Paul A. Ney

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-X L , BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX- and BAK-independent role for a BCL2-related protein in development.

BNIP3 and NIX are proteins related to the BH3-only family, which induce both cell death and autophagy. Consistent with their ability to induce cell death, BNIP3 and NIX are implicated in the pathogenesis of cancer and heart disease. In tumor cells, BNIP3 and NIX are regulated by hypoxia, and the deregulation of BNIP3 or NIX expression is associated with tumor growth. In heart muscle, BNIP3 and NIX are regulated by hypoxia and Galphaq-dependent signaling, respectively, and their expression is associated with decreased myocardial function. Apart from their role in cell death, BNIP3 and NIX are also implicated in the induction of autophagy. In erythroid cells, NIX is required for a specialized type of autophagy that targets mitochondria for elimination (mitophagy). Similarly, BNIP3 regulates mitophagy in response to hypoxia. In this review, we will discuss possible mechanisms by which BNIP3 and NIX induce cell death and mitophagy. We will also consider the potential relationship between cell death pathways and autophagy in development and homeostasis.

Production of a red blood cell's hemoglobin depends on mitochondrial heme synthesis. However, mature red blood cells are devoid of mitochondria and rely on glycolysis for ATP production. The molecular basis for the selective elimination of mitochondria from mature red blood cells remains controversial. Recent evidence suggests that clearance of both mitochondria and ribosomes, which occurs in reticulocytes following nuclear extrusion, depends on autophagy. Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast atg1p, is a critical regulator of mitochondrial and ribosomal clearance during the final stages of erythroid maturation. However, in contrast to the core autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for induction of macroautophagy in response to nutrient deprivation or for survival of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is a component of the selective autophagy machinery that leads to the elimination of organelles in erythroid cells rather that an essential mechanistic component of autophagy.

A powerful enhancer has been mapped to an 18-bp DNA segment located 11 kb 5' to the human epsilon-globin gene within the dominant control or locus-activating region. This enhancer is inducible in K562 human erythroleukemia cells, increasing linked gamma-globin promoter/luciferase gene expression to 170-fold over an enhancerless construct. The enhancer consists of tandem AP-1-binding sites, phased 10 bp apart, which are both required for full activity. DNA-protein binding assays with nuclear extracts from induced cells demonstrate a high molecular weight complex on the enhancer. The formation of this complex also requires both AP-1 sites and correlates with maximal enhancer activity. Induction of the enhancer may have a role in the increase in globin gene transcription that characterizes erythroid maturation. Enhancer activity appears to be mediated by the binding of a complex of proteins from the jun and fos families to tandem AP-1 consensus sequences.

Mitochondrial autophagy (mitophagy) is a core cellular activity. In this review, we consider mitophagy and related cellular processes and discuss their significance for human disease. Strong parallels exist between mitophagy and xenophagy employed in host defense. These mechanisms converge on receptors in the innate immune system in clinically relevant scenarios. Mitophagy is part of a cellular quality control mechanism, which is implicated in degenerative disease, especially neurodegenerative disease. Furthermore, mitophagy is an aspect of cellular remodeling, which is employed during development. BNIP3 and NIX are related multi-functional outer mitochondrial membrane proteins. BNIP3 regulates mitophagy during hypoxia, whereas NIX is required for mitophagy during development of the erythroid lineage. Recent advances in the field of BNIP3- and NIX-mediated mitophagy are discussed.

Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.

Erythroid transcription factor NF-E2 is a tissue-restricted heterodimeric protein which recognizes an extended AP-1 motif [(T/C)TGCTGA(C/G)TCA(T/C)] found in the upstream locus control regions of the alpha- and beta-globin gene clusters. A cDNA clone encoding a cell-type-specific subunit of NF-E2, designated p45 NF-E2, has previously been characterized and shown to encode a basic-leucine zipper DNA-binding protein. Here we describe protein purification and cloning of cDNA that encodes the second basic-leucine zipper subunit of the native NF-E2 heterodimer. This polypeptide, designated p18, is widely expressed. It displays extensive homology to the v-maf oncogene product and a human retinal-specific protein, NRL. Unusual features in the basic region shared by v-Maf, NRL, and p18 place them in a distinct subfamily of AP-1-like proteins.

Increased alpha-synuclein gene (SNCA) dosage due to locus multiplication causes autosomal dominant Parkinson's disease (PD). Variation in SNCA expression may be critical in common, genetically complex PD but the underlying regulatory mechanism is unknown. We show that SNCA and the heme metabolism genes ALAS2, FECH, and BLVRB form a block of tightly correlated gene expression in 113 samples of human blood, where SNCA naturally abounds (validated P = 1.6 x 10(-11), 1.8 x 10(-10), and 6.6 x 10(-5)). Genetic complementation analysis revealed that these four genes are co-induced by the transcription factor GATA-1. GATA-1 specifically occupies a conserved region within SNCA intron-1 and directly induces a 6.9-fold increase in alpha-synuclein. Endogenous GATA-2 is highly expressed in substantia nigra vulnerable to PD, occupies intron-1, and modulates SNCA expression in dopaminergic cells. This critical link between GATA factors and SNCA may enable therapies designed to lower alpha-synuclein production.

Abstract Mitochondrial clearance is a well recognized but poorly understood biologic process, and reticulocytes, which undergo programmed mitochondrial clearance, provide a useful model to study this phenomenon. At the ultrastructural level, mitochondrial clearance resembles an autophagy-related process; however, the role of autophagy in mitochondrial clearance has not been established. Here we provide genetic evidence that autophagy pathways, initially identified in yeast, are involved in mitochondrial clearance from reticulocytes. Atg7 is an autophagy protein and an E1-like enzyme, which is required for the activity of dual ubiquitin-like conjugation pathways. Atg7 is required for the conjugation of Atg12 to Atg5, and Atg8 to phosphatidylethanolamine (PE), and is essential for autophagosome formation. In the absence of Atg7, mitochondrial clearance from reticulocytes is diminished but not completely blocked. Mammalian homologs of Atg8 are unmodified in Atg7−/− erythroid cells, indicating that canonical autophagy pathways are inactive. Thus, mitochondrial clearance is regulated by both autophagy-dependent and -independent mechanisms. In addition, mitochondria, which depolarize in wild-type cells before elimination, remain polarized in Atg7−/− reticulocytes in culture. This suggests that mitochondrial depolarization is a consequence rather than a cause of autophagosome formation in reticulocytes.

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. We have developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 x 10(10) colony-forming units/ml) or low (5 x 10(6) colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.

Purpose of review Reticulocyte remodeling has emerged as an important model for the understanding of vesicular trafficking and selective autophagy in mammalian cells. This review covers recent advances in our understanding of these processes in reticulocytes and the role of these processes in erythroid development. Recent findings Enucleation is caused by the coalescence of vesicles at the nuclear–cytoplasmic junction and microfilament contraction. Mitochondrial elimination is achieved through selective autophagy, in which mitochondria are targeted to autophagosomes, and undergo subsequent degradation and exocytosis. The mechanism involves an integral mitochondrial outer membrane protein and general autophagy pathways. Plasma membrane remodeling, and the elimination of certain intracellular organelles occur through the exosomal pathway. Summary Vesicular trafficking and selective autophagy have emerged as central processes in cellular remodeling. In reticulocytes, this includes enucleation and the elimination of all membrane-bound organelles and ribosomes. Ubiquitin-like conjugation pathways, which are required for autophagy in yeast, are not essential for mitochondrial clearance in reticulocytes. Thus, in higher eukaryotes, there appears to be redundancy between these pathways and other processes, such as vesicular nucleation. Future studies will address the relationship between autophagy and vesicular trafficking, and the significance of both for cellular remodeling.

The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human beta-globin locus control region is required for high level globin gene expression. We investigated interaction between HS2 and the gamma- and beta-promoters using reporter constructs in transient assays in human erythroleukemia (K562) cells. The beta-promoter, usually silent in K562 cells, was activated by HS2. This activity was abolished when a gamma-promoter was linked in cis. Analysis of truncation mutants suggested that sequences conveying the competitive advantage of the gamma-promoter for HS2 included those between positions -53 and -35 relative to the transcriptional start site. This sequence, when used to replace the corresponding region of the beta-promoter, increased beta-promoter activity 10-fold when linked to HS2. The modified beta-promoter was also capable of competing with a gamma-promoter modified internally in the -53 to -35 region, when the two promoters were linked to HS2 in a single plasmid. The corresponding sequences from the Galago gamma-promoter, a species which lacks fetal gamma-gene expression, were inactive in analogous assays. We have identified and partially purified a nuclear protein found in human (fetal stage) erythroleukemia cells, but present in much lower concentration in murine (adult stage) erythroleukemia cells, that binds the -53 to -35 sequence of the gamma-promoter. We speculate that this region of the gamma-promoter functions as a stage selector element in the regulation of hemoglobin switching in humans.

An erythroid specific, inducible enhancer associated with hypersensitive site II (HS II) plays a central role in the function of the human β globin dominant control region. The HS II enhancer consists of tandem AP-1 binding sites and has been shown to bind members of the ubiquitous jun and fos families of proteins. The same sites are now shown to bind the erythroid specific protein, NF-E2. Inducibility of the HS II enhancer depends on NF-E2 binding, even in the presence of another hypersensitive site. Further, increased activity of the enhancer in induced K562 cells correlates with the presence of NF-E2, which appears to be present in a modified form. NF-E2 is distinct from some enhancer binding proteins in K562 nuclear extracts, in that it does not contain Fos or Fra-1 protein. Thus, binding by NF-E2 may be the mechanism, whereby tandem AP-1 binding sites confer erythroid specificity on the HS II enhancer.

The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation.

Abstract ABCG2/BCRP is a member of the adenosine triphosphate–binding cassette (ABC) transporter family and is expressed in intestine, kidney, and liver, where it modulates the absorption and excretion of xenobiotic compounds. ABCG2 is also expressed in hematopoietic stem cells and erythroid cells; however, little is known regarding its role in hematopoiesis. Abcg2 null mice have increased levels of protoporphyrin IX (PPIX) in erythroid cells, yet the mechanism for this remains uncertain. We have found that Abcg2 mRNA expression was up-regulated in differentiating erythroid cells, coinciding with increased expression of other erythroid-specific genes. This expression pattern was associated with significant amounts of ABCG2 protein on the membrane of mature peripheral blood erythrocytes. Erythroid cells engineered to express ABCG2 had significantly lower intracellular levels of PPIX, suggesting the modulation of PPIX level by ABCG2. This modulating activity was abrogated by treatment with a specific ABCG2 inhibitor, Ko143, implying that PPIX may be a direct substrate for the transporter. Taken together, our results demonstrate that ABCG2 plays a role in regulating PPIX levels during erythroid differentiation and suggest a potential role for ABCG2 as a genetic determinant in erythropoietic protoporphyria.

Elimination of defective mitochondria is essential for the health of long-lived, postmitotic cells. To gain insight into this process, we examined programmed mitochondrial clearance in reticulocytes. BNIP3L is a mitochondrial outer membrane protein that is required for clearance. It has been suggested that BNIP3L functions by causing mitochondrial depolarization, activating autophagy, or engaging the autophagy machinery. Here we showed in mice that BNIP3L activity localizes to a small region in its cytoplasmic domain, the minimal essential region (MER). The MER is a novel sequence, which comprises three contiguous hydrophobic amino acid residues, and flanking charged residues. Mutation of the central leucine residue causes complete loss of BNIP3L activity, and prevents rescue of mitochondrial clearance. Structural bioinformatics analysis predicts that the BNIP3L cytoplasmic domain lacks stable tertiary structure, but that the MER forms an α-helix upon binding to another protein. These findings support an adaptor model of BNIP3L, centered on the MER.

The human gamma-globin gene promoter contains a stage selector element (SSE) responsible for preferential interaction of the promoter with a powerful erythroid-specific enhancer in the fetal developmental stage (S.M. Jane, P.A. Ney, E.F. Vanin, D.L. Gumucio, and A.W. Nienhuis. EMBO J. 11:2691-2699, 1992). The element binds two proteins, the ubiquitous activator Sp1 and a protein previously known as -50 gamma and now named the stage selector protein (SSP). Binding of the second protein correlates with SSE activity in transient-transfection assays. We now report that a de novo binding site for the SSP is created by the -202(C-->G) mutation that causes hereditary persistence of fetal hemoglobin (HPFH). This site functions in an analogous manner to the SSE in hybrid beta-promoter/reporter gene constructs transfected into K562 cells. In contrast, the wild-type -202 sequence, which fails to bind the SSP, is incapable of activating the beta-gene promoter. Both the -50 and -202 HPFH sites for SSP binding overlap a consensus sequence for the transcriptional regulator Sp1. In addition, both sites contain CpG dinucleotides that are contact bases for SSP. Since the gamma promoter is known to be hypomethylated in fetal cells but fully methylated at CpG residues in adult erythroid cells, we examined the effects of this DNA modification on protein binding to the two regions. Gel mobility shift assays with nuclear extract from K562 cells (which contain both Sp1 and SSP) demonstrate preferential binding of SSP to the SSE and HPFH sites under conditions in which probe was limiting. Methylation of the CpG residues reverses this preference only in the SSE site, with a marked increase in the binding of Sp1 at the expense of the SSP. Purified Sp1 binds with 10-fold higher affinity to the methylated than to the nonmethylated -50 probe but with the same affinity to the -202 HPFH probe. The methylation-induced preferential binding of Sp1 to the SSE at the expense of SSP may be part of the mechanism by which the gamma genes are repressed in normal adult erythroid cells. In cells containing the -202 HPFH mutation, the inability of Sp1 to displace SSP in the methylated state may explain the persistence of gamma-promoter activity and gamma-gene expression observed in adults with this mutation.

The locus activating region (LAR), contained within 30 kb of chromatin flanking the human beta-globin gene cluster, has recently been shown to be essential for high level beta-globin gene expression. To determine the effect of fragments containing LAR sequences on globin gene expression, mRNA from a marked gamma-globin gene linked to LAR fragments was assayed in stably transfected K562 erythroleukemia cells. DNaseI hypersensitive site II (HS II), located 10.9 kb upstream of the epsilon-globin gene, was required for high level gamma-globin gene expression. We also showed that a 46 bp enhancer element within HS II was necessary and sufficient for the increased gamma-globin gene expression observed with hemin induced erythroid maturation of K562 cells. These results localize a distant regulatory element important for activation of globin genes during human erythroid cell maturation.

The controlled elimination of defective mitochondria is necessary for the health of long-lived post-mitotic cells, like cardiomyocytes and neurons. Mitochondrial elimination also occurs during the course of normal development, in lens epithelial and erythroid cells. Strikingly, at the final stage of erythroid cell maturation, newly formed erythrocytes, also known as reticulocytes, eliminate their entire cohort of mitochondria. We have employed this model to investigate the mechanism of programmed mitochondrial clearance. NIX (BNIP3L) is a Bcl-2-related protein that is upregulated during terminal erythroid differentiation. NIX-deficient reticulocytes have a significant defect of mitochondrial clearance. Consistent with the ability of NIX to cause mitochondrial depolarization, we show that mitochondria are depolarized in wild type but not NIX deficient reticulocytes. NIX does not function through established proapoptotic pathways, nor does it mediate the induction of autophagy in erythroid cells. Rather, NIX is required for the selective incorporation of mitochondria into autophagosomes. Elucidation of the mechanism of this effect will improve our understanding of the role of autophagy in the maintenance of cellular homeostasis.

Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation and chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late-stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.

Hereditary porphyrias are caused by mutations in genes that encode haem biosynthetic enzymes with resultant buildup of cytotoxic metabolic porphyrin intermediates. A long-standing open question is why the same causal porphyria mutations exhibit widely variable penetrance and expressivity in different individuals. Here we show that severely affected porphyria patients harbour variant alleles in the ABCB6 gene, also known as Lan, which encodes an ATP-binding cassette (ABC) transporter. Plasma membrane ABCB6 exports a variety of disease-related porphyrins. Functional studies show that most of these ABCB6 variants are expressed poorly and/or have impaired function. Accordingly, homozygous disruption of the Abcb6 gene in mice exacerbates porphyria phenotypes in the Fech(m1Pas) mouse model, as evidenced by increased porphyrin accumulation, and marked liver injury. Collectively, these studies support ABCB6 role as a genetic modifier of porphyria and suggest that porphyrin-inducing drugs may produce excessive toxicities in individuals with the rare Lan(-) blood type.

Platelet activation requires fully functional mitochondria, which provide a vital energy source and control the life span of platelets. Previous reports have shown that both general autophagy and selective mitophagy are critical for platelet function. However, the underlying mechanisms remain incompletely understood. Here, we show that Nix, a previously characterized mitophagy receptor that plays a role in red blood cell maturation, also mediates mitophagy in platelets. Genetic ablation of Nix impairs mitochondrial quality, platelet activation, and FeCl3-induced carotid arterial thrombosis without affecting the expression of platelet glycoproteins (GPs) such as GPIb, GPVI, and αIIbβ3 Metabolic analysis revealed decreased mitochondrial membrane potential, enhanced mitochondrial reactive oxygen species level, diminished oxygen consumption rate, and compromised adenosine triphosphate production in Nix-/- platelets. Transplantation of wild-type (WT) bone marrow cells or transfusion of WT platelets into Nix-deficient mice rescued defects in platelet function and thrombosis, suggesting a platelet-autonomous role (acting on platelets, but not other cells) of Nix in platelet activation. Interestingly, loss of Nix increases the life span of platelets in vivo, likely through preventing autophagic degradation of the mitochondrial protein Bcl-xL. Collectively, our findings reveal a novel mechanistic link between Nix-mediated mitophagy, platelet life span, and platelet physiopathology. Our work suggests that targeting platelet mitophagy Nix might provide new antithrombotic strategies.

Tandem binding sites for the hematopoietic transcription factor NF-E2 in the beta-globin locus control region activate high-level beta-globin gene expression in transgenic mice. NF-E2 is a heterodimer consisting of a hematopoietic subunit p45 and a ubiquitous subunit p18. Gavva et al. [Gavva, N. R., Gavva, R., Ermekova, K., Sudol, M., and Shen, J. C. (1997) J. Biol. Chem. 272, 24105-24108] reported that human p45 contains a PPXY motif that binds WW domains. We show that murine NF-E2, which contains two PPXY motifs (PPXY-1 and -2) within its transactivation domain, differentially interacted with nine GST-WW domain fusion proteins. Quantitative analysis revealed high-affinity binding (KD = 5.7 nM) of p45 to a WW domain from a novel human ubiquitin ligase homologue (WWP1) expressed in hematopoietic tissues. The amino-terminal WW domain of WWP1 formed a multimeric complex with DNA-bound NF-E2. A WWP1 ligand peptide, isolated by phage display, and a peptide spanning PPXY-1 inhibited p45 binding, whereas an SH3 domain-interacting peptide and a peptide spanning PPXY-2 did not. Mutation of PPXY-1, but not PPXY-2, inhibited the transactivation function of NF-E2, providing support for the hypothesis that WW domain interactions are important for NF-E2-mediated transactivation.

In 1957, Charlotte Friend described a novel retroviral disease in mice characterized by splenic enlargement, erythroleukemia, and death.[1][1] This rapidly progressive disease, now known as Friend disease, has provided a powerful tool for the study of multistage carcinogenesis. Transformed murine

Abstract CREB-binding protein (CBP) and its para-log p300 are transcriptional coactivators that physically or functionally interact with over 320 mammalian and viral proteins, including 36 that are essential for B cells in mice. CBP and p300 are generally considered limiting for transcription, yet their roles in adult cell lineages are largely unknown since homozygous null mutations in either gene or compound heterozygosity cause early embryonic lethality in mice. We tested the hypotheses that CBP and p300 are limiting and that each has unique properties in B cells, by using mice with Cre/LoxP conditional knockout alleles for CBP (CBPflox) and p300 (p300flox), which carry CD19Cre that initiates floxed gene recombination at the pro–B-cell stage. CD19Cre-mediated loss of CBP or p300 led to surprisingly modest deficits in B-cell numbers, whereas inactivation of both genes was not tolerated by peripheral B cells. There was a moderate decrease in B-cell receptor (BCR)–responsive gene expression in CBP or p300 homozygous null B cells, suggesting that CBP and p300 are essential for this signaling pathway that is crucial for B-cell homeostasis. These results indicate that individually CBP and p300 are partially limiting beyond the pro-B-cell stage and that other coactivators in B cells cannot replace their combined loss.

The sickle gene has remarkably pleiotropic effects. In an attempt to understand the complex pathobiology of sickle disease, we have searched for potential relationships between various cellular abnormalities. In the present studies, we treated normal erythrocytes (RBCs) with phenazine methosulfate to stimulate intracellular generation of superoxide and thereby stimulate the excessive autoxidation characteristic of sickle RBCs. In response, RBCs show a dose-dependent increase in adherence to endothelium, easily achieving the degree of abnormal adherence typical of unmanipulated sickle RBCs. This is mediated by adverse effects of oxidant on cellular hydration, as evidenced by amelioration of abnormal adhesivity if the drug's dehydrating effect is inhibited. Consistent with potential relevance of this to sickle pathobiology, the abnormal endothelial adherence of sickle RBCs can be worsened or improved by dehydrating or hydrating these cells, respectively. These data suggest an intimate relationship between three abnormalities of sickle RBCs previously thought to be unrelated (autoxidation, dehydration, and adhesivity). Although multiple mechanisms may contribute to abnormal cell-cell interactions in sickle disease, the potentiation of sickle RBC adhesivity by cellular dehydration may help explain why clinical dehydration has been identified as a precipitant of vasocclusive crisis. Insofar as abnormal adhesivity participates in the sickle vasocclusive process, these data provide further rationale for attempting to manipulate RBC hydration as a therapeutic maneuver in this disease.

B-cell leukemia/lymphoma 2 (BCL-2)/adenovirus E1B interacting protein 3 (BNIP3) and Nip-like protein X (NIX) are atypical BCL-2 homology domain 3-only proteins involved in cell death, autophagy, and programmed mitochondrial clearance.BNIP3 and NIX cause cell death by targeting mitochondria, directly through BCL-2associated X protein-or BCL-2-antagonist/killer-dependent mechanisms, or indirectly through an effect on calcium stores in the endoplasmic reticulum.BNIP3 and NIX also induce autophagy through an effect on mitochondrial reactive oxygen species production, or by releasing Beclin 1 from inhibitory interactions with antiapoptotic BCL-2 family proteins.BNIP3 downregulates mitochondrial mass in hypoxic cells, whereas NIX is required for mitochondrial elimination during erythroid development.BNIP3 and NIX have an emerging role in human health.Cell death mediated by BNIP3 and NIX is implicated in heart disease and ischemic injury.Cancer progression is linked to loss of the prodeath function of BNIP3, but also to induction of its prosurvival activity.Finally, BNIP3 and NIX are implicated in mitochondrial quality control, which is important in aging and degenerative disease.Elucidation of the mechanisms by which BNIP3 and NIX regulate cell death, autophagy, and mitochondrial clearance may lead to treatments for these conditions.

Although BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other antiapoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 deletion results in embryonic lethality beyond embryonic day 13.5 as a result of severe anemia caused by a lack of mature red blood cells (RBCs). Mcl1-deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that MCL-1 is only required during early definitive erythropoiesis; during later stages, developing erythrocytes become MCL-1 independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development because codeletion of the proapoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these 2 antiapoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

Abstract NF-E2 binding sites, located in distant regulatory sequences, may be important for high level α- and β-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2–related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2–deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of α- and β-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2–deficient mice is not the result of compensation by Nrf-2.

During the initial stage of Friend virus-induced erythroleukemia in mice, interaction of the viral protein gp55 with the erythropoietin receptor, and other host factors, drives the expansion of erythroid precursor cells. Recently, we demonstrated that the Friend virus susceptibility locus, Fv2, which is required for the expansion of infected cells, encodes a naturally occurring, N-terminally truncated form of the Stk receptor tyrosine kinase (Sf-Stk). Here we show that in vitro expression of Sf-Stk confers Friend virus sensitivity to erythroid progenitor cells from Fv2(rr) mice. Furthermore, our data reveal that Sf-Stk kinase activity and Y436, but not Y429, are required for Epo-independent colony formation following Friend virus infection. Introduction of a mutation that results in failure to bind Grb2 abrogates the ability of Sf-Stk to induce colonies in response to Friend virus, while the Grb2 binding site from EGFR restores this response. Consistent with the ability of Grb2 to recruit SOS and Gab1, the Ras/MAPK and PI3K pathways are activated by Sf-Stk, and both of these pathways are required for gp55-mediated erythroblast proliferation. These data clearly demonstrate a requirement for signaling through Sf-Stk in the Epo-independent expansion of Friend virus-infected cells, and suggest a pivotal role for Grb2 in this response.

Interaction between the stage selector element (SSE) in the proximal γ-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the β-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5′-untranslated region of the γ-gene also contributes to this competitive silencing of the β-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal γ-promoter in the absence of the SSE, completely silencing a linked β-promoter. Mutation of a 20-base pair sequence of the γ-gene 5′-untranslated region (UTR) led to derepression of β-promoter activity. A marked activation of γ-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5′-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5′-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5′-UTR of the γ-gene contains sequences that may be important for its transcriptional and developmental regulation. Interaction between the stage selector element (SSE) in the proximal γ-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the β-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5′-untranslated region of the γ-gene also contributes to this competitive silencing of the β-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal γ-promoter in the absence of the SSE, completely silencing a linked β-promoter. Mutation of a 20-base pair sequence of the γ-gene 5′-untranslated region (UTR) led to derepression of β-promoter activity. A marked activation of γ-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5′-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5′-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5′-UTR of the γ-gene contains sequences that may be important for its transcriptional and developmental regulation.

The human β-globin gene is expressed at high levels in erythroid cells and regulated by proximal and distal cis-acting DNA elements, including promoter, enhancer, and a locus control region (LCR). Transcription complexes are recruited not only to the globin gene promoters but also to the LCR. Previous studies have implicated the ubiquitously expressed transcription factor USF and the tissue-restricted activator NF-E2 in the recruitment of transcription complexes to the β-globin gene locus. Here we demonstrate that although USF is required for the efficient association of RNA polymerase II (Pol II) with immobilized LCR templates, USF and NF-E2 together regulate the association of Pol II with the adult β-globin gene promoter. Recruitment of Pol II to the LCR occurs in undifferentiated murine erythroleukemia cells, but phosphorylation of LCR-associated Pol II at serine 5 of the C-terminal domain is mediated by erythroid differentiation and requires the activity of NF-E2. Furthermore, we provide evidence showing that USF interacts with NF-E2 in erythroid cells. The data provide mechanistic insight into how ubiquitous and tissue-restricted transcription factors cooperate to regulate the recruitment and activity of transcription complexes in a tissue-specific chromatin domain. The human β-globin gene is expressed at high levels in erythroid cells and regulated by proximal and distal cis-acting DNA elements, including promoter, enhancer, and a locus control region (LCR). Transcription complexes are recruited not only to the globin gene promoters but also to the LCR. Previous studies have implicated the ubiquitously expressed transcription factor USF and the tissue-restricted activator NF-E2 in the recruitment of transcription complexes to the β-globin gene locus. Here we demonstrate that although USF is required for the efficient association of RNA polymerase II (Pol II) with immobilized LCR templates, USF and NF-E2 together regulate the association of Pol II with the adult β-globin gene promoter. Recruitment of Pol II to the LCR occurs in undifferentiated murine erythroleukemia cells, but phosphorylation of LCR-associated Pol II at serine 5 of the C-terminal domain is mediated by erythroid differentiation and requires the activity of NF-E2. Furthermore, we provide evidence showing that USF interacts with NF-E2 in erythroid cells. The data provide mechanistic insight into how ubiquitous and tissue-restricted transcription factors cooperate to regulate the recruitment and activity of transcription complexes in a tissue-specific chromatin domain.

Mitochondria are the primary site of energy production in animal cells. In mitochondria, the flow of electrons through the electron transport chain creates a potential difference across the inner membrane, which is utilized for ATP production. However, due to inherent inefficiencies in electron transport, reactive oxygen species are also produced, which damage mitochondrial proteins and nucleic acids, and impair mitochondrial function.1 Decreased mitochondrial function causes increased reactive oxygen species generation, a decline in cellular function, and potentially cell death.2 Therefore, to maintain cellular homeostasis, mechanisms have evolved to selectively eliminate defective mitochondria.3 Mitochondria are constantly undergoing cycles of fission and fusion, and this process appears to have a role in mitochondrial quality control. Following fission, daughter mitochondria are produced, which can differ in their membrane polarization. Depolarized mitochondria are less likely to undergo subsequent fusion, and more likely to undergo autophagic clearance.4 As would be predicted, given the potential for cytochrome c release, depolarization is a powerful stimulus for mitochondrial clearance. Depolarization causes recruitment of the E3 ubiquitin ligase Parkin to mitochondria, which is required for their subsequent engulfment by autophagosomes.5 Macroautophagy pathways also appear to have a role, as hepatocytes deficient for the E1-like enzyme Atg7 accumulate abnormal mitochondria.6 Finally, recent studies in a developmental model have yielded insight into this process. Newly-formed erythrocytes, also known as reticulocytes, eliminate their entire cohort of mitochondria during development.7 This process depends on the mitochondrial protein NIX, is partially dependent on autophagy, and is independent of mitochondrial depolarization.8-10 Here we describe the use of reticulocytes to study mitochondrial clearance.

Expression profiling is a powerful technique to sample cell state. This review shows how expression profiling is being applied to the study of erythroid differentiation.Expression-based studies of multipotential hematopoietic progenitor cells has shown that these cells express lineage-restricted genes from multiple lineages at low levels, and that they are in effect 'primed' to develop into all hematopoietic cell types. Expression profiling of oligopotent and committed progenitor cells has further shown that commitment to the erythroid lineage is associated with a progressive decline in the number of expressed genes. Lineage commitment is regulated by lineage-restricted transcription factors, and studies show that the erythroid transcription factor GATA1, in addition to activating a subset of genes, has global repressive effects on gene expression. Terminal erythroid differentiation is associated with further reduction in the number of expressed genes. The erythroid program is defined by those genes that are still expressed, and their high-level expression depends on specific epigenetic modifications, recruitment of transcription factors, and posttranscriptional effects.Expression profiling provides the means to identify novel targets for the therapy of erythrocytes disorders, and to obtain insights into the mechanisms of cellular differentiation.

Regulated expression of genes in the β-globin cluster depends upon sequences located between 5 and 20 kb upstream of the ε gene, known as the locus control region (LCR). β-Globin expression in murine erythroleukemia (MEL) cells depends on NF-E2, a transcription factor which binds to enhancer sequences in the LCR. To gain insight into the mechanism of globin gene activation by NF-E2, an NF-E2 null MEL cell line was used to map regions of NF-E2 required for β-globin expression. Within the transactivation domain, two discrete proline-rich regions were required for rescue of β-globin expression. The first was located at the N-terminus of NF-E2, while the second was located N-terminal of the cap 'n collar (CNC) domain. Other proline-rich sequences were dispensable, indicating that proline content per se does not determine NF-E2 activity. Mutations within the conserved CNC domain markedly diminished rescue of β-globin expression. This domain was required, in addition to the basic leucine zipper domain, for DNA binding activity. The requirement for discrete proline-rich sequences within the transactivation domain suggests that globin gene expression in MEL cells depends on specific interactions between NF-E2 and downstream effector molecules.

Transgenic mice homogeneously expressing enhanced green fluorescence protein (EGFP) in primitive hematopoietic cells and all blood cell progeny, including erythrocytes and platelets, have not been reported. Given previous data indicating H2Kb promoter activity in murine hematopoietic stem cells (HSCs), bone marrow (BM), and lymphocytes, an H2Kb enhancer/promoter EGFP construct was used to generate transgenic mice. These mice demonstrated pancellular EGFP expression in both primitive BM Sca-1+Lin-Kit+ cells and side population (SP) cells. Additionally, all peripheral blood leukocytes subsets, erythrocytes, and platelets uniformly expressed EGFP strongly. Competitive BM transplantation assays established that transgenic H2Kb-EGFP HSCs had activity equivalent to wildtype HSCs in their ability to reconstitute hematopoiesis in lethally irradiated mice. In addition, immunohistochemistry revealed EGFP transgene expression in all tissues examined. This transgenic strain should be a useful reagent for both murine hematopoiesis studies and functional studies of specific cell types from particular tissues.

Mitochondria are the site of oxidative phosphorylation in animal cells and a primary target of reactive oxygen species‐mediated damage. To prevent the accumulation of damaged mitochondria, mammalian cells have evolved strategies for their elimination. Autophagy is one means for the controlled elimination of mitochondria; however, although there has been considerable progress in defining the requirements for nonselective autophagy, relatively little is known about the genes that regulate selective autophagy of organelles. To improve our understanding of mitochondrial autophagy in mammals, we have undertaken a genetic analysis of mitochondrial clearance in murine reticulocytes. Reticulocytes provide an ideal model to study this process, because mitochondria are rapidly cleared from reticulocytes during normal development through an autophagy‐related process. Here we describe several methods for monitoring mitochondrial clearance and autophagy in reticulocytes, and show that in reticulocytes these processes require genes involved in both nonselective and selective autophagy.

Erythrocyte formation involves the elimination of mitochondria at the reticulocyte stage of development. Nix(-/-) reticulocytes fail to eliminate their mitochondria at this step due to a defect in the targeting of mitochondria to autophagosomes. To determine the role of autophagy in this process, we generated Atg7(-/-) transplant mice. Atg7(-/-) reticulocytes exhibit a partial defect in mitochondrial clearance, demonstrating that there are both autophagy-dependent and -independent mechanisms of mitochondrial clearance. We used Atg7(-/-) autophagy-defective reticulocytes to study temporal events in mitochondrial clearance. Mitochondrial depolarization precedes elimination, but in Atg7(-/-) reticulocytes the depolarization event is markedly delayed. Since Atg7 regulates autophagosome formation, we infer that mitochondrial depolarization occurs downstream of autophagosome formation in reticulocytes. We propose that there are two mechanisms of mitochondrial clearance: one that is triggered by mitochondrial depolarization, and a second NIX-dependent mechanism, which is not. The NIX-dependent mechanism remains to be elucidated.

Abstract Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed. Subsequently, we showed that a truncated form of the stem-cell kinase receptor (sf-STK) is required for susceptibility to Friend disease. Given the requirement for sf-STK, we sought to establish the in vivo significance of gp55-mediated activation of the EPOR. We found that the cytoplasmic tyrosine residues of the EPOR, and signal transducer and activator of transcription-5 (STAT5), which acts through these sites, are not required for Friend virus-induced erythroblastosis. The EPOR itself was required for the development of erythroblastosis but not for gp55-mediated erythroid proliferation. Interestingly, the murine EPOR, which is required for gp55-mediated Ba/F3-cell proliferation, was dispensable for erythroblastosis in vivo. Finally, gp55-mediated activation of the EPOR and STAT5 are required for Friend virus-induced polycythemia. These results suggest that Friend virus activates both sf-STK and the EPOR to cause deregulated erythroid proliferation and differentiation.

In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific gene p45(NFE2). Frequent disruption of p45(NFE2) due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV in p45(NFE2) mutant mice. Since p45(NFE2) homozygous mice mostly die at birth, erythroleukemia was induced in +/- and +/+ mice. We demonstrate that +/- mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/- mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/- and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/- mice. Establishment in culture was associated with the loss of the remaining wild-type p45(NFE2) allele in 9 of 10 of these cell lines. The loss of a functional p45(NFE2) in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-type p45(NFE2) in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression of p45(NFE2) in these cells also slows tumor growth in vivo. These results indicate that p45(NFE2) functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.

Abrogation of Ron receptor tyrosine kinase function results in defects in macrophage activation and dysregulated acute inflammatory responses in vivo. Several naturally occurring constitutively active alternative forms of Ron have been identified, including from primary human tumors and tumor cell lines. One of these alternative forms, short-form (SF) Ron, is generated from an alternative start site in intron 10 of the Ron gene that eliminates most of the extracellular portion of the receptor and is overexpressed in several human cancers. To test the physiological significance of SF-Ron in vivo, mice were generated that solely express the full-length form of Ron (FL-Ron). Our results show that elimination of the capacity to express SF-Ron in vivo leads to augmented production of IFN-γ from splenocytes following stimulation ex vivo with either concanavalin A or anti-CD3/T cell receptor monoclonal antibody. Moreover, in a concanavalin A-induced murine model of acute liver injury, FL-Ron mice have increased production of serum INF-γ and serum alanine aminotransferase levels and worsened liver histology and overall survival compared with wild-type control mice. Taken together, these results suggest for the first time that SF-Ron impacts the progression of inflammatory immune responses in vivo and further support a role for the Ron receptor and its various forms in liver pathophysiology.

Abstract High-level α-globin expression depends on cis-acting regulatory sequences located far upstream of the α-globin cluster. Sequences that contain the α-globin positive regulatory element (PRE) activate α-globin expression in transgenic mice. The α-globin PRE contains a pair of composite binding sites for the transcription factors activating protein 1 and nuclear factor erythroid 2 (AP1/NFE2). To determine the role of these binding sites in α-globin gene transcription, we mutated the AP1/NFE2 sites in the α-globin PRE in mice. We replaced the AP1/NFE2 sites with a neomycin resistance gene (neo) that is flanked by LoxP sites (floxed). Mice with this mutation exhibited increased embryonic death and α-thalassemia intermedia. Next, we removed the neo gene by Cre-mediated recombination, leaving a single LoxP site in place of the AP1/NFE2 sites. These mice were phenotypically normal. However, α-globin expression, measured by allele-specific RNA polymerase chain reaction (PCR), was decreased 25%. We examined the role of the hematopoietic-restricted transcription factor p45Nfe2 in activating expression through these sites and found that it is not required. Thus, we have demonstrated that AP1/NFE2 binding sites in the murine α-globin PRE contribute to long-range α-globin gene activation. The proteins that mediate this effect remain to be determined. (Blood. 2003;102:4223-4228)

Retinoblastoma-1 (RB1), and the RB1-related proteins p107 and p130, are key regulators of the cell cycle. Although RB1 is required for normal erythroid development in vitro, it is largely dispensable for erythropoiesis in vivo. The modest phenotype caused by RB1 deficiency in mice raises questions about redundancy within the RB1 family, and the role of RB1 in erythroid differentiation. Here we show that RB1 is the major pocket protein that regulates terminal erythroid differentiation. Erythroid cells lacking all pocket proteins exhibit the same cell cycle defects as those deficient for RB1 alone. RB1 has broad repressive effects on gene transcription in erythroid cells. As a group, RB1-repressed genes are generally well expressed but downregulated at the final stage of erythroid development. Repression correlates with E2F binding, implicating E2Fs in the recruitment of RB1 to repressed genes. Merging differential and time-dependent changes in expression, we define a group of approximately 800 RB1-repressed genes. Bioinformatics analysis shows that this list is enriched for terms related to the cell cycle, but also for terms related to terminal differentiation. Some of these have not been previously linked to RB1. These results expand the range of processes potentially regulated by RB1, and suggest that a principal role of RB1 in development is coordinating the events required for terminal differentiation.

In this issue of Blood, Mankelow et al link phosphatidylserine (PS) exposure in sickle erythrocytes to a physiological event in reticulocyte maturation. This discovery has implications for efforts to prevent thrombosis in sickle cell disease (SCD).

Annals of the New York Academy of SciencesVolume 612, Issue 1 p. 141-151 Localization and Characterization of the DNase I–Hypersensitive Site II (HS II) Enhancer A Critical Regulatory Element within the β-Globin Locus–Activating Region BRIAN P. SORRENTINO, Corresponding Author BRIAN P. SORRENTINO Clinical Hematology Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892Address correspondence to Brian Sorrentino, M.D., Clinical Hematology Branch, NHLBI, NIH, Building 10 7C-210, 9000 Rockville Pike, Bethesda, MD 20892.Search for more papers by this authorPAUL A. NEY, PAUL A. NEY Clinical Hematology Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorARTHUR W. NIENHUIS, ARTHUR W. NIENHUIS Clinical Hematology Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892Search for more papers by this author BRIAN P. SORRENTINO, Corresponding Author BRIAN P. SORRENTINO Clinical Hematology Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892Address correspondence to Brian Sorrentino, M.D., Clinical Hematology Branch, NHLBI, NIH, Building 10 7C-210, 9000 Rockville Pike, Bethesda, MD 20892.Search for more papers by this authorPAUL A. NEY, PAUL A. NEY Clinical Hematology Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorARTHUR W. NIENHUIS, ARTHUR W. NIENHUIS Clinical Hematology Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892Search for more papers by this author First published: December 1990 https://doi.org/10.1111/j.1749-6632.1990.tb24300.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Tuan, D. Y. H., W. Solomon, Q. Li & I. M. London. 1985. Proc. Natl. Acad. Sci. USA. 82: 6384–6388. 10.1073/pnas.82.19.6384 CASPubMedWeb of Science®Google Scholar 2 Forrester, W. C., S. Takegawa, T. 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Nucleic Acids Res.. 17: 37–54. 10.1093/nar/17.1.37 CASPubMedWeb of Science®Google Scholar Citing Literature Volume612, Issue1Sixth Cooley's Anemia SymposiumDecember 1990Pages 141-151 ReferencesRelatedInformation

Erythropoietin and the erythropoietin receptor have been cloned and studied in a lower vertebrate species. Key aspects of their structure and function have been evolutionarily conserved.

The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation.

NF-E2 binding sites, located in distant regulatory sequences, may be important for high level α- and β-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2–related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2–deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of α- and β-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2–deficient mice is not the result of compensation by Nrf-2.

Verwandte Abkürzungen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2. Grundlagen der Röntgenbeugungsanalyse 6 3. Aufnahmetechnik . . . . . . . . . . . . . . . . .

In this issue of Blood , Hegde et al show that a novel and naturally produced eicosapentaenoic acid–derived cyclopentenone prostaglandin has antileukemic activity.[1][1] Δ12-PGJ3–mediated apoptosis of leukemic stem cells is mediated at least in part by ATM-p53 signaling. ![Figure][2]

Review on BNIP3L (BCL2/adenovirus E1B 19kDa interacting protein 3-like), with data on DNA, on the protein encoded, and where the gene is implicated.

In this issue of Blood , Kumkhaek et al demonstrate a role for the Ras-like protein MASL1 in erythroid development. MASL1 acts at least in part by stimulating the Raf mitogen-activated protein kinase kinase (MEK) extracellular signal-regulated kinase (ERK) pathway.[1][1] Ras proteins are molecular

In this issue of Blood , Kusakabe et al make a compelling case that the transcription factor c-Maf is critical for erythroblastic island formation and fetal erythropoiesis. 1 The cell adhesion molecule VCAM-1 is a potential mediator of c-Maf activity.

The hematopoietic system is a dynamic multilineage organ, from which we can gain insights into the physiologic roles of cell death pathways. The role of hematopoiesis is to produce blood cells under normal and stress conditions throughout the lifespan of an organism. In humans, effective function of the hematopoietic system entails the steady and regulated production of erythrocytes, which carry oxygen; platelets, which prevent bleeding; and granulocytes, monocytes, and lymphocytes, which are required for host defense. Remarkably, these varied cell types are derived from a common ancestor, the hematopoietic stem cell (HSC). HSCs are known for certain key properties: they are long-lived and self-renewing and give rise to a steady supply of multipotential progenitor cells (MPPs) that are committed to undergo differentiation. MPPs in turn give rise to progenitors with a progressively restricted developmental potential; these undergo regulated expansion and, eventually, commitment to terminal differentiation. The terminal differentiation program is characterized by decreased proliferation or mitotic arrest, global changes in gene expression, and cellular remodeling. Finally, cells that have fulfilled their purpose, or senescent cells, are cleared from the body to make room for new cells, or after a stress response to re-establish homeostasis.

The human gamma-globin gene promoter contains a stage selector element (SSE) responsible for preferential interaction of the promoter with a powerful erythroid-specific enhancer in the fetal developmental stage (S.M. Jane, P.A. Ney, E.F. Vanin, D.L. Gumucio, and A.W. Nienhuis. EMBO J. 11:2691-2699, 1992). The element binds two proteins, the ubiquitous activator Sp1 and a protein previously known as -50 gamma and now named the stage selector protein (SSP). Binding of the second protein correlates with SSE activity in transient-transfection assays. We now report that a de novo binding site for the SSP is created by the -202(C-->G) mutation that causes hereditary persistence of fetal hemoglobin (HPFH). This site functions in an analogous manner to the SSE in hybrid beta-promoter/reporter gene constructs transfected into K562 cells. In contrast, the wild-type -202 sequence, which fails to bind the SSP, is incapable of activating the beta-gene promoter. Both the -50 and -202 HPFH sites for SSP binding overlap a consensus sequence for the transcriptional regulator Sp1. In addition, both sites contain CpG dinucleotides that are contact bases for SSP. Since the gamma promoter is known to be hypomethylated in fetal cells but fully methylated at CpG residues in adult erythroid cells, we examined the effects of this DNA modification on protein binding to the two regions. Gel mobility shift assays with nuclear extract from K562 cells (which contain both Sp1 and SSP) demonstrate preferential binding of SSP to the SSE and HPFH sites under conditions in which probe was limiting. Methylation of the CpG residues reverses this preference only in the SSE site, with a marked increase in the binding of Sp1 at the expense of the SSP. Purified Sp1 binds with 10-fold higher affinity to the methylated than to the nonmethylated -50 probe but with the same affinity to the -202 HPFH probe. The methylation-induced preferential binding of Sp1 to the SSE at the expense of SSP may be part of the mechanism by which the gamma genes are repressed in normal adult erythroid cells. In cells containing the -202 HPFH mutation, the inability of Sp1 to displace SSP in the methylated state may explain the persistence of gamma-promoter activity and gamma-gene expression observed in adults with this mutation.

Die Bildsamkeit (Plastizität) der durch Löschen von Branntkalken in überschüssigem Wasser oder durch Einsumpfen von Trockenhydraten erhältlichen Kalkteige ist eine ihrer wichtigen bautechnologischen Eigenschaften, ermöglicht sie doch erst die vorteilhaften Verarbeitungseigenschaften und die Geschmeidigkeit der Kalkmörtel. In einer Zeit der Rationalisierung und Normierung der Baustoffe kann man sich weder mit der empirischen Feststellung zufriedengeben, daß die Bildsamkeit von Kalkteigen je nach ihrer Herkunft bzw. Herstellungsweise verschieden sein kann, noch sollte man es versäumen, alle im Baustoff Kalk liegenden Möglichkeiten auszuschöpfen.

Abstract Recent studies have suggested that the serine-threonine kinase Ulk1, a mammalian homologue of the yeast autophagy regulator, Atg1, plays an important role in mitochondrial clearance during reticulocyte maturation. In order to gain insight into the regulation and activity of Ulk1, we used an unbiased proteomics approach to identify Ulk1-interacting proteins. Here, we demonstrate that Ulk1 interacts with heat shock protein 90 (Hsp90) and Cdc37, and is among a growing list of proteins whose steady state levels are regulated by this kinase-specific chaperone complex. Treatment of murine embryonic fibroblasts (MEFs) with the HSP90 inhibitor, 17AAG, inhibits the AKT/Tor pathway and induces autophagy despite the decreased steady state levels of Ulk1. By contrast, Ulk1-deficient MEFs are defective in autophagy induction, as measured by LC3 puncta formation (but not LC3 conversion) and ultrastructural analysis. Exposure of terminally differentiating erythroid cells to HSP90 inhibitors prevents the normal accumulation of Ulk1 protein and results in impaired autophagic clearance of organelles, similar to the effect of Ulk1 gene deletion. These findings highlight the importance of Ulk1 protein levels in terminal erythroid maturation and induction of autophagy and its regulation by the Hsp90/Cdc37 chaperone complex.

Abstract Mature erythrocytes are under tight homeostatic control with the need for constant replacement from progenitors to replace damaged or obsolete red blood cells (RBCs). This process is regulated largely by erythropoietin (Epo) which promotes the survival of erythroid progenitors and facilitates their differentiation and proliferation. Ablation of Bcl2l1 (which encodes BCL-xL) results in embryonic lethality with a lack of mature erythrocytes but does not perturb erythroid progenitors. Similarly, conditional Bcl2l1 -deletion results in severe anemia with the death of late erythroid progenitors and induction of extramedullary erythropoiesis. While BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other anti-apoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 -deletion results in embryonic lethality due to severe anemia caused by a lack of mature RBCs. Mcl1 -deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that the dependence on MCL-1 is only during early erythropoiesis, whereas during later stages the cells become MCL-1-independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development since co-deletion of the pro-apoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these two anti-apoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

Die im Rahmen der vorliegenden Forschungsaufgabe durchgeführten Versuche haben gezeigt, daß unter den angewandten Versuchsbedingungen in erster Linie bestimmte Eigenschaften des Rohgesteins darüber entscheiden, ob ein Kalkhydrat hoher oder niedriger Emley-Plastizität erhalten werden kann. Bei einem Rohgestein mit günstigen Eigenschaften ist es durch Wahl geeigneter Brennbedingungen in den meisten Fällen möglich, die Plastizität naßgelöschter Hydrate zu verbessern. Ohne eine relativ kostspielige Nachbehandlung, im wesentlichen Vermahlung, der trockengelöschten Kalkhydrate ist es nicht möglich, deren an sich stets nur mäßige Plastizität zu verbessern. Die Zusammensetzung der flüssigen Phase beim Löschvorgang ist nur in wenigen Fällen von nachhaltiger Wirkung auf die Plastizität; die Beeinflussung der Emley-Plastizität durch Zusatzstoffe ist jedoch wirtschaftlich kaum tragbar.

Homogene Gemische von etwa gleichen Gewichtsteilen Kalkhydrat [im wesentlichen Ca(OH)2] und Wasser besitzen mit Hilfe des Emley-Plastizimeters gemessene empirische Plastizitätswerte von 100 bis ca. 750. Handelsübliche deutsche Weißkalkhydrate besitzen Plastizitätswerte, die in Ausnahmefällen bis 200 reichen. Nach der in den USA geltenden Ansicht bedeuten erst Plastizitätswerte über 200 »gute« Plastizität. Es wurde versucht, die Plastizität von aus deutschen Rohgesteinen gewonnenen Kalkhydraten durch Abänderung der Menge und Zusammensetzung der flüssigen Phase beim Löschvorgang zu beeinflussen bzw. zu verbessern. Dabei mußten nicht nur die Löschbedingungen in Betracht gezogen werden, sondern auch die Eigenschaften der Branntkalke, die Nachbehandlung der Hydrate und die Art und Menge von Zusatzstoffen.

ZusammenfassungEin großer Teil der Versuche wurde mit einem Branntkalk durchgeführt, der aus einem besonders reinen Kalkstein [1, S. 12] im Laboratorium hergestellt wurde. Je nach den Brennbedingungen und der weiteren Behandlung (Mahlen, Sieben) besaß dieser Branntkalk unterschiedliche, aber stets nur geringe Gehalte an CO2 und H2O. Der Kalkstein war von Natur aus frei von Schwefel; auch beim Brennen im elektrischen Widerstandsofen oder im mit Leuchtgas beheizten Ofen hatte der Kalk keine Gelegenheit, Schwefel aufzunehmen.

Im täglichen Leben und in der Technik begegnet man zahlreichen, meist weichen Stoffen, welche sich durch verhältnismäßig geringen Kraftaufwand verformen lassen und welche ihre bei der Verformung erworbene äußere Gestalt beibehalten. Solche plastischen Stoffe sind z. B. Kuchenteig, Mörtel, Ton und Lehm, Dichtungsmassen und stichfeste Kalkbreie. Jeder, der Gelegenheit hatte, mit mehreren plastischen Stoffen umzugehen, macht die Erfahrung, daß die Plastizität bei den einzelnen Stoffen nicht nur in ihrem Ausmaß, sondern auch in ihrer »Art« irgendwie verschieden ist. Diese Verschiedenheit drückt sich auch in den unterschiedlichen Begriffsbestimmungen und Prüfverfahren für die Plastizität aus, die in den Gewerbezweigen und Industrien, welche sich mit plastischen Stoffen beschäftigen, aufgestellt oder vorgeschlagen werden.

20 Kalksteinsorten, deren chemische Zusammensetzung auf Tab. 1 und deren lithologischen Eigenschaften an anderer Stelle [4] ausführlich beschrieben sind, wurden bei 1000, 1150 und 1300°C im Gasbrennofen gebrannt. Aus dem Branntkalk wurden durch Brechen und Mahlen eine Kornfraktion 2-5 mm (im folgenden als »Stückkalk« bezeichnet) und eine Kornfraktion — 0,09 mm (als »Feinkalk« bezeichnet) gewonnen. Je 200 g dieser Kornfraktionen wurden zu Teig gelöscht und nach der Messung der Ergiebigkeit, also im Alter von 7 bis 14 Tagen, auf ihre Emley-Plastizität geprüft. Dabei wurden die in Tab. 2 vermerkten Werte gemessen.

Es erübrigt sich, an dieser Stelle auf die theoretischen Grundlagen der Beugung von Röntgenstrahlen an Kristallen einzugehen, weil dieses Thema Gegenstand zahlreicher Veröffentlichungen ist. Besonders sei hier auf folgende deutschsprachige Arbeiten hingewiesen (die Ziffern beziehen sich auf das Literaturverzeichnis):

Die Tatsache, daß mit Wasser angemachte breiförmige Mischungen von ge löschtem Kalk und Zuschlagstoffen wie etwa Sand nach dem Austrocknen all mählich fest werden und dann dauernd fest bleiben, war sc

ZusammenfassungDie Angaben über die verwendeten Auswertungsmethoden können kurz gefaßt werden, da weder für die qualitative noch für die quantitative Bestimmung der Phasenzusammensetzung neue Methoden entwickelt wurden.

ZusammenfassungDie Röntgenbeugungsanalyse zur Ermittlung der Phasenzusammensetzung von Feststoffgemischen wird seit längerer Zeit auch schon in einigen Industrielaboratorien durchgeführt. Einem verbreiteteren Einsatz in der Industrie stehen einige Schwierigkeiten entgegen: a) Die Anschaffungskosten der Geräte sind hoch, und auch ihre Bedienung muß qualifizierten Kräften überlassen bleiben. b) Die Leistungsfähigkeit des Analysenverfahrens, seine Möglichkeiten und Grenzen für die speziellen Probleme des Industriezweiges sind ohne eingehende Erprobung nicht leicht zu erkennen und werden ebenso oft überschätzt wie unterschätzt. Meist ist es nicht ohne weiteres möglich, aus den Erfahrungen verwandter Industrien zutreffende Schlüsse für die eigene Industrie zu ziehen.

In diesem Abschnitt werden die wichtigsten Stoffe besprochen, die für die Kalkindustrie als Rohstoff, Erzeugnis, Hilfsstoff oder Weiterverarbeitungserzeugnis von Bedeutung sind. Die meisten dieser Stoffe bestehen zwar zum überwiegenden Teil aus einer einzigen Kristallart, aber ihre spezifischen Eigenschaften und ihre Verwendbarkeit werden sehr oft gerade durch die in kleinerer Menge enthaltenen Fremdstoffe wesentlich beeinflußt. Abgesehen von dem Aufwand, den eine quantitative chemische Analyse verursacht, ist auch deren Aussage oft nicht eindeutig, so daß zur sicheren Ermittlung der Phasenzusammensetzung die Röntgenanalyse ohnehin herangezogen werden muß. Um die rasche Erkennung von häufiger vorkommenden Fremdstoffen und bei Produkten gänzlich unbekannter Zusammensetzung auch der Hauptbestandteile zu erleichtern, wird im folgenden bei jedem Stoff ein Diagramm des reinen Hauptgemengteils gezeigt und außerdem Beispiele mit den möglichen und üblichen Gehalten an Fremdstoffen, wie sie in natürlichen und technischen Produkten auftreten.

ZusammenfassungJeweils 480 g eines Weißkalkhydrates und 4920 g Albertysand wurden zur Herstellung eines Mörtels verwendet (Bokumischer; 5 min bei Stufe III). Die folgende Tab. 3 gibt die Versuchs- und Meßdaten.

ZusammenfassungAls Mörtel bezeichnet man breiartige Gemische aus Bindemittel, mehr oder weniger feinkörnigem Zuschlagstoff und Wasser (Frischmörtel), welche nach teilweiser oder vollständiger Abgabe des Anmachwassers erhärten (Festmörtel) und dabei die Fähigkeit besitzen, in relativ dünner Schicht Bausteine oder andere saugfähige Bauelemente dauerhaft miteinander zu verbinden (Mauermörtel) oder festhaftend zu umhüllen (Putzmörtel). Bindemittel für Mörtel sind die verschiedenen Baukalke [1], entweder für sich allein oder zusammen mit Portlandzement [2] bzw. für Innenputze auch mit Baugips [3]. Zuschlagstoffe sind überwiegend natürliche Gruben- oder Floßsande, seltener Brechsande oder feinkörnige Hochofenschlacke. Ein wesentlicher Bestandteil sowohl der Frisch- wie auch der Festmörtel sind feine Luftporen.

Es erscheint uns zweckmäßig, auf die Aufnahmetechnik etwas näher einzugehen, obwohl einerseits jedem Röntgengerät eine ausführliche Gebrauchsanweisung beigegeben wird und andererseits im Schrifttum viele einschlägige Vorschriften zu finden sind. Die von uns geübte Aufnahmetechnik hat sich aus Vergleichsversuchen mit anderen Laboratorien entwickelt.

Mit dieser Arbeit wurde der Versuch unternommen, die Bedeutung der röntgenographischen Phasenuntersuchung für die einzelnen Abschnitte der technischen Kalkherstellung und -verarbeitung nachzuweisen. Aus dem vorhandenen Material wurden mit der Auflage größtmöglicher Beschränkung die vorliegenden Beispiele ausgewählt. Bei der Textfassung und Gliederung wurde berücksichtigt, daß eine Anleitung für die praktische Durchführung von qualitativen und quantitativen Röntgenbeugungsuntersuchungen von Kalkprodukten als weiteres Ziel angestrebt war.

Als die wichtigste Eigenschaft eines Frischmörtels wird seine Verarbeitbarkeit betrachtet. Um die vielfältigen Einflüsse der Mörtelbestandteile und Mörtelherstellung auf die Verarbeitbarkeit zu untersuchen, war es notwendig, zuerst ein Prüfverfahren für die Verformbarkeit von Mörteln zu entwickeln. Die mit Hilfe verschiedener modifizierter Wuerpel-Geräte meßbare Verformungsarbeit eines Mörtels wird als Bewertungsmaßstab für seine Verarbeitbarkeit betrachtet. Die Versuche zeigen vorläufig nur qualitativ, wie sich die Mörtelbestandteile — Bindemittel, Sand, Wasser, Luftporen, Plastifizierungsmittel — nach Art und Menge auf die Verformungsarbeit auswirken können und in welchem Maße ihre Wirkung vom Mischvorgang abhängt. Um einen sowohl prüftechnisch leicht erreichbaren wie auch mit der Baupraxis in Einklang befindlichen Ausgangspunkt zu schaffen, wurden sämtliche Mörtel beim Ausbreitmaß 18 cm verglichen. Die Erprobung des hier vorgeschlagenen Prüfverfahrens an möglichst vielen Stellen wird bei Berücksichtigung der gleichfalls ausführlich diskutierten Einflußgrößen oder Fehlerquellen im Laufe der Zeit die Zahlenwerte bringen, welche den Einfluß der Mörtelbestandteile und Mörtelherstellung auch quantitativ überblicken lassen und die heute noch fehlenden Abgrenzungen verschiedener Mörtelqualitäten erlauben. Das Ergebnis derartiger quantitativer Versuche auf breiter Basis wird aber nur dann den Herstellern von Fertigmörteln, Bindemitteln, Mörtelsanden, Plastifizierungsmitteln und den Bauträgern zugute kommen, wenn diese sich in gemeinsamen Versuchen entschließen könnten, stichhaltige Belege für die Überlegenheit der besser verformbaren Mörtel beizubringen. Erst wenn unter sonst gleichen Bedingungen an geeigneten Bauobjekten einwandfrei bewiesen wird, daß Mörtel mit niedriger Verformungsarbeit, also besserer Verarbeitbarkeit, wesentliche Einsparungen an Muskelkraft bringen und damit größere Leistungen von Putzern oder Maurern oder, daß sie die Verwendung besonders kostensparender Maschinen oder Techniken erst ermöglichen, wird ein Anreiz für die Verwendung qualitativ besserer Mörtel gegeben sein. Erst dann ist eine systematische Verbesserung der Bindemittel und eine engmaschige Kontrolle der Frischmörtelqualität überhaupt sinnvoll.

Abstract This study examined the roles of the Stat5a and Stat5b gene products in growth and differentiation of mouse erythroid cells. BALB/c mice bearing inactivating mutations in both Stat5a and Stat5b were infected with the anemia-inducing strain of Friend virus. The infected mice developed erythroblastosis in the spleen typical of the early phase of Friend disease. Early erythroblasts purified from the spleens of infected mice (Stat5a/b mutants and BALB/c wild type controls) were cultured in medium containing 2 U/mL of erythropoietin (EPO). During the culture period of 60 h, a significant fraction of the Stat5a/b(−/−) erythroblasts differentiated into reticulocytes. However, the population exhibited several properties that were different from controls. Control cell numbers increased 6-fold during the 60 h culture period and about 90% of the resultant population formed reticulocytes. The Stat5a/b(−/−) cells increased in number only 3.5-fold. About 30% of the cells never initiated hemoglobin synthesis, and about one-half of the remainder did not mature into reticulocytes. After 36 h of culture, the Stat5a/b(−/−) erythroblast population contained a higher fraction than normal of cells which were positive for DNA degradation by a flow cytometric TUNEL assay although morphological apoptosis was not pronounced. This pleotropic phenotype does not resemble the type or timing of apoptosis induced by EPO deprivation. The combined result of the above factors was inefficient erythropoiesis by Stat5a/b(−/−) cells in vitro . Several molecular regulators of apoptosis and of cell cycle were examined. Normal levels of Bcl-x production were observed and no activation of caspase 3 or cleavage of PARP was detected. p27 and p21 were significantly elevated in Stat5a/b(−/−) cells. The fractions of cells in G1 and S phases of the cell cycle were different in Stat5a/b (−/−) and control cells throughout the culture period.

Activating transcription factor-4 (ATF4) is a member of the ATF/CREB subfamily of basic region-leucine zipper proteins. ATF4 forms homodimers or heterodimers by interacting with itself or other proteins through its leucine zipper motif. Potential partners of ATF4 include members of the AP-1 and C/

No abstract available.