Erythropoiesis and red cell function in vertebrate embryos.

All vertebrate embryos produce a specific erythroid cell population--primitive erythrocytes--early in development. These cells are characterized by expression of the specific embryonic haemoglobins. Many aspects of primitive erythropoiesis and the physiological function of primitive red cells are still enigmatic. Nevertheless, recent years have seen intensive efforts to characterize in greater detail the molecular events underlying the initiation of erythropoiesis in vertebrate embryos. Several key genes have been identified that are necessary for primitive and the subsequent definitive erythropoiesis, which differs in several aspect from primitive erythropoiesis. This review gives in its first part a short overview dealing with comparative aspects of primitive and early definitive erythropoiesis in higher and lower vertebrates and in the second part we discuss the physiological function of primitive red cells based mainly on results from mammalian and avian embryos.

Absence of mutations in the CDKN2 binding site of CDK4 in childhood acute lymphoblastic leukemia.

The cell cycle regulatory circuit resulting in phosphorylation of the retinoblastoma protein (pRB) is frequently altered in human cancers. Several mechanisms of disruption are known in that pathway. In childhood acute lymphoblastic leukemia (ALL), the main disrupting mechanism is the homozygous deletion of the CDKN2 (cyclin dependent kinase inhibitor 2) genes: p16CDKN2a, p15CDKN2b, and p19ARF. Another pRB pathway disturbance is a previously described point mutation in the exon 2 of CDK4, a pRB phosphorylating enzyme, which abrogates binding of the latter to its inhibitors, p16CDKN2a and p15CDKN2b. Here we report the absence of point mutations in the CDKN2-binding site of CDK4 in 100 cases of childhood ALL, 2 cases of childhood chronic myeloid leukemia and 9 hematologic cell lines screened by PCR-SSCP (polymerase chain reaction single stranded conformational polymorphism gel electrophoresis), thereby minimizing the possibility of the existence of these specific CDK4 mutations in childhood ALL.

Erythroid carbonic anhydrase and hsp70 expression in chick embryonic development: role of cAMP and hypoxia.

In the second half of avian embryonic development cAMP affects major aspects of red blood cell (RBC) function. At day 13/14, progressive developmental hypoxia causes the release of norepinephrine and erythroid beta-adrenergic receptor stimulation initiates the coordinate induction of adaptive key events of erythroid differentiation like carbonic anhydrase (CAII) and 2,3-biphosphoglycerate synthesis. Although cAMP-dependent regulation of CAII protein synthesis has been described in detail, no data exist about the transcriptional regulation in embryonic RBC. Here we report that after day 12 of embryonic development, the caII mRNA is accumulating. Hypoxic incubation at day 10 as well as in vitro incubation of isolated RBC with cAMP-elevating agonists strongly induces erythroid caII expression. The induction of caII occurs fast and does not require new protein synthesis. By screening several late erythroid genes, we could identify hsp70 as another cAMP-induced gene in definitive RBC. Because caII (but not hsp70) is also induced by cAMP in primitive RBC, the signal may regulate key events of late primitive and definitive erythropoiesis.

Cyclic adenosine monophosphate regulates the expression of the intercellular adhesion molecule and the inducible nitric oxide synthase in brain endothelial cells.

The authors studied whether cyclic AMP (cAMP), a widespread regulator of inflammation, modulates the cytokine-mediated expression of the intercellular adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and the inflammatory nitric oxide synthase 2 (NOS-2), in primary and immortalized brain endothelial cell cultures (GP8.3 cell line). When measured by enzyme-linked immunosorbent assay (ELISA), ICAM-1 was constitutively expressed and was up-regulated twofold by interleukin-1beta, with no effect of interferon-gamma. The NOS-2 activity, assessed by nitrite accumulation, was absent from untreated cultures but was induced by interleukin-1beta and interferon-gamma acting synergistically. Stimulation of cAMP-dependent pathways with forskolin or dibutyryl cAMP decreased ICAM-1 protein expression, whereas it increased NOS-2 protein expression. For both ICAM-1 and NOS-2, mRNA expression correlated with protein expression. Blockade of NOS activity with L-N-monomethylargiuine (L-NMMA) did not alter ICAM-1 expression, indicating that the nitric oxide released by NOS-2 did not cause the down-regulation of ICAM-1. Analysis of NFKB activation indicated that cAMP acted through a mechanism other than inhibition of nuclear translocation of NFKB. The authors conclude that cAMP modulates the expression of proinflammatory molecules in brain endothelium. This suggests that inflammatory processes at the blood-brain barrier in vivo may be regulated by perivascular neurotransmitters via cAMP.

Ontogeny of catecholamine and adenosine receptor-mediated cAMP signaling of embryonic red blood cells: role of cGMP-inhibited phosphodiesterase 3 and hemoglobin.

We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, beta-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and beta-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

Effect of high altitude and in vivo adenosine/(&bgr;)-adrenergic receptor blockade on ATP and 2,3BPG concentrations in red blood cells of avian embryos

In chick embryos, developmental changes of the blood oxygen tension control hemoglobin (Hb) oxygen affinity via modulation of ATP and 2, 3BPG concentrations in red blood cells. Hypoxia, which is a normal developmental condition for late chick embryos, causes a decrease of the red cell ATP concentration (and increase of red cell oxygen affinity) as well as activation of 2,3BPG synthesis via cyclic AMP-dependent signaling. Adenosine and catecholamines have been implicated as signaling substances in these red cell responses. To assess the extent to which adenosine and catecholamines are involved in vivo in the control of red cell ATP/2,3BPG concentrations, day 13 chick embryos were treated for 24 h with adenosine A(2) and/or (&bgr;)-adrenergic receptor blockers and red cell ATP and 2,3BPG levels were determined. The data suggest that adaptive effects later in development in chick embryos induced by adenosine and catecholamines are vital. We have also tested whether avian embryos of the free-living, high-altitude, native white-tailed ptarmigan (Lagopus leucurus) alter their organic phosphate pattern in red cells in response to incubation at different altitudes. Embryos incubated at 3600-4100 m decrease their red cell ATP concentration much more rapidly than embryos of the same clutch incubated at 1600 m. From these data it can be inferred that the oxygen affinity of high altitude embryos will be adjusted to the altitude at which the eggs are incubated.

Adenosine 3':5'-cyclic monophosphate (cAMP)-inducible pyrimidine 5'-nucleotidase and pyrimidine nucleotide metabolism of chick embryonic erythrocytes.

Terminally differentiating erythrocytes degrade most of their RNA with subsequent release of mononucleotides. Pyrimidine mononucleotides are preferentially cleaved by an erythrocyte-specific pyrimidine 5'-nucleotidase; deficiency of this enzyme causes hemolytic anemia in humans. Details of the regulation of its activity during erythroid differentiation are unknown. The present study arose from the observation that the immature red blood cells (RBCs) of mid-term chick embryos contain high concentrations of uridine 5'-triphosphate (UTP) (5 to 6 mmol/L), which decline rapidly from days 13 to 14 onward. We analyzed two key enzymes of RBC pyrimidine nucleotide metabolism: pyrimidine nucleoside phosphorylase (PNP) and pyrimidine 5'-nucleotidase (P-5'-N), to evaluate if changes of enzyme activity during embryonic development are correlated with changes of RBC UTP. Secondly, we tested if these enzymes are under hormonal control. The results show that embryonic RBCs contain only minimal activity of PNP. In contrast, P-5'-N increases from day 13 on, suggesting that the enzyme is a limiting factor in UTP degradation. Activation of beta-adrenergic and A2A-adenosine receptors causes transcription-dependent de novo synthesis of P-5'-N. Because beta-adrenergic and adenosine receptors are also found on adult erythroid cells, P-5'-N might be an enzyme of differentiating RBCs whose expression is in part controlled by adenosine 3':5'-cyclic monophosphate (cAMP).

Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR.

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.

Adenosine causes cAMP-dependent activation of chick embryo red cell carbonic anhydrase and 2,3-DPG synthesis.

In late chick embryos, coordinate activation of red cell carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis is initiated by hypoxia. The effects are mediated by unidentified hormonal effectors resident in chick plasma. In the present investigation, we have analyzed the effect of adenosine receptor stimulation on embryonic red cell CAII and 2,3-DPG synthesis. We find that primitive and definitive embryonic red blood cells from chick have an A2a adenosine receptor. Stimulation of the receptor with metabolically stable adenosine analogues causes a large increase of red cell adenosine 3',5'-cyclic monophosphate (cAMP) and subsequent activation of red cell CAII and 2,3-DPG production in definitive red blood cells and of CAII synthesis in primitive red blood cells. Direct stimulation of adenylyl cyclase with forskolin has the same effect. Analysis of red cell protein pattern after labeling with [35S]methionine shows that stimulation of red cell cAMP levels activates synthesis of several other proteins aside from CAII. Presence of actinomycin D inhibits cAMP-dependent changes of protein synthesis, indicating that cAMP-dependent transcriptional activation is required. In contrast to the stable adenosine receptor analogues, adenosine itself was a very weak agonist, unless its metabolism was significantly inhibited. Thus, besides adenosine, other effectors of the adenylyl cyclase system are likely to be involved in the O2 pressure-dependent regulation of red cell metabolism in late development of avian embryos.

Norepinephrine-mediated hypoxic stimulation of embryonic red cell carbonic anhydrase and 2,3-DPG synthesis.

Hypoxia is the stimulus for activation of red cell carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis of chick red blood cells during late embryonic development. We have tested whether plasma catecholamines are involved as hormonal mediators, because hypoxia is a well-known stimulus for catecholamine release in mammalian fetuses. Plasma catecholamines were measured in 8- to 16-day-old chick embryos. Plasma levels of norepinephrine (NE) were initially low, but its concentration increased rapidly from 2.7 nM (day 12) to 13.4 nM at day 13 and 25.5 nM at day 16. Epinephrine (E) was not detectable before day 13. Short-term hypoxic exposure of day 11 embryos (1-h incubation at 13.5% O2) increased plasma NE concentration fivefold compared with the controls but had no effect on E. During 15-h in vitro incubation of red blood cells from day 11, addition of 1 microM NE to the incubation medium increased the red cell 2,3-DPG concentration nearly threefold and CAII activity sixfold compared with the control. The CAII activity and 2,3-DPG concentration were also increased when cells were incubated with plasma from late chick embryos. The activation was induced by beta-adrenergic stimulation of adenylyl cyclase. Atenolol and propranolol blocked the effects of NE and embryonic chick plasma. Analysis of de novo protein synthesis ([35S]methionine incorporation) demonstrated that catecholamines stimulate the synthesis of several proteins besides CAII. The results indicate that developmental changes of plasma NE concentration are instrumental in the adenosine 3',5'-cyclic monophosphate-dependent activation of CAII and 2,3-DPG synthesis of red blood cells from late chick embryos.

Control of red cell function of late chick embryos: role of extracellular ATP/AMP and egg size.

Hypoxia is the alleged stimulus for initiation of increase of carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis of red blood cells from late chick embryos. The PO2-dependent regulation of red cell metabolism is mediated by unknown humoral factors [Million et al., Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1188-R1196, 1991]. In the present investigation we have analyzed whether interindividual differences in egg size (which result in different surface area-to-mass ratios) affect the timing of initiation of 2,3-DPG and CAII synthesis in late chick embryos. We also investigated the effect of extracellular adenine nucleotides on red cell organic phosphate pattern and O2 affinity to test whether the inhibitory effect of normal or elevated PO2 on 2,3-DPG synthesis and the concomitant increase of ATP (and O2 half-saturation pressure) can be mimicked by these agents. The results show that differences in egg size affect the timing of CAII and 2,3-DPG synthesis, indicating that PO2-dependent regulation of red cell function allows adjustment to the properties of the individual egg. We also found that extracellular ATP, which is rapidly degraded to AMP by red cell ectoenzymes, can alter the red cell phosphate pattern and O2 affinity, i.e., significantly increase red cell ATP, decrease red cell 2,3-DPG and O2 affinity, and thus mimic the effect of normoxia and hyperoxia. These findings suggest that extracellular adenine nucleotides may be involved in the PO2-dependent regulation of embryonic red cell metabolism.