose-dependent manner in Epo-dependent cell line UT-7 cells and in normal human erythroid progenitor cells from cord blood (CD36+) and required de novo protein synthesis. TIMP-1 was not expressed in the absence of Epo. Inhibition of the mitogen-activated protein kinase pathway by the specific inhibitors PD98059 and U0126 and of phosphatidylinositol 3-kinase by LY294002, strongly inhibited Epo-induced TIMP-1 expression and secretion. In the absence of Epo, both latent and active forms of matrix metalloproteinase-9 (MMP-9) were secreted into media. Upon Epo stimulation, MMP-9 and pro-MMP-9 secretion was inhibited in a dose-dependent manner parallel to TIMP-1 induction. The addition of PD98059, U0126, and LY294002 in the presence of Epo restored MMP-9 production in UT-7 and CD36+ cells. Our findings strongly suggest an inversely coordinated regulation of the TIMP-1 gene and MMP-9 production by Epo via mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

Erythropoietin induces glycosylphosphatidylinositol hydrolysis. Possible involvement of phospholipase c-gamma(2).

We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-gamma(2) in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-gamma(2) was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-gamma(2) hydrolyzed purified [(3)H]GPI indicating that GPI hydrolysis and PLC-gamma(2) activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-gamma(2), whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-gamma(2) activation.

Evidence of the role of protein kinase C during aclacinomycin induction of erythroid differentiation in K562 cells.

At subtoxic concentrations, aclacinomycin is effective in controlling erythroid differentiation of K562, a human erythroleukemic cell line. To better understand early events implicated in this process, we have used bisindolylmaleimide (GF109203X), an inhibitor with a high selectivity for protein kinase C (PKC). Our data show that GF109203X inhibits aclacinomycin effects on K562, evidenced by a strong reduction of hemoglobinized cells and a marked decrease of mRNA rates of erythroid genes. To establish firmly PKC involvement, we also verified that aclacinomycin stimulates its rapid translocation, from the cytosolic to the membrane compartment. By Western blot analysis, we also show that after short induction times, PKCalpha was the most implicated.

Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine following treatment of UT-7 cells with erythropoietin. We have investigated the expression of IRS-1 and IRS-2 in several cell lines with erythroid and/or megakaryocytic features, and we observed that IRS-2 was expressed in all cell lines tested. In contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine-phosphorylated IRS-2 was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase, SHIP. Moreover, IRS-2 was constitutively associated with the erythropoietin receptor. We did not observe the association of IRS-2 with JAK2, Grb2, or PTP1D. Using BaF3 cells transfected with mutated erythropoietin receptors, we demonstrate that neither the tyrosine residues of the intracellular domain nor the last 109 amino acids of the erythropoietin receptor are required for erythropoietin-induced IRS-2 tyrosine phosphorylation. Altogether, our results indicate that erythropoietin-induced IRS-2 tyrosine phosphorylation could account for the previously reported activation of phosphatidylinositol 3-kinase mediated by erythropoietin receptors mutated in the phosphatidylinositol 3-kinase-binding site (Damen, J., Cutler, R. L., Jiao, H., Yi, T., and Krystal, G. (1995) J. Biol. Chem. 270, 23402-23406; Gobert, S., Porteu, F., Pallu, S., Muller, O., Sabbah, M., Dusanter-Fourt, I., Courtois, G., Lacombe, C., Gisselbrecht, S., and Mayeux, P. (1995) Blood 86, 598-606).

Activation of Raf-1 and mitogen-activated protein kinases by erythropoietin and inositolphosphate-glycan in normal erythroid progenitor cells: involvement of protein kinase C.

In this work, we show that erythropoietin and inositolphosphate-glycan activate Raf-1 and the mitogen-activated protein kinases (MAP kinases) in normal erythropoietin-responsive cells. Using a protein kinase C (PKC) activator such as the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate and the PKC inhibitor GF109203X, we investigated a possible involvement of PKC during activation of Raf-1 and MAP kinase by erythropoietin or inositolphosphate-glycan. We found that erythropoietin increased MAP kinase level with a maximum stimulation reached at 5-10 min. Inositolphosphate-glycan and 12-O-tetradecanoyl-phorbol-13-acetate increased MAP kinase activity in the same manner. This activity was inhibited by cell preincubation with GF109203X. Two MAP kinase isoforms were present in erythroid progenitor cells, the 44 and 42 kDa proteins. We report here that erythropoietin, inositolphosphate-glycan, and 12-O-tetradecanoyl-phorbol-13-acetate activated only the p44 form (erk-1) of MAP kinase and the Raf-1 protein. GF109203X was used at a concentration which inhibited by 50% erythroid colonie (CFU-E) proliferation and differentiation induced by erythropoietin or inositolphosphate-glycan. These results support the hypothesis that erythropoietin and inositolphosphate-glycan activate Raf-1 and MAP kinases in normal erythroid progenitor cells and suggest that this activation involves PKC.

Erythropoietin stimulates glycosylphosphatidylinositol hydrolysis in rat erythroid progenitor cells and inositolphosphate glycan modulates their proliferation.

The involvement of a glycosylphosphatidylinositol/inositolphosphate glycan (GPI/IPG) system in the erythropoietin (Epo) signal transduction was investigated. Endogenous GPI was evidenced in extracts of normal Epo-responsive cells after incorporation of [3H]glucosamine, [3H]inositol and [32P]orthophosphate. Incubation of these cells with Epo produced a rapid and transient hydrolysis of GPI with parallel release of IPG. IPG production was Epo dose dependent and the maximal effect was obtained with the same concentration of Epo which gave the maximal mitogenic effect, i.e. 1 U/ml. The number and size of erythroid colonies (CFU-E) were increased by the addition of purified rat erythroid IPG to the culture medium, but not to the same extent as with a maximal Epo treatment. Exogenous IPG effect was dose dependent. In the presence of suboptimal Epo concentrations, IPG has been found to potentiate Epo-induced CFU-E growth. These results support the hypothesis that a GPI/IPG based signal transduction system may be involved in Epo-induced cell proliferation.

Glucose uptake by rat erythroid cells: the effects of erythropoietin and dexamethasone.

Erythropoietin (Ep) stimulated glucose uptake by erythroid progenitor cells in the liver of fetal rats, as measured by [3H]2-deoxy-D-glucose uptake. This dose-dependent stimulation was maximal at 0.2 U/ml Ep and decreased during cell differentiation. Dexamethasone (DEX) inhibited glucose uptake by erythroid progenitors; this dose-dependent effect was maximal at 10(-7) M DEX. Ep partly counteracted the inhibitory effect of DEX. This antagonism may contribute to the overall antagonism between Ep and glucocorticoids in the development of erythroid tissue in vivo and in vitro.

Erythropoietin induces the tyrosine phosphorylation of its own receptor in human erythropoietin-responsive cells.

Using the human erythropoietin-responsive hematopoietic cell line UT-7, we showed that erythropoietin (Epo) rapidly and specifically induced the tyrosine phosphorylation of its own receptor (M(r) 75,000) and increased the tyrosine phosphorylation of other proteins of M(r) 140,000, 120,000, 95,000, 60,000, 57,000, and 42,000. Neither granulocyte-macrophage colony-stimulating factor, interleukin 3, interleukin 6, nor the kit ligand induced the phosphorylation of the M(r) 75,000 receptor protein, although these growth factors induced the phosphorylation of other proteins. Cross-linking experiments using 125I-Epo indicated that the UT-7 cells expressed three Epo receptor subunits, of M(r) 100,000, 85,000, and 75,000, among which only the M(r) 75,000 subunit was tyrosine-phosphorylated following activation with Epo.

Effects of pertussis toxin on the erythropoietin-stimulated proliferation and differentiation of erythroid-responsive cells.

Erythropoietin (Ep) stimulates the proliferation and differentiation of erythroid progenitor cells. We have investigated the possibility that guanyl nucleotide regulatory (G) proteins may be involved in Ep signal transduction. Pertussis toxin (PT) was found to inhibit Ep-stimulated [3H]-thymidine incorporation and large erythroid colony formation in a dose-dependent fashion, but had no effect on small colony formation or on Ep-induced differentiation. PT specifically adenosine diphosphate-ribosylated a major protein with an apparent molecular mass of 41 kD in erythroid progenitor cell membranes. These findings indicate that the transduction of the Ep signal for proliferation could be mediated in the early steps by a PT-sensitive G protein.

In vivo effects of glucocorticoid excess on the contents of the rat fetal liver in erythroid progenitors.

Following a laparotomy of the pregnant rat at 12 days of gestation, erythroid cell suspensions prepared from the fetal livers at 14 days contained an increased proportion of progenitor cells forming colonies after 2 or 7 days of culture. When laparotomy was performed at 14 days and the fetal livers were sampled at 16 days, the opposite effects were observed. Injection of 0.25 mg/kg dexamethasone to the 12 days pregnant rat increased the proportion of erythroid progenitors in suspensions from 14 days fetal livers; injection of 10 mg/kg (a long-acting dose) produced the opposite effects. Both the composition of the erythroid cell line and its environment change between 12 and 14 days, and these modifications might explain the inversion of glucocorticoid effects between these two stages.

The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage.

Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.

Murine erythroleukaemia cells (Friend cells) possess high-affinity binding sites for erythropoietin.

Murine erythroleukaemia cells represent erythroid precursors blocked near the CFU-E or proerythroblast stage. In contrast to their non-leukaemic equivalents, neither their proliferation nor their differentiation seems to be affected by erythropoietin. However, we show in this paper that both uncommitted and committed, benzidine-positive, cells bind iodinated erythropoietin. The binding is of high affinity (Kd = 490 +/- 160 pM) and reversible with a half-life of the complex of 77 +/- 19 min. The number of binding sites is low (300-600 per cell). In contrast the haematopoietic non-erythroid cell lines HL 60 and L 1210 and the myeloid-erythroid human cell line K 562 do not exhibit specific binding. If these binding sites represent true hormone receptors, their presence on a permanent cell line should facilitate erythropoietin receptor purification.

Induction by hemin of proliferation and of differentiation of progenitor erythroid cells responsible for erythropoietin.

Erythroid progenitor cells were obtained from rat fetal liver by immunolysis of the whole erythroid population with an antiserum directed against adult rat erythrocytes, followed by separation on a density gradient. Immediately after their isolation, these cells contained only minute amounts of globin mRNAs and their heme synthesis was negligible. In the absence of erythropoietin (Epo), they did not proliferate or differentiate. In the presence of Epo, they proliferated, synthetized heme and globins actively, accumulated large amounts of globin mRNAs, and developed hemoglobinized colonies in methylcellulose. Hemin, in concentrations of 5-100 microM, induced, in the absence of Epo, the proliferation and differentiation of these cells (e.g., accumulation of globin mRNAs, synthesis of heme and globins, and increased density of membrane antigens characteristic of the erythrocyte). Nevertheless, Epo and hemin actions were not superimposable: in methylcellulose, Epo induced the appearance of large (greater than or equal to 32 cells) hemoglobinized colonies in 48 h, whereas hemin induced smaller and fewer colonies in only 24 h. Succinylacetone (SA, inhibitor of heme synthesis) mostly prevented the effects of Epo on cell proliferation and differentiation; SA inhibition was relieved by hemin. Thus, hemin seems to intervene in erythroid differentiation as a factor of both proliferation and maturation.

Mode of action of erythropoietin and glucocorticoids on the hepatic erythroid precursor cells: role of prostaglandins.

The hypothesis that prostaglandins, and especially PGE2, are the second messengers of erythropoietin (Ep) and that glucocorticoids inhibit Ep action by inhibiting PG synthesis was tested on the erythroid cell line from fetal rat liver. The optimal (10(-9) M) stimulatory concentration of PGE2 did not reproduce, by far, the maximal effect of Ep on the growth of CFUE erythroid colonies. Ep did not increase PGE2 release in liquid culture media of cell suspensions made of the whole erythroid line or enriched (over 85%) in precursor cells. Ep did not modify the turnover rate of arachidonate. Nevertheless, indomethacin partially inhibited Ep effect on CFUE development, and this inhibition was abolished by PGE2. These results suggest that PGE2 potentiates Ep action but is not its second messenger. Spontaneous PGE2 release in liquid culture media brought about concentrations of the order of 10(-9) M, and 10(-7) M dexamethasone completely inhibited this release. Part of (but not all) the anti-Ep effects of glucocorticoids might thus be mediated this way. Dexamethasone effects required previous protein synthesis.

Effect of the antiglucocorticoid agent RU 38486 on the dexamethasone inhibition of Friend cell differentiation.

Glucocorticoid hormones are known to inhibit the erythroid differentiation of Friend cells. The mechanism of action of these hormones has been questioned, and results suggesting an action not involving the nuclear binding of the receptors have been published. We have used the antiglucocorticoid RU 38486 to block the inhibitory effect of dexamethasone on the induced differentiation of Friend cells. Our results strongly suggest a glucocorticoid action involving the binding of classical receptors to the cell nucleus.

Kinetics of dexamethasone binding to the glucocorticoid receptor of intact erythroid cells from rat fetal liver.

The erythroid cells from the rat fetal liver have been shown to possess a receptor for glucocorticoids. In the present work, the characteristics of [3H]dexamethasone binding have been studied on intact cells, in order to minimize receptor degradation, and at 4 degrees C, in order to prevent the activation of the hormone-receptor complex. Dissociation kinetics were those of a first-order reaction and the value of the rate constant of dissociation was similar to the values available in the literature. When studied at low concentrations of the ligand and using short-term incubations, association kinetics were apparently those of a simple bimolecular reaction. But at high ligand concentrations and/or using long-term incubations, association kinetics indicated a more complex reaction. Our results were compatible with the model proposed by Pratt W.B., Kaine J.L. and Pratt V.D. (J. Biol. Chem. 250 (1975) 4584-4591) for cytosolic preparations. This model implies the rapid formation of a transient unstable form of the complex, further converted into a stable form with slower kinetics. Equilibrium dissociation constant of the first (rapid) reaction was 80 microM and the rate constant of 'stabilization' was of the order of 70 X 10(-3) min-1. These values agree with the results of Pratt et al. relative to a cytosolic preparation from rat thymocytes.